key: cord-0027393-6fum7fcy authors: nan title: SRI 2022: Scientific Abstracts date: 2022-02-18 journal: Reprod Sci DOI: 10.1007/s43032-022-00883-5 sha: 1d10cfe1ca4602880694b27dfbec948bc67512d9 doc_id: 27393 cord_uid: 6fum7fcy nan The Early Career Investigator Award The Early Career Investigator Travel Award, with support from the National Institutes of Health, was established to enhance the career development of early investigators by facilitating interaction with other scientists in the field, development of research collaborations, and exposure to the latest technologies and scientific developments in reproductive biology and medicine. Early career investigators are defined as individuals who have completed their terminal research degree or end of post-graduate clinical training, whichever date is later, within the past 10 years and who has not previously competed successfully as PD/PI for a substantial NIH independent research award. Up to four $1,000 travel awards and certificates will be given beginning in 2019 to awardees who will be selected by competitive review of abstracts submitted to each annual SRI meeting by the Program Committee, with special regard paid to representation of women and minorities. The Laxmi Baxi Award Established in 2013 and will be awarded to PhD individuals only, who are either graduate students still in training or postdoctoral fellows within five years of their PhD degree. Two $1,000 travel awards will be given to the top abstracts in basic reproductive science and in translational reproductive science. The awardees will receive a plaque, award and they will be honored at the SRI Annual Meeting Awards Ceremony. This award is made possible by a generous, long-standing member of SRI, Dr. Laxmi Baxi, and was created to encourage young PhD trainees to present their research at our meeting. Acknowledges the highest ranked abstract by an in training investigator within the field of fetal neuroscience. This award honors the legacy of Dr. McDonald, whose immense contributions to the field of obstetrics and gynecology focused upon the neuroendocrinology of the developing fetus, placental function, fetal brain development, and the uterine contractility. The $750 monetary award and certificate will be presented at the Award Ceremony during the SRI Annual Scientific Meeting. The Giorgio Pardi Foundation Awards Provides a $1,000 monetary award to the three best young worthy investigators coming from Italian Universities. The award winners will be presented at the Award Ceremony during the SRI Annual Scientific Meeting. Meeting, SRI offers Awards to allow discounted membership and Annual Meeting registration for 25 individuals who are underrepresented in their country or originate from a low-income, lower-middle-income, or upper-middle-income country. The awardees will be recognized at the Annual Meeting. Tiffany Habelrih †, 1, 2 Sarah-Eve Loiselle †, 1, 2 David-Étienne Tremblay †, 1, 2 Erica Di Battista †, 2 France Côté †, 1, 2 Xin Hou * , 2 Christiane Quiniou * , 2 Sylvain Chemtob * . 1 Introduction: Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality. Studies have shown that interleukin-1 (IL-1) plays a major role in the pathophysiology of PTB as it participates in inducing the production of pro-inflammatory mediators and uterine activating proteins (UAPs) leading to labour. More importantly, uteroplacental inflammation, associated with PTB's parturition pathways, is detrimental to fragile fetal tissues and leads to long term sequalae. Our group has developed an allosteric antagonist of IL-1's receptor, Rytvela, found to be potent and safe in preventing PTB by suppressing inflammation via the inhibition of the MAP-Kinase pathway while preserving the NF-kB pathway (important in immune vigilance). Rytvela has been shown to inhibit inflammatory upregulation and uterine activation as well as preserving fetal development. The study aimed to further pre-clinical development of Rytvela by evaluating its optimal dose and minimal duration of treatment to inhibit the inflammatory cascade, prolong gestation and promote neonatal outcome. Methods: Pregnant CD-1 mice were injected with LPS (10 ug i.p.) or IL-1b (1 ug/kg i.u.) on gestational day (G) 16 to induce preterm labour. Rytvela was injected at different doses (0.1, 0.5, 1, 2, 4 mg/kg/day s.c.) from G16 to G19. To evaluate the minimal duration of treatment, mice were injected with Rytvela (2 mg/kg/day s.c.) over the course of 24, 36 or 48 hours. Rate of prematurity (35 year old women, found no significant difference for the changes in AMH levels before and after vaccination (Delta AMH) for any of the three groups (p=0.29). Evaluation of the immunity response achieved by the vaccinations in the three subgroups were also comparable (p=0.23). After controlling for age, no association was found between the degree of immunity response (as expressed by anti-covid antibody levels) and AMH levels (partial correlation 0.005 and 0.035 for first and second AMH blood test respectively). Sri V Dangudubiyyam †, Jay S Mishra, Ruolin Song †, Sathish Kumar * . University of Wisconsin-Madison, Madison, WI, United States. Introduction: Pregnancy is associated with adaptive hemodynamic and vascular changes such as enhanced vasodilation, decreased blood pressure, and increased uterine artery blood flow. In contrast, preeclampsia is associated with hypertension and abnormal vascular function. The cause for this abnormal vascular function in preeclampsia remains unclear. Eight out of nine epidemiological studies show that plasma perfluorooctane sulfonic acid (PFOS), a persistent environmental pollutant, is elevated in pregnant women with hypertensive pregnancy disorders Hypothesis: PFOS exposure during pregnancy impairs gestational vascular adaptations via impaired endothelium-dependent vasodilation and heightened angiotensin II-mediated vascular contraction. Methods: Pregnant Sprague-Dawley rats were administered with PFOS (50 μg/mL; n=7) through drinking water from gestational day (GD) 4 until term (GD20). Controls (n=7) received standard deionized waters with no detectable PFOS. Blood pressures were assessed using a non-invasive CODA tail-cuff system, and cardiac function and uterine artery blood flow were determined using Doppler ultrasound. Uterine artery vascular reactivity was assessed with a wire myograph. Total eNOS and AT1R and AT2R mRNA expression and protein levels were also examined in uterine arteries. Placental and fetal weights were also measured. Results: Mean arterial pressures were significantly higher at GD 18 to GD 20 in PFOS-exposed dams compared to controls. Uterine artery blood flow was reduced in PFOS dams. Echocardiographic measurements showed a significant increase in ejection fraction and fractional shortening and increased left ventricular anterior and posterior wall thickness at endsystole in PFOS dams. Endothelium-dependent relaxation responses to acetylcholine were significantly lower, while endothelium-independent contraction to angiotensin II was increased in PFOS dams compared to controls. AT1aR mRNA expression was increased while AT1bR mRNA and AT1R protein expression did not change. AT2R and eNOS mRNA and protein expression were decreased in PFOS dams. Placental weights (control: 0.54 ± 0.01 g; PFOS-exposed: 0.49 ± 0.01 g) and fetal weights (control: 4.09 ± 0.14 g; PFOS-exposed: 3.56 ± 0.09 g) were significantly decreased in PFOS dams compared to controls. Conclusion: Elevated maternal PFOS caused hypertension, cardiac hypertrophy and decreased in uterine blood flow through blunting of endothelium-dependent vasodilation and exacerbation of angiotensin II induced contractions, providing a molecular mechanism linking maternal PFOS exposure and preeclampsia. Glessner-Fischer, Osama Harraz, Nga Ling Ko * . University of Vermont, Burlington, VT, United States. Introduction: Women with preeclampsia and intrauterine growth restriction frequently demonstrate impaired uteroplacental blood flow (UPBF) and inadequate uterine vascular remodeling during pregnancy. We, and others, have found that shear stress secondary to hemochorial placentation is the principal physiological stimulus for both nitric oxide and arterial remodeling. Recently, we have identified Piezo1 channel, a novel mechanosensor for shear stress and linked to eNOS activation, in the uterine circulation. Piezo1 in smooth muscle cells has also been reported to be involved in hypertension-dependent arterial remodeling. Given that maternal cardiovascular prepregnancy physiologic predisposition and superimposed pregnancy are critical features for the development of preeclampsia, we hypothesized that prepregnancy knockout of Piezo1 in the endothelial cells attenuates uterine arterial remodeling. Methods: Nonpregnant conditional endothelial-specific (EC) Piezo1 knockout mice were used for this study. Piezo1 tm2.1Apat /J mice were initially crossed with tamoxifen-inducible cadherin 5 (Cdh5)-Cre-ER(T2) mice and further bred to yield Cdh5Cre Piezo1 -/mice (Piezo1 iEC-KO , n=7). Littermates that were null for the Cre gene (Piezo1 fl/fl , n=5) were used as controls. Piezo1 deletion in adult endothelia was induced by tamoxifen administration to 12-week-old mice (i.p. for 7 days), followed by euthanasia at ~4 weeks after the treatment. Main uterine arteries (MUAs) were isolated, pressurized and evaluated in physiological conditions by pressure myography. The MUA lumen diameter, wall thickness and distensibility as a function of pressure were recorded. MUAs were also collected and processed for Verhoeff Van Gieson and picrosirius red staining to visualize and quantitate elastin and collagen in MUAs, respectively. Results: EC-specific knockout of Piezo1 significantly decreased MUA distensibility (p=0.005) but not lumen diameter nor wall thickness. There was no difference in elastin content in the MUAs from Piezo1 iEC-KO vs. Piezo1 fl/fl mice. Based on measurements of color from polarized light images for collagen analysis, there was a significant increase in the yellow (thick and densely packed, 1.9 ± 0.3 vs. 0.9 ± 0.2%, p=0.047) and green (thin and loosely packed, 1.5 ± 0.3 vs. 0.5 ± 0.1%, p=0.03) collagen fibrils in the MUAs from the Piezo1 iEC-KO mice. Conclusion: Loss of EC-Piezo1 channel to sense shear stress in the uterine arteries results in increases in collagen fibrils (and likely collagen reorganization) and leads to reduced arterial distensibility in nonpregnant mice. We speculate that such prepregnancy changes may contribute to impaired uterine arterial remodeling and UPBF. Further studies on the effect of EC-Piezo1 knockout in the gestational uterine vascular remodeling is warranted. Global Ablation of Adrenomedullin2 Results in Abnormal Glucose Metabolism in Mice Pregnancy. Chandrasekhar Yallampalli * , Akansha Mishra, Ancizar Betancourt, Madhu Chauhan * . Baylor College of Medicine, Houston, TX, United States. Introduction: Adrenomedullin2 (ADM2) appears to play a role in regulating energy metabolism in mice and facilitates placental development in human pregnancy. Therefore, this study was designed to assess the effect of global ablation of ADM2 on the mice pregnancy. Methods: Animal study was approved by animal care and use committee. ADM2 knockout (KO) mouse was generated using CRISPR/Cas9 genome editing in C57BL/6NJ mice by the Mouse Embryonic Stem Cell Core at Baylor College of Medicine. For fertility test, the mating pairs (ADM2-KO females and their wild type littermates (ADM2-WT) crossed with wild type C57BL/6NJ (n=8; one female/ male) were left in cage for a period of 6 months. The gestational age at 1 st delivery, and number of litters / mouse and total dead pups over a period of six months were recorded. Assessment of placental weights and glucose metabolism was performed in ADM2-KO and ADM2-WT females (n=8) that were crossed with ADM2 KO males and ADM2-WT males, respectively (12 weeks old). Placental weights and total insulin content in isolated pancreatic islets were assessed on gestational day (GD) 17.5. Glucose metabolism was assessed by intraperitoneal glucose tolerance test on GD13.5. Data sets are presented as mean ± SEM. Statistical analysis was performed using an unpaired Student t test and significant difference between the groups was defined as P < 0.05. Results: There are no differences in the length of gestational age and number of litters in ADM2-KO mice compared to ADM2-WT, 2) Pup mortality is higher in ADM2-KO compared to ADM2-WT over a period of six consecutive pregnancies (P<0.05), 3) Placental weights tend to be higher in ADM2 KO pregnancy compared to ADM2-WT (p=0.074), 4) ADM2-KO mice develop impaired glucose tolerance during pregnancy with greater area under the curve compared to ADM2-WT (P<0.05); 5) Pregnant ADM2-KO mice appear to have lower total insulin content in pancreatic islets compared to ADM2-WT. Conclusion: Ablation of ADM2 results in complicated pregnancy with impaired glucose metabolism associated with tendency of decreased insulin levels in the pancreatic islets. New Chronic Hypertension 6-12 Months Following a Pregnancy Complicated by a Hypertensive Disorder of Pregnancy. Christina M Ackerman-Banks †, 1 Jessica Pudwell, 2 Lisbet Lundsberg, 1 Graeme N Smith, 2 Heather S Lipkind * . 1 1 Yale University, New Haven, CT, United States; 2 Queen's University, Kingston, ON, Canada. Introduction: Cardiovascular disease (CVD) is the leading cause of death among women. Those who experienced a hypertensive disorder of pregnancy (HDP) have significantly increased risk of CVD, stroke, and mortality later in life. The purpose of this collaborative data sharing study between Yale University, United States (Yale Heart Moms study) and Queen's University, Canada (Maternal Health Clinic) is to better characterize the risk of development of chronic hypertension (cHTN) among women with a pregnancy complicated by a HDP by 6-12 months postpartum using diverse patient populations with a larger sample size. Methods: The combined dataset had 523 women with a HDP (n=288 Preeclampsia, n=235 Gestational HTN) and 104 with an uncomplicated, term pregnancy (non-HDP). Those with a multiple pregnancy or cHTN, CVD, and diabetes (DM) identified prior to pregnancy were excluded. All underwent a complete assessment 6-12 months after delivery. 30year CVD risk estimates and a metabolic syndrome calculation were determined for each patient. Multivariable logistic regression was used to assess the association between HDP and new cHTN, adjusting for relevant medical and sociodemographic variables. Analysis was completed with IBM SPSS Statistics v27. Results: Of the 627 women, 343 (54.7%) had a new diagnosis of cHTN, as defined by the American Heart Association (AHA). Of those with a new cHTN, 26 (7.6%) had no HDP and 317 (92.4%) had a HDP). Those in the non-HDP group were more likely to be multiparous and to breastfeed whereas those in the HDP group were more likely to have a family history of a first degree relative with hypertension, myocardial infarction, or stroke. Participants in the HDP group had significantly increased 30-year CVD risk score and were more likely to have and abnormal lipid profile and metabolic syndrome than their non-HDP counterparts (p<0.05). Participants in the HDP group had a 5.6-fold adjusted odds of a new diagnosis of cHTN 6-12 months after delivery, compared to those in the non-HDP group (aOR 5.61 (95% CI 3.09-10.18)), when adjusting for BMI, parity, age, clinic site, education, and income. Conclusion: Women with HDP and no underlying cHTN, CVD, or DM have a more than five-fold increased odds of new chronic hypertension and are more likely to meet criteria of metabolic syndrome in the first year postpartum. Thus, it is critical to formally implement a standardized initiative for CVD screening in the fourth trimester as primary prevention for maternal cardiovascular morbidity and mortality. Stronger Blood-Brain Barrier Leakage Years After Preeclampsia: A Dynamic Contrast-Enhanced MRI Study at 7 Tesla. Lisanne P.W. Canjels †, 1,2 Chahinda Ghossein-Doha, 1,2 Robert-Jan Alers, 1, 2 3 Eindhoven University of Technology, Eindhoven, Netherlands. Introduction: Preeclampsia, a hypertensive complication of pregnancy, relates to an increased risk of long-term cardiovascular and cerebrovascular disorders. Although the persisting susceptibility to cerebral complications after preeclampsia remains largely unclear, impaired blood-brain barrier (BBB) integrity is often proposed to precede many of these cerebrovascular diseases. Therefore, we investigated the integrity of the BBB years after preeclampsia. Methods: Cerebral MRI at 7 Tesla (Siemens Healthineers, Erlangen, Germany) was performed in 22 formerly preeclamptic women (aged 38 ± 6 years, postpartum time 7 ± 4 years) and 13 parous control women with normotensive pregnancies (aged 41 ± 5 years, postpartum time 9 ± 4 years). The BBB integrity was investigated by assessing the leakage of a gadolinium-based contrast agent (e.g. gadobutrol), measured by dynamic contrast-enhanced MRI. BBB permeability was determined by estimating the leakage rate (K i ) and fractional leakage volume (v L ) of the whole brain and lobular white and gray matter using pharmacokinetic modelling with the Patlak and histogram approach. Multivariable analyses were performed to compare women with and without preeclampsia, adjusted for postpartum time and Fazekas score (e.g. extend of white matter lesions, which are of presumed vascular origin and are related to hypertension, cerebrovascular risk factors, and BBB leakage). Results: K i and v L were significantly higher in formerly preeclamptic women compared to control women in the global white (+625%, p < 0.001) and gray matter (+246%, p < 0.03). Regionally, the temporal (+154%, p < 0.05) and parietal cortical gray matter (+116%, p = 0.02), and the frontal (+571%, p = 0.01), temporal (+515%, p = 0.02) and occipital white matter (+540%, p = 0.001) showed higher leakage measures in formerly preeclamptic women. Conclusion: This observational study shows a globally impaired BBB years after a preeclamptic pregnancy. The subtle, but evident, BBB leakage years after preeclampsia, compared to parous control women, indicates a diffusively spread endothelial dysfunction. This stronger BBB leakage could be one of the pathophysiologic mechanisms in cerebral injury after preeclampsia. Improving the condition of the BBB might, therefore, be a good target for treatment and prevention strategies that are aimed at reducing the psychological complaints and cerebrovascular risks after preeclampsia. Sulfhydration of β1 Subunit Mediates H 2 S-Stimulated Activation of Human Uterine Artery Smooth Muscle BK Ca Channels. Jin Bai †, Fenglong Jiao, Alejandra G Salmeron, Naoto Hoshi, Lan Huang, Dongbao Chen * . University of California Irvine, Irvine, CA, United States. Introduction: Large conductance calcium-activated and voltagedependent potassium (BK Ca ) channels are tetramers of the constitutively expressed α-subunits, whose activity is finely tuned by its tissue/cellspecifically expressed auxiliary β1-4 and γ1-4 subunits. Opening of the β1 containing BK Ca (BKβ1) channels hyperpolarizes smooth muscle (SM) to mediate uterine vasodilation in pregnancy. Hydrogen sulfide (H 2 S) potently stimulates pregnancy-dependent uterine artery (UA) relaxation via activating SM BK Ca channels, but how H 2 S activates the channel is unknown. H 2 S directly converts free thiols (-SH) to persulfides (-SSH) on reactive cysteines thus changing the entire proteome to elicit its biological function. This study was conducted to test a hypothesis that H 2 S stimulates BKβ1 sulfhydration resulting in BK Ca channel activation in human uterine artery smooth cells (hUASMC). Methods: Main UAs were collected from hysterectomies of pregnant and nonpregnant women. SSH-proteins were labeled by Tag-switch; levels of total SSH-proteins and SSH-BKβ1 were quantified by immunoblotting. A low pH quantitative thiol reactivity profiling (LP-QTRP) proteomics platform was developed to analyze SSH-proteome with specific SSHsites (reactive cysteines). Gene ontology and biological pathways of pregnancy-dependent UA and H 2 S-inducible SSH-proteomes were further analyzed by bioinformatics. SSH-deficient BKβ1 mutants were generated by site-directed mutagenesis and used for determining the effects of SSH on BK Ca activation by patch clamp. Results: Levels of total SSH-proteins and SSH-BKβ1 were significantly greater in pregnant vs. nonpregnant UA. Treatment with a H 2 S donor NaSH (300 µM, 30 min) activates BK Ca channel in primary hUASMC in culture, which was significantly inhibited by BKβ1 siRNAs. NaSH increased total SSH-proteins by 1.78-fold and SSH-BKβ1 by 2.17-fold in hUASMC. The SSH-proteomes of UA and hUASMC contain a plethora of proteins with pregnancy-dependent and H 2 S-responsive changes in the degree of SSH in proteins that participate into various biological processes including muscle contraction involving cation channels such as BKβ1. When the H 2 S-inducible SSH-cysteines in BKβ1 were replaced by serine, the C/S mutated SSH-deficient BKβ1 was unable to be sulfhydrated by H 2 S. When these mutants were transfected in hUASMC, H 2 S-induced BK Ca activity was significantly inhibited. The pregnancy-dependent human UA protein sulfhydration ex vivo and H 2 S-induced global hUASMC protein sulfhydration in vitro demonstrate that sulfhydration is an important mechanism for H 2 S to elicit its uterine vasodilatory effects; sulfhydration of BKβ1 results in SM BK Ca activation which may serve as a novel mechanism to mediate the relaxation effect of H 2 S in UA (Supported by NIH grants HD105699, HL70562, and HD097498). Commensal F. nucleatum Bacterium Regulates the Expression of Pro-Angiogenic Factors In Vitro via PAR-1 Pathway. Lea Zemmrich, Damián O. Muzzio, Nicole Normann, Marek Zygmunt * . University Medicine Greifswald, Greifswald, Germany. Introduction: It has been shown that bacteria present in the upper reproductive tract (UTR) may influence early pregnancy events as trophoblast invasiveness and angiogenesis in vitro. F. nucleatum was traditionally associated with poor pregnancy outcomes. However, low abundance of the bacterium has been described in the fertile endometrium. While low concentrations of F. nucleatum improve trophoblast invasiveness, high concentrations are detrimental for trophoblast survival and function. Furthermore, low bacterial loads may improve angiogenic functions of macrophages. We hypothesize that different responses to low and high bacterial loads are associated with the activation of different signalling pathways involved in the release of the pro-angiogenic factors VEGF and IL-8. Methods: THP-1 were differentiated into M2c macrophages in the presence of PMA (10 ng/mL), TGF-β (20 ng/mL) and subsequently stimulated with inactivated F. nucleatum in a multiplicity of infection of 0.1 bacterium per cell (MOI 0.1) and 1 bacterium per cell (MOI 1.0). SCH-79797 (2 µM), Y-27632 (20 µM), DMOG (100 µM) and BMS-345541 (10 µM) were used to inhibit PAR-1, ROCK, TET-2 and NF-κB respectively. The expression of IL-8 and VEGF was assessed by ELISA (n = 7 and n = 5, respectively) while IL8 and VEGF mRNA levels were quantified by qPCR (n = 3). Data were analyzed by one-way ANOVA followed by Dunnett's or Dunn's post-tests. Differences with P adj < 0.05 were considered statistically significant. Results: F. nucleatum induced a significant increase of IL-8 secretion and IL8 expression that was effectively prevented by inhibition of NF-κB at MOI 0.1. VEGF expression was slightly increased after treatment with bacteria and was significantly down-regulated in the presence of all inhibitors at MOI 0.1, while NF-κB was able to significantly reduce VEGF secretion at MOI 1.0. Conclusion: F. nucleatum activates pro-angiogenic pathways at low concentrations in a ROCK and PAR-1 mediated manner. In contrast, higher concentrations might activate further signalling pathways leading to pro-inflammatory responses. These data highlight a unique behavior for low abundance bacteria in the URT supporting early pregnancy events. Pregnancy-Specific Paracrine Factors Change Uterine Vascular Reactivity to Allow Increasing Blood Flow to the Placenta. Hanna H Allerkamp †, Alys R Clark * , Teagan Pole †, Laura Schnauer †, Joanna L James * . University of Auckland, Auckland, New Zealand. Introduction: Failed adaptation of the uterine vasculature is a key pathological contributor to fetal growth restriction (FGR). While inadequate spiral artery remodelling has been a major focus, recent work proposes the upstream uterine radial arteries (RAs) are also key players to enable increased volumetric blood flow to the placenta, and inadequate RA remodelling could contribute to FGR. However, substantial knowledge gaps remain regarding driving mechanisms of RA adaptation. This study combines an ex vivo rat model of RA reactivity with in silico modelling to investigate RA adaptation to pregnancy. Methods: Wall thickness of Sprague-Dawley rat RAs was quantified in tissue sections using custom Matlab scripts. Vessel metrics were retrieved from µCT-scans of vascular casts of rat uteri through custom Python scripts and used to parameterise an in silico model to predict RA flow rates. Pressure myography was used to investigate RA responses from virgin and pregnant (E19.5) rats to increasing flow. Virgin RAs were treated with progesterone, β-estradiol, PlGF and/or VEGF and compared to controls. L-NAME was used to assess nitric oxide synthase (eNOS) involvement. Expression of estrogen receptor α, progesterone receptor, and VEGF receptor-1, and -2 in RAs was investigated by immunohistochemistry. Results: Pregnancy increased RA radii (52.5µm pregnant vs. 34.1µm virgin; p=0.0002, n=6), whilst wall thickness was unchanged (65.9μm pregnant vs. 38.63μm virgin; p=0.07, n=12), implying outward hypertrophic remodelling. Virgin and pregnant RAs showed flowmediated constriction after reaching the in vivo fl ow rates predicted by the in silico model (48µl/min pregnant vs. 11µl/min virgin). All receptors were expressed in RA endothelial and/or smooth muscle cells, with higher VEGFR-1 expression in pregnant RAs (p=0.03, n=10). Progesterone, β-estradiol and PlGF (but not VEGF), and all factors combined, delayed fl ow-mediated constriction in virgin RAs (p=0.04, n=5-8/group). This enabled them to withstand higher fl ow rates before constricting, mimicking behaviour of pregnant RAs. eNOS inhibition by L-NAME partly reduced RA reaction to fl ow induced by single paracrine factors, but did not aff ect RA reactivity when all factors were combined (p=0.95, n=5-10/group), showing eNOS is involved in RA adaptation, but additional mechanisms likely also play a role. Conclusion: Placenta derived paracrine factors progesterone, β-estradiol and PlGF allow RAs to maintain larger diameters under higher fl ow rates, ensuring constant blood fl ow to the placenta. Maladaptation of RAs may lead to smaller vessels that are overly sensitive to fl ow, thereby constricting at lower fl ow rates and limiting utero-placental fl ow. The interdisciplinary modelling approach employed improves translation of in vitro data to human pregnancy, to help better clinically detect and treat FGR. Marisol Castillo-Castrejon †, 1,2 Lana Madi, 1 Kathryn Erickson, 1 Thomas Jansson, 1 Veronique Ferchaud-Roucher, 3 Theresa L Powell * . 1 3 University of Nantes, Nantes, France. Introduction: Infants born to women with metabolic disease are at risk for fetal overgrowth due to excess fat accretion and have an increased risk of metabolic disease in adult life. However, little is known about the interaction eff ect of pre-gestational obesity on placental function in pregnancies complicated by gestational diabetes (GDM). We tested the hypothesis that obese women with GDM have increased expression of placental nutrient transporters and increased fatty acid uptake. Methods: Term placentas were collected from women with normal BMI, (N, n=11), obese (OB, n=10), normal BMI with GDM (N-GDM, n=9) and obese with GDM (OB-GDM, n=10). Insulin, leptin and adiponectin were measured in maternal serum. Syncytiotrophoblast microvillous (MVM) and basal (BM) plasma membranes were isolated and expression of fatty acid, amino acid transporters and mTOR signaling were determined by Western Blot. Primary human trophoblast cells were isolated, cultured and incubated for 2h (FA uptake) or 24h (cellular FA incorporation) in a mixture of stable isotope 13 C labeled fatty acids: palmitic, oleic, linoleic and docosahexaenoic acids. Cellular lipids were extracted and quantifi ed by GC-MS. Statistical signifi cance tested by One-and Threeway ANOVA. Results: Maternal insulin concentration was increased in GDM groups. Overall, leptin (r=0.57, p=0.003), adiponectin (r= -0.42, p=0.01) and placental weight (r=0.28, p=0.03) correlated with BMI. In OB and OB-GDM, expression of 4E-BP1(p=0.03), a component of the mTOR signaling pathway, and fatty acid transporter FAT/CD36 (p=0.001), FATP6 (p=0.01), FATP2 (p=0.05) expression were increased with a signifi cant interaction between GDM+BMI and correlated with birth weight FAT/ CD36 (r=0.79, p=0.002), FATP2 (r=0.69, p=0.01), SNAT4 (r=0.60, p=0.03), LAT2 (r=0.68, p=0.01). Linoleic acid uptake was increased in OB (p=0.03) and palmitic acid cellular incorporation was increased in OB-GDM (p=0.005) compared to normal. No changes were found in N-GDM group. Conclusion: Our results are consistent with an increased placental capacity to transfer amino acids and fatty acids to the fetus in pregnancies complicated by obesity and GDM, but not in GDM alone. These changes may contribute to increased birth weight and neonatal adiposity known to be associated with both obesity and GDM. Exposure of Gut Microbes to Maternal High Fat Diet Impacts Their Eff ect on Enteroids. Erin Bolte †, 1 Mindy Engevik, 2 Jim Versalovic, 3 Kjersti Aagaard * . 1 In a non-human primate model, we have previously shown that off spring exposed to maternal high fat diet (mHFD) display persistent juvenile-onset anxiety and an altered gut microbial community, independent of post-weaning diet. To investigate the relationship between maternal diet and gut microbes on the neurometabolites relevant to anxiety, we aimed to determine if mHFD-exposed microbes are suffi cient to induce altered neurotransmitter activity at the level of the gut. We hypothesized that gut microbes from mHFD-exposed off spring will alter levels of intestinal serotonin in vitro via microbe-host interactions compared to maternal control diet. Methods: Japanese macaques were fed mHFD or control diet during gestation/lactation then weaned onto a control diet. Stool was collected at 3 years (after 2.5 years of control diet feeding). Wild-type mouse jejunal enteroids (n=3) and adult human jejunal enteroids (n=2) were incubated with the macaque stool microbes (see Figure 1 ). The enteroids were analyzed for serotonin secretion and expression of serotonin-relevant proteins. Results: In mouse jejunal enteroids, stool treatment from mHFD animals decreased secreted serotonin levels (52ng/mL vs 250ng/mL, p=0.009), serotonin rate limiting enzyme expression (TPH1, 0.02 vs 0.66 fold change, p=0.03), and serotonin-producing cell marker expression (chromogranin A, 0.05 vs 0.65 fold change, p=0.03). However in human jejunal enteroids, stool treatment from mHFD animals increased TPH1 expression (1.24 vs 0.27 fold change, p=0.003). Our results indicate that stool effl uents from mHFD-exposed off spring induce changes in serotonin via host cells in the gut, even after 2.5 years of control diet feeding. These results were host species dependent. We speculate that interactions between microbes and the host are crucial to primate enteroneuroactivity and programmed development of the gut-brain axis. Outcomes. Amir Lueth † * , Bob Silver, Allshouse Amanda, Maureen Murtaugh, Fernando Wilson. University of Utah, Salt Lake City, UT, United States. Introduction: Women with prior adverse perinatal outcomes (APOs) have a subsequent increased cardiovascular risk, but mechanisms are uncertain. Thus, we assessed the relationship between Allostatic load (AL) and cardiovascular disease (CVD) in a large, diverse longitudinal cohort 2 to 7 years later. Methods: This was a secondary analysis of NuMom2b-HH, a large prospective cohort study. Participants were recruited in their fi rst trimester of pregnancy and followed through delivery, with an additional visit 2 to 7 years postpartum. AL biomarkers were assessed in serum between 6w0d to 13w6d weeks gestation. Our primary exposure was dichotomous high AL defi ned as 4 or more out of 12 biomarkers in the worst quartile: systolic blood pressure (SBP), diastolic blood pressure (DBP), insulin, cholesterol, low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides, body mass index (BMI), high sensitivity C-reactive protein (hsCRP), creatinine, albumin and glucose. The worst quartile was the lowest for HDL, creatinine, and albumin, and the highest for the rest. The primary outcome was incident diagnosis of composite (cCVD) ascertained 2 to 7 years postpartum: new hypertension (HTN), and metabolic disorder (MS) (fasting glucose greater than 126 mg/ dL diagnosis of or medication for diabetes); each component was analyzed as a secondary outcome. A sensitivity analysis was performed to account for prior APOs. Multivariable logistic regression was used to test the association between high AL and cCVD adjusted for potential confounders. Results: Among 4,104 women, cCVD was identifi ed in 1,483 (36 Introduction: Singleton gestations conceived with in-vitro fertilization (IVF) have an increased risk for obstetric complications including low birth weight and preeclampsia (PRE) . Data regarding the complications associated with twins conceived with IVF are limited. The aim of this study was to compare obstetric complications in dichorionic diamniotic (didi) twins conceived with IVF with twins conceived spontaneously in a large geographically diverse population. Methods: This is a planned secondary analysis of data from a retrospective cohort study aimed to evaluate cell-free DNA screening in twins seen at 17 centers between 12/2011 -2/2020. Twins conceived with infertility treatment other than IVF, monochorionic and vanishing twins, cases with structural and chromosomal abnormalities and incomplete pregnancy outcome data were excluded. Univariate analyses were performed using Pearson Chi-square, Fisher's exact and Wilcoxon rank sum tests. Multivariate logistic regression models were used to account for confounding. Results: Of the 1,021 cases included, 549 (54%) were conceived with IVF and 472 (46%) were conceived spontaneously. Older maternal age, lower parity and BMI, white race and a lower incidence of chronic hypertension and tobacco use were associated with IVF conception. Patients who conceived with IVF had an increased incidence of PRE and gestational diabetes (GDM) ( Table 1) . After adjusting for age, BMI and parity the association with PRE was no longer significant (aOR 1.41 95% CI 0.94-2.12). After adjusting for age, race, and BMI patients who conceived with IVF were more likely to be diagnosed with GDM (aOR 1.79 95% CI 1. 11-2.87 ). There were no differences in gestational age at delivery, mean twin birth weight, birth weight <10 th or <5 th percentile in 1 or both twins. Conclusion: In this large cohort of didi twin pregnancies, conception with IVF was associated with an increased risk for developing GDM. Earlier screening may prove to be of value. Unlike the reported risks in singleton IVF conceptions, we did not observe an association with PRE or lower birth weight in IVF twins after adjusting for potential confounders. Epidemiologic associations between PTB and maternal clinical history, demographic characteristics, and social determinants have been extensively described, but have been limited in inference owing to a lack of attendant biological data. Methods: This work employed multiomic profi ling and multivariate modelling to investigate the biological signatures of some of these maternal covariates and gain insight into the epidemiological signature of PTB. Maternal covariates and plasma samples were collected during early pregnancy from a multinational cohort of 13,841 pregnant women (PTB rate of 11.4%) across four low-and middle-income countries. Plasma samples from 231 participants were further analyzed to generate proteomics, metabolomics, and lipidomics datasets. Results: An epidemiological model for the prediction of PTB achieved moderate accuracy (AUROC=0.70, p-value < 10 -146 ), highlighting the diffi culty of the predictive task. Machine learning-based multiomics models had strong performance for the prediction of multiple relevant objectives, including time to delivery (Pearson's R=0.64, p-value < 10 -26 ) and maternal covariates such as age (R=0.57, p-value < 10 -20 ), gravidity (R=0.53, p-value < 10 -17 ), and body mass index (BMI, R=0.80, p-value < 10 -51 ). The biological signature for time to delivery included fetal-associated proteins such as alpha-fetoprotein and immune proteins like PD-L1. Maternal age had a strong negative correlation with type IX collagen conserved across our multinational cohort. Gravidity had an ageindependent association with levels of endothelial NOS and infl ammatory chemokine CXCL13, an observation further validated on an independent Stanford cohort (n=17). BMI positively correlated with adipocyte hormone leptin and fatty acid protein FABP4. Conclusion: These results simultaneously provide the fi rst integrated view of the epidemiological signature of PTB and identify novel biological signatures of various maternal covariates impacting this disease. Furthermore, this work puts forward a conceptual framework for studying the biological mechanisms underlying some of the long-standing observed population-level risk factors of PTB. Finally, this approach sheds light on potential biological and socioeconomic interventions that generalize across multiple populations. Platform. Haruo Usuda * , 1 Shimpei Watanabe * , 2 Masatoshi Saito * , 2 Matthew Kemp * . 3 1 The University of Western Australia, Crawley WA, Australia; 2 Tohoku University Hospital, Sendai, Japan; 3 The University of Western Australia, Singapore, Singapore. Introduction: Artifi cial placenta therapy (APT) is an experimental intensive care strategy for extremely preterm infants born at 21-24 weeks' gestation. In our previous studies, blood taken from the maternal ewe was used as a priming solution for the artifi cial placenta circuit. Although effi cacious in sheep, the use of adult transfusion products as a priming solution in a human clinical setting conveys several challenges including diffi culty in confi rming blood type cross matching prior to delivery, a risk of one or more critical neonatal complications including necrotizing enterocolitis and viral infection with agents such as cytomegalovirus. We explored the use of synthetic erythrocytes (hemoglobin vesicles; HbV) as the basis of a priming solution for APT used to manage extremely early preterm ovine fetuses. Methods: Six ewes with singleton pregnancies at 95d gestation (term=150d) were adapted to APT and maintained for 72h with constant monitoring of key vital parameters. A synthetic red cell solution consisting of HbV, sheep albumin and electrolytes was used as a priming solution for the APT circuit. Fetuses were evaluated on gross appearance, physiological parameters and bleeding after euthanasia. Data were tested for mean diff erences with ANOVA. Results: Two out of six APT fetuses were successfully maintained for 72 h with controllable anemia (> 10 g/dL) and methemoglobinemia (< 10 %) using adult transfusion products and nitroglycerin no sooner than 1hr after APT commencement, suffi cient time to crossmatch blood products and screen for viral agents of concern. A comparative assessment using cord blood gas data before (0h) and after the induction of APT (1h) showed that HCO 3 -, Ca 2 + , tHb and fHb values were signifi cantly decreased (P<0.05), while lactate, MetHb and ACT levels were significantly increased (P<0.05). There were no diff erences in pH, pCO 2 , pO 2 , CtO 2 , BE, Na + , K + , Cl -, Glu, SO 2, COHb, O 2 Hb and HHb values (P>0.05). Conclusion: Extremely preterm sheep fetuses were maintained for a period of up to 72h using APT in combination with circuit priming using a synthetic red cell (HbV) preparation. Our study identifi ed several issues for the future development of this technology: i) the need for a smaller circuit priming volume; ii) the need for refi nement of HbV compounds; iii) better fetoplacental circulatory management or appropriate administration of autacoids for the management of refractory hypotension; and iv) the need for judicious combination of synthetic and adult transfusion products. These fi ndings demonstrated the potential clinical utility of synthetic blood products in the eventual clinical translation of artifi cial placenta technology to support extremely preterm infants. Estradiol Increases Antimicrobial Peptides to Prevent UTI Infections. Nicholas A Zawadzki †, Humberto Escobedo, Kathleen Connell, Joshua Johnson, Michael Schurr, Marsha K Guess * . University of Colorado Anschutz Medical Campus, Aurora, CO, United States. Introduction: Approximately 150 million people worldwide suff er from urinary tract infections (UTIs) annually. UTIs are more common in postmenopausal women, whose lower levels of 17-β-estradiol predispose them to more frequent infections. Estradiol is delivered vaginally as a clinical method of UTI prevention; however, our understanding of the mechanisms behind its contribution to urothelial cell protection from uropathogens is poor. The hypothesis tested: Estradiol stimulates the innate immune response to increase the production of antimicrobial peptides (AMPs) . An in vitro model was used where Escherichia coli, UTI189 (UTI89) were applied to human bladder cell line HTB-9. Methods: Several methods were used to determine estradiol's eff ects on bacterial eradication and urothelial cell survival. Fluorescence microscopy was used to visualize HTB-9 cells treated with 10 -7 M estradiol or vehicle (VEH), for 16 hours before infection with GFP-expressing UT189. After a 1-hour infection, the numbers of colony forming units per milliliter (CFU/mL) were determined. To test whether AMPs were secreted in response to estradiol, concentrated media prepared from 16-hour estradiol or VEH treated HTB-9 cells was evaluated for the ability to kill UTI89. Flow cytometry was used to quantify the number of live HTB-9 cells, pre-treated with estradiol or VEH, following a 1-hour UTI89 infection. qRT-PCR was used to determine the expression of AMPs Defensin Beta 1 (DEFB1) and S100A7 in cells treated with estradiol or VEH. Student's t-test was used to evaluate diff erences between the groups with statistical signifi cance defi ned as a p-value <0.05. Results: Fluorescence microscopy revealed that HTB-9 cells treated with estradiol 16 hours before infection had a 2.4-fold decrease in the number of infected cells relative to VEH (p = <0.0001). Concentrated media from estradiol-treated HTB-9 reduced UTI89 CFU/mL approximately 50%, 30 minutes post-application, compared to VEH (p = 0.022). A corresponding 10.4% increase in cell survival was observed on fl ow cystometry when HTB-9 cells were pre-treated with estradiol relative to VEH (p = 0.011). Finally, qRT-PCR detected a 3-fold and 1.25-fold increased expression of DEFB1 and S100A7, respectively in HTB-9 cells treated with E2 compared to VEH. Conclusion: Estradiol reduced bacterial burden in in vitro infected urothelial cells in a way that corresponded to increased AMP production. The protective eff ect of estradiol included both increased urothelial cell survival and reduced numbers of bacteria. The coincident increased expression of S100A7 and DEFB1 AMPs favors a mechanism where estradiol protects urothelial cells in part by increasing AMP production. As seen in other female reproductive tract tissues, estradiol is an attractive adjuvant for the treatment of UTIs in postmenopausal women due to a protective eff ect upon the bladder urothelium. Racial Disparities in Surgical Management of Endometriosis: Pre-Surgical ED Visits, Pre-Operative Indications, and Fertility-Sparing Surgery. Howard J Li †, Yue Song, Hugh S Taylor, Yonghee Cho * . Yale School of Medicine, New Haven, CT, United States. Introduction: To explore racial disparities in the surgical management of endometriosis among women receiving a primary diagnosis of endometriosis. Methods: Retrospective chart review on women ages 14-50 receiving a primary laparoscopic diagnosis of endometriosis at an academic tertiary hospital system, Jan. 2017 -Dec. 2020. Medical records identifi ed using appropriate CPT codes. Results: 135 patients met inclusion criteria, including 68 (50%) White or Caucasian, 17 (13%) Black or African-American, 37 (27%) Hispanic or Latina, and 10 (7%) Asian. Median diagnostic delay was 25.9 months for White patients, 15.2 (IQR 5.0-29.0) for Black patients, 38.7 (IQR 10.5-97.1) for Latina patients, and 48.1 (IQR 2.4-81.1) for Asian patients. Latina patients had a signifi cantly longer delay compared to White patients (p=0.01). Non-white patients were more likely to have visited the ED for pelvic pain prior to laparoscopy: 70.6% of Black, 54.1% of Latina, and 50% of Asian patients, compared to 27.9% of White patients (p=0.003). Patients who were publicly insured were also more likely to have visited the ED (OR 2.9, 95%CI 1.4-5.7). Non-white patients more often had an additional medical indication (other than pelvic pain) for laparoscopy at the time of endometriosis diagnosis (fi broids, abnormal imaging, infertility, sterilization, etc.): 70.6% of Black, 54.1% of Latina, and 70.0% of Asian patients, compared to 36.8% of White patients (p=0.03). Non-white patients were more likely to undergo hysterectomy at time of endometriosis diagnosis: 29.4% of Black, 16.2% of Latina, 40.0% of Asian patients, compared to 7.4% of White patients (p=0.01). Conclusion: These findings suggest that non-white patients may experience barriers to diagnostic laparoscopy, including a greater clinical threshold for surgery. However, diff erences in ED visits, additional indications for laparoscopy, and rates of hysterectomy may also refl ect diff erences in health-seeking behaviors and prevalence of gynecologic comorbidities between these populations. Factors Associated with HPV Vaccination Among LatinX Females: Insights from the ELLAS Study. Breonna Slocum †, 1 Erica Marsh * , 1 Torie Plowden, 2 Maricela Castillo MacKenzie, 1 Gina Aliste, 1 Anne Waldo. 1 1 University of Michigan, Ann Arbor, MI, United States; 2 Walter Reed Medical Center, Bethesda, MD, United States. Introduction: Human papilloma virus (HPV) is the most common sexually transmitted disease aff ecting approximately 13% of adults in the US, and is the primary known cause of cervical cancer. Despite the availability of an eff ective and safe HPV vaccine, racial disparities exist in vaccine uptake, HPV prevalence, and cervical cancer prevalence. Latinx (LX) women living in the United States are 20% less likely to receive an HPV vaccine compared with White women. Few studies have characterized the factors associated with vaccination status among Latinx females residing in the United States. Methods: The Environment, Leiomyomas, Latinas and Adiposity Study (ELLAS) is a longitudinal prospective cohort study of 21-50-year-old LX females. Participants completed interviews and questionnaires that collected demographic, medical and reproductive health data. Statistical analysis was performed using Chi-squared tests for categorical variables and t-tests or Wilcoxon rank sum tests for continuous variables. A multivariable logistic regression model was created using forward selection with variables that were signifi cant in univariate analysis. Results: HPV and vaccine data were available on 617 participants. The mean age of participants was 37.5 ± 7.0. 73 (11.8%) of participants had a history of an abnormal Pap smear and 34 (5.5%) reported a history of being HPV positive. 105 (17.0%) reported receiving the HPV vaccine and 83 (13.5%) reported that they didn't know if they had received the vaccine. Participants receiving the HPV vaccine were younger (mean age 34.0±7.9yrs vs 38.4±6.6yrs, p<0.001), more likely to attend college (41.0% vs 25.9%, p=0.002), be born in the US (21.9% vs 7.0%, p<0.001), have higher acculturation (29.5% vs 9.1%, p<0.001), have health insurance (55.2% vs 40.8%, p=0.004), and have adequate health literacy (73.3% vs 61.5%, p=0.015). In addition, participants receiving the vaccine were less likely to have had only 1 sexual partner (33.3% vs 51.7%, p<0.001) and be infertile (14.3% vs 28.0%, p=0.004). Participants did not diff er by vaccination status in terms of ever having an abnormal pap smear, ever having an STI, or annual household income. Multivariable model selection for having received the vaccine resulted in a fi nal model signifi cant for age (OR 0.97, 95% CI 0.94-0.99), number of sexual partners (2-4 partners vs 1: OR 1.98, 95% CI 1.12-3.49; ≥5 partners vs 1: OR 2.68, 95% CI 1.44-6.00) and infertility (OR 0.47, 95% CI 0.24-0.92). Conclusion: HPV vaccination status is lower in our study population of LatinX females compared with national vaccination rates. In multivariable analysis, HPV vaccination status was associated with age. Interventions to reduce this disparity will need to include targeted, accessible, literacy appropriate and culturally meaningful outreach and expanded insurance coverage. Biomarkers of Immediate Postpartum Pelvic Floor Injury Correlate to Delivery Type. Linda Burkett, 1,2 Pamela Moalli, 2 Timothy Canavan, 2 Stephanie Glass Clark, 2 Lauren Giugale, 2 Rachel Durst, 2 Amanda Artsen * . 2 1 VCU Health System, Richmond, VA, United States; 2 Magee-Womens of UPMC, Pittsburgh, PA, United States. Introduction: The pelvic fl oor undergoes extensive remodeling during pregnancy in preparation for labor however, postpartum injury occurs in up to 70% of deliveries. Elective induction of labor (eIOL) at 39 weeks gestation age has been associated with decreased maternal and fetal morbidity in the ARRIVE trial and OR 6.0 decreased risk of immediate postpartum injury. We hypothesized that women undergoing eIOL would have decreased biomarkers of pelvic injury, matrix metalloproteinases (MMPs), and elastase compared to women who delivered spontaneously. Methods: A prospective cohort study of primparous women with singleton, vertex, term vaginal deliveries were enrolled for a 4-8 week postpartum pelvic exam. Participants were divided into two groups: eIOL or spontaneous delivery. Swabs of vaginal epithelial cells and secretions were collected in the vaginal fornix prior to pelvic exam. Proteinase inhibitor was added to samples and then concentrated. MMP 2, 3, 8, 9 and 12 concentrations were determined using a bead-based multiplex assay and expressed as pg/ml protein. EnzChek elastase assay kit was used to determine amount of elastase activity in which 1 unit is the amount of enzyme necessary to solubilize 1 mg of elastin. Results: From July 2019 to Oct 2020, 102 participants were screened, 45 completed follow up. Six vaginal samples were excluded for contamination or inadequate protein leaving 20 in spontaneous group and 19 in eIOL group. Mean age was 29.7 years in spontaneous group vs 28.7 years in eIOL group (p=0.2) and there were no significant differences in clinical or sociodemographic characteristics. MMP2 and MMP12 were significantly higher in the spontaneous labor group (Table) . Pelvic organ prolapse on pelvic exam was not different between groups. MMP2 and MMP12 values were highly correlated within samples rho=0.518, p=0.001. Conclusion: MMP2 and 12, which have been previously associated with obstetric injury and pelvic organ prolapse, were significantly higher in spontaneous deliveries compared to eIOL. These findings correlate with previously demonstrated ultrasonic disruption of pelvic floor musculature. Further studies are need to better assess if this relationship is indicative of differential pregnancy adaptations, more severe injury or variable remodeling. Introduction: Endometriosis is a complex condition with a prevalence of ~10% in women of reproductive age and is a leading cause of pelvic pain and infertility. Studies have reported a shared genetic basis between endometriosis and metabolic traits such as obesity, waist to hip ratio and type 2 diabetes. We aimed for the first time to identify the genetic drivers of shared and distinct transcriptomic profiles in both endometrium and adipose tissue from women with and without endometriosis to advance insights into pathophysiology. Methods: We mapped cis-acting eQTLs and splicing (sQTLs) in eutopic endometrium (n=162) and adipose (n=122) using QTLtools, analysing all variants within 1MB of all genes and adjusting for population structure. We also performed a meta-analysis of QTLs using available adipose transcriptome data from the GTEx consortium (n=581) using OSCA software. To understand how loci from genome wide association studies (GWAS) may functionally affect transcription, we performed colocalization analysis using eCAVIAR at 42 GWAS risk loci with QTLs in the two tissues. Additionally, we performed differential expression and pathway enrichment analyses between endometriosis cases and controls in both tissues, stratified by the revised American Society for Reproductive Medicine (rASRM) stages(I/II and III/IV). Menstrual cycle phase was considered through adjustment and stratification (secretory vs. proliferative phase). Results: QTL mapping identified 816 and 683 eGenes (expression affected by a cis-eQTL) and 915 and 820 sGenes (transcript splicing affected by cis-sQTL) in endometrium and adipose tissue respectively (FDR<0.05). From meta-analysis with GTEx adipose data, we identified a total 10,490 eGenes and 5,912 sGenes. Colocalization analysis of the identified QTL maps and endometriosis GWAS revealed a total of 12 putatively causal genes at GWAS loci with eQTLs and sQTLs in both tissues. Differential expression analyses between endometriosis cases and controls identified enriched pathways for stage-based differential expression in both secretory and proliferative phase relating to TNF-alpha signalling, KRAS signalling, inflammatory response, P53 pathway, hypoxia, epithelial to mesenchymal transition and interferon gamma response. Conclusion: While previous studies have identified an epidemiological and genetic correlation between endometriosis and obesity related traits, an in-depth transcriptome analysis comparing the effect of genetic risk variants across tissues relevant to both clinical phenotypes has not been performed. Our data suggests that the integrated 'omics' analysis of disease relevant tissues for endometriosis and related traits will be of great benefit in uncovering key pathways previously unknown as relevant to the pathophysiology of endometriosis. Deepening the Endometrial Preparation Through Systems Pharmacology. Antonio Parraga-Leo †, 1,2 Patricia Sebastian-Leon, 2 Almudena Devesa-Peiro †, 2 Jose Remohi * , 1,3 Antonio Pellicer * , 1, 4 Patricia Diaz-Gimeno * . Introduction: In Assisted reproduction treatments (ARTs) to prepare the endometrium for embryo transfer is a widespread practice despite that the best protocol, dose, and timing for that remain still unclear. Our aim was to understand from a systems pharmacology perspective the molecular complexity of endometrial progression (EP) to propose alternative therapies and discover new potential targets for drug development. Methods: An endometrial gene expression matrix with 109 patients in natural cycle across menstrual cycle obtained from the integration of five endometrial datasets was used to select the genes involved in EP. For gene selection, differential expression analysis and time dose series according to menstrual cycle phases were applied (limma and maSigPro R packages respectively). Hormonal players involved in EP were also included. Selected genes were mapped in the human interactome creating an endometrial progression network (EPN) where two genes were connected based on experimentally proved physical protein-protein interactions. Network parameters were analysed to highlight the main genes in the EPN. Approved drugs from DrugBank with at least one target in our EPN were selected to build the drug-target discovery model. Drugs were selected according to their proximity to EPN and side effects. Results: EPN was composed of 937 proteins which represent menstrual cycle progression. These proteins were highly connected forming a significant large component (P < 0.01) in comparison with what was expected by chance. Eighty-four proteins were prioritized as key players in the molecular network, where 69 of them showed no approved drugs in DrugBank. While ESR1 and PGRMC2 were selected as prioritized proteins; PGR, ESR2 and PGRMC1 were not relevant in EP. We found 90 human approved drugs with at least two targets in our EPN. Proximity analysis revealed 19 drugs as the most ideal drugs to control menstrual cycle progression (FDR<0.05). Proteins targeted by them were ESR1, ESR2, AR and PGR in general, finding ESR1 as a common target for all drugs except copper which did not target any hormonal receptor directly. Conclusion: We created a molecular network applying the latest technologies in systems genomics and pharmacology to model the complexity of EP for the first time in the reproductive field. We proposed 69 new pharmacological targets, highlighting the role of the non-classical progesterone receptor, PGRMC2, in the menstrual cycle progression. Moreover, despite that most drugs targeted hormonal receptors, we found other possible non-hormonal alternatives such as copper. 1 Imperial College London, London, United Kingdom; 2 University West of England, Bristol, United Kingdom; 3 University College London, London, United Kingdom; 4 University of Edinburgh, Edinburgh, United Kingdom. Introduction: Substantial evidence implicates the pregnancy vaginal microbiome with preterm birth (PTB) risk. In research settings, molecular tools (e.g. metataxonomics, metagenomics) can provide cultureindependent characterisation of microbiota but are costly, laborious and fail to capture host response. Clinically, diagnosis of vaginal dysbiosis and infection is largely limited to evaluation of symptoms plus time-consuming culture and subjective microscopy investigation. Here, we describe the development and validation of direct on-swab metabolic profiling by Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) for rapid, sample preparation-free and simultaneous characterisation of the vaginal metabolome and host response. Methods: Vaginal swabs were collected from two independent pregnancy cohorts (VMET, n = 160; 455 swabs; VMET II, n = 205; 573 swabs). Metabolic profiles were directly acquired from swabs in less than 3 minutes and without sample preparation using direct on-swab DESI-MS. Linear mixed effect modelling was used to identify metabolic features associated with vaginal microbiota and inflammatory status . ROC curve analysis and Random Forest classifiers were used to assess the ability of DESI-MS profiles to predict microbiota composition. Results: DESI-MS identified 113 metabolites significantly differing between Lactobacillus dominated and depleted samples in independent analyses of both patient cohorts. These provided robust prediction of genera-level classification across cohorts (VMET/VMET2; AUC 94.1/90.6; sensitivity: 62.0/54.5; specificity: 97.8/96.4). Discrimination between the major vaginal community state types (I, III and IV) could also be achieved (VMET/VMET2; AUC 95.76 (94.3-97.7); sensitivity: 69.74(57.5-80.2); specificity 98.28 (97.7-98.9)). A total of 23 metabolites were correlated with key local immune mediators including IL-1β, IL-8, MBL, C5, C3b/C3bi, IgE, IgG2, IgG3, IgG4, and IgM. In this patient cohort, vaginal microbiota instability and innate immune activation, as predicted using DESI-MS, associated with PTB, including in women receiving cervical cerclage. Conclusion: Our findings highlight direct on-swab metabolic profiling by DESI-MS as an innovative approach for stratification of PTB risk phenotypes through rapid assessment of vaginal microbiota-host dynamics. Novel Demonstration That the Interleukin (IL)- We have demonstrated efficacy for rytvela, an allosteric peptide antagonist of the IL-1 receptor 1, in mouse and sheep in vivo studies in relation to blocking preterm birth or fetal inflammation stimulated by lipopolysaccharide, a Toll-like receptor 4 (TLR4) gram (-) bacterial mimic. TLR4 agonists stimulate increases in IL-1β. However, rytvela has not been tested for efficacy in term pregnant human tissues. Our objective was to test rytvela on antagonizing IL-1β in two human tissues critically important for activation of the uterus for PTB. We hypothesized that rytvela would block IL-1β-stimulated cytokine output and receptors in hFM explants and maternal peripheral leukocytes. Methods: We obtained hFM explants using a 6mm tissue punch from whole placentas and peripheral blood samples collected from term non-labouring women undergoing elective caesareans at the Royal Alexandra Hospital in Edmonton, AB. Explants were acclimated in DMEM F12 containing 15% FBS and 1x antibiotic/antimycotic for 48h before treatment. Leukocytes were isolated, washed, re-suspended in RPMI medium, counted, and diluted to a concentration of 10^6 cells/mL. Explants and leukocyte suspensions were then treated for 6h with 0, 0.1, 1, or 10 ng/mL IL-1β, with or without 1uM rytvela. RNA was extracted and RT-qPCR was used to measure chemokine receptors in leukocytes (n=6). The concentration of up to 40 different cytokines and chemokines was measured in culture medium of hFM explants (n=4). Statistical analysis: t-test, one-way or two-way ANOVA on log-transformed data with Tukey posthoc testing; significance achieved at p<0.05. Results: Twenty-eight cytokines were significantly upregulated by IL-1β stimulation of hFM (p<0.05), and 13 were significantly decreased with rytvela co-administration -often to control levels (p<0.05). IL-1β also increased and rytvela blocked mRNA abundance levels of leukocyte chemokine receptors known to be associated with PTB: CXCR1, CXCR2, CXCR5, and CX3CR1. Conclusion: As delivery approaches, the fetal membranes release chemoattractant that attracts circulating peripheral leukocytes to migrate into the uterus. Rytvela is efficacious at suppressing cytokine and chemokine release from fetal membranes, and reduces mRNA abundance of chemokine receptors involved in leukocyte migration. These data suggest that rytvela has considerable potential to prevent leukocyte migration and thereby block preterm birth and prolong pregnancy in women. Funding: WCHRI, CIHR. Introduction: Preterm birth (PTB) affects 15 million babies a year and is the leading cause of mortality among neonates. The most common cause of spontaneous PTB is inflammation. We have previously reported our discovery that the widely used drug excipient, N,N-dimethylacetamide (DMA), attenuates secretion of pro-inflammatory cytokines and chemokines in in-vitro, in-vivo and ex-vivo models of PTB by blocking activation of the nuclear factor kappa B (NF-κB) pathway. Subsequently, we showed that N,N-diethylacetamide (DEA), an analog of DMA with enhanced lipophilicity, inhibits the secretion of pro-inflammatory cytokines and chemokines in RAW 264.7 cells, HTR-8/SV-neo cells and human placental explants. Based on these initial data, we hypothesized that DEA would prevent inflammation-induced PTB in vivo by inhibiting NF-κB . Methods: Control mice (n=8) were injected with lipopolysaccharide (LPS) (Serotype 026:B6, 40 mg/kg) but not treated with DEA; DEA-rescued mice were injected with LPS and treated with either 750 mg/kg (n=6) or 375 mg/kg (n=6) i.p. DEA 15 min before and 10 h after LPS injection; and negative control mice were injected with either 750 mg/kg (n=3) or 375 mg/kg i.p. DEA (n=3), but not with LPS. For immunofluorescence studies, RAW 264.7 and THP-1 cells (after macrophage differentiation) were stimulated with LPS (1ug/ml) in the absence or presence of 10 mM DEA for 24 h and then stained with p65 antibody to localize NF-κB dimers. Results: All of the mice in the control group delivered within 24 h (8/8). Mice injected with LPS and treated with i.p. DEA were rescued from PTB in a dose dependent fashion. A dose of 750 mg/kg DEA rescued 83% of LPS-challenged mice (5/6, P<.01, ANOVA), while 375 mg/kg DEA rescued 33% of LPS challenged mice (2/6). As expected, drug control mice injected with 750 (n=3) or 375 mg/kg DEA (n=3), but not with LPS, did not deliver prematurely. No congenital anomalies were observed in any of the 18 DEA exposed litters. Immunofluorescence studies revealed that DEA, similarly to DMA, prevents the activation of the NF-κB pathway and translocation of p65 into the nucleus. Conclusion: Taken together, the data indicate that DEA prevents inflammation-induced PTB via inhibition of NF-κB activation in this murine model. DEA is a member of a family of closely related aprotic solvents recently discovered to inhibit NF-κB, which includes DMA, N,N-dipropylacetamide and N-methylpyrrolidone. Given the lack of a Food and Drug Administration approved drug for the prevention of PTB, the identification of novel, safe and efficacious compounds that arrest preterm labor are likely to have a significant impact on maternal and neonatal health. Integrated Analysis Reveals Associations Between Cervical Neutrophil Gene Expression and Cervicovaginal Microbiota in Women at High Risk of Preterm Birth. Alicia P Hadingham †, 1 Amirah Mohd Zaki, 1 Flavia Flaviani, 2 Deena Gibbons, 1 Yasmin Haque, 1 Jia Dai Mi, 1 Andrew Shennan, 1 Mansoor Saqi, 2 James Mason, 1 Rachel M Tribe * . 1 1 King's College London, London, United Kingdom; 2 Guy's and St Thomas' NHS Foundation Trust and King's College London, London, United Kingdom. Introduction: Risk of spontaneous preterm birth (sPTB) has been associated with vaginal microbiota composition, metabolites, and host defence peptides. Research into the contribution of cervical immune cells to sPTB is lacking. Based on research showing associations between the gut microbiota and neutrophil phenotype, we hypothesised that there could be associations between gene expression in cervical neutrophils and the cervicovaginal microbiota. In this exploratory study, we analysed cervical neutrophil transcriptomics and cervicovaginal metagenomics to investigate their relationships. Methods: Cervical cytobrush samples and cervicovaginal samples were obtained from pregnant women at high risk of sPTB from the INSIGHT cohort. Neutrophil cells were isolated by flow cytometry and RNA was sequenced using BGISEQ-500 (n=10). The cervicovaginal microbiome was sequenced by shotgun metagenomics using BGISEQ-500 (n=9). Transcriptomics bioinformatic analysis was performed using hisat2, samtools, featureCounts, sva and DEseq2. Metagenomic bioinformatic analysis was performed using Kneaddata and VIRGO. Genes were clustered into modules using weighted gene correlation network analysis (WGCNA). Associations were determined between module eigengenes (MEs), which represent each module, and selected microbiota using Pearson's correlations. Gene ontology (GO) analysis of modules was performed using anRichment. Results: Several statistically significant associations were found between MEs and the relative abundance of cervicovaginal microbiota taxa. GO analysis revealed one module, of 582 genes, was associated with neutrophil mediated immunity, neutrophil activation and neutrophil degranulation. The ME of this module was negatively correlated with Gardnerella vaginalis abundance (-0.72, p=1 .82 x 10 -2 ) and positively correlated with Lactobacillus iners abundance (0.65, p=4.25 x 10 -2 ). Another module, of 1034 genes, was associated with the type-I interferon signalling pathway and response to type-I interferon, and its ME was positively correlated with L. gasseri (0.98, p=1.58x 10 -6 ) and L. vaginalis (0.97, p=3.72 x 10 -6 ) abundance. Conclusion: This exploratory study found associations between cervical neutrophil gene expression and relative abundance of cervicovaginal microbiota. These results suggest that cervical immune populations need to be further investigated, to explore their relationship with the cervicovaginal microbiome and their possible role in sPTB. Introduction: Mitochondria regulate immune function and a variety of reproductive functions, including oocyte maturation, fertilization, and early embryonic development. Mitochondrial function is also relevant to diseases of pregnancy. Methylation-controlled J protein MCJ (also known as DNAJC15) is a mitochondrial inner membrane protein which is a known endogenous inhibitor of respiratory chain complex I. Loss of MCJ is associated with attenuation of inflammatory responses, and this is related to decreased release of inflammatory cytokines in response to Toll like receptor signaling. We sought to examine the potential role of MCJ deficiency on LPS-induced preterm birth in a mouse model. Methods: C57BL/6 (wild-type, WT) or MCJ deficient (KO, n~8 each) females were mated to same strain males and allowed to litter to observe pregnancy related parameters, including litter size, pregnancy weight gain, and weight of pups (~25 each) from birth through early life. In separate experiments, mice underwent timed mating and on day 14 of gestation, were weighed and given 30 μg LPS (E.coli 055:B5 Sigma-Aldrich) in 200 ul PBS. After 10 hrs., vaginas were observed for moisture (clear fluid, stuck bedding) and swabbed with pH paper. The presence of vaginal moisture that immediately changed pH paper deep blue was interpreted as delineating ruptured membranes (ROM). Mice were observed at regular timepoints for ROM, bleeding or pup delivery. Mice were euthanized at 48 hrs. in order to observe implantation sites with remaining pups and their condition (macerated, necrotic). Regression analysis was used to evaluate differences in weight or food intake over time. Time from injection of LPS to rupture membranes or loss of first pup was used for survival analysis (Mantel-Cox) . Other data was analyzed by t-test. Analyses utilized GraphPad Prism 9.0. Significance was set at p<0.05. Results: During unexposed pregnancy KO and WT mothers experience similar weight gain over time (nonlinear fit, p=0.9) food intake (linear fit slope p=0.6) and litter size (5-6 pups, p=0.3). Although KO pups are larger at birth (p=0.03), they grow at the same rate (linear fit slope p=0.4263) as WT. After LPS injection, KO mice tended to rupture membranes later (median 12 vs.11 hrs. p=0.09) and delivered the first pup later (median 22 versus 17 hrs., p=0.01). Although at 48 hrs., KO and WT mothers had a similar proportion of implantation sites delivered (p=0.8), the proportion of sites with macerated, necrotic pups was lower in KO mice (0.1 vs 0.6, p=0.027) Conclusion: While deficiency in MCJ may not be critical for overall pregnancy metabolism, it is a modulator of specific pathways relevant to preterm birth and intrauterine demise. Supported by NIH R21AI137539 and P30 GM118228. Membrane and Fetal Brain Inflammatory Gene Expression in a Non-Human Primate Model of Choriodecidual Ureaplasma Infection. Sudeshna Tripathy, Yvonne T Dimagiba, Miles J Novy, Victoria HJ Roberts, Meredith A Kelleher. Oregon National Primate Research Center, Beaverton, OR, United States. Introduction: Intrauterine infection is associated with chorioamnionitis, preterm birth, and the fetal inflammatory response syndrome (FIRS). Choriodecidual infection represents an intermediate stage of ascending reproductive infection, providing a model of infection-driven fetal inflammation, without direct microbial exposure of the fetus. The current study aims to examine the inflammatory responses in the placenta and fetal brain in response to choriodecidual Ureaplasma infection. Methods: Chronically catheterized pregnant rhesus monkeys with choriodecidual and intra-amniotic catheters were assigned to control (n=5) and choriodecidual infection (CDI; n=4) groups. Animals received inoculation with sterile media/saline or Ureaplasma parvum (Serovar 1, 10 5 CFU/mL) starting at 118±1 dGA every 5 days until C-section delivery at 136±2 dGA (Term=167d). Placenta, amnion, chorion and fetal brain tissues were collected at the time of delivery and immediately frozen. Expression of inflammatory, intracytoplasmic cytokeratin and metalloproteinase genes, along with genes associated with brain glial cells were determined by RT-qPCR. Immunohistochemical staining of corresponding proteins in maternal and fetal tissues was also performed. Statistical significance (p&It;0.05) was assessed by Student's t-test or Mann-Whitney U-test after testing for normality (Shapiro-Wilk) . Results: Expression of pro-inflammatory genes were upregulated with CDI, including the chemokine Cxcl2 in the amnion (p=0.0357) and chorion. Membranes also showed an increase of Il-1 isoforms which mediate the activation of enzymes involved in prostaglandins synthesis (e.g., Ptgs2) involved in myometrial activation and labor. Ptgs2 (p=0.0025) was upregulated in the chorion of CDI animals in tissue adjacent to the area of choriodecidual catheter placement but not in other regions of the chorion. Timp3, a tissue inhibitor of metalloproteinases, which regulates degradation of extracellular matrix, had significantly reduced expression in the amnion and catheter site chorion tissue. An increase in Cxcl2 and Ptgs2 expression in CDI brains shows a fetal neuroinflammatory environment without direct infection of the fetus. The expression of myelin gene Plp- 1(p=0.0028) was increased in the frontal cortex of CDI fetal brains, which may be linked to increased staining of activated microglia. Conclusion: Utilizing a translational NHP model, our findings indicate that choriodecidual Ureaplasma infection activates maternal and fetal inflammatory responses related to the onset of the preterm labor syndrome (pro-inflammatory signaling, membrane weakening) and fetal neuroinflammation, even at this early stage of ascending uterine infection prior to microbial invasion of the amniotic cavity and fetal infection. Exosomes Introduction: Using a microfluidic cervix-on-a-chip (COC), we determined if exosomes from ectocervical epithelial cells (Ecto) infected with Ureaplasma parvum (UP Ecto Exo) can cause cell death and inflammation at the feto-maternal interface and preterm birth (PTB) in a mouse model of pregnancy. Methods: A COC with 6 culture chambers interconnected by microchannels containing cells from vagina (VEC), cervical epithelium and stroma, transition zone, and decidua were used to mimic the female reproductive tract (Fig 1A&B) . Cells in COC were characterized by morphology and expression of cell-specific markers. Ecto cells in the COC Low (2.2E6 exosomes) were treated with and high (2.2E8 exosomes) doses of UP Ecto Exo for 48 h. Cytotoxicity (LDH assay) and inflammation (cytokine assays) in each cell type were determined. For physiological validation of in vitro data, high dose UP Ecto Exo was delivered in the vagina of pregnant CD-1 mice on E15. IVIS imaging was done to ensure exosomes propagation to the intrauterine cavity. PTB (log2-fold. KEGG pathway analysis showed genes in vascular development signaling pathways were significantly enriched among the differentially expressed mRNA. Gene ontology (GO) enrichment analysis showed cardiovascular system development, vasculature development, and blood vessel development were the top 3 significantly enriched pathways, consistent with our placental histopathology data. Conclusion: Chronic maternal THC use resulted in abnormal placental villous development in a NHP model. Histologic changes were consistent with gene expression enrichment in vascular developmental pathways, suggesting that chronic maternal THC use may affect placental vascular development. Given the increased prevalence of THC use in pregnancy, further mechanistic studies are needed to understand the implications of our findings. Single Cell Transcriptome Description of Early Development Haploid Androgenotes and Parthenotes. Pedro de Castro †, 1,2 Laura Escrich, 3 Noelia Grau, 3 Nuria Soler †, 3 Alicia Quiñonero, 1 Roberto González †, 1 María José Escribá * , 3,1,2 Francisco Dominguez * . Introduction: Uniparental parthenotes and androgenotes contains each a unique set of maternal or paternal chromosomes. They are a useful model to investigate each parental participation. Studies on early uniparental embryo development have been done using diploid or diploidized androgenotes and parthenotes. However, studies with haploid embryos are scarce. This research aimed to compare the transcriptome of haploid androgenotes and parthenotes throughout early stages of development using a single cell approach. Methods: For uniparental production, fresh MII oocytes were donated from 18-35 years old women after controlled ovarian stimulation and subsequent ovum pick up. Parthenotes were produced by artificial oocyte activation, using ionomycin (10µM; A23187) and puromycin (8µM) in sequential incubations. To produce androgenotes, MII oocytes were enucleated and then an intracytoplasmic sperm injection (ICSI) was performed. At different developmental stages, every uniparental embryo was disaggregated into constituent blastomeres wich were used for RNAseq. Briefly, libraries were created for each single cell and RNAseq was performed according to established protocols of Illumina NovaSeq, followed by the analysis of the results using FASTQ. Functional enrichment of Biological pathways were performed using g:Profiler, Reactome and Cytoscape. Developmental stages analysed were: pronuclear stage (n=6), 4/5-cell stages (n=5); 7/8-celll stages (n=4) and one 10-cell stage parthenote. Results: As embryo development progressed, differences in transcriptome were observed in both uniparentals. At pronuclear and 4-cell stages, androgenotes showed a significantly higher number of downregulated biological processes such as "RNA splicing", "Cellular Metabolic" and "Transcription Regulation" compared to parthenotes. However, at 7 to 10-cell stages, profiles found were the opposite. Further, Heatmaps and PCA analysis of the studied development stages showed that within each uniparental there was a variable number of cells that tend to behave differently, showing a similar transcriptome to other less advanced stages. Conclusion: In our knowledge, this is the first single cell research carried out on human haploid uniparental embryos. Longitudinal comparison of the genome expression profile between both parental origins showed that transcriptome maturity might be reached differentially according to the origin. Furthermore, within the same uniparental, individual cells showed different degrees of transcriptome maturity. These preliminary results support the suitability of the haploid androgenotes and parthenotes as biological models to explore the involvement of each gamete in early development. Support: ISCIII FIS proyect (FI20/00086). Archana K Ayyar †, Amy K. Schutt, William E. Gibbons, Chellakkan S. Blesson * . Baylor College of Medicine, Houston, TX, United States. Introduction: Bisphenols are widely used in the manufacturing of various common objects and are ubiquitously present in water, food, and plastics. Restrictions were placed on Bisphenol A (BPA), due to its estrogenic activity and possible carcinogenic potential. This resulted in the substitution of BPA with other bisphenols such as BPAF, BPS, and BPF in "BPA free" labeled products. Studies show that BPAF has even more estrogenic activity than BPA and is a selective estrogen receptor modulator. The aim of this study is to evaluate the impact of BPAF on the developing mouse embryo. Methods: Cryopreserved single cell mouse (C57/BL6) embryos were thawed and cultured in KSOM media containing BPAF 10 nM (n=69), 1uM (n=42), or vehicle (n=65). Embryos were cultured in the Embryoscope TM , an incubator equipped with time lapse imaging system for 6 days. Time-lapse analysis was conducted to determine the time to different cell stages of development. Randomly selected day 6 blastocysts from each group (n=18-20) were stained with DAPI and cells were counted. Further, day 6 embryos from BPAF 10 nM and control groups were collected to investigate the expression of Esr1, Esr2, Pgr using qPCR. Appropriate statistical methods were used to compare the various groups (mixed model analysis, ANOVA, Chi Square and t-test). Results: Day 6 blastocysts exposed to BPAF showed increased number of cells in both low (77.35±4.2, p=0.013) and high (79.39±5. 1, p=0.008) concentrations when compared to control (59.1±4.4) group. However there were no differences between the groups in blastulation rates for starting blastocysts, expanded blastocysts and hatching blastocysts between the three groups or for any of the developmental stages in the morphokinetic analysis between the groups. Interestingly, BPAF exposure increased the expression of Esr1 compared to the control (1.83±0.3 vs 1.00±0.17, p=0.048), but decreased the expression of Esr2 (0.39±0.06 vs 1.0±0.25, p=0.019) and Pgr (0.45±0.04 vs 1.00±0.21, p=0.005). Conclusion: Our results show that BPAF exposure increased the embryo cell numbers in physiologically relevant concentrations and this could be due to increased cell division or reduced elimination of abnormal cells. Further, BPAF regulates the gene expression of estrogen and progesterone receptors indicating its modulating action on estrogen receptor signaling. Although the mechanisms are unknown, our study show that BPAF regulates embryo growth and cell signaling. BPAF could potentially impact human embryos and caution should be taken when using BPAF. ERβ Regulated Indian Hedgehog Signaling in the First Wave of Ovarian Follicles. Iman Dilower †, Eun B Lee †, Saeed Masumi †, Hindole Ghosh †, Richita Roy †, Anohita Paul †, Ayushi Kothari †, Subhra Ghosh †, V. Praveen Chakravarthi †, Michael Wolfe * , MA Karim Rumi * . University of Kansas Medical Center, Kansas City, KS, United States. Introduction: The first wave primordial follicles are activated immediately after their formation; in contrast, the majority of second wave primordial follicles remain in a dormant state and serve as the ovarian reserve. As the first wave follicles deplete, a small number of the second wave primordial follicles gradually undergo activation to replenish the pool of activated follicles. The mechanism that maintains the dormancy of second wave primordial follicles, and allows the steady activation of selected follicles, plays a key role in determining the reproductive longevity. We identified that the loss of estrogen receptor β (ERβ) leads to an excessive activation of primordial follicles in Erβ knockout (Erβ KO ) rats. The canonical transcriptional regulatory function of ERβ is essential for the regulatory mechanism. Methods: To identify the ERβ-regulated genes that control the primordial follicle activation, we performed RNA-sequencing of ovaries collected from wildtype and Erβ KO rats on postnatal day (PND) 4, 6, and 8. The transcriptome data were further validated by RT-qPCR, in situ hybridization, and immunofluorescence staining. Results: Among the differentially expressed genes in Erβ KO developing ovaries, indian hedgehog (Ihh) displayed the highest downregulation among the ovary enriched genes. IHH-regulated genes such as Hhip and steroidogenic enzymes Cyp11a1, Cyp19a1, and Hsd17b1 were also markedly downregulated in Erβ KO rat ovaries. This phenomenon was found to be specific for transcriptional regulation by ERβ and responded to the treatment with selective ERβ-agonists and -antagonists. However, the expression of Ihh did not correlate with the expression levels of Gdf9 and Bmp15, which are known regulators of Ihh expression in granulosa cells. More strikingly, the downregulation of Ihh gene started disappearing after PND12, and there was no difference between the wildtype and Erβ KO ovaries at PND21, when a significant number of second wave primordial follicles are activated. Conclusion: Our findings indicate that the expression of Ihh during early postnatal period is ERβ-dependent only within the first wave of ovarian follicles. In wildtype ovaries, IHH produced by the first wave follicles may act as an ERβ-regulated inhibitory signal to maintain the dormancy of second wave primordial follicles. (epididymosomes) are crucial in shaping the molecular landscape of sperm. Studies have demonstrated the importance of epididymosomes for sperm maturation and offspring health. It has been suggested that the miRNA cargo within epididymosomes becomes altered after environmental adversity and this altered miRNA complement once delivered into sperm, can influence gene expression within the embryo. However, it is currently unclear where in the epididymis these epididymosomes are altered and whether they are responsible for influencing sperm miRNA after synthetic glucocorticoid (sGC) exposure. We hypothesized that the miRNA profile of caput and cauda epididymosomes will be altered following sGC exposure, paralleling changes that we have previously demonstrated in sperm. Methods: Adult male guinea pigs were exposed to the sGC dexamethasone (Dex) in drinking water (3mg/kg) or normal water (Ctrl) (N=6/group) every other day for 48 days. Caput and cauda epididymal fluid was ultracentrifuged to isolate epididymosomes. Epididymosomes were imaged with a transmission electron microscope and size/concentration were evaluated with NanoSight. Total RNA was extracted from caput and cauda epididymosomes with Trizol. miRNA from caput and cauda epididymosomes was assessed by miRNA 4.0 microarray and data were processed with TAC 4.0.1. miRNA expression was evaluated using a one-way ANOVA and p-values were FDR adjusted by the Benjamini-Hochberg algorithm. Fold-Change (FC) >+-1.5, and FDR corrected p-value <0.05 was considered significant. Results: Dex-treatment did not affect epididymosome size or concentration. However, Dex-treated animals exhibited reduced concentrations of RNA in cauda epididymosomes. miRNA expression analysis revealed no significant changes in the caput following Dextreatment. In the cauda, 125 miRNA were down-regulated while 157 were up-regulated. Examples of miRNA that were significantly downregulated by Dex included miR-125 (FC=-4.3) and miR-199 (FC=-2.72). These miRNAs were also affected in the cauda sperm of Dex-treated animals. Conclusion: These findings demonstrate that Dex-exposure influences the miRNA cargo of epididymosomes in the cauda of the epididymis. Further, miRNAs altered in sperm exhibit a similar profile as those observed in epididymosomes. These data highlight the importance of epididymosomes in influencing the miRNA profile of sperm. This is highly clinically relevant as a deeper understanding of the molecular mechanisms responsible for the transmission of paternal experiences could help in the development of new approaches to break the cycle of intergenerational transmission, and identify novel predictive biomarkers of embryo, fetal and postnatal health. Introduction: The first week post-fertilization is critical to human development but is poorly characterized. Current knowledge pertaining to preimplantation development is primarily based on mouse models. However, differences have been identified between the mouse and human and caution is warranted when translating findings. For example, although the involvement of Hippo signaling in inner cell mass (ICM)trophectoderm (TE) segregation is clear in mice, it remains controversial in humans. Recent studies suggest that the Wnt pathway may be involved in human lineage segregation. Here, we seek to better understand the role of the Wnt pathway during lineage segregation in both the human and the mouse preimplantation embryo. We hypothesize that 1) inhibition of the Wnt/β-catenin pathway is involved in ICM-TE segregation, and 2) the Wnt/Planar Cell Polarity pathway is required for blastocoel formation and TE specification. Methods: We re-analyzed our human scRNA-seq dataset (E3-E7) to specifically look at the expression of Wnt signaling components. We performed a comparative analysis of human (OVO Fertility) and mouse embryos treated with small molecule inhibitors (CHIR99021 and IWP2) to inhibit/activate the Wnt pathway. Embryos (n=2-10/group) were imaged by immunofluorescence using lineage markers (NANOG, GATA6, CDX2) and components of the Wnt pathway (B-catenin, pJNK). Results: We found a switch in the expression (on/off) of genes involved in Wnt signaling at late embryonic day (E) 5; corresponding to the segregation of epiblast (EPI), primitive endoderm (PE) and TE. Further, many of the genes involved in the Wnt/β-catenin pathway were downregulated in TE cells compared to EPI and PE cells. Inhibiting the Wnt pathway led to developmental arrest of human embryos around E4-E5 as expected but did not prevent mouse development. Activating the Wnt pathway decreased CDX2 expression in both human and mouse embryos, suggesting a retainment of pluripotency and failure to form TE. Conclusion: Together our data suggests that Wnt signaling plays an important role in TE formation. Our results suggest species differences in the pathways driving ICM-TE segregation, emphasizing the need for human focused studies. Knowledge from these studies will shed light onto early human embryogenesis and may be translated to the field of Stem Cell Biology. Mechanistic Opposition of TNFα and AMH upon Integrated Stress Response Activity in Granulosa Cells. Evelyn Llerena Cari †, 1 John Rushing, 1,2 Ivy Lersten, 1, 2 1 signaling and its control of protein translation likely regulate ovarian primordial follicle (PF) growth activation (PFGA), subsequent granulosa cell (GC) proliferation, and the selection of individual follicles for survival. 2 Treatment of GCs with the PFGA activator Tnfα leads to ISR activation as measured by significantly elevated Serine 51 phosphorylation of eukaryotic Initiation Factor 2 alpha subunit (P-eIF2α). We hypothesized that Tnfα upregulates the ISR at least in part by upregulating intracellular reactive oxygen species (ROS), and that the PFGA suppressor anti-Müllerian Hormone (AMH) 3 would block ISR activation by Tnfα. Methods: Stable knockdown (KD) OV3121 cells were generated using lentiviral transduction, targeting Tnfα receptor 2 (Tnfr2, 73% KD) or Amh receptor 2 (Amhr2; 81% KD). Parent or KD cells were optionally pretreated with 10 ng/ml AMH or vehicle (VEH) for 3 hours (h), and then were treated with Tnfα or VEH for an additional 4 h. ROS were measured using a biochemical assay kit. Western blots were performed for total and P-eIF2α, and densitometric analysis was performed by normalizing to the total protein. The significance threshold was p<0.05, Welch's t test. Results: Our hypotheses were supported, as Tnfα treatment significantly elevated cellular ROS (p<0.01), which was abrogated by AMH pretreatment. AMH pretreatment also significantly decreased P-eIF2α levels in parent cells treated with Tnfα (p < 0.05), entirely blocking an approximate 2.5-fold increase in the ratio of P-eIF2α:eIF2α with Tnfα treatment alone. This was dependent upon AMHR2 levels as AMH pretreatment of Amhr2 KD cells did not block the Tnfα-induced rise in P-eIF2α. In contrast, P-eIF2α did not differ between Tnfr2 KD and parent cells, revealing that ISR upregulation by Tnfα does not depend upon TNFR2 binding. Conclusion: Activation of the ISR by cellular stressors, including ovarian Tnfα, is blocked by AMH signaling through its receptor AMHR2. While information is available about how these factors regulate PFGA and GC growth separately 3, 4 , these findings shed light on a mechanistic interaction between the two signaling pathways that converges upon the ISR, and begin to reveal the requirement(s) for ligand:receptor binding. AMH blocking ISR activation by Tnfα reveals a new control mechanism for the GC cell cycle, and potentially, for the growth activation of primordial follicles in vivo. 1. Pakos-Zebrucka et al. (2016) . EMBO Rep 17, 1374 -1395 . 2. Llerena Cari, et al. (2021 . Mol Hum Reprod, 27, gaab050.3. Meinsohn et al. (2021) . PNAS, 118, 2100920118.4. Greenfeld et al. (2007) . Biol Reprod. 76, Novel Anti-Mullerian Hormone Receptor 2 Binding Peptide (AMHR2BP) Modulates Preantral Follicle Development In Vitro. Laura Detti, 1 Vaani Nanavaty, 2 Arsela Gishto, 2 Nina Desai * . 2 1 Cleveland Clinic, Cleveland, OH, United States; 2 Cleveland Clinic, Beachwood, OH, United States. Introduction: Anti-Müllerian hormone (AMH) plays a major role in the ovary, regulating primordial follicle recruitment and progression of follicles from primary to antral stages. Recently a novel anti-Mullerian hormone receptor 2 binding peptide (AMHR2BP) has been shown to modulate follicular development in vivo by inhibiting granulosa cell replication and decreasing hormone production. This potentially "protective" effect on ovarian reserve is intriguing. This study investigates the direct effect of AMHR2BP on preantral follicle growth and oocyte maturation in vitro. Methods: Ovaries from 14-16 day-old B6D2 mouse pups were removed and enzymatically digested with collagenase. Pre-antral follicles with 2-3 layers of granulosa cells were collected. Follicles (4 per well) were randomized between three treatments groups :(A) Control-follicle culture medium (FCM) alone; (B) Early AMHR2BP supplementation; (C) Late AMHR2BP supplementation. The basal follicle culture medium was α-MEM with 5% FBS, 100 mIU/ml FSH and insulin-transferrin-selenium (ITS). In the early AMHR2BP group, 50 µg/ml was added to medium from outset, versus in the late AMHR2BP treatment it was added to medium from day 10 until maturation day. Culture medium (100 µl) was added to each well. Final ovulation and oocyte maturation was triggered with hCG (1.5 IU/ml) and EGF (5 ng/ml) after ~12 days of growth. Ovulated complexes were recovered and oocyte nuclear status evaluated. Outcome parameters were follicle morphology, antrum formation, ovulated cumulus:oocyte complexes after hCG trigger, germinal vesicle breakdown and oocyte maturation to the metaphase II stage. Differences between control and AMHR2BP treatments were compared using Chi square and ANOVA for multiple comparisons. P values ≤0.05 were considered to be statistically significant. Results: Data from three replicate experiments is shown in Table 1 . Early-AMHR2BP administration resulted in significantly higher antrum formation (91.7%) as well as oocyte maturation (49.8%) . Late-AMHR2BP only enhanced oocyte maturation rate, when compared to the control. Conclusion: This study showed that AMHR2BP supplementation of culture media enhanced secondary follicle growth and oocyte maturation in vitro. This peptide may serve as a survival factor promoting in vitro growth of preantral follicles. Further testing of oocytes for functional competence is needed. Murine Oocyte Maturation and Developmental Competence Using Biocompatible Hydrogel Based on Decellularized Bovine Ovarian Cortex. Rosalba Lopez †, 1, 2, 3 Emilio Francés-Herrero †, 2,3 Adolfo Rodríguez-Eguren †, 1,2 Luís Miguel Del Castillo †, 2,3 Amparo Faus, 2 Antonio Pellicer * , 3, 4 Sonia Herraiz * , 1,2 Irene Cervelló * . 1 Establishing a suitable xenogeneic hydrogel could provide a valuable and versatile biomaterial for translational research. The goal of this study was to evaluate murine oocyte competence and developmental potential following in vitro follicle growth (IVFG) using a biocompatible decellularized bovine ovarian cortex extracellular matrix (ECM) hydrogel. Methods: Secondary follicles (108-215 µm) were mechanically isolated from 14-16 day old B6CBAF1/J female mice and individually cultured in vitro during 8 days: (I) on an ECM hydrogel coating (10 mg/mL; n=99), (II) with ECM hydrogel supplemented medium (0.1 mg/mL; n=194) or (III) standard medium (n=184). Ovulation was induced and cumulus-oocyte complexes (COCs) were recovered. Denuded oocytes were assessed for polar body extrusion and either (1) parthenogenetically activated and cultured in vitro for 5 days, assessed for (2) meiotic spindle assembly, or (3) mitochondrial activity. COCs from the ampullae of hormonally stimulated females were used as an in vivo control group (IV). Data were analyzed by ANOVA and Tukey's range tests (p<0.05 statistically significant). Results: Follicles grown on the hydrogel coating had similar COC recovery rates (34.69 ± 47.84% vs 32.07 ± 46.8%; p>0.05) but higher oocyte maturation rates (30.30 ± 46.31% vs 22.22 ± 41.71%; p>0.05) compared to standard culture. Although both significantly outperformed the supplemented group (2.58 ± 15.89% and 10.07 ± 30.19%, respectively; p<0.05), oocyte maturation was significantly higher in vivo (57.50 ± 50.06%; p<0.0001). Out of the 14, 5, 21 and 5 parthenotes from groups I-IV, respectively: 11, 4, 13 and 3 advanced to the 2-cell stage; 6, 1, 5 and 2 to the 4-cell stage; 3, 0, 1 and 1 to the 8-cell stage; 1, 0, 0 and 0 to the morula stage (p>0.05). Normal meiotic spindle formation and homogeneous mitochondrial distribution within the ooplasm were evidenced in all 4 conditions. Notably, mitochondrial activity was lower for the hydrogel coating and in vivo conditions oocytes. Conclusion: These preliminary findings highlight the potential value of using a xenogeneic bovine ovarian cortex ECM hydrogel as a coating in IVFG to generate mature and competent murine oocytes. *RL and EF-H contributed equally. FUNDING: GRISOLIAP/2018/029; FPU18/06327; FPU19/04850; F P U 1 6 / 0 5 2 6 4 ; P I 1 7 / 0 1 0 3 9 ; C P 1 9 / 0 0 1 4 9 ; C P 1 9 / 0 0 1 4 1 ; PROMETEO/2018/137. Pain. Pamela Stratton * , 1 Hannah K Tandon †, 1,2 Vy Phan †, 1, 3 Jacqueline V Aredo †, 1, 4 Ninet Sinaii, 1 Jay P Shah, 1 Barbara I Karp * . 1 1 NIH, Bethesda, MD, United States; 2 U Nebraska Med Center, Omaha, NE, United States; 3 Georgetown University, Washington, DC, United States; 4 University of California, San Francisco, CA, United States. Introduction: Women with endometriosis-associated chronic pelvic pain (endo-CPP) often have pain that persists despite optimal surgical and hormonal management of lesions. Pelvic floor muscle spasm may contribute to pain persistence. Here, we evaluated the longitudinal efficacy of botulinum toxin (BoNT) in relieving endo-CPP. Methods: Women (18-50yrs) with endo-CPP (surgically diagnosed, optimized hormonal treatment) in a randomized, placebo-controlled, double-masked trial received 100U onabotulinumtoxinA or placebo into pelvic floor muscles with spasm (NCT01553201). An optional open BoNT (2 nd ) injection was offered any time 1-12 months after masked injection at subject's request. Baseline, 1 month, 6 month and 1 year evaluation included # pelvic floor muscles in spasm, current benefit, pain rating, Endometriosis Health Profile (EHP), SF36 and Oswestry Disability Index. Subjects rated response to injection as benefit (benefit/no benefit) and benefit duration. Repeated measures mixed-effects models analyzed EHP and SF36 scores over time, adjusting for benefit, # active injections, disability, and current # pelvic floor muscles in spasm. Results: 29 women were randomized; all identified pelvic floor spasm as a major focus of endo-CPP. No participant dropped out. At 1 month, 12/15 (80.0%) in BoNT group vs 5/14 (35.7%) in placebo group had benefit (p = 0.025). Over 1 year, 27/29 requested open BoNT; 2 (both BoNT group) had masked injection only. Overall, 4 patients (2 placebo, 2 BoNT) had no durable benefit after either injection. Ongoing benefit at 1 year was reported by 6/14 in placebo group and 10/15 in BoNT group. At 1 year, persistent EHP pain relief (baseline vs 1 year, p < 0.001) was associated with ongoing benefit (p = 0.008), current fewer # pelvic floor muscles in spasm (p < 0.001) and lower baseline disability (p < 0.001). Improvement in EHP sexual intercourse was associated with current fewer # pelvic floor muscles in spasm (p = 0.032) and lower current disability (p = 0.016). Lower current disability was associated with improvement in SF36 physical functioning, bodily pain, general health, fatigue, and mental health scores (all p < 0.001). By 1 year, those in BoNT group used less pain medication than placebo group (p = 0.037). We demonstrate pelvic floor muscle spasm as part of endo-CPP and relief of pain/spasm from BoNT compared to placebo that persisted over time. BoNT was well tolerated. Baseline and current disability scores predicted improvement in pain, sexual intercourse, functioning, fatigue and mental health scores. Introduction: Uterine leiomyosarcoma (LMS) is an aggressive tumor associated with high recurrence rates, metastasis and poor prognosis. It is the most common type of uterine sarcoma. Uterine fibroids (UFs) are benign neoplasms of the myometrium, representing the most common tumors in reproductive age women globally. Notably, uterine LMS and UFs share several common clinicopathological and imaging features that complicate their differential diagnosis. Currently, LMS is often diagnosed postoperatively, and reliable pre-op diagnostic measures differentiating LMS from UFs are lacking. There is an urgent unmet need to develop early diagnostic tools to distinguish these two uterine tumors as UF can be treated conservatively, allowing patients to avoid invasive surgery and preserve their fertility while LMS requires urgent surgery (hysterectomy) and adjuvant chemotherapy. The aim of this study is to identify novel serum biomarkers specific for LMS using quantitative proteomics. Methods: Nude mice were inoculated with 2x10 7 of SK-UT1 human LMS cells (n=5) or UFs cells (n=5) or control with no cells (n=5). After 4 weeks the animals' blood was collected. Serum was depleted of the top 3 most abundant blood proteins and analyzed using quantitative mass spectrometry-based proteomics. Raw data was searched against a concatenated mouse/human proteome database and human-specific proteins were identified by ≥ 2 human-specific peptides. Differential analysis was performed using LIMMA and significantly (p<0.05) altered candidates exhibiting a fold-change ± 1.5 were prioritized for downstream analysis. The global proteomic analysis quantified 194 human-specific proteins across samples. Differential analysis identified six significantly altered proteins were shared in common between LMS and UFs, while 12 were unique to UFs, and 10 were unique to LMS. Notably, three proteins altered in LMS alone encoded signal peptide sequences targeting proteins for secretion or to the cellular membrane where they may also be shed to the extracellular space. Specifically, transthyretin (TTR) and dual oxidase 2 (DUOX2) proteins were upregulated, and G-protein coupled receptor 179 (GPR179) was downregulated in LMS relative to control. In utero exposure of a maternal high fat diet (mHFD) results in the targeted epigenetic reprograming and disruption of circadian clock genes in the fetal liver. However, it is unclear if this disruption of the circadian clock exacerbates the effect of the offspring's post-weaning high fat diet (oHFD) on the function of its gut microbiome. To understand the interaction of the circadian clock and the offspring gut microbiome with HFD feeding, we disrupted liver Npas2 expression and analyzed the resultant microbiome in light and dark cycles of feeding. Methods: Npas2 cKO (n=146) and wild-type control (WT, n=155) mice were fed either a HFD or a control diet (CD). At 16 weeks post-weaning, mouse feces were collected at 4-hour intervals. DNA was extracted and 16S V4 amplicons were sequenced (Illumina) followed by taxonomic analysis and functional pathway abundance prediction using DADA2 and PICRUSt2 bioinformatic software packages. Comparisons were subjected to ANOVA and Posthoc Tukey-Kramer (multiple), or Welch's t-test (2-way) with 95% CI. We have previously shown that Npas2 cKO mice fed a HFD gained signifi cantly more weight when compared with controls (WT), which correlated with an increased food consumption at nighttime. Npas2 cKO mice fed an oHFD also had a signifi cant phase shift in the circadian rhythmicity of the taxonomic abundance of the gut microbiome. Few changes were observed in the function of the off spring gut microbiome between circadian disrupted (Npas2 cKO mice) and WT controls when fed a CD (Fig.1 ). When fed an oHFD, the Npas2 cKO mice gut microbiome during dark cycles was diff erent in circadian disrupted mice but not WT controls (Fig.2) . We have demonstrated that the loss of a single circadian gene in the liver signifi cantly alters the circadian entrainment of the gut microbiome under the metabolic stress of HFD feeding. Our fi ndings collectively suggest that other disruptions to Npas2 hepatic expression, such as fetal exposure to a maternal HFD, signifi cantly alters the gut microbiome which may play a role in disrupting metabolic adaptation rendering obesity. Maternal Clinical studies suggest that cannabis use in pregnancy leads to fetal growth restriction (FGR), and fetal growth defi cits are associated with an increased risk of developing cardiovascular disease in postnatal life. We have previously demonstrated that maternal exposure to 3 mg/kg/day of Δ9-tetrahydrocannabinol (THC, the major psychoactive constituent in cannabis) from gestational day 6 to term results in FGR along with impaired cardiac function and cardiac remodeling (e.g., increase in collagen type 1 and 3) in 3-week-old rats. Given omega-3 (n-3) fatty acids (i.e., DHA and EPA) have been shown to play an important role for both development and cardiovascular health, we hypothesize that supplementation with a n-3 enriched maternal diet will ameliorate the cardiac defi cits in off spring exposed to THC in utero. Methods: Pregnant Wistar rats were administered vehicle saline (VEH) or 3 mg/kg THC i.p daily from gestational day (GD) 6 to term. For each treatment, dams were either fed with control diet or n-3 enriched diets from GD5 to postnatal day (PD) 21. MALDI-IMS was used to image and measure hepatic free fatty acid content at PD21. Off spring were followed up with echocardiogram analysis for cardiac function at PD21. Results: MALDI-IMS indicates that in utero THC exposure leads to depleted hepatic DHA in PD21 off spring. We further confi rmed that THC results in signifi cant (p<0.05) decreases in birth weight and cardiac function at PD21, however, in off spring where dams were fed with the n-3 enriched diet, THC-exposed off spring did not exhibit FGR. Moreover, at PD21, maternal supplementation with the n-3 diet ameliorated THCinduced defi cits in cardiac function (e.g., cardiac output, stroke volume, ejection fraction) in off spring. These data suggest that maternal n-3 supplementation may be benefi cial to reduce the fetal and postnatal cardiac defi cits associated with in utero THC exposure. Considering that n-3 fatty acids are important for development and have potential cardioprotective eff ects, we speculate it improves both placental effi ciency and reduces the incidence of postnatal cardiac remodelling. Future studies are warranted to determine whether shorter windows of intervention (i.e., lactation only) rescues cardiac defi cits in off spring. Given the high rate of maternal cannabis consumption and legalization in North America, understanding the eff ects of THC on the off spring is of great relevance. Moreover, this study provides a promising intervention for babies exposed to THC in utero. (Nuzzo et al., 2018; Ganguly et al., 2021) . ER stress activates the unfolded protein response (UPR) to modulate ER homeostasis; however, whether ER stress activates UPR in a sex-specifi c manner in placenta from hypoxic male and female off spring is not known. We hypothesize that hypoxia-induced ER stress activates the placental UPR in a sexually dimorphic manner, leading to sex-dependent diff erences in the placental response to hypoxia. Methods: Pregnant rats were exposed to hypoxia (11% O 2 ) or normoxia (21% O 2 ) from gestational day (GD) 15-21 (term=GD 22). On GD 21, whole placentae (including junctional and labyrinth zones) from both sexes were collected and snap frozen (n=7/group/sex) for Western blot analysis. Placental expression of UPR-related markers, glucose-regulated protein 78 (GRP78), phosphorylated and total eukaryotic initiation factor subunit 2 α (eIF2α) and inositol requiring endonuclease 1 (IRE1), and NOD-like receptor pyrin-containing 3 (NLRP3) infl ammasome were determined. Total amount of protein per sample was used to normalize the data. Data were analyzed by two-way ANOVA, with Sidak's posthoc test; presented as Mean ± SEM; p<0.05 was considered signifi cant. Results: Gestational hypoxia increased GRP78 protein levels in male placentae (normoxia-males: 0.06 ± 0.01 vs. hypoxia-males: 0.10 ± 0.01; p=0.001) but not in female placentae. Total eIF2α expression was higher in normoxic female compared to normoxic male placentae (normoxiafemales: 0.19 ± 0.02 vs normoxia-males: 0.10 ± 0.01; p=0.01), while prenatal hypoxia increased total eIF2α expression only in the male placentae (normoxia-males: 0.10 ± 0.01 vs. hypoxia-males: 0.16 ± 0.02; p=0.05). No diff erences in peIF2α/eIF2α were observed among the groups. Hypoxia had no eff ect on either IRE1 or pIRE1/IRE1 and NLRP3 expression compared to normoxic controls. Conclusion: GRP78 was increased by gestational hypoxia only in male but not female placentae, suggesting a sex-specifi c divergence in the placental adaptive response to gestational hypoxia. Increased GRP78 and total eIF2a levels, without evidence of UPR activation, suggest a sexually dimorphic adaptation in male hypoxic placentae to maintain ER homeostasis. Sex-specifi c diff erences in the placental capacity to respond to stressful environments could account for the sexual dimorphism observed in placental function and pregnancy outcomes in complicated pregnancies. , 2018) . However, it remains unknown: 1) whether gestational hypoxia aff ects the cardiac mitochondrial respiratory chain in the fetus; 2) if so, whether this eff ect is sex-dependent, and 3) whether this is modifi ed by antioxidants. Therefore, using an established rat model, we measured sex-dependent eff ects of gestational hypoxia and maternal melatonin treatment on fetal cardiac mitochondrial respiration, ROS production and lipid peroxidation. Methods: Pregnant Wistar rats were subjected to normoxia or hypoxia (13% O 2 ) during gestational days 6-20 (term ~ 22 days) ± melatonin treatment (5µg/ml in maternal drinking water). On day 20, aerobic respiration, ROS production, lipid peroxidation and citrate synthase activity were determined in fetal cardiac mitochondria. Statistical signifi cance was assessed using a nested two-way ANOVA. Results: Cardiac mitochondria of male but not female fetuses of hypoxic pregnancy showed reduced oxygen consumption. Despite this, fetal hearts of both sexes in hypoxic pregnancy showed increased ROS production, without changes in lipid peroxidation or citrate synthase activity (Fig. 1 ). Maternal melatonin treatment had no independent eff ect on mitochondrial function. Gestational hypoxia reduced maternal body weight, despite a transient reduction in food intake. Fetal weight was unaff ected by hypoxia, but placental weight was signifi cantly increased (data not shown). We show that gestational hypoxia affects cardiac mitochondrial function in the fetus in a sex-dependent manner, reducing oxygen consumption in males but not females. We confi rm that ROS production is increased in hearts of both male and female fetuses, and show that the site of production is the electron transport chain in hypoxic pregnancy. We show that maternal melatonin treatment had no eff ect on these relationships, which has implications for the development of future therapies for pregnancies aff ected by fetal hypoxia. Supported by the BHF. .1 A) . This effect was also observed in HUVEC exposed to hypoxia in vitro (83 out of 146, p < 0.01, FDR < 0.05). Analysis of Piezo1 protein in the endothelium of umbilical vessels showed decreased levels in FGR compared with control pregnancy (Fig. 1B) . The effect of hypoxia on Piezo1 expression was confirmed by qPCR, showing that decreased oxygen levels result in short-(4 hours) and long-term (up to 24 hours) down-regulation of Piezo1 (Fig. C (n=8) were divided into 2 groups, control (CON, n=3) and marijuana-exposed (THC, n=5). All animals were maintained on a standard chow diet with the THC group receiving an additional THC edible daily. THC animals were titrated to 2.5mg/7kg/ day of THC (equivalent to a heavy human medical marijuana dose) over 4 months to model established medical marijuana acclimation guidelines. All animals underwent time-mated breeding with the THC group maintained at their THC dose throughout pregnancy. The fetal brain was assessed by MRI at gestational day 85 (G85), G110, G135, and G155. All animals were delivered by cesarean section at G155 (term is ~168 days), and fetal tissue was collected for histological analysis. Results: Maternal age, parity and weight, and fetal weight were not significantly different between CON and THC groups. Fetal brain MRI showed no regional volume differences between CON and THC groups (p=0.42). On brain histology, all THC-exposed fetal brains (n=5) demonstrated hyperacute ischemic changes in the cerebellum Fig. 1A and findings suggestive of some degree of ischemic white matter injury, including reactive astrocytosis and increased microglial clusters Fig. 1B particularly within the temporal and parietal lobes. Conclusion: In our study, THC exposure did not demonstrate adverse short-term outcomes, including gross fetal anomalies, growth restriction, or pregnancy loss. However, THC-exposed fetal brains were notable for microscopic findings on histology suggestive of brain dysregulation that may have long-term implications for offspring development. Due to its highly lipophilic profile, THC can bind to cannabinoid receptors and modulate cellular responses within the brain. Further studies are needed to determine whether these histologic changes correlate with long-term offspring cerebrovascular health. by ~20% compared with control fetal sheep (CON; intratracheal room air, n=7) in late gestation. We used 13 C-glucose tracer infusion to measure whole-body glucose oxidation rates and lactate production from glucose. Signaling pathways and targets regulating glucose utilization, glucose and pyruvate oxidation, and lactate production were measured in liver, muscle, and adipose tissue using qPCR, western blotting, and enzyme assays. We measured pancreatic tissues and mechanisms that may produce lower insulin concentrations. Significant differences and correlations were declared with P<0.05 by t-test. Results: Lower fetal glucose oxidation rates correlated with the severity of hypoxemia (fetal blood oxygen content, r=0.54) and lower glucose utilization (r=0.71). HOX fetuses had increased lactate production from glucose. In muscle and adipose tissue, expression of the glucose transporter GLUT4 was decreased. In muscle, pyruvate kinase (PKM, muscle isoform) and lactate dehydrogenase B (LDHB) expression was decreased. In adipose tissue, LDHA and lactate transporter (MCT1) expression was increased. In liver, expression of pyruvate kinase (PKLR, liver isoform) and mitochondrial pyruvate transporter (MPC2) were decreased and LDHA was increased. LDH activity was decreased in HOX skeletal muscle, whereas pyruvate dehydrogenase (PDH) activity was decreased in HOX liver. There were no differences in basal insulin signaling across tissues nor deficits in pancreatic measures of beta cell function. The HOX fetus has coordinated tissue responses that constrain glucose oxidation and increase lactate and pyruvate production. These may be mediated by increased norepinephrine and cortisol, which inhibit insulin secretion resulting in lower insulin concentrations, decreased stimulation of glucose utilization and oxidation, and increased lactate and pyruvate production. Increased lactate may be used as a fetal substrate, while pyruvate is shuttled to the placenta. Thus, constrained glucose oxidation and shifts in fetal substrate metabolism and flux between the placenta may represent critical events in the initiation of fetal growth restriction in response to sustained hypoxemia. Results: Significant differences were observed between the two supposedly identical twins of the same mother according to birth order as reported in Table 1 . Second-born twins showed significantly lower birthweight and UVBF. Umbilical O 2 delivery and uptake per kg were also significantly reduced in the second twin, with a significant relationship between O 2 uptake and delivery and between O 2 and glucose uptake. The comparison with previous data obtained by our group in singletons suggests the presence of compensatory mechanisms adopted by MC twins to reduce their metabolic needs and adapt to less favourable intrauterine conditions, resembling the findings reported for singleton fetuses with growth restriction. These innovative findings hint at preexisting conditions possibly deriving by uneven metabolic function of the two placental territories and prompt further physiological studies on MC twins and placenta metabolism. The pathogenesis of endometriosis remains unclear, but enhanced cell invasion and cell viability contribute to development and progression of the disease. Our previous studies found that NOTCH1 is overexpressed in response to Interleukin 6 (IL-6) and is mediated by E-proteins in ectopic epithelial cells of both baboons and women with endometriosis. NOTCH activation can induce epithelial-mesenchymal transition (EMT) to promote cancer metastasis and development. Therefore, this study aimed to investigate the role of NOTCH1 in the regulation of EMT during the development of endometriosis. Methods: Immortalized human ectopic epithelial cells (12Z) were transfected with a vector overexpressing the NOTCH1 intracellular domain(N1ICD) and/or RBPJ siRNA. The expression of EMT markers E-cadherin, α-SMA, Snail and Slug were determined by RT-qPCR and immunofluorescence. Cell migration in a wound healing assay was tracked by time lapse microscopy. A 3D organoid invasion model was used to examine the invasion of endometriotic organoids generated from N1ICD overexpressing 12Z cells and immortalized ectopic stromal cells. Organoid invasion into the Matrigel containing human peritoneal mesothelial cells (LP-9) expressing GFP was imaged by confocal microscopy and further quantified by image analysis software (IMARIS). 12Z cells were treated with recombinant JAG2-Fc (a NOTCH ligand) to activate NOTCH signaling and subsequently incubated with gamma secretase inhibitor (GSI) for 24h, EMT markers were analyzed as described above. Finally, Ltf cre/+ N1ICD f/+ mice, in which N1ICD is specifically overexpressed in endometrial epithelial cells, were utilized to induce endometriosis to study the effect of epithelial specific N1ICD overexpression in endometriotic lesion development. Results: Overexpression of N1ICD in 12Z cells significantly decreased epithelial markers E-cadherin, increased mesenchymal markers α-SMA and transcriptional activators, Snail and Slug. Cell migration was significantly increased when N1ICD was overexpressed in 12Z cells. These effects were reversed following RBPJ knockdown which decreases NOTCH signaling. Endometriotic organoid invasion into the Matrigel-LP9 layer was significantly higher when NOTCH1 signaling was activated in ectopic epithelial cells. E-cadherin was significantly down-regulated while α-SMA, Snail and Slug were up-regulated by JAG2-Fc triggered NOTCH activation. However, this effect was neutralized by GSI inhibition. Finally, Ltf cre/+ N1ICD f/+ mice developed significantly more and larger ectopic Introduction: Endometriosis is a disorder that affects ~10% of females of reproductive age. Decidualization, a progesterone (P4) driven process in which endometrial stromal cells (eSCs) differentiate to support implantation, is impaired in endometriosis. It is proposed that these P4-induced changes are mediated by the transcriptional activity of its canonical receptors (PR-A and PR-B). While prior studies focused on P4 resistance in ectopic endometriosis lesions, we sought to investigate P4 responsiveness using eutopic eSCs obtained from endometriosis cases and controls. Methods: Low passage (p2-3) menstrual effluent-derived eSCs (ME-eSCs) obtained from women with confirmed endometriosis or healthy controls were stimulated with vehicle or MPA, a stable P4 analogue. qPCR and single cell RNA-seq (scRNA-seq, n=4 controls and 4 cases) were used to measure P4-induced gene expression and identify dysregulated pathways. Subcellular localization of PR isoforms and the PR chaperone protein, FKBP52, was assessed using immunofluorescent microscopy and western blotting of total, nuclear, and cytoplasmic preparations of ME-eSCs ±MPA stimulation. Results: MPA-stimulated PLZF and IHH gene expression are significantly blunted in ME-eSCs from cases vs. controls, as determined by qPCR. At baseline, global gene expression by scRNA-Seq of control ME-eSCs significantly differed from case ME-eSCs; MPA stimulation induced differentially expressed genes, including CEBPD, DKK1, FABP4, MGP, and NNMT which were significantly upregulated in controls only and IGFBP5, MALAT1, DIO2, and KRT17 that were significantly downregulated in controls only. PR localization studies support differential subcellular localization of PR isoforms and chaperone FKBP52 in cases and controls under baseline and MPA-stimulation conditions. Conclusion: ME-derived eSCs provide a model system for studying eutopic P4 sensitivity. In endometriosis, eutopic ME-eSCs show significantly blunted responsiveness to P4 stimulation. This attenuated P4 response may be, in part, due to altered FKBP52 and PR expression and nuclear localization. Better understanding the mechanism(s) underlying P4 resistance will help develop improved treatments for endometriosis. the 18 endometriosis drugs with variants were analgesics, 3 aromatase inhibitors, and 3 hormonal treatments. CYP19A1 (aromatase), one of the main targets of endometriosis, was on the top 5 of genes with more variants affecting drug efficacy (4 variants) and producing toxicity (4 variants). Drug-drug interaction analysis showed 368 interactions between endometriosis drugs producing side effects (13.8%) or affecting drug efficacy (5.8%). Pregabalin increases the efficacy of 5 endometriosis drugs when combined, and Human Chorionic Gonadotropin (hCG) and Dydrogesterone were the safest according to the analysis of variability in drug response. Conclusion: This work highlights a considerable difference on drug response influenced by a collection of genomic variants that diminish the efficacy and/or produce toxicity depending on the patient genome and reports important information for tailoring endometriosis treatments. Analgesics reported the higher number of variants being a source for personalizing the endometriosis-related pain in the population. Additionally, we reported a considerable percentage of drug-drug interactions between commonly prescribed drugs in endometriosis that could diminish the effect or produce side effects, while others such as the combination with Pregabalin could increase treatment efficacy. This study provides the genomic system with the list of variants and the best drug combinations towards precision medicine for developing more targeted, accurate and safer therapies in endometriosis. The Introduction: Uterine leiomyomas (fibroids), the most common tumors of the female reproductive system, are known to have a hypoxic microenvironment, which is a major source of oxidative stress. However, it is unclear if hypoxia contributes its pathobiology. The objective of this study is to investigate how hypoxia produces reactive oxygen species (ROS) and leads to cellular growth in leiomyomas. Methods: Under informed consents, primary leiomyoma cells were isolated from leiomyoma. Cells were cultured in a hypoxic chamber (2% O2). Proliferation was valued by MTT assay. Changes in intracellular ROS levels were determined using CM-H2DCF-DA. The level of apoptosis was evaluated by Caspase-3 activity. The protein expressions of NADPH oxidase 4 (NOX4), transforming growth factor (TGF)-β3 and smad3 were measured by western blotting. To explore hypoxia induces ROS generation via TGF-β3 and NOX4, cells were treated with a specifi c NOX4 inhibitor (GLX351322) or TGF-β type1 receptor/Smad inhibitor (SB431542) under hypoxic condition. Results: We found that hypoxia induced proliferation and inhibited apoptosis in human leiomyoma cells. This was accompanied by an increase in ROS generation and the expression of NOX4, an enzyme that produces ROS. We showed that NOX4 is implicated in hypoxiainduced proliferation since the addition of GLX351322 reduced this eff ect. To understand the mechanism of NOX4 mediated proliferation, we treated leiomyoma cells grown in normoxia with media from hypoxic leiomyoma cells. This resulted in elevated ROS generation and NOX4 expression, suggesting the NOX4-induced proliferation is mediated by an autocrine fashion. Previous studies showed that TGF-β3, a secreted growth factor, increases NOX4 expression. The expressions of TGF-β3 and pSmad3, its downstream signaling target, were increased in hypoxic leiomyoma cells, and the increased TGF-β3 expression was inhibited by HIF-1α inhibitor (KC7F2). Pharmacologic inhibition with TGF-β/Smad inhibitor (SB431542) reduced hypoxia-induced NOX4 expression and ROS generation and attenuated cell proliferation. Our results show that under hypoxia, leiomyoma cells produce TGF-β3 that acts in an autocrine fashion to induce NOX4 expression, resulting in ROS generation and cell growth. These observations add to our understanding of the role of hypoxia in leiomyoma pathobiology. We used an anisotropic fi brous model to capture tissue behaviors under loading and assessed the locking stretch (collagen fi ber extensibility), initial fi ber stiff ness (collagen fi ber stiff ness), bulk modulus (ground substance stiff ness), and fi ber dispersion (collagen fi ber orientation). Results: As gestation progresses: (1) the locking stretch increases signifi cantly; (2) the initial fi ber stiff ness decreases signifi cantly; (3) the early second and the early third trimester present a higher bulk modulus compared to NP and the late third trimester; (4) the fi ber concentration (the reciprocal of dispersion) decreases from NP to the late third trimester; (5) values of the bulk modulus, the fi ber stiff ness, and the locking stretch for each subject become less distinctive between the internal and external os. Conclusion: Our results suggest with the progression of pregnancy, cervical collagen fi bers become more extensible and compliant. This is likely due to the increase in the ratio of immature crosslinks in the tissue. The ground substance stiff ness increases from the beginning to mid-gestation and decreases into late-gestation. Throughout pregnancy, the collagen fi ber dispersion increases, indicating its arrangement moves from aligned to randomly oriented; the bulk modulus, the fi ber stiff ness, and the locking stretch between the internal and external os all converge, indicating a material homogenization of the organ. Introduction: At labour, the myometrium transitions from quiescent to contractile and this switch is thought to be controlled by changes in gene expression. However, the molecular mechanisms underlying these changes, specifically, the epigenetic changes that regulate this transition from the pregnant to the labouring myometrium is understudied. We investigated accessible chromatin regions, shown as peaks, using an Assay for Transposase Accessible Chromatin paired with Sequencing (ATAC-seq) in mouse myometrium tissue at various timepoints. These timepoints included non-pregnant (NP), day 15 (D15), term labour (LAB), and 4 days postpartum (PP). ATAC-seq peaks were subjected to differential peak analysis to identify peaks specific to each timepoint. These differential peaks were subsequently analyzed for underlying motifs, using HOMER, to uncover timepoint-specific accessible transcription factor (TF) binding sequences. Results: Comparison between different timepoints revealed that approximately 38% of all peaks are shared in the myometrium across all timepoints including the non-pregnant sample. Further analysis revealed that 13.8% of peaks are specific to pregnancy with 21.5% specific to the labouring myometrium. Interestingly, the most dramatic change in chromatin accessibility occurred not between non-pregnant and pregnant samples but between labouring and postpartum samples, with a decrease in 52,172 regions at PP. To identify any specific proteins that may be driving these changes in the accessibility of the genome, the sequence at accessible regions was evaluated for motif enrichment. Motif analysis revealed differential motif enrichment during gestation and labour, with several TF binding sites belonging to labour associated TFs enriched at labour. Interestingly, HOX motifs were enriched at the PP timepoint compared to labour. Conclusion: Though there are many shared accessible chromatin regions across all timepoints, there were several timepoint-specific regions. The analysis also shows differential motif enrichment at each timepoint, suggesting changes in the TFs that regulate chromatin accessibility, and the switch to contractility in the myometrium. We observed reduced chromatin accessibility at the PP stage, which may be associated with the uterine involution process and suggest an involvement of HOX TFs in this process. Together, these data suggest that changes in the accessible chromatin landscape, coupled with the TFs that bind them, underpin the quiescent-to-contractile to postpartum switch in the myometrium. Repurposing Introduction: A lack of FDA-approved tocolytic drugs exists due to adverse effects or lack of efficacy. Repurposing FDA-approved drugs offers a better risk-vs-benefit compared to traditional drug development. Furthermore, drug combinations targeting different molecular pathways involved in myometrial contractility may produce synergistic tocolysis and reduce off-target effects. The goal of this study was to identify: 1) FDA-approved drugs that selectively inhibit Ca 2+ mobilization (CM) in myometrial (myo) cells with a favorable in vitro therapeutic index (TI), 2) synergistic combinations of hit drugs and 3) hit drugs that effectively/ potently inhibit ex vivo mouse and human myometrial contractility without affecting the vasoreactivity of the fetal ductus arteriosus (DA) -a current off-target of some tocolytics. Methods: A phenotypic high-throughput (HT) CM assay using primary term pregnant human myo cells was used to identify antagonists of oxytocin-induced CM from the SelleckChem FDA library of 1180 drugs. Uterine-selectivity of hit antagonists was determined by comparing their concentration-response between myo and vascular smooth muscle cells (VSMCs) -the main off-target limiting the use of current tocolytics. The HT assay was adapted for combination screening in 8x8 dose matrices to detect synergy using Combenefit. A WST1 assay was used to evaluate in vitro TI (ratio of IC 50 values from cell viability and Ca 2+ assays). The tocolytic efficacy and potency of mono and combination drugs were examined using mouse and human myo tissue in an ex vivo isometric contractility assay. The effect of drugs on mouse fetal DA was assessed by concentration-response on vessel diameter using ex vivo myography. Results: Nine FDA-approved drugs effectively and selectively (> 5 fold) inhibited in vitro CM from human primary myo cells compared to VSMCs. Combining these drugs that target different molecular pathways, with one another and 3 currently utilized tocolytics (atosiban, indomethacin and nifedipine), yielded 66 non-redundant combinations that were investigated for synergy. Four drug combinations resulted in both synergistic efficacy and potency, while 12 were synergistically efficacious and 2 potent. Results: In total, we found 1, 13 and 2 genome-wide significant associations for the PE, PE-HTP and PE-SGA phenotypes, respectively. Nine of these loci (eight in PE-HTP and one in PE-SGA) have not been associated with pre-eclampsia in any previous GWAS study. Seven of these associations were near genes previously associated with blood pressure traits. The single most interesting finding was an association near gene 'pregnancy zone protein' (PZP), that has been previously suspected to contribute to PE via its role in stabilizing misfolded proteins, but this connection has not been demonstrated in genetic association studies until now. Also, we replicated all five loci previously associated with PE in the previous large GWA study. The associations discovered in this study close to genes affecting blood pressure support the idea of pregnancy as a window to future cardiovascular health: the increased genetic susceptibility to cardiovascular disease may become evident for the first time during pregnancy. The novel association near PZP sheds more light into molecular mechanisms causing protein instability in maternal PE. Introduction: Beta adrenergic receptor antagonists (beta blockers) are an important option for the management of patients with cardiac dysfunction to achieve heart rate control; bisoprolol is one of the most common cardioselective beta-1 blockers prescribed. Case series have reported increased rates of fetal growth restriction (FGR) in women exposed to beta blockers; the effects of bisoprolol on the fetoplacental circulation are unknown. We tested the hypothesis that in utero exposure to bisoprolol will alter ex vivo human fetoplacental vascular function. Methods: Chorionic plate arteries (CPAs) were obtained from the placentas of normal pregnancies (NP; N=14) and those of women with cardiac dysfunction medicated with bisoprolol (N=12). Constriction to U46619 (10 -10 -2x10 -6 M) and relaxation to sodium nitroprusside (SNP; 10 -10 -10 -5 M) were assessed using wire myography. Primary endothelial (ECs) and smooth muscle (SMCs) cells were isolated from CPAs and expression of beta-1 adrenergic receptors was determined by immunofluorescence. Results: Bisoprolol significantly reduced constriction of CPAs compared to the NP group (P<0.01); maximum constriction showed a trend towards reduction (12.14 ± 1.32 vs 15.04 ± 0.96 kPa, bisoprolol vs NP; P=0.0847), whereas sensitivity to U46619 was not different (-7.516 ± 0.07 vs -7.462 ± 0.05 LogEC 50 , bisoprolol vs NP; P=0.5488) between the groups ( Figure 1A ). In CPAs exposed to bisoprolol relaxation to SNP was significantly reduced compared with the NP group (P<0.001); maximum relaxation was significantly lower (54.33 ± 5.07 vs 71.9 ± 4.89%, bisoprolol vs NP; P<0.05), whereas sensitivity to SNP was attenuated (-7.200 ± 0.14 vs -7.569 ± 0.14 LogEC 50 ; bisoprolol vs NP; P=0.0753) in the bisoprolol group compared with the NP group ( Figure 1B) . Expression of beta-1 adrenergic receptors appeared to be higher in fetoplacental ECs than in SMCs. Conclusion: Chronic exposure to bisoprolol significantly dysregulates the ex vivo reactivity of CPAs. Detection of beta-1 adrenergic receptors in the fetoplacental vasculature suggests that bisoprolol may increase CPA vascular resistance by blocking beta-1 receptors and impeding nitric oxide release. Further study is needed to understand whether increased rates of FGR in women exposed to beta blockers associates with mechanisms mediated by beta-1 adrenergic receptors. Preeclampsia Increases Risk of Maternal Pulmonary Hypertension at High Altitude in Bolivia. O V Patey †, 1 C E Salinas, 2 D A Giussani * . 3 1 Royal Brompton Hospital, London, United Kingdom; 2 IBBA, UMSA, La Paz, Bolivia, Plurinational State of; 3 University of Cambridge, Cambridge, United Kingdom. Introduction: Women with a history of preeclampsia (PE) have more than double the risk of future cardiovascular adverse events compared to women with normotensive pregnancies (Fraser et al. Circulation125:1367 , 2012 . However, whether this predisposition applies to the maternal pulmonary vascular bed has been less well studied. It is also established that pregnancy at high altitude is an independent risk factor for PE (Keyes et al. Pediatr Res54:20, 2003) . However, whether women with PE during high altitude pregnancy are more likely to develop pulmonary arterial hypertension (PAH) during and after pregnancy is unexplored. Here, we tested the hypothesis that during high altitude pregnancy in Bolivia, women who develop PE are at greater risk of PAH compared to women undergoing healthy pregnancy. Methods: The study was conducted at the Instituto Boliviano de Biología de Altura (IBBA) on 140 pregnant patients derived from the Hospital Materno Infantil and the Hospital de La Mujer in La Paz, Bolivia (3640m; 11942ft). Women undergoing healthy pregnancy at high altitude served as controls (C, n=70;29±5 years old, mean+SEM). Women diagnosed with PE, defined as per ACOG and RCOG guidelines, served as the experimental group (PE, n=70, 31±6 years old). Conventional (2D, M-mode, PW Doppler) and modern (pulsed wave tissue Doppler imaging) ultrasound techniques were applied for maternal cardiovascular assessment at a mean gestational age of 35±4 weeks and 6±2 weeks after birth. Results: Relative to controls, PE women had significantlyenlarged right ventricular and right atrial chamber sizes and pulmonary artery dimensions (Fig1.A-C),increased RV global longitudinal contractility and blood flow to the lungs (Fig1.D&E), andelevated systolic pulmonary arterial pressure (Fig1.F).These differences remained post-partum ( Fig.1. A-F Introduction: Preeclampsia (PE) is a major obstetrics complication affecting at 8 % of first-time pregnancies and its origin remains unknown. Recently, we have established an in vivo endometrial transcriptomic signature encoding defective decidualization in women who suff ered severe PE (sPE), reinforcing the maternal contribution to the etiopathogenesis of this condition. Here, we present the transcriptomic atlas of defective decidualization in sPE at the single cell level. Methods: Single cell RNA-seq (scRNA-seq) was performed on 40.000 cells corresponding to late secretory phase from controls (n=2) and women who have suff ered sPE previously (n=4). Endometrial samples were enzymatically digested, and this cell suspension was subjected to single cell isolation in vesicles following the 10x Genomics protocol. RNA retrotranscription takes place in these vesicles to obtain complementary DNA (cDNA), which is then used for the preparation of libraries and sequenced on a NovaSeq 6000. Raw sequence fi les were processed with Cell Ranger (v3.1.0) and aligned to GRCh38-3.0.0 reference. Bioinformatic analysis of single-cell RNASeq (scRNASeq) data was performed by Seurat R package. The cluster annotation was based on the data provided by Wang et al. 2020. Results: Data integrative analysis revealed the presence of main expected cell types: epithelium, stroma, perivascular fraction, endothelium, lymphocytes and macrophages (as reported in Wang et al. 2020) . Interestingly, specifi c gene markers of menstrual cycle progression denoted a shift of stromal subpopulations in defective decidualization present in previous sPE compared to controls. Particularly, a subset of stromal cells characterized by high expression of DKK1, IL15 and WNT5A, together with other decidualization markers -HAND2 and IGFBP3− was greatly reduced in previous sPE specimens. Altogether, the characterization of human endometrium at single-cell resolution revealed striking diff erences in cell composition between control and sPE group during decidualization. Conclusion: Our data reveal for the fi rst time a snapshot of defective decidualization in sPE at single-cell resolution. The imbalance in cell populations subtypes of stroma reveals a disrupted response to decidualization stimulus in vivo. This data might allow the identifi cation of potential molecular mechanisms underlying the uterine/endometrial pathogenesis underlying sPE. Funding: Grant RTI2018-094946-B-100 and PI19/01659 (MCIU/AEI/ FEDER, UE) ᶲBoth authors equally contributed. Deciphering the Role of PGRMC2 in Decidualization and Trophoblast Invasion Using Primary In Vitro Models. Yassmin Medina-Laver †, 1 Andrea Palomar †, 2 Pedro de Castro †, 2 Roberto González-Martín †, 1 Alicia Quiñonero * , 1 Francisco Domínguez * . 1,2 1 IVI Foundation-RMA Global, Valencia, Spain; 2 IIS La Fe, Valencia, Spain. Introduction: PGRMC2, a non classical progesterone receptor, is involved in ovarian and endometrial function, pregnancy maintenance and delivery. Concerning the human endometrium, our previous analysis in human endometrial stroma cells (EnSCs) suggests an important role of PGRMC2 in the implantation process, being signifi cantly upregulated in decidualized EnSCs (dEnSCs) compared to non-decidualized EnSCs (ndEnSCs). The main objective of this work was to study the role of PGRMC2 during decidualization and embryo invasion using a PGRMC2 inhibition approach in EnSCs. Methods: Regarding decidualization, PGRMC2 inhibited (iPGRMC2) and scramble control primary EnSCs obtained from healthy egg donors were in vitro decidualized with a combination of cyclic adenosine monophosphate (cAMP) and medroxyprogesterone 17-acetate (MPA) for 4 days (n=12). EnSCs were transfected with a pool of 3 diff erent PGRMC2 siRNAs (100nM/well) or scramble siRNA (100nM/well), using Lipofectamine 3000. Inhibition was checked by qPCR (n=6) and Western Blot (n=6). For invasion studies, an outgrowth model was carried out where primary EnSCs with iPGRMC2 and scramble control (n=5) were cocultured in vitro with human trophoblastic spheroids (JEG-3; n=30-45) after in vitro decidualization. Trophoblast cells were stained using Cell-Tracker fl uorescent probes. The trophoblast expanded area was observed by optical and fl uorescence microscopy at 24h and 48h and measured by ImageJ software. All decidualization procedures were checked by measuring secreted prolactin (PRL) by ELISA. Results: mRNA and protein analysis confi rm a >80% and >60% reduction of PGRMC2 expression and abundance in EnSCs transfected with PGRMC2 siRNAs. No statistically signifi cant diff erence was found in PRL secretion (decidualization) between non inhibited PGRMC2 dEnSCs (scramble control) and iPGRMC2 dEnSCs after in vitro decidualization for 4 days. However, when we analysed the trophoblast outgrowth, optical and fl uorescence microscopy measures revealed a signifi cantly reduction up to 70% of trophoblast expansion in iPGRMC2 dEnSCs, compared to scramble control dEnSCs (p<0.05). Conclusion: Our data showed that the inhibition of PGRMC2 did not aff ect PRL secretion in EnSCs, and thus, the decidualization process. Nevertheless, inhibition of PGRMC2 in dEnSCs promote a decrease in the area of the trophoblast expansion, suggesting a role of this non classical progesterone receptor during the embryo invasion process. However, further studies are needed to clarify the function of PGRMC2 during embryo implantation. Ovarian stimulation (OS), used for the development of multiple ovarian follicles for IVF, alters the fluctuations of estradiol (E2) and progesterone (P4) levels compared to natural cycles (NC). These hormonal changes presumably cause pathologic decidualization that negatively impacts endometrial receptivity. Endometrial receptivity and decidualization are highly regulated processes, and studies have identified adhesion G proteincoupled receptor G2 (ADGRG2) as a regulator of decidualization and as a marker of endometrial receptivity. Accordingly, ADGRG2 is included on the Endometrial Receptivity Array (ERA). Further investigation of ADGRs offers the potential to expand the panel of ADGRS, that can be used to improve IVF outcomes. Methods: Endometrial biopsies (EMB) were obtained from 17 subjects in natural cycles (n=8) in the early secretory phase (LH+1, n=4) and window of implantation (WOI: LH+8, n=4) and in OS cycles with a GnRH antagonist/hCG trigger protocol (n=9) in the early secretory phase (hCG+2, n=5) and WOI (hCG+9, n=4). Serum E2 and P4 levels were found to be significantly increased at hCG+2 versus LH+1 (p=0.002 and p=0.02, respectively). When comparing hCG+9 versus LH+8, only E2 was significantly increased (p=0.03). RNA was isolated from each biopsy and next generation sequencing and bioinformatics were conducted (Genomics Core, Albert Einstein College of Medicine) to identify differentially expressed genes (DEGs) between natural and OS cycles. P <0.05 was considered significant. Results: Among the DEGs, we observed significant changes in genes encoding four ADGRs (ADGRL1, ADGRL2, ADGRG1and ADGRG2). Among these ADGRs, there was significantly different expression between the early secretory phase and the WOI when comparing NC to OS cycles (Table 1) . that participants remained weight stable throughout the study. FSH and LH were measured by immunoassay (Siemens Centaur XP), during 2, 6-hour frequent blood sampling (q10 min) sessions in the early follicular phase (day 2-5) of the participants menstrual cycles, before and after the diet. At 4hrs, a 75 ng/kg GnRH bolus was administered, and sampling continued until 6hrs. A paired analysis was used to assess the impact of dietary intervention on baseline and GnRH-stimulated gonadotropin secretion. Results: As shown in the figure, average early follicular phase LH serum concentration at baseline was significantly lower after one month of high fat diet compared to pre diet levels (P < .001). Similarly, mean serum FSH concentration was significantly reduced after the dietary intervention (P < .003). Total serum LH response to exogenous GnRH treatment was also significantly attenuated post dietary intervention (P < .005). FSH also exhibited a reduced response to GnRH stimulation after high fat diet, but this did not achieve statistical significance (P = .08). Conclusion: Exposure of eumenorrheic, normal weight women to a one month eucaloric high-fat diet was sufficient to induce the hypogonadotrophic reprometabolic syndrome characteristic of obesity. These findings imply that specific circulating factors in response to a high fat diet are critical to the development of obesity-related reproductive endocrine dysfunction and its sequelae, and thus may be amenable to discovery and treatment by pharmacologic or dietary intervention. The pregnancy rate in MSC treated group was 60% to 100% after treatment and highest (100%) in low dose and mid dose group. The average number of pups were 4.5±2.9 in low dose group and 6.0±3.7 in mid dose group which are almost equal to that of healthy control (4.2±2.9). On the other hand, pregnancy rate in Exosome treated group was 30% to 50% after treatment, and highest in the high dose exosome group. The average number of pups were 5.5±0.7 in high dose exosome group. Interestingly, in the long-term effect, MSC treated mice still shows 60% to 80% of pregnancy rate in second round breeding. However, exosome treated group became infertile again in second round breeding. Conclusion: Our study provides a preliminary baseline data between MSC treatment and exosome treatment in POI mouse model. Our study revealed that exosome treatment is still promising but the sustenance of treatment is shorter than that of MSC. Our data support that the multi-dose regimen in exosome treatment is essential for long-term treatment effects. Introduction: Prorenin is present at high concentrations in ovarian follicular fluid and secreted by theca cells into the circulation. As it is the precursor of renin, we hypothesize that prorenin contributes to angiotensin synthesis, which is relevant in reproduction given the role of angiotensin in follicular development and oocyte maturation. Here we investigated whether periconceptional maternal prorenin levels associate with oocyte morphology, preimplantation embryo quality, as assessed by developmental time-lapse parameters and clinical treatment outcomes Methods: 309 couples with an indication for in vitro fertilization (IVF) treatment or intracytoplasmic sperm injection (ICSI) were included in the VIRTUAL EmbryoScope study, embedded in the Rotterdam Periconception Cohort. Embryos (n= 1,024) were cultured in a timelapse incubator. From embryos that were selected for transfer or cryopreservation, we retrospectively recorded the time of fertilization (t0), pronuclear appearance (tPNa) and fading (tPNf), as well as the exact timing of reaching the 2, 3, 4, 5, 6, 7-and 8-cell stage (t2-t8) , the start of blastulation (tSB) and reaching the full (tB) and expanded blastocyst stage (tEB). Embryo quality assessment was based on these morphokinetic features using the KIDScore algorithm. Oocyte morphology was determined by measuring the oolemma area at T0, tPNa and tPNf. Clinical treatment outcomes included pre-and post-transfer outcomes, such as number of follicles, fertilization, and implantation. Maternal prorenin plasma levels were determined at the day of embryo transfer (day 3 n=222 and day 5 n=87). We have previously shown that upregulation of miR-150-5p at as early as 12 weeks gestation can be used as a predictor of subsequent cervical shortening and/or preterm birth (PTB). We herein demonstrate the use of a Lateral Flow Assay (LFA) for the detection of miR-150-5p directly from patients' plasma as a tool to stratify those at risk of PTB, early in pregnancy. Methods: Oligonucleotide Templated Reactions (OTRs) were used whereby the miR of interest acted as a template for a highly sequence-specific reaction between two bespoke peptide nucleic acid (PNA) probes that result in the formation of a characteristic fluorescent signal. The PNAs were printed on the LFAs and the signal was detected using a bench-top fluorescence scanner (ESEQuant LFR, Qiagen). The plasma samples used were all collected between the 12 th to 14 th weeks of pregnancy, in pregnancies where outcomes were subsequently recorded (n=5 term, n=2 pre-term, n=3 short cervix who delivered at term following cervical cerclage). To improve miR extraction, a denaturing hydrogel-cellulose composite sample pad was developed to promote the on-chip miR release from the clinical samples. Data were compared using two-tailed student t-tests, where p<0.05 was considered statistically significant. Results: Using just 3µL of plasma directly on the LFA with a cellulose sample pad, the difference in prevalence of miR-150-5p in the plasma samples from patients at low and high risk of PTB was detected (p=0.0178). The area under the receiver operator characteristic curve (AUC) using this method was 0.692. Both the statistical significance when comparing the two groups and the AUC improved (p<0.0001 and 0.972 respectively) when the hydrogel-cellulose composite sample pad was incorporated. This was comparable to the results from RT-qPCR, the current gold-standard for miR detection, where p<0.0001 and AUC=1.000. Conclusion: This proof-of-concept study and preclinical validation represent the first steps towards a bedside test for the early prediction of preterm birth to identify women most at risk and to allow administration of outcome-modifying interventions. Introduction: Pregnancy includes complex cascades of events which need to be properly orchestrated for optimal health of the baby. Preterm birth (PTB) is a complication affecting 8-10% of pregnancies making it the main cause of infant mortality and morbidities. Inflammation is a key regulator of both term and preterm birth, however heterogeneity related to PTB has stunted advancements in identifying the critical immune and inflammatory changes involved. In order to better understand the maternal and placental contribution to PTB, we aimed to acquire an integrated view of the PTB syndrome via studies of immune and inflammatory profiles in women with term and preterm deliveries. We collected blood and placenta from 79 pregnancies (term and preterm) and characterized the immune and inflammatory profiles through FACS and ELISAs. Furthermore, we performed histopathological analysis of the placenta to identify structural/inflammatory lesions. To obtain an unbiased view of the transcriptome we performed placental RNA-sequencing of term and preterm placentas. Statistical analyses were done using GraphPad. Results: Maternal circulating immune cells were altered in preterm compared to term births with increase in proinflammatory monocytes and CD4+ lymphocyte (Th1) cells and a decrease in natural killer T cells. This pro-inflammatory shift was supported by elevated CRP with decreased cortisol and progesterone levels in the maternal circulation. Additionally, the placenta showed increases in TNF-a and IL-6. Placental histopathology revealed increased incidence of lesions (structural/inflammatory) in preterm versus term placentas. Transcriptomic changes in the placenta showed 331 genes that were differentially expressed (p<0.05, logFC 1<>1), of which 102 were up regulated in PTB. Using gene ontology enrichment analyses, these genes were found to be linked to inflammatory and immune biological processes. Conclusion: A clear pro-inflammatory shift was observed in the maternal circulation and placenta in association to PTB. Unbiased analysis of the placental transcriptome revealed a predominance of inflammatory pathways altered in PTB which, in combination with the changes observed in the maternal circulation, is key to identifying novel candidate markers and potential therapeutic targets for PTB. In light of these new findings and to further address the gaps in our understanding, we aim to further integrate our results to understand the contribution of each compartment (i.e. mother, placenta) and link our findings to infants' development. This would facilitate the identification of women/infants who could benefit from targeted anti-inflammatory therapeutic interventions. Single Second Trimester Cervical Length is Predictive of Preterm Delivery Among Patients with History-Indicated Cerclage. Sunitha C Suresh †, 1 Caitlin MacGregor, 2 Annie Dude * , 1 Emmet Hirsch * . 1 1 University of Chicago, Chicago, IL, United States; 2 NorthShore University Health System, Evanston, IL, United States. Introduction: Progressive shortening of the cervix in patients with prophylactic cerclage in place has been linked to early gestational age (GA) at delivery. Cervical length (CL) at anatomic survey is routinely obtained. The utility of a single CL measurement at second trimester ultrasound among those with history-indicated cerclage in place is unknown. Methods: This is a secondary analysis of a retrospective study of patients with two pregnancy outcomes at a single institution whose initial delivery both occurred preterm (< 37 weeks) and had placental pathology. Patients with a history-indicated cerclage in their subsequent pregnancy and a recorded CL at second trimester anatomy scan were included. Baseline characteristics were compared by short cervix (< 2.5 cm) using Fisher's exact and Wilcoxon rank sum as appropriate. Primary outcome was delivery < 32 weeks in subsequent pregnancy. Logistic regression was used to assess the risk of delivery at < 32 weeks by short cervix adjusting for GA at delivery in the index pregnancy Results: 77 pregnancies had any cerclage placement in the subsequent pregnancy. 24 were excluded due to ultrasound vs exam-indicated cerclage or abdominal placement. 3 did not have a 2 nd trimester CL. A total of 50 unique pregnancy pairs were included, representing 48 patients, of which 7 had a short cervix and 43 had a normal cervical length. Baseline characteristics were similar between groups (Table 1) . 57.1% of those with a short cervix delivered < 32 weeks compared with 2.3% of those with a normal CL (p= 0.001, Table 2 ). Median GA at delivery was 31.4 weeks in those with short cervix vs 37.1 weeks in those normal CL (p=0.03, Table 1 ). After adjustment for GA at initial delivery, short cervix at anatomy US was associated with an adjusted odds ratio of 48.2 for delivery at < 32 weeks (95% CI 3.9, 599.9). Conclusion: A short cervix at time of 2 nd trimester anatomy US among patients with history-indicated cerclage in place is associated with a significantly increased risk of delivery < 32 weeks. Future work is needed to determine optimal management strategies in these patients Results: The number of cardiac myocytes that exhibited spontaneous calcium release events with isoprenaline was increased in off spring of hypoxic pregnancy (Figure 1a ). There were more events per minute in cardiac myocytes collected from adult off spring of hypoxic pregnancy ( Figure 1b ). When measuring calcium wave amplitude, cells isolated from adult off spring of hypoxic pregnancy also had larger waves relative to those of normoxic controls ( Figure 1c ). Conclusion: Abnormal calcium handling was observed in off spring of hypoxic pregnancy indicating an increased risk of developing arrhythmia. Here we show that cardiac myocytes from adult off spring of hypoxic pregnancy have increased susceptibility to generate larger arrhythmogenic calcium waves and spontaneous calcium release events. Therefore, the data support the developmental programming of an increased risk of cardiac arrhythmia in adult off spring of hypoxic pregnancy. Mitochondrial health is crucial for normal glucose metabolism in skeletal muscles. In this study we sought to analyze mitochondrial structure, function and associated genes in skeletal muscles to explore molecular mechanisms of insulin resistance LP programmed female offspring. Methods: On day four of pregnancy, rats were randomly assigned to a control diet containing 20% protein, or an isocaloric 6% protein-containing diet. Standard laboratory rat chow was given to the dams after delivery until the end of weaning, and pups were given the standard laboratory rat chow after weaning. Mitochondrial ultra-structure, function, copy number and the expression of associated genes were analyzed from four months old female skeletal muscle samples. Results: Gestational LP diet led to changes in mitochondrial ultrastructure in the gastrocnemius muscles including, a 9-fold increase in the presence of giant mitochondria along with loss of cristae. Further, functional analysis using seahorse analyzer show that the LP programmed females have impaired mitochondrial function in flexor digitorum brevis muscles with a decrease in maximal respiration ( The fetal liver (FL) receives first pass nutrient and oxygen supply from the placenta, making it vulnerable to adverse intrauterine exposures from maternal western diet (mWD). Fetuses exposed to mWD suffer hepatic steatosis and oxidative stress. However, the substrate delivery and metabolic pathways that drive these FL effects, or effects on gluconeogenesis (GNG) and nutrient sensing pathways, are unknown. We hypothesized that alterations in umbilical fuel mix underlie key metabolic changes in the FL due to mWD. Methods: Adult non-human primate females were fed a control (CD) or WD for >3 yr before and during pregnancy. C-sections were done at 0.65 gestation to collect umbilical vein (UV) and artery (UA) blood and FLs. UV and UA were analyzed for blood gas (iSTAT), insulin (ELISA), amino acids (AA, HPLC) and metabolites (UHPLC-MS). In FL tissue and isolated hepatocytes, gene (qPCR) and protein expression (western blot) were measured. Significance was declared as P<0.05 by t-test. Results: Glucose and markers of insulin sensitivity were similar in CD and mWD fetuses, and mWD fetuses had lower UV and UA pO 2 and sO 2 . mWD fetuses had decreased total AA uptake driven by lower uptake (UV-UA difference) of Leu, Ile, Val, Asn, Pro, Phe, Glu, and Ser, representing branched chain, glucogenic, and AAs used for antioxidant synthesis. In UV, a-ketoglutarate and antioxidant pathway metabolites (dehydroascorbate, GABA, Cys-Gly) were decreased. In UA, oxidative stress metabolites (lactate, urate, uracil, 5,6-dihydrouracil) were increased. mWD FLs had decreased expression of genes driving glutathione synthesis from Glu and Ser (GLUD1, PSAT1, SHMT, PSPH, GGT1, GSS), and anaplerosis (PC). Further, isolated mWD hepatocytes had increased GNG gene expression (PCK1, G6PC, PGC1A) and glucose production. Hypoxic treatment further increased GNG gene expression in CD and mWD hepatocytes. mWD hepatocytes had decreased S6 protein phosphorylation, a target of the nutrient sensor mTOR, which was further decreased with hypoxia treatment. In the fetus, mWD decreases AA uptake and produces mild hypoxia. We speculate that lower AA uptake restricts FL antioxidant synthesis, anaplerosis, and substrate supply for GNG in vivo. In addition, we speculate that mWD-associated hypoxia coupled with lower AA uptake decreases FL mTOR and S6 activity, which in turn activates PGC1A, potentiating GNG in vitro. These findings support that mWD impacts FL metabolism and may increase postnatal liver disease risk. Heterogeneity in the Genetic Basis of Preterm Delivery and Gestational Duration and their Links with Other Pregnancy and Reproductive Traits. Bo Jacobsson * , on behalf of the EGG Consortium. Gothenburg University, Gothenburg, Sweden. Introduction: The timing of parturition is crucial for neonatal survival and infant health. Despite the clinical relevance of preterm delivery, the biological mechanisms behind the timing of parturition remain largely unresolved, partly due to the diversity in the biological and mechanical triggers involved. Adding to the complexity, while gestational duration is a major determinant of birth weight, this relationship can be affected by conflicts between the maternal and fetal genomes. Methods: To understand the genetic heterogeneity of gestational duration and preterm delivery, and their association with other reproductive and pregnancy traits, we performed the largest GWAS meta-analysis of gestational duration (n= 192 085) and preterm delivery (n= 279 43, cases= 18 797) using maternal samples. We then compared the GWAS to each other and to the genetic effects on birth weight and other female reproductive outcomes. The genetic effects on other traits were obtained from previously published GWAS meta-analysis. Results: We identified 19 independent loci not previously reported, one of which is exclusively linked to preterm delivery. Index SNPs were in close proximity to loss-of-function intolerant genes (p= 8.1e-13), and tissue-specific gene enrichment analysis highlighted the endometrium and smooth muscle. Reproductive traits genetically correlated with gestational duration were also correlated with preterm delivery, while the opposite was not true. We observed a sex-specific genetic correlation and causal effect of testosterone, bioavailable testosterone and sex-hormone binding globulin on preterm delivery. We finally show a complex genetic relationship between the maternal and fetal effects on gestational duration and birth weight. Maternal and fetal effects on gestational duration showed positive genetic correlation with maternal only effects on birth weight (p= 3.4e-31 and 6.1e-8, respectively); no correlations were observed with fetal only eff ects on birth weight (p= 0.216 and 0.761, respectively). Using a haplotype-based polygenic score to instrument fetal growth (n= 16 246 trios), the paternal transmitted polygenic score was negatively associated with gestational duration (beta= -3.2, p= 4.1e-4), suggesting that fetal growth has a negative impact on pregnancy duration. The maternal eff ects on birth weight were partly mediated by the maternal eff ects on gestational duration, and the maternal gestational duration increasing alleles had negative fetal eff ects on birth weight. Conclusion: We confi rm genetic heterogeneity between gestational duration and preterm delivery, which is mirrored in their links with other female reproductive traits, particularly, sex hormones. The results show a complex genetic relationship between the maternal and fetal eff ects on gestational duration and birth weight, likely infl uenced by co-adaption between the two genomes. which may underlie the intrauterine origins of obesity and diabetes. The extent to which diet quality is associated with placental signaling, and which specifi c pathways are impacted is unknown. The objective of the current study was to examine the sex-specifi c association of maternal diet quality according to the Healthy Eating Index (HEI) -developed to align with recommendations from the Dietary Guidelines for Americanswith placental proteins involved in metabolism and environmental stress/ infl ammation/growth factor mediators. Methods: Among 108 women from the Healthy Start cohort with a mean (SD) age of 29.0 (6.1) and pre-pregnancy BMI of 24.8 (5.3) we conducted multivariable linear regression analysis stratifi ed by off spring sex. We adjusted for maternal race or ethnicity, age, education, prenatal smoking habits, and physical activity and tested for an association of maternal HEI>57 vs. ≤57 and the abundance and phosphorylation of key proteins involved in insulin/growth factor signaling, mediators of environmental stress/infl ammation/growth factors, mechanistic target of rapamycin signaling proteins, and energy sensing in placental villus samples. HEI> 57 was chosen given its prior relevance among Healthy Start mother-child dyads. Conclusion: Higher quality diet was associated with placental protein abundance and phosphorylation in a sex-specifi c manner. Given that these proteins have been correlated with neonatal anthropometry, our fi ndings provide insight into modifi able factors and placental pathways that should be examined in future studies as a potential links between maternal diet and off spring metabolic health. Introduction: Selective serotonin reuptake inhibitor (SSRI) exposure during pregnancy may be associated with increased risk of small-forgestational-age (SGA; birth weight <10th percentile for gestational age). Previous studies may not have adequately accounted for potential confounding-by-indication and associations may vary by timing of SSRI exposure. We aim to evaluate associations of SSRI exposure and timing of SSRI exposure in pregnancy with the risk of SGA among women with depression prior to becoming pregnant. Methods: We conducted a retrospective cohort study among women enrolled in Providence St. Joseph Health throughout their pregnancy and delivered singleton live births from 2013 -2020 (n=284,436). Among women with a history of depression, we identified women with depression diagnosis or ≥1 antidepressant prescription order ≤180 days before pregnancy (n=6,118). Women with no SSRI order during pregnancy (n=2, 980) constituted the unexposed group (no SSRI exposure group). The late SSRI exposure group consisted of women with an SSRI order after the first trimester (n=2,488). The early-only SSRI exposure group consisted of women with SSRI orders only in the first trimester (n=650). We used logistic regression to calculate odds ratios (ORs and corresponding 95% confidence intervals, CI) of small-for-gestational age using propensity score adjusted models. We also addressed the confounding impact of depression severity and concomitant use of SSRI and non-SSRI antidepressant. Results: We identified 6,118 study participants; 12.4% of babies born were small-for-gestational age, mean gestational age at birth was 271.9 days, mean maternal age was 30.1, 76.1% were white, and 60.1% were Medicaid or Medicare insured. The late SSRI exposure group was not associated with increased risk for small-for-gestational age (OR=1.2 [0.9,1.5], p=0.2), relative to the no SSRI exposure group. Early-only SSRI exposure was at equal risk for small-for-gestational age (OR=1.0 [0.6, 1.5], p=0.9) when compared to the no SSRI exposure. Conclusion: To our knowledge, this is the first study to minimize confounding by indication (e.g. depression), characterize impact of exposure timing, and account for depression severity using standardized assessment (Patient Health Questionnaire; PHQ). Our findings suggest that intaking SSRI during pregnancy does not increase the risk for small-forgestational age. The next steps include investigation on fetal sex-specific differences and drug-specific analysis. ancillary study visit 2 from 2015-2018. At visit 2, HDP occurring prior to visit 1 were retrospectively assessed and defined as: hypertension or high blood pressure first diagnosed in pregnancy (GH), preeclampsia (PE), or eclampsia. Change in cognitive function was defined as the difference in cognitive score from HCHS/SOL visit 1 to visit 2 in the following tests: Brief-Spanish English Verbal Learning Test (B-SEVLT), Digit Symbol Substitution (DSS) and Word Fluency (WF). We used linear regression analysis to determine whether HDP was associated with change in cognitive function adjusting for: socio demographic characteristics, clinical and behavioral risk factors, follow-up time and baseline cognitive score. All analyses accounted for the complex survey design of the HCHS/SOL. Results: At visit 1, mean age was 56.2 (SE: 0.2) years old with 13.5% of participants reporting at least one HDP, with GH being the most common (11.1%) followed by PE (4.9%) and eclampsia (1.2%). After an average of 7 years of follow-up, executive function (measured by DSS) decreased an average of 2.2 units, with greater decreases among participants with GH versus without GH (ß:-1.02, 95% CI:-1.94, -0.10). Fully adjusted associations were not significant for other cognitive domains or with PE or eclampsia. Conclusion: Decreases in Cognitive function as measured by DSS were greater among US Hispanics/Latinas with GH. Our results suggest that GH, the most common identified HDP, may be an independent risk factor for cognitive decline later in life. A larger sample size may be needed to establish the relationships between PE and E and cognitive decline and reduce the risk of a type II error. Batoul Hojeij †, 1 Sam Schoenmakers, 1 Lenie Van Rossem, 1 Sten P. Willemsen, 1 Andras Dinnyes, 2 Melek Rousian, 1 Régine Steegers-Theunissen. 1 1 Erasmus MC, Rotterdam, Netherlands; 2 BioTalentum, Godollo, Hungary. Introduction: Lifestyle behaviours, including diet, contribute to the achievement of a successful pregnancy. Assessment of the perceived attitudes and practices can aid in identifying gaps in unhealthy behaviours and intervention effectiveness. The coaching program www. SmarterPregnancy.co.uk (SP) provides information, raises awareness and empowers the user to adopt and maintain healthy lifestyle behaviours. Therefore, we aim to investigate the impact of SP coaching on lifestylerelated attitudes and practices during the periconception period. Methods: 1,691 women contemplating pregnancy were selected from a randomized controlled trial in an assisted reproductive technology (ART) population and a periconceptional cohort and survey in natural conceived and ART pregnancies. To study attitudes, women were analyzed for their intention to increase fruit and vegetable intake, as well as smoking cessation. Actual practices were studied in terms of fruit and vegetable intake, smoking, alcohol and folic acid supplement use. Outcomes were compared at 12 and 24 weeks of the intervention (coaching period) in the ART population with (ART-I) and without SP intervention (ART-C) and between the ART-I and natural conceived pregnancies with SP intervention (NAT-I). Analysis was performed using regression models adjusted for maternal covariates and relative baseline attitudes and practices. Results: Compared to ART-I, ART-C had higher odds for negative attitude towards vegetable intake and showed a relatively lower intake at weeks 12 (OR adj 4.13, p<.001; β adj -25.719, p<.001, respectively) and 24 (OR adj 3.60, p<.001; β adj -23.836, p<.001, respectively), indicating less improvement, whereas no difference was observed for the other lifestyle behaviors. Considering NAT-I, differences were only observed in alcohol use at 24 weeks, being higher compared to ART-I (OR adj 2.76, p=.001). Compared to overall SP users with positive attitude, SP users with negative attitude towards vegetable and fruit intake at week 12, had lower intakes at week 24 (β adj -0.494, p<.001; β adj -30.065, p<.001, respectively), whereas no difference was observed for smoking. Conclusion: SP coaching improved vegetable intake-related attitudes and practices in subfertile women undergoing ART treatment. We recommend Introduction: Early in pregnancy, placental cells known as trophoblast stem (TS) cells differentiate and invade into the uterus, helping to facilitate spiral artery remodeling. In the human, invasive trophoblast cells are termed extravillous trophoblast (EVT) cells. When trophoblast invasion is inadequate, pregnancy complications such as intrauterine growth restriction, preeclampsia, and preterm birth can occur. Little is known about regulatory mechanisms controlling TS cell differentiation into EVT cells. Cyclin dependent kinase inhibitor 1C (CDKN1C) has been implicated in the regulation of placentation and its dysregulation linked to placental disease, including those associated with impaired EVT cell function. Human TS cells represent an excellent model for investigating regulatory mechanisms controlling EVT cell development. We hypothesized that CDKN1C influences human EVT development. Methods: CDKN1C expression was evaluated in first trimester human placental tissue using in-situ hybridization. Expression was also measured in human TS cells maintained in the stem state and following EVT differentiation using RT-qPCR and western blotting. Roles for CDKN1C in human TS cell development were investigated using a loss-of-function approach with short hairpin RNAs. Trophoblast cell responses to CDKN1C silencing were determined through assessments of cell morphology, transcript profiles, and DNA content. Results: CDKN1C mRNA and protein expression were mapped to invasive trophoblast cells in first trimester human placental tissue and in human TS cells. EVT cells exhibited dramatically higher levels of CDKN1C mRNA and protein compared to TS cells. Short-hairpin RNAs were effectively used to knockdown CDKN1C in human TS cells. Knockdown EVT cells also exhibited increased proliferation and impairments in EVT cell differentiation. Elongated cell morphology, which is a hallmark of EVT cell differentiation, was observed in control cells but not in CDKN1C knockdown EVT cells. RNA (Tong et al. SRI 2021) . Here, we tested the hypothesis that hypoxic pregnancy in sheep alters placental mitochondrial function, structure and metabolism. Methods: Pregnant ewes were exposed to normoxia (N; n=10) or 10% O 2 hypoxia (H; n=7) from 105 to 138 days of gestational age (dGA; term ~ 145 dGA) in isobaric chambers. At 138 dGA, placentomes were collected for molecular, transmission electron microscopy and respirometry analyses. Data were compared statistically via the Student's t test. Results: Chronic hypoxia activated indices of the mitochondrial unfolded protein response, showing increased levels of ATF5, HSP60 and TID1 ( Fig.1A-C) . Relative to N controls, mitochondria from H placentae were smaller, and showed decreased markers of mitochondrial fusion OPA1 and MFN2 (Fig.1D) , with loss of the characteristic cristae structure (Fig.1 E&F) . Relative to N controls, respirometry showed that H placentomes maintained total OXPHOS capacity but had a decreased flux control ratio for fatty acid oxidation(FCR F ), and a decreased capacity for octanoyl carnitine oxidation relative to pyruvate (SCR FA/P ; Fig.1G&H ). Relative to N controls, H placentomes also showed increased IGF2 and GLUT3 transcription and activation of AKT signalling through PDK1 and AKT phosphorylation ( Fig.1I-L) . Finally, relative to N controls, H placentomes showed increased levels of the adipokine adiponectin and fatty acid transporter CPT1, and decreased levels of the fatty acid synthesis enzyme ACC ( Fig.1M -O). The data support the hypothesis that hypoxic pregnancy in sheep alters placental mitochondrial metabolism and morphology. These alterations are compensatory, maintaining cellular respiration by switching from ß-oxidation to glucose metabolism, and triggering adaptive responses to modulate placental lipid and glucose metabolism. Placental Endothelial Cells in Severe Fetal Growth Restriction Exhibit Aberrant Adhesion to Collagen IV. Lauren Sayres †, Shuhan Ji, Carla Rey Diaz, Emily J Su * . University of Colorado, Aurora, CO, United States. Introduction: Severe fetal growth restriction with abnormal Doppler velocimetry (FGRadv) leads to significant morbidity and mortality. Impaired placental vascular development is a central finding in these cases, but its underlying mechanism is poorly understood. We have previously demonstrated that extracellular matrix (ECM) generated from placental stromal fibroblasts contains high abundance of fibronectin, collagen I and IV, laminin, and fi brinogen and that the ECM regulates endothelial cell (EC) angiogenic properties. Our objective was to determine how each individual ECM substrate impacts EC adhesion, which is a critical fi rst step in the process of angiogenesis. We hypothesized that adhesion of ECs to collagen IV and laminin, essential basement membrane proteins, is impaired in the setting of FGRadv. Methods: ECs were isolated from human placentae, either term controls (n=6) or those aff ected by FGRadv (n=6), immediately after delivery. ECs were cultured for one hour in wells individually coated with fi bronectin, collagen I, collagen IV, laminin, or fi brinogen. Cell staining and colorimetry were used to determine the degree of adhesion of ECs to each ECM substrate. Optical densities at 560nm were normalized to negative control wells containing bovine serum albumin (BSA). Technical triplicates were conducted. Parametric t tests were performed for statistical comparison. Results: Adhesion to collagen IV of ECs from FGRadv placentae was signifi cantly decreased as compared to adhesion of control ECs (p=0.049). In contrast, there were no diff erences between FGRadv and control ECs in adhesion to fi bronectin, collagen I, laminin, or fi brinogen. Conclusion: EC adhesion to collagen IV is impaired in FGRadv and could be an underlying mechanism for defi cient angiogenesis in this setting. One explanation is diminished EC affi nity for integrins that specifi cally bind this critical basement membrane protein, which represents an avenue for further study. Future research will also investigate the eff ects of individual ECM proteins on other EC angiogenic properties such as migration and apoptosis. By advancing our knowledge of EC-ECM interactions, we may uncover interventional targets in pregnancies at risk of or aff ected by FGRadv. Circadian Rhythms in the Fetus and the Placenta Develop and Change During Gestation. Keenan Bates †, Elizabeth Sapiro, Jacob Amme, Ron McCarthy, Sarah K Engalnd, Erik D Herzog * . Washington University in St. Louis, Saint Louis, MO, United States. Introduction: Circadian rhythms in gene expression and hormones are ubiquitous across species and across cell types. Circadian rhythms depend on a transcription-translation negative feedback loop that drives many daily rhythms in behavior and physiological processes. In mammals, a master circadian pacemaker in the hypothalamus regulates circadian clocks throughout the brain and body. Mammalian pregnancy presents a unique situation with circadian rhythms in the mom and, eventually, circadian rhythms in the fetus. We and others recently found that circadian rhythms arise in the fetal mouse SCN around embryonic day 14.5 (E14.5, where E0 is the day of conception). We hypothesized that fetal circadian rhythms arise during pregnancy and synchronize to the mother prior to birth via maternal-to-fetal circadian communication. All prior studies examining fetal clock gene expression were either limited to in vitro physiology from tissue harvested at identifi ed embryonic stages or to in vivo histology from tissues pooled from pups within a litter. Methods: In order to test our hypothesis, we monitored the development of daily rhythms in the fetus in utero using mice expressing a luciferase reporter for PERIOD2, a core clock protein (PER2::Luc). We imaged PER2::Luc fetuses at least twice a day from E8.5 to E17.5 across the same pregnancy. This allowed us to determine the gestational stage at which clock gene expression begins, starts its daily cycle, and if it changes (e.g. the amplitude or time of daily peak) in utero during gestation. Additionally, we examined whether circadian rhythms in the placenta change over the course of pregnancy because of its likely role in facilitating maternal-tofetal communication. Using PER2::LUC tissue explants, we examined the circadian rhythms in placental cross-sections and isolated explants of the three layers of the placenta (decidua, junctional zone, and labyrinth zone) at diff erent stages of gestation, E9, E15, and E18. Results: We found that the bioluminescence derived from the embryos was detectable from the fi rst day of imaging, E8.5, before the fetal SCN forms. Fetuses exhibited consistent daily rhythms beginning on E12 and those that failed to sustain circadian rhythms were aborted. We found that all three layers of the placenta exhibited circadian rhythms at each stage of pregnancy, but each layer had a diff erent phase for the timing of their peak signal. Daily period length decreased, and amplitude increased between E15 and E18 in cross-sections of the placenta but not in individual layers. Conclusion: We conclude that diurnal variation of fetal PER2 expression is detectable around E11.5 and that each layer of the placenta exhibits circadian rhythms at E15 and E18 but with diff erent phases for their peak expression. This work was supported by the NIH F31HD104307. Placenta Specifi c Knockdown of MFSD2a, a Lysophosphatidylcholine Transporter, Results in DHA Defi ciency in the Fetal Brain. Theresa L Powell, Kenneth Barentsen, Owen Vaughan, Charis Uhlson, Karin Zemski-Berry, Kathryn Erickson, Kelsey Barner, Thomas Jansson * . Univ of Colorado, Aurora, CO, United States. Introduction: Docosahexaenoic acid (DHA) is a n-3 long chain polyunsaturated fatty acid critical for fetal brain development. DHA is not produced in the fetus or placenta and must therefore be transported across the placenta from the mother. The mechanisms by which DHA is transferred to the fetus are unclear. We reported that cord blood lysophosphatidylcholine (LPC)-DHA correlates with syncytiotrophoblast basal plasma membrane expression of the LPC transporter, MFSD2a (Major Facilitator Superfamily Domain Containing 2a). This data suggests that the placenta delivers DHA to the fetus as LPC-DHA, which is the form in which DHA is transported across the blood-brain barrier. We hypothesized that placenta specifi c knockdown of MFSD2a reduces DHA accumulation in the fetal brain. Methods: Mouse blastocysts collected at E3.5 were transduced (4 hr) with an EGFP-expressing lentivirus containing shRNA targeting MFSD2a or a scrambled control shRNA (SCR), then transferred to pseudopregnant females (20 per recipient). At E18.5, conceptuses were weighed, then placenta, and fetal brain, liver and plasma were collected. MFSD2a mRNA expression was determined by qPCR and phospholipids were extracted and quantifi ed by LCMS. Results: Implantation rate was within the normal range for experimental embryo transfer (~ 32%, n=27 dams). EGFP was expressed in the placenta but not the fetus. Compared to SCR (n=34 pups), the MFSD2a-targeting shRNA knocked down placental MFSD2a expression by 38% (n=45, P<0.008) but did not aff ect MFSD2a expression in fetal brain or liver. Fetal brain weight was reduced by 14% (p=0.007) whereas placental and liver weights were not impacted. Placental MFSD2a mRNA expression correlated with phosphatidyl choline DHA in the fetal brain (R 2 =0.55, P=0.014). LPC-DHA was low in fetal brain of SCR pups, likely due to rapid re-esterifi cation to phosphatidyl choline, and was undetectable in all pups with placental MFSD2a knockdown. Loss of MFSD2a in the mouse placenta resulted in smaller fetal brains deficient in phospholipid forms of DHA and completely lacking LPC-DHA. This data provides mechanistic evidence that placental MFSD2a mediates maternal-fetal transfer of LPC-DHA. We speculate that placental MFSD2a is critical for normal brain growth and long term cognitive function in the offspring. Varying Degrees of Fetal Growth Restriction in Chorionic Somatomammotropin RNA Interference Pregnancies. Amelia R. Tanner †, 1 Victoria C. Kennedy †, 1 Cameron S. Lynch †, 1 Quinton A. Winger, 1 Paul J. Rozance, 2 Russell V. Anthony * . 1 1 Colorado State University, Fort Collins, CO, United States; 2 University of Colorado School of Medicine, Aurora, CO, United States. Introduction: Previously, we reported varying degrees of fetal growth restriction (FGR) in response to chorionic somatomammotropin (CSH) RNA interference (RNAi) near term, which mirror responses due to CSH gene mutations in humans. The varying phenotypic severities can provide insight into the development of CSH RNAi induced FGR by determining which features are present in all CSH RNAi animals and which are only present in the most severely growth restricted. Thus, we set out to document the distinctions between CSH RNAi pregnancies of differing severity. Methods: To generate the pregnancies, the trophectoderm of hatched sheep blastocysts (9 days of gestational age; dGA) were infected with a lentivirus expressing either a scrambled control (CON RNAi; n = 6) or CSH-specific shRNA (CSH RNAi; n = 8), prior to transfer into recipient sheep. At 126 dGA, pregnancies were fitted with vascular catheters. At 136 dGA, pregnancies underwent steady-state metabolic studies with the 3 H 2 O transplacental diffusion technique. Tissues were subsequently harvested. While all 8 CSH RNAi pregnancies had FGR (fetal weights 2 SD below CON RNAi), a subset of pregnancies (CSH RNAi PI-IUGR) also had placental insufficiency (n = 5; placental weight 2 SD below CON RNAi). Data were analyzed by Student's T-test. Results: Fetal and placental weights were reduced (P ≤ 0.05) by 21% and 37% respectively in CSH RNAi pregnancies, and by 30% and 52% in CSH RNAi PI-IUGR pregnancies. Uterine and umbilical blood flows (mL/min) were reduced (P ≤ 0.05) by 35% and 28% respectively in CSH RNAi pregnancies, and by 41% and 42% in CSH RNAi PI-IUGR fetuses, resulting in reduced umbilical uptakes (P ≤ 0.05) of oxygen, glucose, lactate, and amino acids in both CSH RNAi groups. Fetal liver, heart, and kidney mass were reduced (P ≤ 0.05) in both CSH RNAi groups, however, the CSH RNAi PI-IUGR group also had reduced (P ≤ 0.05) crown-rump length, lower leg length, and spleen weight. Cotyledonary CSH protein concentrations were reduced (P ≤ 0.05) 24-30% in both CSH RNAi groups. However, only the CSH RNAi PI-IUGR group had reduced (P ≤ 0.05) uterine vein CSH concentrations. Furthermore, while both CSH RNAi subgroups had reduced umbilical insulin concentrations (P ≤ 0.05), only CSH RNAi PI-IUGR fetuses had elevated (P ≤ 0.05) cortisol. Conclusion: It appears that the direct actions of CSH are to promote blood flow and nutrient uptake by the uteroplacental unit as present regardless of degree of severity of placental insufficiency. However, in cases where CSH RNAi has a greater impact on placental weight, fetal distress is greater, and fetal growth is more impaired. This suggests that the actions of CSH RNAi on the placenta determines the severity of growth restriction. Supported by NIH HD093701. The Guinea Pig Embryo: A Potential New Model for Human Development. Jesica Canizo †, 1 Vandal Katherine †, 1 Biondic Savana †, 1 Sophie Petropoulos * . 1,2 1 University of Montreal, Montreal, QC, Canada; 2 Karolinska Institutet, Stockholm, Sweden. Introduction: In Canada, 1 in 6 couples experience infertility. Knowledge of the molecular and cellular processes that control human preimplantation development would help to better understand and treat infertility. Mouse embryos have historically been used to model reproduction, but caution is warranted as several discrepancies with the human exist during preimplantation. Guinea pigs have been a long standing model of human development and are known to better recapitulate placentation and brain development compared to the mouse. We now speculate that guinea pigs may also represent a superior animal model to study preimplantation development. However, to date, guinea pig preimplantation embryos remain to be investigated in great detail. We now aim to assess the developmental dynamics of the guinea pig preimplantation embryo with cross-species comparisons to both human and mouse. Methods: In vivo guinea pig embryos were flushed on embryonic days (E)3-E6. Immunofluorescence of Sox2 (epiblast (EPI) marker), Sox17 and Gata6 (primitive endoderm (PE) marker) and Cdx2 and Gata3 (trophectoderm (TE)) was performed. Cells were counted and the dynamics of lineage marker expression and blastocyst formation were determined. In parallel, scRNA-seq of guinea pig embryos was performed. Results: At the compacted morula stage (late E4.25-E4.75), similar to the human and in contrast to the mouse, we observe co-expression Sox2, Gata6 and Gata3, with no detection of Sox17 and Cdx2. Similar to the human, blastocyst formation begins between E5.0-E5.25. By mid-blastocysts (E5.25-E5.5), cells are poised for lineage specification with a high number of cells co-expressing Sox2/Sox17, and no cells coexpressing markers of all three lineages. By late blastocyst, E5.5-E5.75, specification of all three lineages is achieved and no cells co-expression Sox2/Sox17/Cdx2 or Sox2/Gata6/Gata3. Further, similar to the human and in contrast to the mouse, there is a disconnect between morphology and lineage specification in the early blastocyst. These data suggest that signaling pathways governing lineage specification in the human and guinea pig differ compared to the mouse. Finally, cross-species analysis of scRNA-seq revealed conserved pluripotency gene signatures between the human and guinea pig embryos, suggesting that the guinea pig may also contain naive stem cells and that plasticity of these lineages may be more similar to the human compared to the mouse. Conclusion: Preimplantation guinea pig embryos appear to better recapitulate the human embryo compared to the mouse and as such offer a promising new animal model to study preimplantation insults and longterm consequences, naive pluripotency and infertility. Insights from this study have broad application in the fields of Reproduction, Development, ART and Stem cell biology. Untargeted Metabolomic Identification of Diagnostic Biomarkers in Ectopic Pregnancy. Onur Turkoglu †, 1 Robert Quinn * , 2 Stewart F Graham * , 1 Ray Bahado-Singh * . 1 1 Beaumont Health / Oakland University School of Medicine, Royal Oak, MI, United States; 2 Michigan State University, Lansing, MI, United States. Introduction: Ectopic pregnancy (EP) is a potentially life-threatening condition and early diagnosis still remains a challenge, causing a delay in management leading to tubal rupture. Targeted metabolomics has been shown to identify novel biomarkers for the detection of ectopic pregnancy. Using untargeted metabolomics approach, we sought to identify putative plasma biomarkers for the detection of tubal EP compared to intrauterine pregnancies. Methods: This case-control study included prospective recruitment of 50 tubal EP cases and 50 early intrauterine pregnancy controls. Plasma samples were biochemically profiled using tandem liquid chromatographymass (MS/MS) spectrometry with untargeted metabolomics approach. To avoid over-fitting, datasets were randomly divided into a discovery group (30 cases vs 30 controls) and a test group (20 cases and 20 controls). Logistic regression models were developed in the discovery group and validated in the independent test group. Molecular networking and metabolite identification was employed using Global Natural Products Social Molecular Networking (GNPS) data base. Univariate and multivariate analysis were performed via MetaboAnalyst 5.0. Results: In total 585 molecular features were identified. 46 metabolite concentrations were significantly altered in EP plasma (p<0.05). Metabolomic profiling yielded significant separation between EP and controls (p<0.05) ( Figure 1 ). Independent validation of a two-metabolite model consisting of D-erythro-sphingosine and oleoyl-carnitine, achieved an AUC (95% CI) = 0.962 (0.910-0.1) with a sensitivity of 100% and specifi city of 95.9%. Molecular feature networking revealed signifi cant alterations in glycerol phosphocholine pathway and depleted levels of sphingolipids. Conclusion: We report novel untargeted metabolomic biomarkers with a high accuracy for the detection of EP for the fi rst time. Accurate biomarkers could potentially result in improved early diagnosis and better understanding the metabolism of tubal EP cases. Introduction: Early pregnancy loss is a common complication of human gestation, occurring in over half of all women trying to conceive. Although most pregnancy loss is unexplained, dysregulation of the immune system has emerged as a contributing factor for some women. Glucocorticoids are master regulators of the immune system via their genomic eff ects, and we determined that the uterine-specifi c eff ects of glucocorticoids are critical for the establishment of pregnancy in mice, using a targeted deletion model. Importantly, we found signifi cant disruption to the genes essential for the infl ammatory response and immune cell traffi cking in the mice defi cient for uterine glucocorticoid receptor (GR) signaling. The purpose of this study was to evaluate the conserved functions of GR in the human endometrium during essential processes at the establishment of pregnancy. Methods: To identify glucocorticoid target genes, primary human endometrial stromal cells (ESC) were treated for 6 hours with glucocorticoids (100nM dexamethasone). To determine the role of GR in human stromal cell decidualization, ESCs were transfected with non-targeting control (NTC) or siRNA targeting the GR gene (NR3C1; GRKD) and decidualized with 10nM estradiol, 100nM progesterone, and 0.5mM dibutyryl cAMP. Transcriptional changes were evaluated by microarray analysis and validated with independent biological replicates. Morphological changes induced by decidualization were monitored by phase contrast microscopy and biomarker assessment. Results: In human ESC, glucocorticoids signifi cantly regulate 1,483 genes, while knockdown of GR in ESC leads to signifi cant changes in the transcript levels of 3,815 genes. Gene ontology analysis of both gene lists identifi ed "Reproductive System Disease" as a highly enriched canonical pathway. In response to the decidualization stimulus, 1479 genes were signifi cantly regulated in NTC control cells (p<0.05), of which only 721 genes were also regulated when GR was knocked down in ESC. The top diff erentially regulated genes were independently validated and found to have functions related to the immune system (somatostatin-SST and interleukin 15-IL15) and cellular diff erentiation (twist-related protein 2-TWIST2 and heart and neural crest derivatives expressed 2-HAND2). Moreover, the morphological changes associated with decidualization and the induction of decidualization markers were largely absent in GRKD ESC (induced prolactin-PRL was <5% in GRKD ESC compared to NTC ESC; p<0.0001). Conclusion: These studies demonstrate that physiological uterine glucocorticoid signaling is required for the critical fi rst steps of pregnancy in human endometrial stromal cells by regulating the expression of key target genes. Exposure Previous studies have shown that bone marrow-mesenchymal stem cells (BM-MSCs) secrete various bioactive products that regulate cellular processes essential for tissue regeneration. We have previously shown that exposure to the BM-MSC secretome upregulates human endometrial stromal cell (HESC) migration and invasion in vitro; however, the mechanisms responsible remain unknown. We hypothesize that the secretome of human BM-MSCs activates genes important for HESC cell motility, including those involved in epithelial-mesenchymal transition (EMT). Methods: MSCs were cultured from the bone marrow aspirate of a healthy female donor (BM-MSC-1) or purchased from ATCC (BM-MSC-2). Telomerase-immortalized HESCs (T-HESCs) were exposed to the BM-MSC secretome via indirect co-culture for 24hrs, total RNA was extracted from T-HESCs and mRNA sequencing (mRNA-Seq) performed to identify diff erentially expressed genes (DEG) and altered pathways. MSigDB was used to identify the top 15 enriched biological pathways (padj < 0.05). RT-qPCR was performed to validate changes in mRNA expression of DEG common to both BM-MSC exposures and statistical signifi cance was defi ned as p<0.05. Results: Exposure of T-HESCs to BM-MSC secretome altered expression of 6099 genes in T-HESCs exposed to BM-MSC-1 and 8281 genes in T-HESCs exposed to BM-MSC-2 (padj < 0.05), with 4351 genes overlapping. Pathway analysis revealed that 9 of the top 15 signifi cantly enriched pathways in T-HESCs exposed to BM-MSC were common to BM-MSC-1 and BM-MSC-2, including the EMT pathway. Within the top 20 up-and down-regulated signifi cant DEG, 3 cell motility genes, HGF and CCL-2 (up), and GDNF (down), were common to both BM-MSC exposures. RT-qPCR confi rmed that T-HESC expression of HGF and CCL-2 was signifi cantly increased by BM-MSC-1 (1.8-fold and 7.7fold, respectively) and BM-MSC-2 (1.3-fold and 5-fold, respectively) secretomes, whereas expression of GDNF was unchanged. Conclusion: Exposure to the BM-MSC secretome increases T-HESC expression of HGF and CCL-2, which are cytokines that induce cell motility via EMT in multiple cell types. Our data support upregulated HGF and CCL-2 as a mechanism by which BM-MSC induce endometrial stromal cell motility, a process crucial for cyclic regeneration. Future studies will determine how loss of HGF and CCL-2 impacts endometrial stromal cell motility. Entosis Act Through the Rho-ROCK Signalling Pathway During Human Embryo Implantation. Andrea Palomar †, 1 Alicia Quiñonero, 2 Yassmin Medina-Laver †, 2 Roberto González-Martín †, 2 Stefania Salsano, 2 Francisco Dominguez * . Valencia, Spain; 2 IVI Foundation, Valencia, Spain. Introduction: Entosis is a non-apoptotic cell-in-cell invasion process driven through Rho-ROCK signaling, that clear endometrial epithelial lining during embryo implantation in mice (Li et al., 2015) . Data from murine models suggest that lysophosphatidic acid (LPA) released by autotaxin (ATX) could activate the Rho-ROCK pathway (Aikawa et al., 2017) . We previously reported that entosis occur during human implantation. The aim of this research was to characterize key participants of entosis and explore the effect of inhibiting ROCK and ATX on this mechanism using in vitro models of human embryo implantation. Methods: In vivo ATX and LPA receptor (LPA3) localization and abundance along the human menstrual cycle were determined by immunohistochemistry (IHC) in human endometrial tissue obtained from healthy donors (n=16). ATX and LPA3 expression were also confirmed in cultured endometrial epithelial cells (hEEC) obtained from endometrial biopsies by immunocytochemistry (ICC; n=5). LPA (10-100 uM) was supplemented to hEEC for 48h. LPA3, RhoA and ROCK abundance and expression were quantified by western blot (WB; n=5) and quantitative PCR (qPCR; n=5). hEEC were cocultured in vitro with human trophoblastic spheroids (JEG) and human blastocysts discarded for clinical use to assess internalization of hEEC inside trophoblast cells. hEEC and trophoblasts were differentially stained (Cell-Tracker fluorescent probes). Entosis was evaluated by confocal microscopy and 3D reconstruction. hEEC were treated with ROCK (Y-27632, 10uM) and ATX (S32826, 90 nM) inhibitors. Inhibited and control hEEC monolayers were cocultured with trophoblast spheroids. Entosis in treated hEEC using adherent cocultures was also assessed at different timepoints (2h, 6h and 24h). Results: In vivo IHC of endometrial tissue revealed that ATX and LPA3 were found mostly in human luminal and glandular epithelium throughout the human menstrual cycle. This fact was confirmed by ICC of ATX and LPA3 in primary hEEC. Besides, WB results confirmed that LPA3 was found in both, control and LPA-treated hEEC. Interestingly, WB and qPCR analysis evinced an increase in RhoA abundance and ROCK gene expression in LPA-treated hEEC. Confocal microscopy analysis and 3D reconstruction confirmed entosis of hEEC by trophoblastic cells (JEG) and human trophoectoderm. Furthermore, ROCK and ATX inhibition in hEEC prevent the internalization of hEEC inside trophoblast cells. Conclusion: Our study confirmed the in vivo expression of ATX and LPA3 along human menstrual cycle and its implication during the entosis process. In vitro studies confirmed that LPA increases RhoA and ROCK abundance in primary hEEC. Then, our findings suggest that entosis during human implantation occur through the Rho-ROCK signalling pathway. Support: ISCII (PI/00009; CPII18/00002 and PI17/00931). three times a day. Evaluations at baseline and at the end of ibuprofen treatment consisted of determinations of total and free testosterone, DHEAS, SHBG, gonadotropins, fasting glucose, fasting insulin as well as assessment of insulin sensitivity (ISI: Matsuda-DeFronzo Index) by determinations of glucose and insulin during 2-hour glucose tolerance test. Primary outcome was the change of total testosterone. Statistical analysis was performed using JMP-Pro (v.15.0). Pre-and post-treatment values were compared using the Wilcoxon signed-rank test. Multiple linear modeling of the predictors of the primary outcome was carried out. The study was registered at ClinicalTrials.gov (NCT04485403). Results: Ibuprofen treatment was followed by a change of total testosterone from 0.75±0.06 ng/ml to 0.59±0.05 ng/ml (decrease by 21%; P=0.008). Free testosterone declined from 1.22±0.14 ng/dl to 0.88±0.09 ng/dl (decrease by 28%; P=0.01). There was no significant change in the levels of DHEAS, gonadotropins, glucose, or insulin ISI. Multiple linear modeling revealed that the change of total testosterone correlated negatively with both baseline testosterone (coefficient -0.53; P=0.004) and baseline ISI (coefficient -0.04; P=0.03). Conclusion: Present findings demonstrate that the inhibition of inflammatory pathways by a non-selective cyclooxygenase inhibitor selectively reduces testosterone levels. Since the decline of testosterone level was not paralleled by significant changes in DHEAS, gonadotropins or parameters of insulin sensitivity, it is likely that the effects of ibuprofen were due to the direct inhibition of testosterone production by the ovaries. These observations provide rationale for search of novel treatments of hyperandrogenism focused on inhibition of pro-inflammatory pathways. Introduction: Circulating gonadal steroids have sex specific effects on the brain. Change in hormone levels after menopause has implications for cognition in women. The cholinergic system influences memory and cognition by acetylcholine (ACh) in the septohippocampal pathway and may have a role in sex specific effects on cognition. The only endogenous source of choline is de novo synthesis of phosphatidylcholine, catalyzed by phosphatdylethanolamine-N-methyltransferase (PEMT). The PEMT gene has several estrogen-responsive components in its promoter region and is induced by estrogen. Postmenopausal (hypoestrogenic) women with loss of function mutations in PEMT have been found to exhibit end-organ damage typical of choline deficiency. This study examined the impact of a single nucleotide polymorphism (SNP) in PEMT on brain structure and cognition in postmenopausal women. Methods: Ninety-three postmenopausal women who had previously undergone genetic, hormonal, imaging and cognitive testing underwent genotyping for the PEMT SNP rs4646343. MRI was used for structural brain assessment, specifically quantifying four nuclei of the basal forebrain cholinergic system (BCFS): the medial septal nucleus (Ch1); the vertical nucleus of the diagonal band of Broca (Ch2); the horizontal limb of the diagonal band of Broca (Ch3), and; the nucleus basalis of basal Meynert (Ch4). Cognitive testing included the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and the letter number sequencing test from the Wechsler Memory Scale. Two-sample T-tests assuming unequal variance were performed to compare subjects with or without variant alleles. Results: Participants with at least one variant allele were found to have reduced BCFS volume, specifically in the right Ch 1, 2, 3 nuclei (p = 0.016) and left Ch1, 2, 3 nuclei (p = 0.019), but not the bilateral Ch4 nuclei. Baseline measures of neuropsychological function and working memory did not detect discernible differences. Conclusion: Single nucleotide variants in the estrogen-responsive promotor region of the choline-synthesizing gene PEMT were associated with reduced BCFS volumes in postmenopausal women. BFCS volumes have been studied as an indicator of health of the cholinergic system and as a biomarker for developing cognitive impairment and Alzheimer's disease, though we did not find an association between variant alleles and baseline measures of cognition. Cognitive performance may be influenced by other protective or harmful SNPs in PEMT and genes responsible for choline metabolism (including CHKA, MTHFD1, MTHFR and CHDH), or by dietary choline intake. A holistic approach to investigating choline intake, production and metabolism should be undertaken to more clearly define these relationships and their influence on the brain after menopause. Novel Actions of Chemotherapy on Extracellular Vesicle Release and Their Impact in the Prepubertal Testis. Michael Philip Rimmer †, Christopher D Gregory, Rod T Mitchell * . University of Edinburgh, Edinburgh, United Kingdom. Introduction: Extracellular vesicles (EVs) are small membrane-delimited structures released by cells to communicate with other cells throughout the body. They interact with recipient cells through EV uptake into the cell, cargo release at the membrane surface and interaction with surface receptors. Following EV uptake by recipient cells, their cargoes (lipids, proteins, RNA, DNA, metabolites) can alter the function of recipient cells. Cargo loading into EVs is dynamic and altered by the state of the parent cell, releasing the EV. Known EV functions are wide ranging and include induction of apoptosis, angiogenesis, acquisition of sperm motility, development of the pre-metastatic niche and conferring chemotherapy resistance. In the prepubertal testis, EV release is poorly characterised. Whether chemotherapy alters their release or their impact on other cells in the testis is unknown. The release and subsequent uptake of EVs in the testis may mediate further damage to healthy testicular cells following chemotherapy, as would occur in a pre-pubertal boy receiving chemotherapy. This damage may then contribute to the patients' risk of male infertility in later life. Methods: EV characterisation from mouse spermatogonial stem cells (GC1-spg) and Sertoli (TM4) cells was undertaken, growing cells in EVdepleted cell culture media, comparing cisplatin-treated cells to controls. Change in EV number was quantified using nanoparticle tracking analysis and impact on treatment naïve recipient cells was assessed using in vivo cell imaging and detection of cleaved caspase. EV uptake and movement within cells was examined using super-resolution microscopy and live cell imaging. Results: GC1-spg and TM4 cells treated with cisplatin release twice as many EVs than control cells (p=0.004). EVs released by cisplatin treated cells have greater uptake in treatment-naïve cells (p=0.002) and move faster when inside these cells. We identified higher rates of apoptosis in treatment-naïve TM4 cells when incubated with EVs released by cisplatin-treated TM4 cells vs. control EVs (p=0.03). This effect was not seen in GC1-spg cells. Conclusion: We characterised EVs from prepubertal spermatogonial stem cells (GC1-spg) and Sertoli cells (TM4), identifying significantly altered release when treated with chemotherapy. Altered release, uptake and intracellular speed of EVs released by cisplatin-treated cells, as well as their impact on treatment-naïve cells in the testis, are novel findings. Damage to these cells in the testis may be a contributing factor to future male infertility and represent additional off target effects of chemotherapy. During this time, embryonic cells specify into the fi rst distinct cell populations; the trophectoderm (TE) and inner cell mass (ICM). In mice, the leading model of ICM-TE specifi cation involves diff erential activation of Hippo signaling and subsequent transcriptional regulation (CDX2 and SOX2 restriction to the TE and ICM, respectively). However, our lab's recent work with human embryos suggests that this mechanism may involve an additional molecular component. In murine embryos and human embryonic stem cells, it has been shown that miRNAs, which regulate transcription, play a role in TE specifi cation. To identify miRNAs of interest in the mammalian embryo, we leveraged human embryo smallseq data spanning development (embryonic days (E)3-E7) previously collected by our lab. At E5, coinciding with lineage specifi cation, 439 miRNAs were signifi cantly diff erentially expressed between the ICM and TE in the human embryo. Examining the expression dynamics of these miRNAs before and after segregation. miR-24-3p (highly expressed in the ICM and known to target TE marker, CDX2) and miR-200c-3p (highly expressed in the TE and known to target pluripotency markers, SOX2) were of particular interest due to their progressive increase in lineagespecifi c expression. Here, we aim to begin defi ning the role of miRNAs in lineage specifi cation. We hypothesize that miRNAs are important upstream regulators of lineage specifi cation. Methods: Murine embryos (C57BL/6 x DBA, n=3-8/group) were cultured to the 8 cell stage, at which time concentrations (1-20 uM, dose response) of miRNA mimics or a negative control oligo were added to the media. Embryos were further cultured with treatment until the 32 blastocyst stage when they were collected, stained and analyzed via confocal microscopy. Results: Addition of miR-200c-3p to murine embryos led to dosedependent decreases in both CDX2 (-65%, p≤0.01, ANOVA with Tukey post-hoc) and SOX2 (-72%, p≤0.001) in their respective lineages relative to control blastocysts. Addition of 2uM of miR-24-3p trends toward a decrease in expression of SOX2 while CDX2 remained unchanged compared to controls. At higher concentrations (5uM and 10uM) a decrease in the expression of both SOX2 and CDX2 was observed. For both miRNAs, doses of 20uM resulted in embryo death. Conclusion: This preliminary data suggests diff erences exist in miRNA biogenesis between TE and ICM lineages in embryos. Further, miRNAs may drive lineage specifi cation in the embryo by regulating the expression of key genes known to be important in establishing lineage. Changes throughout the menstrual cycle make it extraordinarily challenging to identify markers or cell types associated with uterine pathologies since disease-state alterations in gene and protein expression are convoluted with cycle phase variations. We hypothesized that using an integrated, data driven proteomic and single-cell RNA-sequencing (scRNA-seq) approach would allow us to overcome these cycle phase challenges to look for intrinsic diff erences in eutopic endometrium of control and endometriotic patients. We collected uterine biopsy samples from a total of 17 patients; 8 for proteomics, 6 for scRNA-seq, and 13 for histology with some samples used for multiple analyses. Using a linear mixed effect model, we identified proteins associated with cycle stage and disease to assess for pathway analysis. We then analyzed our scRNA-seq data to identify cell types expressing cycle and disease-associated genes identified in our proteomics data. Using these identified cell types of interest, we looked at cell-to-cell communications through ligand and receptor pairs which revealed the Axl/Tyro3 -Gas6 as an axis of interest which was then confirmed through histological staining of paraffin-embedded patient endometrial biopsy samples. Results: Protein-based pathway analysis highlighted several extracellular vesicle-associated pathways that directed our attention to cell-to-cell interactions as an important aspect of disease. Using the scRNA-seq data, we found that the macrophage ligand Gas6 to endometrial stromal cell receptors Axl and Tyro3 scored highly as a likely interaction occurring in all patients we analyzed. Transcriptomically, Tyro3 expression was significantly decreased (p<0.05) in endometriotic patients compared to controls. This decrease in expression was confirmed through histological staining for Tyro3 in endometrial biopsy samples (p<0.01). Conclusion: Our integrated proteomic and scRNA-seq approach provides a framework for integrating LMMs, proteomics and RNA-seq data to deconvolve the complexities of complex uterine diseases and identify novel genes and pathways underlying endometriosis. Using this method, we identified Tryo3 as a novel diagnostic biomarker or treatment avenue for patients with endometriosis. Microphysiological systems present the next exciting evolution of in vitro or ex vivo culture as they allow for the dynamics and interaction of multiple organ mimics. Methods: To study PCOS, we developed LATTICE, a cost-effective and user-friendly multi-organ microfluidic platform. The media in LATTICE is transported between culture wells with a high correlation (R 2 = 0.9826) between the pump commands (10-35μl per step) and flow rate. Results: To test the effects of dynamic flow, 30 adipocyte spheroids were cultured in static (n=3) or dynamic conditions in LATTICE (40 μl/hr, n=3) for 14 days. Glucose consumption and L-lactate measurements showed higher levels of glucose (p<0.001) and decreased L-lactate (p<0.01) in LATTICE, demonstrating the replenishment of nutrients and elimination of waste. Next, murine ovarian explants in LATTICE were able to respond physiologically to the treatment of an adjusted 13-day human-like ovarian cycle using exogenous gonadotropins, including the estradiol peak in FSHinduced follicular phase and progesterone secretion in the hCG-induced luteal phase. When ovarian explants (n=4) were exposed to PCOS-like high levels of hCG (30 mIU/ml), ovarian explants produced higher levels testosterone (T, 23591.18 pg/ml) compared to the control (3826.17pg/ml; P<0.01) in a 8-day follicular phase culture regimen. Finally, treatment of human fallopian tube explants or murine oviductal epithelial cells with high T in LATTICE resulted in increased proliferation and a significantly decreased cilia beating frequency without affecting viability. There were no significant differences in the rates of readmission at the 2 or 6-week time points between pre-IPAC and post-IPAC. There was 1 (0.08%) death in the cohort. In total from 2018-2020, 34 (3%) of readmissions occurred within 2 weeks, and 17 (2%) before 6 weeks. There was no significant difference in adherence to a 6 week PP visit (79% vs 76% p=0,301). There was also no significant difference between referral to post-partum mental health services (6% vs 6%, p=0.672), if a 2 week visit was completed. It was noted that at the 6-week visit, more individuals in the post-time point discussed contraception compared to the pre-time point (97% vs 93%, p=0.004). Conclusion: A significant difference in readmission rates as a reflection of morbidity post IPAC implementation was not seen. In the post implementation cohort where a 2 week visit was scheduled for the patient there was not a significant increase in adherence to either the 2-week or 6 week visit. We suspect socioeconomic barriers contributed to this, with further investigation planned. Dispelling To date, it is still unclear to which extent placental damage could be responsible for this, as no histopathological "footprint" has been described in association with SARS-CoV-2. The purpose of our study was to investigate to which extent the virus affects placental tissue. Methods: We prospectively collected placentas from 56 women with SARS-CoV-2 infection during pregnancy. In addition, RT-qPCR was performed for SARS-CoV-2 in vaginal fluid, placental and fetal membranes. SARS-CoV-2 IgG antibodies were measured in the umbilical cord blood. Placentas were examined histopathologically and immunohistochemically with antibodies for: SARS-CoV-2 proteins, ACE2, TMPRSS2, Il6, TNFa, Il1b, M30 CytoDEATH, CD3, CD8, CD68 and CD163. Further, in situ hybridization was performed by using the V-nCoV2019-S probe. Six controls matched for maternal age, gestational age at delivery, and presence of comorbidities were similarly stained. IRB approval and informed consent were obtained. Results: Seven women had a severe course of the disease and three were admitted at the ICU, followed by premature delivery. In no case was microbiological evidence of SARS-CoV-2 found in the placenta or vaginal fluid. Strong SARS-CoV-2 signal was detected in placentas of women with severe symptoms. Compared to controls, CD8+ cells were increased in the decidua and 20/56 cases showed an infiltration into the villous tissue. Chorionic villi showed increased numbers of CD68+ cells in 44/56 cases. No apoptosis or inflammatory marker and no severe histopathological changes were detected. Conclusion: Our experiments show clear evidence of SARS-CoV-2 in the placenta after infection during pregnancy, particularly when symptoms were severe. Increase in CD8+ and CD68+ cells further underlines the results by demonstrating viral infection. Mechanisms of stillbirth in association with SARS-CoV-2 are not yet elucidated, but our data strongly suggest placental implication. Therefore, appropriate counseling and surveillance is mandatory in these pregnancies. Evaluating 1A ). Genes coding for type 1 interferons and interleukins 2, 10, 17 and 23 were among those substantially upregulated in the intervillous. The pattern of expression corresponded with the activation of 35 pathways corresponding to interferon, TLR and interleukin signaling and inactivation of IL1RN in the intervillous (Fig. 1B) . In the decidua genes related to interferon signaling including STAT1 and 5 were among those significantly upregulated. Pathways in the decidua also related to interferon, TLR and interleukin signaling with notable differences such as the activation of CCL5 and IL6 signaling, that was not present in the intervillous. Conclusion: A robust upregulation of innate immune genes was detected in all placenta samples from SARS-CoV-2 infected pregnancies. The activation of innate immune genes and related pathways persisted to term even when infection occurred early in pregnancy. Interestingly, the presence of viral RNA corresponded with interferon production in the intervillous. In contrast, the lack of viral RNA in the decidua was associated with the activation of interferon signaling. This finding suggests crosstalk between placental compartments, wherein infected intervillous cells elicit antiviral responses in the decidua. This may represent a novel mechanism of viral containment that prevents SARS-CoV-2 from reaching the fetus. The clinical severity and lifelong morbidity of sickle cell disease (SCD) and β-thalassemia, the major disorders of β-globin gene mutations, can be ameliorated by production of fetal hemoglobin (HbF). In fetal life, γ-globin (encoded by 2 duplicated genes in the β-globin locus) does not use the mutated adult β-hemoglobin (HbA). The HbF to HbA transition is regulated by a repressor, Bcl11a, which silences fetal γ-globin. Given our overarching goal to develop a fetal gene therapy for SCD which "unsilences" γ-globin, we aimed to disrupt the Bcl11a erythroid-specifi c enhancer region using three diff erent synthetic single guide RNA (sgRNA) sequences with CRISPR/Cas9 in murine erythroleukemia (MEL) cells ( Fig. 1) . Methods: Three distinct sgRNA sequences (sg1, sg2, sg3) were designed to target the murine Bcl11a erythroid-specifi c enhancer region. The sgRNAs were complexed with Cas9 mRNA using Lipofectamine MessengerMAX. Transfection effi ciency was measured by a nuclear fl uorescent reporter siRNA and Bcl11a expression was quantitated with qRT-PCR after normalization to GAPDH. Unpaired, two-tailed t-tests were performed using Prism (v9.2 GraphPad), with signifi cance determined as p<0.05. Results: All wells treated with Lipofectamine MessengerMAX and the fl uorescent reporter siRNA showed nuclear uptake of the siRNA after a media change at 24 hours, indicating transfection effi ciency >90% (Fig. 1) Introduction: Maternal marijuana use is associated with off spring neurodevelopmental disruption. Altered microRNA (miRNA)-mediated gene regulation has been observed in neurodevelopmental disorders. Our study's objective was to investigate molecular regulatory mechanisms of fetal responses to marijuana. We will assess its impact on miRNA cargo within extracellular vesicles (EVs) of fetal cerebral spinal fl uid (CSF) to identify targets that regulate post-transcriptional gene expression in a rhesus macaque model. Methods: Female rhesus macaques (n=8) were divided into 2 groups: control (CON, n=4) and marijuana-exposed (THC, n=4). All animals were maintained on a standard chow diet, but the THC group had an additional daily edible with 2.5mg/7kg/day of delta-9-tetrahydrocannabinol (THC, equivalent to a heavy human medical marijuana dose). Subjects underwent time-mated breeding; the THC group was continued on THC during during pregnancy. Animals were delivered by cesarean section at gestational day 155 (term is ~168 days). Fetal tissue was collected for histology and fetal CSF was collected for analysis of miRNA expression (n=7, 3 CON and 4 THC). Size exclusion chromatography (SEC) was used to isolate EVs from fetal CSF. Total RNA was isolated and converted to cDNA for miRNA expression analysis using TaqMan Advanced miRNA quantitative PCR arrays. Results: Fetal brain histology revealed hyperacute ischemic changes and ischemic white matter injury in THC-exposed fetuses only. miRNA expression analysis found a signifi cant (p=<.05) greater than 2-fold reduction in mir-448 and a greater than 2-fold increase in mir-199a-3p ( Fig. 1 ) in THC exposed vs CON fetal CSF. Ingenuity Pathway Analysis (IPA) indicates that the messenger RNAs targeted by these 2 miRNAs are in canonical pathways associated with axonal guidance signaling, netrin signaling, and cardiac hypertrophy signaling. These miRNAs are also implicated in developmental disorders and neurological and reproductive system diseases. Additionally, they are involved in gene expression and cellular development, growth, assembly, and function. Conclusion: Our study identifi ed two EV associated miRNAs with a greater than 2-fold change in fetal CSF with maternal THC use. This data is indicative of subtle molecular changes consistent with the observed histological data and implicate a potential role for miRNA regulation by THC in fetal off spring. Effects Introduction: Maternal obesity is associated with increased risk of poor pregnancy outcomes, including preeclampsia, preterm birth and fetal growth restriction (FGR). The mechanisms underlying these associations remain poorly defi ned. Previous studies in mice have demonstrated that a maternal high-fat diet fed before and during pregnancy can impact placental development, evidenced by changes in the placental transcriptome, altered labyrinthine zone vascularity and reduced fetal and placental weights. Herein we aimed to further characterise the maternal and fetal phenotype associated with maternal obesity in mice, including feto-placental blood fl ow parameters. In addition, we aimed to determine whether maternal beetroot juice (BRJ) supplementation, which provides a source of dietary nitrate and has been shown to improve vascular function in other models, can improve pregnancy outcomes in this mouse model of maternal obesity. Methods: Female C57BL/6 mice were fed either a standard chow or high fat diet (HFD; 60% fat) from 5 weeks of age for 10-12 weeks prior to mating. Following mating (identifi cation of vaginal plug designated embryonic day (E)0.5), pregnant females were continued on the same diets for the duration of pregnancy. At E12.5, mice were randomly allocated to receive BRJ in the drinking water (delivering a nitrate dose of ~1mmol/ kg) or remain on their normal drinking water. At E18.5, umbilical artery (UmbA) and vein (UmbV) blood fl ow velocities were determined in vivo using high-frequency ultrasound (Visualsonics) and peak systolic velocity (PSV), end diastolic velocity (EDV) and resistance index (RI) calculated. Following ultrasound, dams were sacrifi ced and maternal and fetal tissues collected and weighed. Results: Maternal bodyweight and adipose tissue weights were signifi cantly increased in HFD-fed dams compared to chow-fed control animals (p<0.001). Litter size was unaff ected by maternal diet, but fetal and placental weights were significantly reduced (p<0.01 for both) in litters from HFD dams compared with control dams. HFD significantly increased UmbA EDV compared to control dams (P<0.05). Maternal BRJ supplementation increased UmbA EDV in both chow-fed and HFD dams (overall effect of treatment, p<0.01), and reduced UmbA RI (P<0.05), potentially reflecting reduced placental vascular resistance in treated animals. UmbV parameters were unaffected by maternal diet or treatment. Despite these effects on blood flow velocities, maternal BRJ treatment did not alter fetal or placental weights at E18.5, in either control or HFD dams. Conclusion: Maternal obesity significantly reduces fetal and placental weights in mice. Our findings suggest that short-term maternal BRJ treatment may alter fetoplacental vascular function in late pregnancy, but this did not translate to an increase in fetal growth. Here, we tested the hypothesis that H 2 S protects cardiac function during hypoxic development using the chicken embryo. This model system permits isolation of the direct effects of therapy on the developing heart, independent of effects on the mother and placenta. Methods: Fertilized Bovans Brown eggs were incubated under normoxic (21% O 2 ) or hypoxic (14% O 2 ) conditions from day 1 (term is 21 days). Chicken embryos were treated with NaHS (H 2 S donor, 10 µmol/kg/day) or vehicle from day 13 to day 18. Using sterile techniques, NaHS was injected daily through the air cell onto the chorioallantoic membrane via a 1-mm hole in the eggshell. On day 19, following cervical transection, the heart was excised, and cardiac function determined via a Langendorff preparation. Results: Relative to normoxic controls, embryos incubated under hypoxic conditions were growth restricted and they showed a significant elevation in left ventricular end diastolic pressure (LVEDP), a marker of pronounced diastolic dysfunction ( Fig. 1) . Treatment with H 2 S during hypoxic incubation rescued the cardiac dysfunction but did not protect against embryonic growth restriction. Treatment with H 2 S in normoxic embryos had no effect (Fig.1) . The data support the hypothesis tested that H 2 S protects cardiac function during hypoxic development. Further, the data dissociate protective effects of H 2 S on embryonic growth and cardiac dysfunction. Therefore, fetal growth restriction in hypoxic pregnancy is not a prerequisite for programmed heart disease. Supported by The British Heart Foundation. Introduction: Fetal growth restriction (FGR) is a poorly understood pregnancy complication, defined as fetal growth ≤ 10th percentile. One cause of FGR is utero-placental insufficiency. In growth restricted female guinea pig (GP) fetuses, we previously showed increased expression of the inflammatory marker Tgf-β in brain when compared to male fetuses. This difference was ameliorated with treatment of the placenta with nanoparticle containing human insulin-like 1 growth factor gene. Our aim was to analyze Tgf-β signaling in fetal brain to understand its role in blood brain barrier (BBB) integrity and homeostasis in FGR with and without treatment. Methods: Female GPs were assigned to a control (C, n=11) or 30% restricted diet (MNR, n=12) with a subset of each group undergoing ultrasound-guided intraplacental nanoparticle (NP: C=6 and MNR=7) or saline (S: C=5 and MNR=6) treatment. Five days later, fetal brain tissue was collected and mRNA expression of two Tgf-β signaling pathway members: occludin (Ocln) and tight junction protein 1 (Tjp1) was analyzed by qPCR. Immunohistochemistry was used to visualize protein expression of two other Tgf-β signaling pathway members: claudin-5 (Cldn5) and P-glycoprotein (P-gp). Data was analyzed using generalized estimating equations including diet, treatment, and fetal sex as main effects, maternal ID as a random effect and gestational day as a covariate. Statistical significance was considered at P<0.05. Results: Tjp1 and Ocln mRNA was increased in MNR-S female brains as compared to C-S female brain (P=0.049 and P<0.0001, respectively). Furthermore, in MNR-NP females, Ocln and Tjp1 expression was similar to C-S females (P>0.05, for both respectively), and Ocln was lower compared to MNR-S females (P<0.0001). In males, Ocln expression was not significantly different in MNR-NP brain compared to C-S and MNR-S (P>0.05 for both, respectively), whilst Tjp expression was higher in MNR-S and MNR-NP when compared to C-S (P=0.01 and P=0.041, respectively). Cldn5 protein was localized to the blood vessels throughout the brain, whilst P-gp was expressed in the chondrocytes. Claudin-5 staining was greater in MNR-NP female fetuses as compared to Cl-S males. Conclusion: Placenta nanoparticle treatment normalized mRNA expression of Ocln and Tjp1 in brain tissue of growth restricted females but not males, and similar to that of normal growing fetuses. This suggests that in FGR, Tgf-β signaling may function in a sex-dependent feedback manner to maintain BBB integrity. Further investigation into the blood brain barrier proteins may help improve understanding of its potential disruption in FGR. Homologous (Fig 1a and 1b) . To determine if IFN signaling alone can reduce cell growth, PARPi-resistant cells were treated with recombinant IFNalpha (IFNa) and PARPi. Similar to EHMTi/PARPi treated cells, cells treated with IFNa/PARPi had reduced cell growth compared to controls. This eff ect was rescued with Ruxilitinib (an inhibitor of IFNa signaling) (Fig 1c and 1d ). TE transcripts can form dsRNA and induce an IFN response. EHMTi has been reported to promote transcription of TE, thus using TEtranscripts, the data also revealed re-activation of several TE Introduction: Targeted therapy for high-grade serous ovarian cancer (HGSOC) is challenging as the disease is characterized by copy number variation and not recurrent somatic mutations but displays well-conserved epigenetic features such as expression of PAX8. We propose employing therapies that capitalize on interactions between PAX8 and specifi c DNAbinding sites to develop therapeutic approaches to target HGSOC and we present a novel gene-editing system that combines polymeric delivery of base-editors with lineage-restricted tissue targeting. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-null cell lines including MCF-7 and HeLa were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices and surface zeta potentials were measured. Polyplexes were imaged for uptake and functionality using fl uorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. To monitor in vivo transfection effi ciencies, we delivered fl uorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We synthesized polyplexes using the polyamine PPLG-gazidopropylamine (PPLG) and base editor (BE) or guide-RNA plasmids. We observed that the BE and the gRNA can be effi ciently packaged into two distinct polymeric nanoplexes and co-delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of PLK1 or ATR, which is also synthetically lethal with mutated tp53, as observed from cancer cell death compared to controls. In vivo, the mice receiving polyplex particle treatment lived signifi cantly longer and had a visible reduction in tumor burdens as observed during necropsies compared to the no treatment tumor-bearing mice. Conclusion: Combining nanotechnology and gene editing can improve specifi city toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Due to the synthetic lethality nature of ATR and tp53, the addition of a combinatorial PARP inhibitor to the proposed therapy will reduce tumor burden. While resistance can develop to ATR inhibitors, ovarian cancer cells are unable to downregulate the expression of PAX8, thus ensuring continued response to the PAX8-directed therapy. The Introduction: Placental dysfunction is a common cause of pregnancy complications like intrauterine growth restriction (IUGR) and preeclampsia (PE). Importantly, a heightened inflammatory environment is often involved in the pathogenesis of these pathologies. An understudied shared feature of IUGR and PE is the widespread loss of microvilli (MV) from the syncytiotrophoblast (ST) apical membrane. MV are supported by a filamentous actin (F-actin) core and are a hallmark of epithelial polarity. Importantly, active maintenance of cell polarity is crucial to maintain microvillar structure. However, the mechanisms regulating ST microvilli maintenance are unknown. Atypical protein kinase C (aPKC) isoforms are evolutionarily conserved apical polarity regulators. In other tissues, aPKCs regulate microvilli via actin. Thus, we hypothesized that aPKC isoforms maintain the ST apical surface and polarity. Methods: 10-12 wk. and term human placental floating explants were cultured +/-5μM myristoylated aPKC inhibitor for 6 hrs. and fixed for immunostaining and scanning electron microscopy (SEM). Explants were stained with phalloidin (F-actin) and Hoechst (nucleus) and imaged with confocal microscopy. 10-12 wk and term explants and in vitro differentiated primary ST were treated with TNF-α (0-10ng/ml) for 4-6 hrs. and collected for western blotting analysis (anti-aPKC isoforms) and fixed and stained as above. The purpose of our analysis was to investigate whether inflammatory biomarkers in maternal serum during pregnancy were associated with inflammatory and vascular lesions in the placenta. Methods: Study participants were drawn from a randomized controlled trial (RCT) of group vs individual prenatal care. Participants completed blood draws in the second and third trimester and biomarkers of inflammation, including CRP, IL-10, IL-1Ra, IL-6, and TNFα, were measured in serum. Biomarkers were log-transformed, standardized, and summed to create a composite for each trimester. Additionally, average values across the trimesters were calculated for the individual biomarkers. Placentas were collected following delivery and evaluated by a single perinatal pathologist. Placental lesions were categorized according to Amsterdam Consensus Criteria: acute inflammation, chronic inflammation, maternal vascular malperfusion, and fetal vascular malperfusion. Associations between placental lesions and inflammatory biomarkers during pregnancy were estimated using logistic regression. Models were adjusted for gestational age at the blood draw, maternal age, race/ethnicity, body mass index, infant sex, and type of prenatal care (RCT group). Results: Data from 677 study participants who completed at least one blood draw and whose placentas were collected were included in the analysis. The most prevalent placental lesion category was acute inflammation (60.1%), followed by chronic inflammation (48.6%), maternal vascular malperfusion (46.8%), and fetal vascular malperfusion (17.0%). In an adjusted model, a one standard deviation increase in the inflammatory composite during third trimester was associated with increased odds of chronic inflammation in the placenta ( Controls remained normotensive throughout pregnancy. Genomic DNA was isolated from maternal peripheral venous blood and umbilical venous blood obtained at delivery. Genotyping was performed using Taqman assays. Clinical characteristics were obtained from maternal and perinatal medical records. Binary logistic and linear regression were used to test for associations between rs235011 and PE or birth weight, respectively. Results: Genotype distributions were as follows: maternal T/T (64%), C/T (33%) and C/C (3%) and infants T/T (65%), C/T (29%) and C/C (6%). Using an additive inheritance model, maternal rs235011 was positively associated with PE (p=0.019) even after adjusting for maternal age, body mass index, and primiparity (p=0.022). While not associated with PE, infant rs235011 correlated with lower birth weight using an additive model after correction for gestational age (p=0.050). We identified a NF-κB variant that is strongly associated with PE and located within a genomic region where Andeans are genetically differentiated from lowland populations. We speculate that rs235011 contributes to individual differences in PE risk in this population by modifying immune function at the maternal-fetal interface. Studies to determine the functional relevance of rs235011 for PE are underway. Introduction: Hyperactivation of the innate immune system is associated with pregnancy complications such as preeclampsia. Toll-like receptor 9 (TLR9) is a DNA sensor of the innate immune system, and TLR9 expression and activity are elevated in women with preeclampsia. Immune system molecules affect circadian clock function, and circadian rhythm desynchrony has been proposed as a risk factor for preeclampsia. The purpose of this study was to assess the effects of TLR9 stimulation on maternal heart rate and blood pressure dynamics, including circadian rhythms, during gestation. We hypothesized TLR9 activation would impair maternal cardiovascular dynamics and disrupt circadian rhythms in pregnant rats. Methods: Maternal resistance mesenteric arteries were isolated on gestational day (GD) 16 (term = 22-23) from healthy pregnant Sprague-Dawley rats and treated with a TLR9 agonist (ODN2395) or vehicle (Krebs-Henseleit buffer) for 24 hours ex vivo before assessing release of endothelin-1 (ET-1) using ELISA. Radiotelemeters were implanted in virgin female rats to monitor temporal blood pressure dynamics prior to mating and throughout gestation. Intraperitoneal injections of 1 mg/ kg ODN2395 or vehicle (saline) were administered to pregnant rats on GD 14, 16, and 18. On GD 20, rats were euthanized, and maternal serum was collected to assess circulating ET-1. Temporal data were analyzed via Lomb-Scargle periodogram to detect changes in circadian rhythms. Disruption of circadian rhythmicity was defined as the absence of a statistically significant oscillation over a 24-hour period. Results: ODN2395-treated maternal mesenteric arteries released elevated levels of ET-1 compared to vehicle-treated arteries (ODN2395: 1.268 ± 0.25 pg/mg, Vehicle: 0.240 ± 0.10 pg/mg, p = 0.0031). Yet, there was no difference in circulating ET-1 in ODN2395-treated pregnant rats compared to controls (p > 0.05). Heart rate during gestation was elevated in ODN2395-treated rats compared to controls during both light (resting) and dark (active) phases (p ≤ 0.0091). No differences in maternal blood pressure were detected between groups (p > 0.05). Circadian rhythms for diastolic and systolic blood pressure were disrupted after ODN2395 injection on GD 14 (p ≥ 0.097). Circadian rhythmicity was restored for both systolic and diastolic blood pressure after ODN2395 injection on GD 16 (p < 0.0001), but diastolic circadian rhythmicity was disrupted again following the final ODN2395 injection on GD 18 (p = 0.072). Conclusion: TLR9-mediated innate immune system activation during pregnancy potentiates maternal heart rate and disrupts circadian rhythmicity of maternal blood pressure. Innate immune system-mediated disruption of the light/dark cycle in pregnancy may increase risk of pregnancy complications. Hege N Skytte †, 1 Jacob J Christensen * , 2 Nina Gunnes * , 1 Kirsten B Holven * , 2 Tore Henriksen * , 1 Trond M Michelsen * , 1 Marie Cecilie P Roland * . 1 1 Oslo University Hospital, Oslo, Norway; 2 University of Oslo, Oslo, Norway. Introduction: There are well-known associations between maternal metabolic changes and preeclampsia, but broader longitudinal metabolomic studies are lacking. Methods: We performed comprehensive metabolic profiling (91 metabolic measures) by high-throughput nuclear magnetic resonance (NMR) spectroscopy at three time points during pregnancy in 108 pregnant women from a Norwegian longitudinal cohort study. We fitted a linear regression model for each metabolic measure to compare the plasma metabolome in women who later developed preeclampsia (n= 38) and healthy controls (n=70). The associations of body mass index (BMI) and preeclampsia with the various metabolic measures were also compared. Multiple testing correction was based on principal component analysis, and inferred statistical significance at p < 0.006. Results: Among women who developed preeclampsia, 92% had late onset preeclampsia (birth > 34 weeks). Compared to women with healthy pregnancies, very-low-density lipoprotein (VLDL) was significantly increased in women with preeclampsia both at gestational age (GA) 14-16 weeks and 30-32 weeks ( Figure 1 ). The difference was most pronounced for VLDL triglyceride concentrations. Unsaturated fatty acids and proportion of polyunsaturated fatty acids to total fatty acids were lower in women with preeclampsia compared to healthy pregnancies at GA 22-24, while docosahexaenoic acid was lower in week 30-32. After adjustment for age, BMI and parity, all metabolic differences were attenuated at GA 14-16 and 22-24 weeks, but less affected by the adjustment at week 30-32. Cholesterol in HDL and proportion of linoleic acid to total fatty acid were lower in preeclamptic than healthy pregnancies at week 30-32, although not significant after correction for multiple testing. Concentration of glycine tended to be lower in pregnancies with preeclampsia compared to healthy pregnancies at all time points, and the association was almost unaffected by BMI. Conclusion: In early pregnancy, many of the metabolic differences between healthy and preeclamptic pregnancies disappeared after adjustment for BMI, probably representing the women's pre-pregnancy metabolic status. In early third trimester, several weeks before clinical manifestation, the differences were less influenced by BMI indicating preeclampsia-specific changes. These changes were most marked in the triglyceride carrying VLDL particles. can be useful in predicting cardiovascular disease such as congestive heart failure or atrial fibrillation from ECGs, often regardless of symptoms and even if the ECG is characterized by physicians as normal. Cardiovascular dysfunction is common in preeclampsia, and EKG abnormalities are more common at the time of diagnosis among women with preeclampsia compared to healthy pregnancies. We proposed that deep and machine learning on ECGs obtained early in pregnancy could help identify women at high risk for preeclampsia later in pregnancy. We studied a total of 128 women with preeclampsia (77% African-American; average age 29 yrs) from a medical center in Memphis, TN, who had received a clinical evaluation (including ECG) earlier during pregnancy for symptoms such as chest pain, syncope, shortness of breath, etc. Controls were 252 women without preeclampsia, matched by age and race, but who also received EKGs for similar reasons. ECGs taken at least 3 months prior to diagnosis of PE (for cases) or delivery (for controls) were used for analyses. Signal processing methods were applied to extract features from ECG data. A stochastic search algorithm was used for feature selection and light gradient boosting was used to predict preeclampsia during pregnancy. Model performance was evaluated by five-fold stratified cross validation. The mean time between ECG and delivery was 5.6 months for cases and controls. In the model based solely on 31 ECG features, the cross-validation AUC was 0·87 (95% CI 0.83-0.90), with a sensitivity of 77% and specificity of 77%, positive predictive value 63% and a negative predictive value of 87%. With specificity set to 90% (to reduce false positives), model sensitivity was 60% and PPV was 63.5%. The final model predicted early onset preeclampsia (onset <34 wks) and preterm preeclampsia (delivery <37 weeks), with cross-validation AUCs of 0.88 and 0.89, respectively. Model performance was similar regardless of trimester when ECGs were obtained. Conclusion: ECGs early in pregnancy may be able to predict preeclampsia later in pregnancy, enabling earlier initiation of preventive therapies. This study is a critical step to develop AI based tools for 12-Lead ECGs to predict risk for preeclampsia, and single lead versions for smartwatch deployment in low resource environments. The balance between T helper (Th)1/Th2 immunity has been reported to be critical for a successful pregnancy, and unbalanced Th1/Th2 cell immunity was associated with obstetrical complications such as recurrent pregnancy losses (RPL) and preeclampsia. In this study, we aim to investigate Th17, regulatory T (Treg), and NK cell immunity during pregnancy in women with reproductive failure (RF) including, ≥2 RPL and ≥2 repeated implantation failures (RIF). We performed a prospective cohort study in 26 non-pregnant and 52 pregnant women with RF, including 12 women who miscarried and 40 ongoing or delivered. Controls were 21 non-pregnant and 26 healthy pregnant women. Peripheral blood was collected before pregnancy and during the first trimester. Intracellular cytokine analysis, immunophenotype, and NK cell cytotoxicity (NKC) were performed by flow-cytometry using peripheral blood. Statistical analysis was performed by using the student t-test and the one-way ANOVA test. Results: Non pregnant women with RF have significantly higher NKC (E:T ratio, 50:1) (25.7 ± 4.6%) and CD19+/5+ B-1 cells (7.2 ± 3.0%) compared to non-pregnant controls (13.5 ± 5.7%, 15.3 ± 9.8%) (P <0.001 respectively). NKC and CD19+/5+ B-1 cells of pregnant controls (12.6 ± 5.1%, 12.9 ± 7.8%) were significantly different from women who miscarried (21.2 ± 4.2%, 4.4 ± 3.5%) (P<0.001) and delivered (22.1 ± 4.5%, 2.7 ± 2.9%) (P<0.001). TNF-alpha/IL-10 of pregnant controls were significantly different from women who miscarried (P<0.05). IFNgamma/IL-10 and Th17 of pregnant controls were significantly different from women who delivered (P<0.05 respectively). Treg and Th17/Treg ratio of pregnant controls were significantly different from women who miscarried (P<0.001 respectively ) and delivered (P<0.001 respectively). Th17/Treg ratios of women who miscarried were significantly different from those of women who delivered (P<0.05). Conclusion: Our findings highlight that women with RF have shifted Th1 and Th17 immunity and up-regulated NKC compared to normal controls. The balance between Th17 and Treg, Th1 and Th2 cells, and down-regulated NKC play a critical role in early pregnancy success in women with RF by mediating maternal immune tolerance to the fetus. Human Our previous studies have showed that transplantation of human bone marrow-derived mesenchymal stem cells (hBM-MSC) and MSC derived exosomes in chemotherapy-induced POI mouse ovary can reverse POI phenotype and achieve pregnancy. This study aims to understand the molecular mechanism underlying the regenerative effect of hBM-MSC exosomes using an in vitro POI model. Methods: We used the human granulosa cells (HGrC1) to create a POI model by inducing apoptosis using 100 µg/ml of cyclophosphamide for 24 h and treated these damaged HGrC1 cells with a dosage of 1.50X109 commercially available exosomes derived from hBM-MSC at day 24 hours post cytotoxicity. Cell proliferation, cell viability and apoptosis were studied. Relative gene expression was studied using three sets of genes which include i) markers of cell proliferation and survival (AKT, Ki67 and TK1) ii) markers of apoptosis (Cas3, Bcl-2 and Bax) iii) makers of steroidogenesis (StAR, CYP19A1 and FSHR). RNA sequencing and Immunoblot analysis were performed to study the AKT pathway and apoptotic protein expression. The exosome treated HGrC1 group showed improved cell proliferation (p<0.05) and compared to untreated damaged cells and increased cell viability was observed in exosome treated group (95.72%) compared to the damaged cells (92.01%). Among the genes tested, the relative gene expression of Bcl-2, an anti-apoptotic marker in exosome treated group was significantly upregulated (p<0.001) compared to untreated damaged cells. RNA sequence data and downstream apoptotic markers at the protein level using immunoblot analysis revealed presence of key miRNAs responsible for granulosa cell regeneration and activation of AKT pathway respectively. We showed that treatment of HGrC1 cells with hBM-MSC exosomes stimulates cellular proliferation and cell survival with the concurrent upregulation of Bcl-2. These preliminary findings encourage us to study the role of specific miRNA molecules identified and downstream anti-apoptotic pathways such as activation of AKT signaling responsible for the regenerative potential of hBM-MSC derived exosomes which warrant as a novel therapeutic option for POI patients. Murine Fetal Lung-Derived Amniotic Fluid Macrophages Are Polarized to a TAM-like State Prior to Labor at Term. Alina P Montalbano, Carole R. Mendelson * . UT Southwestern Medical Center, Dallas, TX, United States. Introduction: Preterm birth remains the leading cause of death in newborns and children under 5 years worldwide. This is due to our incomplete understanding of the integrated immuno-endocrine mechanisms that maintain uterine quiescence throughout pregnancy and those that trigger the inflammatory cascade leading to labor. We previously reported that surfactant protein A (SP-A) and platelet activating factor (PAF), secreted by the maturing fetal lungs beginning at 17.5 days post-coitum (dpc) (term = 19 dpc) into amniotic fluid (AF), serve as fetal hormonal signals for labor in mice. Based on these, and other findings, we propose that SP-A and PAF polarize AF macrophage (Mϕ) to a proinflammatory state and induce their migration into the maternal uterus where an increase in inflammatory mediators is temporally associated with NF-κB activation and parturition. Herein, we define the regulatory mechanisms by which SP-A and PAF polarize AF Mϕ and promote transmission of the fetal lung signal for labor at term. Methods: CD1 females were time-mated to like males and AF Mϕ were isolated at the indicated gestational age. Phenotypic changes in AF Mϕ were evaluated using multiparameter flow cytometry, fluorescence activated cell sorting (FACS), RT-qPCR, Illumina microarray technology and Ingenuity Pathway Analysis (IPA) software. Statistical analyses were performed using Graphpad Prism 9.2. Results: Flow cytometric analysis of 15.5, 17.5 and 18.5 dpc AF Mϕ revealed a significant increase in F4/80 + Mϕ (p<0.05) within the CD45 + AF compartment, enhanced expression of CD54, a marker of cell activation, and a tandem increase in proinflammatory/M1 (i.e., IL-1β, IL-6) and anti-inflammatory/M2 (i.e., ARG1, FIZZ1) activation markers beginning at 17.5 dpc. Notably, dual M1/M2 activation is reminiscent of tumor-associated Mϕ (TAM). Microarray transcriptome profiling of FACS isolated 15.5, 17.5 and 18.5 dpc F4/80 + AF Mϕ confirmed a dual increase in M1 and M2 marker expression and revealed enhanced levels of carbonic anhydrase 4 (Car4), a lung Mϕ-specific marker at 17.5 and 18.5 vs. 15.5 dpc. IPA identified 'Th1 and Th2 Activation' and 'Granulocyte Adhesion and Diapedesis' among the top enriched pathways at 18.5 dpc. Upstream regulator analysis predicted PAF (p<5.30E -06 ) among the potential activators responsible for the coordinated and enhanced expression of differentially expressed genes between 15.5 dpc and 18.5. Conclusion: These data demonstrate that SP-A and PAF polarize F4/80 + AF Mϕ to a TAM-like state and modulate chemotaxis just prior to labor. Collectively, this study provides the first comprehensive analysis of the dynamic phenotypic changes in F4/80 + AF Mϕ from a period of myometrial quiescence to contractility and reveal the major molecular pathways by which this immune cell population may transmit the signal for labor. Introduction: Placental invasion of the uterine wall is a crucial process for establishing a pregnancy. The inadequate invasion often leads to placental complications, such as preeclampsia and intrauterine growth restriction. During early pregnancy, extravillous trophoblasts (EVT) invade the maternal endometrium and spiral arteries, contributing to placental anchorage, spiral artery remodeling, and immunological defense. Despite their essential roles, molecular mechanisms underlying the regulation of EVT specification have been elusive. So far, only a few key transcription factors (TFs) are known in this context and their contributions to EVT function remain poorly characterized. As cell-type-specific TFs are often associated with multiple cell-type-specific enhancers, we sought to identify previously unknown key EVT-specific TFs by mapping enhancers in EVT and validate their roles by using human trophoblast stem cells (hTSC) to EVT differentiation as a model system. Methods: hTSC were differentiated into EVT as previously reported, and the activation of EVT marker genes was confirmed. To identify key TFs responsible for EVT functions, we mapped EVT-specific enhancer usage by ChIP-seq combined with global gene expression profiling during hTSC to EVT differentiation. Among multiple candidate TFs, we validated five TFs by loss-of-function approaches followed by monitoring changes in morphology, marker expression, and invasion ability. Changes in the transcriptome were also analyzed upon knockdown (KD) of putative key EVT-TFs by RNA-seq to understand how the candidate TFs regulate gene expression programs responsible for EVT functions. Results: After mapping cell-type-specific enhancer usages in hTSC and EVT, we identified a handful of novel EVT-specific key TFs. Individual KD of the candidate TFs during hTSC to EVT differentiation showed morphological defects and impaired activation of EVT markers, suggesting that these candidate TFs are crucial for EVT specification. KD also resulted in invasion defects, further supporting the necessity of candidate TFs for proper EVT function. Additional transcriptome analysis upon KD of candidate TFs revealed that KD cells show impaired activation of EVT-specific genes with delayed downregulation of hTSC markers. Interestingly, we found commonly down-regulated genes among KD cells, implying an interconnected regulatory circuitry of the tested TFs in EVT specification. Conclusion: Altogether, we have identified novel TFs regulating EVT specification and function. Elucidation of the underlying molecular level regulation mediated by these TFs will enhance our knowledge of placentation and inform efforts to treat pregnancy disorders. Introduction: Ovarian cancer is a rare, but deadly disease. As the deadliest gynecologic cancer, ovarian cancer is also the fifth leading cause of cancer death among women in the United States. Greater than 60% of women are diagnosed with serous ovarian tumors, of which, high-grade serous ovarian cancers (HGSC) are the most deadly. A substantial subset of HGSC are homologous-recombination (HR) proficient and resistant to poly (ADP-ribose) polymerase inhibitors (PARPi). Here, we've created a biobank of patient-derived ovarian organoids for the functional analysis of chemo-sensitivity, HR proficiency, and the screening of therapeutic combinations such as HDACi and PARPi. Methods: Tumor biopsies and ascites fluid were collected and used to generate ovarian cancer organoids for the establishment of a "living biobank". We used an adapted form of the previously published organoid media from the D'Andrea lab (Harvard). Primary tissue was also banked for histological analysis, and primary cell cultures were generated from ascites fluid for further in vitro analysis. Following organoid propagation, we performed histological analysis using fixed and embedded samples. We assessed HR proficiency by irradiating organoids, and staining for Rad51 and yH2AX. Lastly, we performed cell viability assays to assess tumor organoid sensitivities to carboplatin and olaparib. Results: To date, we have successfully generated nine organoid cultures from patient biopsies and ascites. We have successfully cultured 100% of our patient-derived ovarian organoids. Organoids derived from chemoresistant patients, showed resistance to carboplatin. In the organoid lines tested for HR proficiency, we did not see a significant difference. Conclusion: Our preliminary results have shown that functionally, patientderived ovarian organoids can be used for the screening of therapeutic drugs and HR proficiency. With the ever-increasing need for personalized medicine for cancer patients, the establishment of a living organoid biobank will aid the discovery novel therapeutics and the identification of biomarkers. Future studies will continue to screen for drug sensitives and the further assessment of HR proficiency. (P2X7R) is a ligand-gated cation channel that triggers the activation of the NLRP3 inflammasome and subsequent release of pro-inflammatory cytokine IL-1β. This study explored if genetic blockade of P2X7R plays a key role in mCMV-mediated placental damage, and whether blockade of IL-1β decreased viral load in placental tissue. Methods: CD-1 (n=40), C57BJL (WT; n=40), and C57BJL P2X7R -/-(KO; n=40) mice were inoculated with 5 x 10^5 murine CMV (mCMV) or vehicle (mock) at embryonic day (E) 10 in the uterine muscle. Dams were harvested at 96, 120, 144, 168, and 192 hours post injection (hpi). Placental IL-1β concentration was determined by ELISA across gestation. mCMV titers at 120 and 192hpi were determined by plaque assay. Immunohistochemistry staining was performed on placental samples at 192hpi to evaluate trophoblast and endothelial damage. Standard statistics were employed. Results: Between 96hpi, and 168hpi, IL-1β showed a temporal increase in expression in placental samples following mCMV injection in CD-1, decreasing at 192hpi (P<0.05 at E16 and P<0.01 at E17; Figure 1 -A). KO placentas, at 120hpi and 192hpi, showed a significantly decreased number of plaques as compared to WT (P<0.001). Cytokeratin (trophoblast) and Vimentin (endothelial) staining in KO, following mCMV infection resulted in a statistically significant decrease in placental damage as compared to WT (P<0.001; Figure 1 -B, 1-C Introduction: Controversies surrounding the existence of the placental microbiome and its origins persist. We tested the hypothesis that the outer membrane vesicles (OMVs) from Gram-negative bacteria serve as an alternate source of microbial DNA, regardless of the existence of a microbial community in the placenta. Methods: Placenta (n=35) were collected under sterile conditions from term, not in labor women undergoing Cesarean delivery. OMVs were isolated from 10 g tissues using density gradient ultracentrifugation and size exclusion chromatography. OMVs were characterized based on morphology (immunogold transmission electron microscopy), size and quantity (nanoparticle tracking analysis), expression of OMV, and tetraspanin markers (immunoprecipitation-Western blot for ompA and ALIX). The bacterial origin of OMVs was determined using 16S rRNA sequencing. The bacterial content of corresponding placental samples and a pool of all reagent controls were evaluated by 16S rRNA sequencing. Culture media from THP-1 macrophages treated with a low dose (10 7 ) and high-dose (10 10 ) OMVs were analyzed for IL-1β, IL-6, TNF-α, and VEGF to determine their functional properties. Results: We developed a protocol and isolated OMVs from the placenta with amplifiable DNA. The median size of OMVs was 130-140 nm, and the mean concentration was 1.8-5.5 × 10 11 OMVs/g of the wet placenta. OMVs have circular morphology and contain LPS and ompA but no human exosome markers. IP-WB analysis confirmed ompA but not ALIX and flagellin. The OMVs had higher bacterial diversity than placental tissues and were dominated by Streptococcus, Pelomonas, Pseudomonas, Enterobacter, and Anaerobacillus. The placental samples had a much lower alpha diversity with merely any reads belonging to Escherichia-Shigella, Anaerobacillus, and Bacillus. Corynebacterium, Pseudomonas, Streptococcus were primarily identified in OMV samples, with barely any signal detected in the placenta group. Lastly, OMVs significantly increased IL-1β and IL-6 in THP-1 macrophages in a dose-dependent manner. Conclusion: This is the first report to develop a protocol for isolating OMVs from any human tissue capable of promoting inflammation in immune cells. Placental OMVs represents a distinct microbiome. OMVs and other microbial vesicles can reach the placenta through hematogenous spread from both maternal and fetal colonizers, intercellular paracrine way, or from the placental microbial colony, if any. OMVs can potentially confound placental microbiome studies, and their low biomass in the placenta is unlikely to have any immunologic impact. Introduction: Group B streptococcus (GBS) is a bacterium that asymptomatically colonizes the vaginal tract of ~30% of women. During pregnancy, colonized women are at risk for developing invasive disease, leading to adverse pregnancy outcomes such as preterm birth, stillbirth, and neonatal disease. Annually, up to 3.5 million preterm births, 55,000 stillbirths and 90,000 infant deaths are attributable to GBS invasive disease, thus developing effective therapeutics remains urgent. Current animal models to study GBS invasive disease inoculate pregnant animals late in gestation to cause clinically relevant outcomes, such as chorioamnionitis, fetal demise, and preterm birth. However, this approach does not reflect the true course of GBS pathogenesis, as women are colonized with GBS prior to pregnancy. To adequately determine factors unique to pregnancy that promote invasive disease, we developed a mouse model of long-term vaginal colonization prior to pregnancy. Methods: On Day 0, 6-8-week old female C57Bl/6J mice were injected with β-estradiol (0.1mg/ml), and inoculated 24 hours later with 10 7 CFU of invasive (GB411; n=6) or colonizing (GB653; n=6) GBS strains for comparison to mice inoculated with PBS (n=3). Colonization was quantified by intravaginal swabs every 3 days. To monitor pregnancy outcomes, on colonization day 21 dams were paired with sires and insemination, weight gain, gestational length, neonatal viability, and GBS colonization of the reproductive tract were assessed. Results: Mice induced into estrus sustain GBS colonization for >30 days. All dams were inseminated at similar rates regardless of colonization status. During pregnancy, 85% of dams cleared GBS to undetectable levels by gestational day 10, which contrasts significantly with non-pregnant dams where 90% were colonized at the equivalent time point. Neonatal viability was similar amongst GBS colonized dams compared to PBS controls, except one GB653 colonized dam with dystocia; all pups died in utero due to GBS endometritis. Dams with GBS vaginal colonization throughout gestation tested positive in the uterus, cervix, and vagina 1 day post-partum. One dam inoculated with GB653 had no detectable GBS throughout pregnancy, but tested positive for GBS in the uterus and cervix at 1 day post-partum. This dam's pups were GBS positive in the thoracic cavity. GBS-positive dams had increased CD4+ and CD8+ T cell infiltrates in reproductive tissue compared to PBS controls. Conclusion: Taken together, we established long-term GBS vaginal colonization and subsequent pregnancy in mice, where >15% of pregnant dams tested positive for GBS colonization at parturition. Of these 1 dam vertically transmitted GBS to the litter and 1 dam had pups died in-utero. This model will allow us to assess the GBS colonization dynamics throughout gestation to define the role of adaptive immunity in GBS colonization and invasive disease. Introduction: Spontaneous preterm birth (sPTB) impacts newborn morbidity and lifelong health. The placenta is a key regulator of the developing newborn, and pathological changes related to parturition may be reflected in the placental transcriptome. This analysis aimed to identify genes whose placental expression was associated with sPTB (including early sPTB and late sPTB) and labor status and delivery method in two distinct cohorts. The ECHO PATHWAYS consortium extracted RNA from placental samples collected from 788 women enrolled in the CANDLE study (Shelby County Tennessee, USA), and 271 women enrolled in the GAPPS study (Seattle and Yakima, WA, USA). Transcriptomic data were obtained using paired-end RNA sequencing. Each cohort was analyzed separately. Linear models were fit to estimate separate associations between (A) placental gene expression and sPTB, adjusted for confounding variables and excluded patients with induced labor, and (B) placental gene expression and labor status and delivery method, adjusted for confounding variables including gestational length. Biological pathways were identified using self-contained gene set testing. Genes and pathways were considered differentially expressed at a Benjamini-Hochberg false discovery rate p <0.05. Results: We identified 942 genes and 51 pathways within CANDLE and 840 genes and 47 pathways within GAPPS that were significantly associated with spontaneous preterm delivery. We observed a greater number of transcriptomic differences in early preterm infants (<34 weeks) than late preterm infants (<37 weeks) compared to term. We observed increased expression of genes involved in metabolism and replication and decreased expression of genes in the Notch signaling pathway. We identified 1,264 genes that were significantly associated with C-section no labor deliveries in CANDLE. Increased expression of genes within signaling, immune, and endocrine pathways was associated with spontaneous C-sections, but decreased expression of genes within the same pathways was associated with no labor C-sections. Conclusion: These data strengthen our understanding of the interplay between placental physiology, parturition, and birth outcomes, which may inform advances in clinical obstetric practice. Blunted Introduction: Stress during pregnancy can detrimentally impact maternal and neonatal outcomes and may underlie health disparities seen in the human population. However, stress can be difficult to measure in humans due to variability in stress perception. Stress questionnaires may be subjective, and many do not take into account other types of factors that influence stress such as racial discrimination. Salivary cortisol awakening response (CAR) has been associated with stress levels, with blunted levels indicating higher psychosocial stress. The best measure of stress during pregnancy is unclear. We hypothesized that CAR correlates with stress questionnaire scores. Methods: Stress was assessed in 15 women using 1) perceived stress questionnaire (PSS); 2) perceived ethnic discrimination questionnaire (PEDQ); 3) salivary CAR in early (12-16 weeks) and late pregnancy (34-36 weeks). Maternal pregravid BMI, age and education level were recorded. CAR area under the curve (AUC) was calculated for early (n=13) and late (n=12) pregnancy and Spearman rank R values were calculated for all other maternal variables. Results: CAR or baseline cortisol measured at wakeup were not associated with maternal BMI, age or PPS in early or late pregnancy. There was a trend for an association of CAR with PEDQ in early pregnancy (r = -0.49, P = 0.09) that became significant in late pregnancy (r = -0.9, P < 0.0001). Baseline wakeup cortisol was associated with PEDQ at early (r = -0.55, P = 0.035,) and late (r = -0.7, P=0.007) pregnancy. CAR AUC was also associated with education level in both early (r = 0.54, P=0.04) and late (r = 0.8, P = 0.0035) pregnancy. There was no association between PPS and PEDQ. The PEDQ may be a more sensitive predictor of stress in pregnancy than PSS, as it is associated with a blunted CAR which can indicate chronic stress exposure. Questions targeted to experiences of racial discrimination may be a better reflection of stress impacting biological processes in diverse populations of pregnant women. Introduction: There is significant variability around the age at which women reach the end of their reproductive potential. We hypothesize that this is due to epigenetic age acceleration, which will subsequently associate with lower ovarian reserve and poor cycle outcomes. Methods: Women 18-50 years of age undergoing oocyte retrieval were approached to participate in this study. Cycle characteristics and demographics were abstracted from the medical record by a qualified physician, including Anti-Mullerian hormone (AMH) level, antral follicle count (AFC), number and maturity rate of oocytes retrieved, and successful fertilization. Follicular fluid samples were collected from 70 participants. Granulosa cells were isolated and DNA was assayed with the MethylationEPIC array. DNA methylation age (GrimAge) and age acceleration (AA) were calculated using the method described by Horvath. Linear regression was used to determine associations between age acceleration and baseline measures of ovarian reserve (AFC, AMH) and cycle outcomes, such as total number and number of mature oocytes retrieved, and successful fertilization. Sensitivity analyses were conducted controlling for chronological age. Results: Participants were a mean of 37 ± 4 years old and 54% were white, 19% Black, and 27% other. AA was calculated as the residual of GrimAge and chronological age. AA is associated with baseline characteristics of ovarian reserve measured prior to cycle initiation, operationalized as AMH (t=-3.2, p=.002) and AFC (t=-4.2, p=7e-5). Results indicate that increased AA is correlated with lower ovarian reserve. Additionally, AA was associated with cycle outcomes. Both the total number of oocytes (t=-3.9, p=.0002) and the number of mature oocytes (t=-3.8, p=.003) were negatively associated with AA. Higher AA was also associated with having fewer oocytes successfully fertilized (t=-2.7, p=.008). These associations remained significant in models adjusting for chronological age. Conclusion: Our data suggests women with increased reproductive AA have lower ovarian reserve at the start of IVF and have poorer cycle outcomes. Future studies will be needed to determine if advanced reproductive aging may be an early indicator of other age-related health problems. Knowledge of reproductive aging may allow clinicians to better counsel patients about treatment prognoses, and may identify patients with increased risk for other adverse health outcomes associated with having a diminished ovarian reserve. Silencing P38 MAPK Reduces Cellular Senescence in Human Fetal Membrane Cells. Brett Goldman †, Enkhtuya Radnaa, Ramkumar Menon * . University of Texas Medical Branch at Galveston, Galveston, TX, United States. Introduction: Signals of cellular senescence have been identifi ed within the chorioamniotic membrane and linked to term and preterm parturition, and preterm premature rupture of membranes. Activation of cellular stress associated p38 mitogen activated protein kinase (MAPK) in fetal membrane cells is associated with senescence. This study tested the hypothesis that elimination of p38MAPK will reduce oxidative stress (OS)-induced cellular senescence. Methods: p38 MAPK was silenced in amnion epithelial cells (AECs) and chorion trophoblast cells (CTCs) using CRISPR/Cas9 technology by using multi-guide RNA (p38MAPK KO). p38MAPK KO was confi rmed by western blot. OS was evoked by exposing AEC and CTC wild type (WT) and p38 KO cells to cigarette smoke extract (CSE, an OS inducer). Cellular senescence was measured using senescence-associated β-galactosidase (SA β-gal) staining assay. T-test was used to assess statistical diff erences between WT and p38 KO AECs and CTCs. Results: Western blot analysis confi rmed p38MAPK KO in both AECs and CTCs. CSE exposure increased mean percentage of SA β-gal positive WT AECs from 4.93% (control) to 15.33%, resulting in a 210.95% increase (p<0.05). Senescence was reduced to 7% in p38MAPK KO AECs cells (a 119% diff erence, p<0.05). SA-β-Gal positive cells were 25.9%, 27.4% and 24.1% in WT CTCs (control), CSE exposed WT CTCs and p38MAPK KO CTCs respectively (p=ns). Conclusion: Confi rming our hypothesis, senescence in human fetal amnion epithelial cells is mediated by p38MAPK. OS induced senescence was minimal in CTCs and therefore p38MAPK KO had no impact on senescence suggesting refractoriness of CTCs to senescence. This is likely to protect the integrity of feto-maternal interface. Lack of senescence in p38MAPK KO AECs suggests that an alternate mechanism of senescence induction (e.g. p53 or other signaler mediated) is unlikely when amnion is stressed. Reducing p38MAPK activation may minimize amnion layer senescence and membrane dysfunction predisposing to preterm birth and pPROM. To determine p38MAPK's functional role, we first created cell free amnion membrane extracellular matrix (nude membrane) by treating normal term not in labor FM using Urea (5M). Wild type (WT) and p38 KO hAEC (using CRISRP/Cas9 approach) were treated with TGF-b (EMT inducer; 20 ng/ml) or cigarette smoke extract (CSE, OS inducer). Development of EMT, EMT-induced cellular migration and matrix invasion were tested after seeding either WT or KO hAEC over the nude membrane. EMT (vimentin staining) and inflammation (multiplex assay of IL-6, IL-8, IL-10 in culture media) were tested. FM specific cKO mice were achieved by injecting Cre recombinase encoded exosomes intraamniotically into each amniotic sac of p38α loxP/loxP mice. FM collected on E15 was tested for P-p38, total-p38 and EMT marker vimentin by WB, and senescence by histochemical staining. Results: Western blot showed successful deletion of the p38MAPK by CRISPR/Cas9 approach in hAECs ( Fig.1 A) . TGF-β induced EMT in WT hAEC but not in p38 KO (via morphological analysis; Fig 1B- OS induced inflammation in WT cells, but not in p38KO cells, as indicated by lower concentrations of IL-6 and IL-8 in media (P=0.005 and P=0.007, respectively) ( Fig.1 D-E) . FM from p38 cKO mice had reduced senescence and vimentin expression compared to WT animals ( Fig.2 A-B) . Conclusion: By disrupting p38 MAPK, a critical role played by this stress signaler in EMT, senescence and inflammation in both human and mice FM tissues were determined. Lack of senescence, EMT and inflammation in KO models supports that FM's integrity in utero is manifested by p38MAPK and lack of compensation by other signalers such as p53. Organs. Jiahui Ding †, Anthony Maxwell, Nicholas Adzibolosu, Marianna Sadagurski, Lucas K Debarba, Douglas M Ruden, Michael C Petriello, Gil Mor. Wayne State University, Detroit, MI, United States. Introduction: Benzene is a colorless, flammable liquid with a gasolinelike odor, which is a major source of environmental pollution. Benzene exposure during pregnancy in human is associated with several adverse outcomes, such as preterm birth, low birth weight and childhood leukemia in the offspring. However, the mechanism associated with these detrimental outcomes induced by benzene exposure during pregnancy is poorly understood. Here we tested the hypothesis that exposure to benzene during pregnancy induces maternal inflammation, which will lead to abnormal placental function and restricted fetal growth. More importantly, we aimed to evaluate the effect of benzene on the transcriptome changes in fetal immune organs, such as thymus and spleen. Methods: Female C57BL/6 pregnantmicewere exposed to benzene (50 ppm) for 5h/day from E0.5 to E17.5 usinginhalation chambers. On E17.5, mice were sacrificed, pregnancy outcomes, fetal growth measurement and placenta pathology were evaluated. Cytokine expressions were determined by Luminex. Placenta and fetal tissues were collected for RNA sequencing. Results: Exposure of benzene during pregnancy leads to maternal inflammation characterized by increased concentrations of circulating pro-inflammatory cytokines, including IL-3, IL-12 (p40), IL-12 (p70), IL-17, Eotaxin, TNFα and GCSF. Fetal resorption, impaired placental vascularity, and intrauterine growth restriction (IUGR) were all observed in the benzene-exposed group. These phenotypes correlated to our RNA sequencing data, which showed a severe inflammatory response in the placenta and significant transcriptome changes in the fetal thymus and spleen. We report the characterization of a mouse model of maternal inflammation associated with the exposure to benzene during pregnancy. Our findings correlate with human studies describing the association between maternal exposure to benzene and pregnancy complications, such as preterm birth and IUGR. Our data suggests that benzene-induced maternal and placental inflammation may be the mechanism responsible for these pregnancy complications. Moreover, RNA sequencing data suggest that benzene exposure during pregnancy causes significant transcriptome changes in the placenta and fetal immune organs, "reprograming" fetal immunity in utero and consequently predisposing the offspring to bacterial and/or viral infection postnatally. transcription factor activation. Our previous data has shown that small pieces (<1000bp) of cell-free fetal DNA (cffDNA) is able to cause the translocation of this transcription factor, but that the resultant IL-6 and Matrix Metalloproteinase (MMP) secretion from FM explants appears to occur with a sex specific pattern. Therefore, we aimed to characterize these sex specific differences and hypothesized that the differences between the fetal sexes may be due to the activation of different receptors capable of NF-κB activation. Methods: Human FM were collected from term repeat Cesarean sections (≥ 39 weeks) at Kapiolani Medical Center for Women and Children with IRB approval. FM explants were placed into a Transwell to create a two-compartment system and the fetal side treated with either whole (>1000bp) or 4 min sonicated (100-1000bp) cffDNA for 24hrs (n=9). IL-6 (R&D Systems), MMP levels were measured by ELISA and zymography respectively, from conditioned media collected and normalized to total secreted protein. Fetal sex was determined by SRY and ALT1 expression. Immunohistochemistry (IHC) was performed on FM paraffin embedded sections for the receptors: TLR-9 (ab37154, 1:25), STING (ab189430, 1:25) and RAGE (ab3611, 1:100) using the Vectastain Elite ABC-HRP, DAB Peroxidase HRP Substrate (n=8) with citrate buffer antigen unmasking treatment for RAGE and STING. Results: Treatment of male tissue with 4 min cffDNA increased IL6 secretion 5.38 (p=0.02) and 1.27 (p=0.01) fold in fetal and decidual compartments respectively whereas whole cffDNA did not have an effect on IL-6 secretion. Female tissue responded similarly to both whole and 4 min cffDNA having a 40-80% increase (n/s) in IL-6 secretion from the top and bottom wells. Interestingly, the female explants only also had a 30% increase in proMMP9 secretion fold (n/s) from the basal side of the explants. We then confirmed the expression of the three receptors TLR9, RAGE and STING in the human FM. We saw expression of all three receptors in all cells of the amnion, chorion and decidua and saw qualitative expression level differences between patient tissues (n=8). Conclusion: This data suggests that males and female FM explants respond differently to cffDNA. Many other studies, have also demonstrated differential inflammatory responses that are dependent on fetal sex, although the mechanisms for this are not understood. It is known that small pieces of DNA interact with TLR9 causing inflammation, however, larger pieces can be sequestered by HMGB1 and then activate RAGE or activate the STING receptor. Therefore, ongoing studies are aimed at understanding the status of these DNA sensing receptors to determine any sex specific expression or activation pattern. The maintenance of coordinated powerful episodic contractions of the uterus is the crucial factor for normal labor. The uterine contractility is gradually enhanced with the progression of labor, which is related to the gene expression of myometrium, and endogenous RNA (ceRNA) can also regulate the gene expression. However, a comprehensive analysis of the role of ceRNA network in labor has been rarely reported. Methods: Transcriptome sequencing was performed on the myometrium of 17 parturients at different labor duration, and identified mRNA, long non-coding RNA (lncRNA), circular RNA (circRNA), and microRNA (miRNA) which correlated with their expression levels and labor duration. Then, targeting relationships between mRNAs, lncRNAs, circRNAs and miRNAs were predicted, and the ceRNA regulatory network was established. The mRNA expression patterns associated with cervical dilation and the function of these mRNAs were further investigated. Results: Through correlation calculation, a total of 934 RNAs positively correlated with labor duration (859 mRNAs, 28 lncRNAs, 45 circRNAs, and 2 miRNAs) and 157 RNAs negatively correlated with labor duration (122 mRNAs, 28 lncRNAs, and 7 miRNAs) were identified. These mRNAs were involved in protein metabolism, transport and cytoskeleton functions. According to the targeting relationship between the 9 miRNAs and other RNAs, a ceRNA network consisting of 9 miRNAs, 122mRNA, 2 circRNAs and 1 lncRNA was established. In addition, two mRNA expression patterns were established by time-series analysis of mRNA expression in different stages of cervical dilation. Conclusion: This is the first study to identify human myometrial transcriptome in different labor duration, and to establish the regulatory network of ceRNA in labor. At the genetic level, it provides new insights for maintaining of uterine contractions during labor. Cervicovaginal However, the molecular mechanisms regulating these effects are unknown. The objective of this study was to identify transcripts and functional clusters altered by host-microbe interactions using whole genome RNA sequencing. Methods: Human ectocervical (Ecto), endocervical (Endo) and vaginal (VK2) epithelial cells were co-cultured with live LC (ATCC 33197) or GV (ATCC 14019) (~1x10 6 -1x10 4 CFU/well) or their supernatants (1% v/v) for 24 hrs. Bacterial abundance was quantified by CFU. RNA was extracted and sequencing was performed. PCA plots were created using pcaExplorer. Analysis was done using DESeq2 and a Log2FoldChange cutoff of ≥1 and ≤-1 and an adjusted p-value<0.01 were established. Functional pathway analysis was performed with Metascape and DAVID. Results: PCA revealed gene expression differences between CV cells exposed to live microbes and bacteria-free supernatants (SUPS). Adjusting for multiple comparisons and using a strict false discovery rate, many genes were significantly altered; gene expression changes were bacteria and cell specific ( Fig 1A) . Protein-protein interactions revealed unique networks among different CV cells with the same bacterial exposure (Live GV; Fig 1B) . For all CV cells exposed to GV (live or SUPS), cytokine signaling and immune response was the most significant functional annotation cluster (p-value=1x10 -14 -7x10 -16 ) with each cell type having its own unique immune signature. GV also induced significant changes in cell adhesion pathways in cervical cells. Live LC altered very few genes, while LC SUPS altered the greatest number of genes with nucleosome and histone clusters having the highest functional significance (p-value=3x10 -3 -3x10 -13 ). The transcriptomic profile of CV epithelial cells exposed to common microbes is bacteria and cell specific. Live bacteria and their supernatants elicit unique transcriptomes revealing the complexity of host-microbe interactions in the CV space. The perturbation of immune function and cellular adhesion pathways by GV in contrast to the induction of epigenetic machinery by LC suggest novel pathways by which these microbes may regulate reproductive health and disease. (5R01HD098867, 1R01HD102318) The , the cells that are key for the maintenance of the integrity of the FM during pregnancy, has not been studied. Therefore, the aim of this work was to understand HMGB1's effect on AMC and we hypothesized that HMGB1 would increase cytokine production from AMC, leading to the production of the ECM weakening enzymes. Methods: Term human FM were collected from repeat Cesarean sections (≥ 38 weeks) at Kapiʻolani Hospital for Women and Children with IRB approval. FM AMC's were isolated, passed (P2-3), and grown at until 60% confluent (n=6). They were then treated with HMGB1 (1, 5, 10, 50, and 100ng/ml) for 2 hours. RNA was isolated using RNAeasy Mini Kit (Qiagen), cDNA were generated with the High Capacity RNA to cDNA kit (Applied Biosystems) and RT-PCR performed using Taqman Gene Expression assays for Human IL6, IL8, TNFɑ, and GM-CSF and normalized to GAPDH and I8s housekeeping controls. AMC were also placed in 3D cell culture at a density of 1 million cells per scaffold for 5 days. They were then treated with HMGB1, LPS, or Cell-Free Fetal DNA (cffDNA) at 100ng/ml for 24 hours (n=4). Matrix metalloproteinases (MMP) levels were then assessed in conditioned media by zymography and normalized to sample total protein. Results: Cytokine gene expression increased with HMGB1 treatment. IL-8 expression increased with 5ng/ml (not significant (n.s)) and at 10ng/ ml (p<0.05) of HMGB1, but expression gradually decreased to baseline at higher concentrations. The pattern of increased IL-6 was similar with a significant increase at lower concentrations of HMGB1 treatment, 1ng/ml and 5ng/ml (p<0.05). The expression was non-significantly increased at higher HMGB1 concentrations but then returned to baseline at 100ng/ml. TNFɑ expression was only significantly increased at 1ng/ml (p<0.05) of HMGB1. GM-CSF increased at 1ng/ml (n.s), peaking at 5ng/ml (n.s) but then returned to baseline at higher concentrations. cffDNA and HMGB1 treatment did not increase proMMP9 or proMMP2 levels, however, LPS did cause a non-significant increase in proMMP9 secretion. Conclusion: Our data suggests that despite large inter-patient variation in responses, HMGB1 is able to stimulate inflammation in AMC. However, we were not able to determine if it causes changes in the ECM weakening enzymes. This indicates that when cell stress produces DAMPs, like HMGB1, they can stimulate inflammation and this could contribute to fetal membrane weakening. The Introduction: Chorioamnionitis is associated with preterm birth (PTB) and fetal injury, especially before 32 weeks' gestation. Recent studies reported that interleukin (IL)-1 targeting agents may improve outcomes in the setting of intrauterine inflammation. Using an extremely preterm sheep model of pregnancy, we aimed to investigate the anti-inflammatory efficacy of the competitive IL1R agonist, Kineret, in response to IA lipopolysaccharide (LPS) exposure. We hypothesized that intraamniotic (IA) Kineret would reduce fetal inflammation relative to treatment with saline. Methods: Sheep with a single fetus at 105d (term=150d) gestation were randomised to either: 1) IA injection of 10mg E coli LPS and IA injections of saline at 0h and 24h (LPS Only Group, n=11); 2) IA injection of LPS and IA injections of 100mg Kineret at 0h and 24h (LPS + Kineret Group, n=12); or 3) IA injections of saline at 0h and 24h (Saline Only Group, n=12). Animals were euthanized 48h after first IA treatment and subjected to necropsy. Inflammatory responses were characterized by qPCR, ELISA, haematology and biochemistry. One-way ANOVA was used to test group differences with a p value of 0.05 taken as significant. Results: qPCR analysis of fetal lung mRNA showed significant increases in IL-1β, IL-8, MCP-1, and TNFα in the LPS Only and LPS + Kineret Groups compared to the Saline Only Group. IL-6 was significantly increased solely in the LPS Only Group compared to the Saline Only Group. ELISA analysis of fetal lung protein showed significant increases in IL-6 and IL-8 in the LPS Only Group compared to the Saline Group; there were no differences between the LPS + Kineret Group and the Saline Only Group. Cord blood WBC counts were significantly decreased in the LPS Only Group relative to the Saline Group; those in the LPS + Kineret Group were comparable to the Saline Only Group. Cord blood insulin-like growth factor (IGF)-1 concentrations in the LPS + Kineret Group were significantly decreased compared to the Saline Only Group. Conclusion: IA administration of Kineret partially resolved LPS-driven fetal inflammation, but suppressed a key growth factor, IGF-1, in an ovine model of pregnancy. These data highlight the need for carefully targeted anti-inflammatory interventions in the treatment of chorioamnionitis. The Introduction: Uterine contraction is the crucial factor of childbirth, but the mechanism of their regulation is unclear. SERPINE1 was significantly elevated in the proteomics and transcriptome of the myometrium at labor, suggesting that SERPINE1 may be a biomarker of labor. However, the mechanism of SERPINE1 involved in regulating the contraction of the uterus in labor is unknown. Therefore, we investigate the effects and mechanism of SERPINE1 on an important factor of uterine contractility during labor. Methods: We used RNA sequencing (RNA-Seq) to assess genomewide differences in mRNA expression between non-labor and in-labor myometrium. The expression and biological function of SERPINE1 involved in uterine contractions were performed though transcriptome analysis, immunofluorescence, WB, qPCR and tension measurement. We established hypoxia models of uterine muscle strips and human muscularis smooth muscle cells (MSMCs), and tested the regulation of SERPINE1 on the contractile function of uterine smooth muscle cells by cell contraction assay. SERPINE1-siRNA was constructed and transfected to human muscularis smooth muscle cells (MSMCs), and RT-PCR was used to detect the expression of SERPINE1 in smooth muscle cells after transfection. Binding site analysis of ChIP-seq data of transcription factors was used to explore the up-stream of SERPINE1. Results: We found that SERPINE1 was dramatically high expressed both in parturient myometrium and hypoxic uterine myocytes, which were all linked to the increase expression of contraction-associated proteins, myosin light chain kinase (MYLK), the connexin 43(CX43) and cyclooxygenase-2 (COX2). SERPINE1 expression was decreased after siRNA transfection followed the reduction of the contractile activity of smooth muscle cells. Moreover, the expression of HIF-1α and SERPINE1 increased under hypoxia, and the contractile activity of smooth muscle cells also increased. We found that HIF-1α is directly binding on the promoter of SERPINE1 and increased the expression of SERPINE1, and the rescue experiments were performed to identify the regulatory mechanism of HIF-1a/SERPINE1 pathway in contractions of myometrium. Conclusion: In summary, we discovered a HIF-1a/SERPINE1 pathway that plays important roles in contractions of myometrium during labor and provided novel therapeutic targets for the regulation of contractions during labor and postpartum bleeding. Methods: Nineteen subjects participated after written informed consent and divided into endometriosis (N=12) or control (N=7) subjects. All samples were obtained at the time of surgery from women with the confirmed presence or absence of endometriosis. DNA isolated, and modified with sodium bisulfite for DNA methylation. Five regions upstream from the Let-7b coding region on chromosome 22q13.31 were amplified using bisulfite-specific PCR to amplify regions. DNA sequences were analyzed using BiQ Analyzer to determine methylation status at each CpG island (+/-). Also visualized and analyzed using Chromas software and percent methylation was calculated for each CG location by comparing cytosine to thymine peaks to compare methylation. Results: Regions were chosen based on percent (%) GC content and data from Ensembl/ENCODE databases which predict locations of promoters, enhancers, CTCF and transcription factor binding sites as well as candidate cis-regulatory elements. Methylation was calculated from analyzed nucleotide peaks on Chromas software. A region 1,161 basepairs upstream of the Let7b coding region was significantly differentially methylated in ectopic samples compared to eutopic endometrium from patients with endometriosis (ectopic mean 0.835 vs eutopic mean 0.769; p=0.01 t-test). Four specific CG locations within this CG island were further analyzed individually, and CG #2 was found to be significantly methylated in ectopic samples vs eutopic (ectopic mean 0.928 vs ectopic 0.671; p=0.003). We used the Gene Transcription Regulation Database to identify specific transcription factors that may bind this site and found that ZN770, SP1, SP2, SP3, PATZ1, and SP4 were all predicted to bind at CG #2. Conclusion: This data strongly supports that methylation of this location regulates Let7b in endometriosis, demonstrating the epigenetic nature of the disease. Methylation of Let7b upstream regions may decrease transcriptional activation and thereby influence endometriosis pathogenesis. Let7b has been clearly demonstrated to be important in the pathophysiology of endometriosis, and hence understanding the impact of epigenetics on its transcriptional regulation may allow for novel diagnostics and treatments. We previously identified select xenobiotic metabolites in cervicovaginal (CV) fluid as early biomarkers of spontaneous preterm birth (sPTB). Though the biology of sPTB is rooted in a myriad of pathogenic processes, cervical remodeling is believed to be a common antecedent. We posit that xenobiotics are biologically active in the CV space and drive cervical remodeling on the pathway to sPTB. Methods: Vaginal (VK2), ectocervical (ecto), and endocervical (endo) cells were treated with xenobiotics diethanolamine (2.5mM), ethyl glucoside (5mM), or tartrate (2.5mM) for 24-48h. Concentrations were selected based on doses resulting in cytotoxicity equivalent to controls. Proinflammatory markers associated with sPTB (IL-8, IL-1β, MMP-9) were measured by ELISA. Permeability was measured in cervical cells by FITC-Dextran movement across collagen-coated membranes. Standard analytic approaches were performed to establish significance (p<0.05). Results: Diethanolamine increased IL-8 in VK2 and endo (p=0.004 and p=0.001, respectively; Fig. 1 ). Tartrate increased IL-8 in VK2 (p=0.002), IL-1β in ecto and endo (p=0.030 and p<0.001, respectively), and MMP-9 in endo (p=0.049) (Fig. 1 ). Ethyl glucoside decreased IL-8 in VK2 and endo (p=0.038 and p=0.186, respectively), increased IL-1β in endo (p<0.001), and decreased MMP-9 in VK2 (p=0.008) (Fig 1) . Diethanolamine and tartrate increased permeability in ecto (p=0.006 and p=0.020, respectively; Fig. 2 ). Conclusion: Xenobiotic metabolites are biologically active in the CV space and induce epithelial barrier dysfunction. Diethanolamine and tartrate disrupt the CV epithelium and modify local immune output. These data begin to ascribe a mechanistic link between exogenous exposures and cervical remodeling in sPTB. Xenobiotics in the CV space may facilitate sPTB risk stratification and unveil novel modifiable risk factors. . Women with high leukocyte counts (n=572) had higher prevalence of Lactobacillus dominated microbiomes compared to women with low leukocyte counts (n=247), and this was mostly driven by significantly higher relative abundance of L. iners (p<0.01, Mann-Whitney). Lactobacillus spp. depleted microbiomes, high sialidase activity and/or high leukocyte counts was not associated with higher PTB risk. The pregnancy vaginal microbiome of Chinese women is most commonly dominated by L. crispatus. Despite sialidase activity associating with high diversity community composition, the microbiota was not associated with PTB risk in this cohort. This provides further evidence that microbiota-host interactions during pregnancy are likely influenced by ethnicity and/or geographical differences. Differences in host immune response to vaginal microbiota in pregnancy may contribute to lower rates of PTB in this Chinese population. The uterus adapts to pregnancy in support of fetal growth. The uterine myometrium remodels to maintain uterine structural integrity for pregnancy and to provide contractile force at parturition. Failure on this process may lead to pregnancy complications such as intrauterine growth restriction and laboring disorders. Progesterone is required to sustain pregnancy and modulate muscle contraction. Progesterone acts through the progesterone receptor (PGR) to regulate cellular functions and command genome actions. We hypothesize that myometrial PGR confers a unique gene expression program that maintains structure and functionality of the myometrium and supports uterine remodeling during pregnancy. Methods: PGR was ablated in mouse smooth muscles by crossing together Myh11Cre and PGRflox alleles. Fertility was assayed by 6-moth breeding trials. Morphology was assessed by histology, immunohistochemistry, and micro computed tomography. Uterine isometric contractility was measured by wire myograph. Transcriptome, PGR occupancy, H3K27ac histone marks and chromatin conformation of pregnant mouse uterine walls were assessed by RNAseq, ChIPseq and HiC assays. Results: Mutant females exhibited a subfertility phenotype. Mutant oviducts manifested an abnormal spatial organization of tubules and retained embryos at 3.5 days post coitum. Isolated uterine strips showed 76.6% reduction of oxytocin-stimulated isometric contractility in mutant compared with control. At mid-pregnancy, mutant uterine wall showed abnormal muscle and collagen fiber morphology with an altered muscle and extracellular matrix ( The Introduction: Programmed cell death protein 1 (PD-1) and its legend PD-L1 pathway are crucial mediators of immune responses, especially immune tolerance and immune evasion. However, the relationship between PD-1/PD-L1 and macrophages differentiation during maternal inflammation has not been fully elucidated. We hypothesized the PD-1/PD-L1 pathway expression associated with macrophages changes following sub-chronic inflammation. Methods: At embryonic (E) day 14, CD-1 dams (n=25) were randomly allocated into two groups: PBS and IL-1β groups. Dams received intraperitoneal injection (IP) of 0.5µg rIL-1β in 100 µL PBS or 100 µL of PBS for four consecutive days. PTB and viability were observed. Maternal blood, decidua and placental labyrinth were harvested at 24-or 48-hour post injection (hpi). Flow cytometry was performed to characterize CD45 + leukocyte, monocyte or macrophage, and PD-1/PD-L1 expression. Standard statistics were employed. Results: In maternal peripheral blood, the monocytes were increased following rIL-1β exposure. In decidua and placental labyrinth, the CD45 + leukocyte infiltration was significantly increased and the PD-1 expression on CD45 + leukocyte was decreased (Table 1 -2). In decidua, the PD-L1 expression on CD45 + leukocyte was increased, while in placental labyrinth was decreased following rIL-1β exposure (Table 2 ). In decidua, total macrophages significantly increased with decreased PD-L1 expression at 24hpi or increased PD-1 expression at 48hpi. In the placental labyrinth, while macrophage number did not change, their PD-1 and PD-L1 expression was decreased (Table 1 -2). Our study provides evidence of dynamic immunological alteration in the proportions of immune cells, especially macrophages in the maternal blood, decidual, and labyrinth following sub-chronic maternal inflammation. We demonstrated that IL-1β mediated PD-1/ PD-L1 pathway expression and altered the homing and differentiation of placental immune cells. Introduction: Preterm birth is the leading cause of infant morbidity and mortality and currently available tocolytics are ineffective. The non-desensitizing ꞵ3AR has been well studied in urinary bladder and the ꞵ3-agonist mirabegron (Myrbetriq™) is available for treatment of overactive bladder. Recent reports indicate that ꞵ3AR are also expressed in myometrium and relax the tissue. Earlier studies imagined a therapeutic potential of ꞵ3AR stimulation but did not explore signaling beyond assumptions that myocyte cAMP sub-served relaxation. Because nitric oxide (NO) relaxes myometrium, we hypothesized that ꞵ3 receptors may be present in both endothelial and muscle compartments with the effect of ꞵ3 receptor stimulation a result of both endothelial and myocyte signaling. Methods: Myometrial tissue from term non-laboring patients (TNL; n=3) were finely dissected into strips secured in a horizontal tissue bath system (Danish Myo Technology) for physiological measurements. Mounted strips were bathed in oxygenated Krebs buffer at 37 o C and exposed to 60 mM KCl (3min) followed by washout and addition of oxytocin (8 nM, 30 min). Tissues were then exposed to mirabegron (MBG, 100µM), MBG plus L-NNA (100µM), MBG plus the selective ꞵ3 antagonist SR59230 (10µM), or DMSO control for 60 min followed by washout. Digital recordings were analyzed using via LabChart (ADInstruments). Isolated human myometrial cells were derived by enzymatic digestion of isolated myometrial strips and grown in primary culture (72 hr). Primary cells were detached and selected as endothelial versus myocyte with CD31 +antibody binding. Cells were plated on a 96 well plate with 4,000 cells per a well, pre-incubated with 100µM DAF-FM diacetate (Ex/Em of 495/530) to detect NO generation, and then treated with MBG (100µM) or DMSO control for 120 min. Data was generated using a multimode microplate reader. Results: Immunofluorescence staining of isolated myometrial cells revealed β3AR in both endothelial and smooth muscle cells compartments. Relaxation of muscle strips by MBG was abolished by SR59230A (p<0.001), while 15 min pretreatment of strips in the bath with the eNOS blocker 100µM L-NNA only partially inhibited MBG-mediated relaxation (p<0.05). MBG-stimulated NO production by endothelial cells was blocked by L-NNA. As expected, NO production was not seen in isolated myocytes. The partial reduction of MBG-mediated relaxation by L-NNA demonstrated that NO-mediated signaling is only partially, if significantly responsible for ꞵ3AR-mediated relaxation. These data suggest that both endothelial and smooth muscle cells contribute to relaxation through disparate signaling pathways. The failure of ꞵ2AR agonists as tocolytics raises the possibility that ꞵ3AR signaling in the myocyte may involve a non-cyclic nucleotide pathway. These data argue that further examination of ꞵ3AR signaling in myometrium may reveal MBG as a useful tocolytic in a combination tocolysis regimen. Organ-on-Chip. Lauren Richardson, 1 Sungjin Kim †, 2 Arum Han, 2 Ramkumar Menon * . 1 1 UTMB, Galveston, TX, United States; 2 TAMU, College Station, TX, United States. Introduction: Infection and inflammation during pregnancy contributes to spontaneous preterm birth (PTB). Two distinct inflammatory pathways can lead to PTB: ascending maternal infection (maternal-fetal), intraamniotic infection, followed by a fetal inflammatory response (fetal-maternal). However, propagation of infectious and inflammatory mediators across the feto (fetal membrane)-maternal (decidua) interface (FMi) (i.e., amnion [mesenchyme and epithelium], chorion, decidua) is difficult to study due to the lack of appropriate in vitro models and limitations of animal models. To study infectious agent's propagation kinetics, the development of inflammatory responses, and pro-inflammatory cellular changes like Epithelial-to-Mesenchymal Transition (EMT), we developed a novel microfluidic, interconnected, organ-on-chip (OOC) device to mimic the FMi. The feto-maternal interface OOC (FMi-OOC) is a circular, four chamber, device containing cells and collagen matrix from the FMi connected by Type IV collagen coated microchannels that mimics the FMi in vivo. Lipopolysaccharide (LPS, 100 ng/mL) was added to either the inner decidua chamber or outer amnion epithelium chamber, to model maternal infection or intra-amnionic infection respectively. Cellular (immunocytochemistry) and inflammatory (multiplex cytokine immunoassay) changes were monitored. EMT induction defined as a change (p<0.05) in the Vimentin/Cytoketatin-18 ratio in amnion epithelial cells was also determined. Results: LPS propagated from decidual cell (mimicking maternal ascending infection) through the chorion and amnion mesenchyme in 48h, and reached the amnion epithelium within 72h. Similarly, LPS exposure of amnion epithelium (mimicking intraamniotic infection), propagated through the amnion mesenchyme and chorion in 48h, and reached the maternal decidua within 72h. Compared to untreated controls, both decidual and amnion exposures of LPS induced time-dependent and cell-type-specific IL-6 and GM-CSF in FMi cells ( The myometrium contracts strongly during labor, but the underlying mechanisms are unclear. Our previous studies have found that autophagy is activated in the human myometrium during labor, and autophagy may be a new mechanism for regulating myometrium contraction. The role of golgi reassembly stacking protein 2 (GORASP2) in promoting autophagy has been demonstrated in tumor cells. This study was aim to explore the role of GORASP2 in regulating autophagy of uterine smooth muscle cells and its effect on uterine contraction. Methods: The mRNA and protein expression level of GORASP2 and autophagy markers (LC3B, P62, and Beclin-1) were detected in myometrium tissue from women that in labor and not in labor. Primary uterine smooth muscle cells (USMCs) were cultured under hypoxic conditions to simulate the hypoxic environment during labor. siRNA was used to inhibited GORASP2 expression in USMCs, and autophagy levels of the cells were measured to assess the effect of GORASP2 on autophagy. RNA sequence was performed to explore the pathways and molecular mechanisms of GORASP2 involved in autophagy regulation. Results: Both mRNA and protein expression of GORASP2 in the myometrium in labor was significantly higher than that not in labor. The autophagy level in USMCs was significantly inhibited after reduced the expression of GORASP2. The were 357 genes altered their expression significantly in USMCs as GORASP2 down-regulated. These differential expressed genes involved multiple functions and pathways that related autophagy. The autophagy of human myometrium was increased during labor, and that may promoted by GORASP2. GORASP2 can be a potential regulates for myometrial contraction to be used for labor management. Maternal Platelet Activation and Preterm Birth. Susan E. Introduction: Studies have reported reductions in the risk of preterm birth (PTB) with use of low dose aspirin (LDA), yet the mechanism of this association is unclear. We examined whether maternal plasma platelet factor 4 (PF4) concentration, a biomarker of platelet activation, is associated with PTB, and whether preconception treatment with LDA modifi es this association. Methods: This study is a secondary analysis of the Eff ects of Aspirin in Gestation and Reproduction (EAGeR) trial, which randomized participants to LDA or placebo prior to conception. Participants who conceived and carried a pregnancy to at least 16 weeks were included. The exposure of interest was maternal PF4 concentration, measured at preconception, 12, and 28 weeks gestation. PF4 concentrations were analyzed in tertiles, with the 1st tertile as the referent group. The primary outcome was any PTB before 37 weeks gestation. Secondary outcomes included spontaneous and medically indicated PTB. Log-binomial regression estimated relative risks for the association between PF4 and outcomes of interest, adjusted for maternal age, BMI, smoking, education, income, and previous PTB. Results: Analyses included 581 participants, of which 51 (8.7 percent) had a PTB. Participants with PF4 in the 3rd tertile had higher BMI and lower income compared to participants with PF4 in the 1st tertile. We did not detect an association between maternal preconception PF4 and PTB (Table 1) . Results were similar using maternal PF4 at 12 and 28 weeks. Stratifi ed analyses by treatment group also did not reveal an association between maternal PF4 and PTB (Table 2) , although sample size was limited. We did not detect an association between maternal platelet activation and PTB among EAGeR trial participants, although power was limited. Associations between LDA and PTB may be driven by mechanisms other than platelet activation. In fertility, it is common for patients to decide to use egg donors to supplement their treatment. A primary reason for using egg donors is that the female patient/partner is unable to conceive using their own eggs. This can be due to age, menopause, or poor-quality eggs. On the other hand, for same-sex male couples, it is the only option. Egg donors undergo culture and blood testing, infectious disease testing, and a physical. One of the hormones included in the blood testing is the Anti-Müllerian hormone (AMH). AMH is a protein hormone produced by maturing follicles in the ovaries. It is often described as an endocrine marker for predicting the maturation potential, or fertility potential, in a woman's eggs. Although AMH levels are measured in egg donors, there is no established cut-off in place, which allows all eggs, including from donors with low AMH levels, to be used in fertility treatment. The purpose of this study is to determine the relationship between AMH hormone levels in egg donors and treatment outcomes in order to establish an appropriate AMH cut-off in patients undergoing assisted reproductive technology (ART) with the use of donated oocytes. Methods: A retrospective chart review at a private, multi-site fertility clinic was conducted. Twenty-fi ve patients between the ages of 18-34 who used an egg donor during their treatment were included. Based on the Advanced Fertility Center of Chicago and additional fertility literature, AMH levels were categorized into low (1.5 ng/mL and lower), normal (1.6-3.9 ng/mL), and high (4.0 ng/mL and above). Donor AMH levels and frozen embryo transfer (FET) cycle outcomes, including clinical pregnancy, biochemical pregnancy, or miscarriage, were analyzed. Chisquare analysis was used to analyze the data. Results: Twenty-fi ve cases of FET using donated egg(s) were evaluated. There were a total of 3 individuals in the low-level group, 7 individuals in the normal-level group, and 15 individuals in the high-level group. The AMH level in egg donors and treatment outcome did not result in a statistically signifi cant relationship (p>0.05). However, all egg donors with an AMH level of 6.22 ng/mL and higher only resulted in clinical pregnancies. Studies have shown that the levels of AMH are not representative of or telling of an individual's egg quality. This assumption has led to a lack of an established cut-off for individuals who are able to donate eggs for fertility treatments. Although the data did not show a signifi cant relationship between the two variables, the data in egg donors with an AMH level of 6.22 ng/mL and higher showed to lead to more clinical pregnancies compared to egg donors with lower AMH levels. These results demonstrate the need to further investigate the relationship between AMH levels in egg donors and treatment outcomes. Introduction: Preterm birth (PTB) is one of the main causes of neonatal mortality and morbidity. Current studies have shown that neonate morbidity in PTB is linked to increased levels of IL-6 in amniotic fl uid, fetal blood and gestational tissues (GT) and that IL-6 increases uterine activating proteins (UAP) expression leading to PTB. A small peptide, HSJ633, developed in our lab inhibits selectively IL-6-induced STAT3 phosphorylation and LPS-induced PTB in mice. We hypothesize that IL-6 can induce damages to fetal tissues, and that inhibiting the IL-6 receptor using our nanopeptide, HSJ633, will improve birth outcome and maintain the integrity of fetal tissues. Methods: An established LPS-induced PTB model on timed pregnant mice was used to evaluate the amount of infl ammatory modulators in GT, maternal plasma and amniotic fl uid. Pregnant mice were injected with LPS (10μg/kg i.p.) at gestational day (GD) 16 in presence or absence of HSJ633 (1mg/kg/12h), Tocilizumab (TOC; 10mg/kg/12h), or vehicle. HEK-Blue IL-6 cells were treated with IL-6 (0,1μg/ml) in presence or absence of HSJ633 (1μg/ml) and TOC. In addition, we injected FITC-HSJ633 (1mg/kg/12h) in our PTB murine model at GD18 and tissues (placenta and fetus) were collected 4h after injection. Fluorescence was evaluated to determine the localization of HSJ633. The analysis conducted on the fetus is ongoing. We already showed that HSJ633 and to a lesser extent TOC, reduced the gene expression of pro-inflammatory cytokines and UAPs and expression of pro-inflammatory proteins in GT, maternal plasma and amniotic fluid. We then investigated the mechanism of action of HSJ633 in HEK-Blue IL-6 cells treated with IL-6 and HSJ633. We demonstrated that HSJ633 reduced the activation of STAT3 but not p38, AKT and ERK. In addition, the injection of a STAT3 inhibitor in a LPS-induced PTB mouse model reduced PTB by at least 50%. Thus, according to in vitro and in vivo results, we can assume that HSJ633 reduced PTB by inhibiting STAT3 activation. Fluorescence analysis of HSJ633-FITC revealed its presence in the placenta on both fetal and maternal sides when inflammation was present (inducted by LPS). Conclusion: Collectively, our data shows that HSJ633 antagonized the activity of IL-6R in a LPS-induced PTB model by inhibiting STAT3 activation, and improved birth outcome by increasing survival and preserving neonatal organ integrity. These findings highlight the importance of IL-6 in PTB and uncover in vivo pharmacological efficacy of a novel IL-6R modulator. HSJ633 is a promising new therapeutic prototype in the prevention of PTB. Introduction: Sterile inflammation is one of the mechanism implied in the pathophysiological rupture of the fetal membrane (FM) with an important actor the receptor of advanced glycation end products (RAGE). Its regulation by phosphorylation is still poorly understood. Lastly, it has been shown that Casein kinase 2 (CK2), regulates placental proliferation, migration, and syncytialization of trophoblast cells. Thus, we hypothesize that CK2 could also regulate RAGE by phosphorylation in FM. The first step of this project is to precisely detail the expression levels of CK2 in FM at term with or without labor. Methods: Human fetal membranes were obtained from women with healthy pregnancy during the three trimesters of pregnancy and at term undergoing planned caesarean section or after labor. mRNA quantifications and protein levels of α, α', β CK2 subunits were determined by RT-qPCR and Western-blot in amnion and choriodecidua explants. Localization of CK2 subunits was done by immunofluorescence using confocal microscopy. Statistical analysis were performed by non parametric tests using GraphPad PRISM software. Results: CK2 ontogenesis shows that the expression of α, α' mRNA CK2 subunits increase throughout pregnancy in amnion and this particularly between the 2 nd and the 3 rd trimester. At term, CK2 protein expression is higher after labor compare to before. Moreover, we observe that α and β subunits expression of CK2 protein are higher in the amnion compared to the choriodecidua after labor. Finally, confocal analyzes reveal a global localization of all CK2 subunits in different cells of amnion and choriodecidua. Conclusion: For the first time, we fully characterized at term without or with labor the CK2 expression in FM during the pregnancy. We demonstrate that CK2 is particularly present in the amnion at term with labor in an inflammatory context. Such results paves the way to study the specific regulation of the RAGE signaling pathway by CK2 phosphorylation in the pathophysiological context of fetal membrane rupture. inflammation and subsequently may lead to preterm labor. Programmed cell death protein 1 (PD-1) is a transmembrane immune checkpoint receptor which contributes establishment of maternal-fetal tolerance and maintenance of pregnancy by modulation of T cells homeostasis. We hypothesized that maternal inflammation imbalances T cell homeostatic and PD-1 expression on T cells in a mouse model of cMI. Methods: At embryonic (E) day 14, CD-1 dams (n=25) were randomly allocated into two groups: PBS, rIL-1β. Dams received intraperitoneal injection (IP) of 0.5µg rIL-1β in 100 µL PBS or 100 µL of PBS for four consecutive days. The preterm birth rate and viability were observed. Maternal blood, decidua, and placental labyrinth were harvested at 24 or 48 hpi. Flow cytometry was performed to characterize immune cells and PD-1 expression. Standard statistics were employed. Results: At 24hpi, rIL-1β significantly decreased the number of total CD3 + T cells and CD4 + T in maternal peripheral blood and placental labyrinth, with no changes in decidua. Total CD8 + T cells significantly decreased in maternal peripheral blood and placental labyrinth, then increased in decidua following rIL-1β exposure. The ratio of CD4/CD8 significantly increased in maternal peripheral blood at 24hpi while decreased in the labyrinth at 48hpi following rIL-1β exposure. (Tab 1). In maternal peripheral blood, rIL-1β significantly up-regulated PD-1 expression on CD3 + T cells at 24hpi then decreased at 48hpi. In decidua, PD-1 expression increased on CD3 + T cells while decreased on CD8 + T cells following rIL-1β exposure. In placental labyrinth, rIL-1β significantly down-regulated PD-1 expression on CD3 + T cells, CD4 + T cells, and CD8 + T cells (Tab 2). Conclusion: Our study demonstrated that T cell homeostasis was altered following cMI. Maternal cMI alters PD-1 expression differential on T cells and subsets, correlated strongly with CD4/CD8 ratio and immune activation. The homeostatic change during cMI may play a key role in preterm labor and perinatal sequelae. Tab.1 Different immune cell phenotypic population following cMI. Fig 1A) . 3 The vagina was exposed to 4.7 (basal), 20, and 40 mM of potassium chloride (KCl). Pressure was increased to the in vivo intra-vaginal pressure (7 mmHg) and held constant for 100 seconds, then removed for 1000 seconds for recovery. 3 Egtazic acid removed smooth muscle tone then the protocol was repeated. Introduction: Endometriosis affects 10% of women of reproductive age, and the incidence increases to 50-60% of women with chronic pelvic pain and infertility. However, current medical and surgical treatments are prone to substantial risks and side effects, are associated with a high rate of disease recurrence, and are best considered temporizing rather than curative. Forkhead box a2 (FOXA2) regulates biological pathways and genes in the glands that influence uterine receptivity. Of import, FOXA2 expression is decreased or lost in the glands of both eutopic and ectopic endometrium of women with endometriosis compared to women without endometriosis. However, the pathophysiological role of FOXA2 in endometriosis has not been studied. We evaluated the effect of FOXA2 loss and leukemia inhibitory factor (LIF) treatment on endometriosis development using a mouse model of endometriosis as well as Foxa2 uterine specific knock-out mice. . Using a human SE-lncRNA microarray the SE-lncRNAs in eight pairs of leiomyoma and matched myometrium was analyzed. The analysis indicated 7680 SE-lncRNAs were expressed, of which 721 SE-lncRNAs were increased, while 247 SE-lncRNAs were decreased by 1.5-fold or greater in leiomyoma as compared to myometrium. Thirteen novel SE-lncRNAs and their corresponding protein coding genes were selected from these differentially expressed SE-lncRNAs and their expression was confirmed in sixty-six paired leiomyoma tissues by qRT-PCR. The thirteen SE-lncRNAs and their corresponding protein coding genes included RP11-353N14.2/CBX4, SOCS2-AS1/SOCS2, RP1-170O19.14/HOXA11, CASC15/PRL, CTB-113P19.1/SPARC, RP11-225H22/NEURL1, RP5-1086K13.1/CD58, AC092839.3/SPTBN1, RP11-69I8.3/CTGF, EGFLAM-AS1/EGFLAM, TM4SF1-AS1/TM4SF1, RP11-399K21/COMTD1 and RP11-373D23/ FOSL2. Among these SE-lncRNAs, the expression of RP11-353N14.2/ CBX4, SOCS2-AS1/SOCS2, RP1-170O19.14/HOXA11 and RP11-225H22/NEURL1 was significantly higher in AA, while the expression of RP11-353N14.2/CBX4, SOCS2-AS1/SOCS2, RP1-170O19.14/HOXA11, CASC15/PRL, CTB-113P19.1/SPARC and EGFLAM-AS1/EGFLAM significantly correlated with MED12 mutation status in leiomyomas. In summary, our results provide the first evidence for differential expression of SE-lncRNAs in leiomyomas. This data indicates that SE-lncRNAs play an important role in leiomyoma pathogenesis through their regulation of protein-coding genes. and women taking hormonal medication (EMO-H) share features with standard endometrial organoids (EMO) and therefore have potential for modelling disease states and for precision medicine. MFO and EMO-H have the advantage of being derived 1) non-invasively or 2) from 50% of the population. Therefore MFO and EMO-H can be acquired from greater numbers of women, enabling population-wide assessment of endometrial epithelial behaviour. MFO and EMO-H are likely to prove invaluable tools for gynaecological research, enabling assessment of endometrial health from adolescents with presumptive endometriosis and without the need to cease hormonal medication, a mainstay of endometriosis treatment. Pathways Important for Uterine Fibroid Disease. Emmanuel N. X. Paul †, Tyler J. Carpenter, †, Joshua A. Grey †, Jose M. Teixeira * . Michigan State University, Grand Rapids, MI, United States. Introduction: Uterine fibroids (UFs) are the most common benign tumor of the human female reproductive tract with an incidence of 70-80% by age 50. Black/African American (B/AA) women are 2-3 times more likely to develop UF compared to White (W) women and often have more severe symptoms, i.e. heavy menses and pain. The racial disparity of the disease leads to a lower quality of life for B/AA women and a greater incidence of surgical intervention. Hysterectomy, resulting in the loss of fertility, is the only effective treatment and UFs are the most common indication for hysterectomy in the US. Understanding the pathogenesis of this disease could lead to alternative and less invasive therapies. Although Vitamin D deficiency and psychological stress have been associated with the racial disparity in UFs, little is known about the genetic and epigenetic differences between B/AA and W. Previous studies focused on the genetic profile of the fibroid tissues have not definitively identified the cause of the disparity. We decided to take a different approach and study the adjacent myometrial tissue to determine gene expression changes that could play a role in the establishment of the disease and the race disparity. Methods: Fibroid and matching myometrium tissues were collected from 15 B/AA and 19 W Med12 patients. RNA was isolated with the TRIzol method and submitted for library preparation and RNA-sequencing. Quality trimmed reads were mapped with STAR and differential expression (DE) analysis was completed using edgeR. Results: Unsupervised hierarchical K-means clustering analysis of all expressed genes showed that B/AA and W myometrial samples were significantly (p = 0.0013) grouped by race. In contrast, the fibroid samples were randomly scattered among all branches of the dendrogram (p = 1). Similarly, in the principal component (PC) plot myometrial samples were separated by race on PC3. In the myometrium, 1318 DE genes were found between B/AA and W, including brain-derived neurotrophic factor (BDNF), a gene associated with pain and stress (FC > 2, FDR < 0.05). By comparison, only 273 genes were DE in the fibroid samples. The top enriched GSEA pathways in the myometrium were stress or fibroidsrelated pathways, including oxidative phosphorylation, reactive oxygen species, Wnt/β-catenin and TGFβ signaling. Conclusion: Transcriptional differences in B/AA versus W that may play a role in UFs pathogenesis were found in the myometrium but not in the fibroid tissue, suggesting that once the tumor is formed, the transcriptome is independent of race. Investigating the myometrial transcriptome could elucidate molecular mechanisms underlying the racial disparity in UFs. Identification of FOXC1 as a Key Transcriptional Regulator in Endometriotic Lesions. Gregory W Burns †, Yong Song †, Niraj R. Joshi, Asgerally T. Fazleabas * . Michigan State University, Grand Rapids, MI, United States. Introduction: The molecular mechanisms underpinning the pathophysiology of endometriosis are poorly understood. Endometrial organoids comprised of epithelial and stromal cells offer an exciting model to study endometriotic lesion development. This study sought to identify changes in gene expression in organoids containing endometriotic stromal cells compared to uterine fibroblasts and identify central regulators through combined transcriptomic analyses with endometriotic lesions. Methods: Organoids were generated with endometriotic epithelial cells (12Z) combined with immortalized endometriotic stromal cells (iEc-ESC) or immortalized uterine fibroblasts (iHUF) in a scaffold-free agarose microwell culture system and cultured in MammoCult medium supplemented with heparin and hydrocortisone for 4 days. Total RNA was isolated for high-throughput sequencing from 12Z+iEc-ESC and 12Z+iHUF organoid samples (n=3) or eutopic endometrium and matched spontaneous endometriotic lesions from baboons (n=4). Baboon genes (Papio anubis) were converted to human orthologs using the biomaRt package (Ensembl v101). Differential expression (edgeR-robust) and gene set enrichment (clusterProfiler) analyses were conducted in R. Results: Transcriptomic analyses found 4,522 differentially expressed genes (DEG; q<0.05) in organoids and 4,171 DEG in baboon lesions compared to eutopic endometrium. An overlap of orthologous DEG from both experiments was highly significant (p=7.5x10 -25 ). FOXC1 was identified as an upstream transcriptional regulator of 7 upregulated transcription factors. Increased expression of FOXC1 in human lesions compared to eutopic endometrium was confirmed by qPCR (p<0.0001). Additionally, gene set enrichment analyses identified coordinately upregulated TNF signaling via NFKB, inflammatory response, and hypoxia pathways. IL6, a proinflammatory cytokine, was a central hub in a network of thegene sets. In iEc-ESC, plasmid-based overexpression of FOXC1 upregulated the expression of IL6 (p=0.02) and MMP2 (p=0.01). We identified significant overlap in differential gene expression in organoids and spontaneous endometriotic lesions from baboons. Further, combined transcriptomic analyses identified FOXC1 as an upstream transcription factor andIL6 as a central cytokine in inflammatory pathways. Increased IL6 in endometriotic stromal cells has been hypothesized to play a key role in lesion pathophysiology by affecting immune cells and local inflammatory signaling. FOXC1 upregulated the expression of IL6 and MMP2, supporting its centrality in proinflammatory signaling and lesion development. These results strongly support a role for FOXC1 in endometriotic lesions and the value of organoids to investigate the molecular pathology of endometriosis. Supported by NIH HD083273, HD099090, F32HD104478, and Endometriosis Foundation of America Introduction: Endometriosis is a complex disorder characterized by progesterone resistance. Roughly 30-50% of endometriosis patients experience infertility. The cause of endometriosis and its link to infertility is not completely understood. It has been shown that endometrial stromal cells (eSCs) from patients with endometriosis show decreased decidualization, a progesterone-driven process critical for implantation. Quercetin is a flavonoid with anti-inflammatory and immunomodulatory activities. In a rat model of endometriosis, quercetin inhibited the growth of endometrial implants and exerted anti-inflammatory effects. We assessed the effect of quercetin on decidualization and progesterone sensitivity using eSCs. Methods: eSCs were isolated from menstrual effluent (ME) obtained from healthy controls and surgically diagnosed endometriosis cases. To induce decidualization, passage 2-3 eSCs were treated with dibutryl-cAMP (cAMP, 0.25-0.5mM) ±10 -7 M medroxyprogesterone acetate (MPA) for 48hr. eSCs were pre-treated with vehicle or quercetin (25µM) 4hr prior to decidualization. PRL and IGFBP1 protein and mRNA in the culture supernatants and cells, respectively, were assessed by ELISA and RT-qPCR, respectively. Progesterone sensitivity was also assessed by measuring progesterone-induced genes, PLZF and IHH. Two sample t tests (unpaired or paired) were used to determine significance. Results: Quercetin significantly enhanced cAMP-induced decidualization of control-eSCs (p<0.02) and case-eSCs (p≶0.05), although eSCs from cases showed reduced decidualization. Addition of MPA potentiated cAMP-induced decidualization in control-eSCs, and, to a lesser extent case-eSCs and this was significantly enhanced by pre-treating with quercetin (p<0.05 ). When quercetin alone was added to controland case-eSCs, MPA-induced PLZF and IHH mRNA expression was significantly increased over vehicle (p<0.05). Conclusion: Endometriosis-eSCs exhibit decreased decidualization compared to control-eSCs. MPA potentiates decidualization when added with cAMP and quercetin further enhances control-eSC and case-eSC decidualization and MPA responsiveness. These data support further studies investigating the role of quercetin in promoting progesterone sensitivity in endometriosis. Macrophages. Kanako Hayashi * , Liang Zhao †, Mingxin Shi †, Sarayut Winuthayanon †, James A. MacLean * . Washington State University, Pullman, WA, United States. Introduction: Our recent findings indicate that macrophages, which are considered critical regulators for peritoneal inflammation to induce endometriosis-associated pain, could be targeted by niclosamide, an FDAapproved anti-helminthic drug. As the changes of macrophage populations dynamically accommodate the inflammatory conditions in the peritoneal cavity, a better understanding of the recruitment and maturation of macrophages is needed to elucidate the molecular mechanisms underlying the regulatory functions of niclosamide in endometriosis. Methods: We took advantage of scRNA-seq to resolve the dynamic progression of macrophages and to understand their stage-specific changes caused by the induction of endometriosis-like lesion (ELL) using a mouse model of endometriosis. The efficacy of niclosamide on the ELL-induced alterations to this biological progression of macrophages was also investigated. Results: scRNA-seq captured the heterogeneity of peritoneal macrophages with three novely identified intermediate types, which are difficult to distinguish by flow cytometry. Gene set enrichment analysis (GSEA) of macrophage populations showed enhanced inflammatory conditions in the ELL group, which were largely reversed by niclosamide treatment. Moreover, genes that were enhanced by ELL, such as Retnla, Ccr2 and Mrc1, were found to be mostly distributed in the lineages of recruited small peritoneal macrophages (SPMs). Lineage tracing studies indicated these genes function in the differentiation of SPMs from dendritic cells. Thus, ELL are expected to promote the recruitment of SPMs from dendritic cells, and this process is suppressed by niclosamide. On the other hand, most genes that were down-regulated by ELL, such as Hp, C3 and Cfb, restricted their expression to the large peritoneal macrophage (LPM) lineages. Trajectory analysis showed that decreased expression of those genes facilitates the maturation of intermediate LPM. Importantly, this promoted maturation was inhibited by niclosamide. Moreover, a decreased expression of Timd4, a marker for residential LPMs, was found in the ELL group, suggesting a loss of this population in numbers. Introduction: Endometriosis (EM), a chronic estrogen dependant inflammatory condition, remains to be understood from a pathophysiological perspective. A novel target that may contribute to disease progression is Leukemia Inhibitory Factor (LIF). LIF is responsible for successful implantation, facilitating decidualization and immune modulation within the endometrium. To date, LIF has only been studied in EM within the context of infertility. However, LIFs capacity to influence cellular changes as well as its immunomodulatory effects, make it an attractive player for study in EM pathophysiology. We hypothesize that LIF will maintain ectopic tissues by promoting cell proliferation, vascularization, as well as modulating immune phenotypes within the microenvironment through macrophage polarization and Treg stimulation. Methods: To quantify ectopic LIF, a tissue microarray (TMA) of eutopic and ectopic tissues (EM patients) and matched healthy endometria (controls) were stained for LIF using IHC. To understand the role of LIF family members in the EM lesion microenvironment RT 2 qPCR was conducted for LIF, its receptor (LIFR) and downstream targets in ectopic, eutopic and normal samples. To understand the influence of LIF on EM pathophysiology, endometriotic epithelial (12Z) and endothelial (HUVEC) cell lines were treated with varying LIF concentrations (1-200ng/mL) to determine effects on cell proliferation (WST-1), apoptosis (Caspase 3/7) and cytokine secretion. To gain mechanistic insight on heightened LIF on EM associated inflammation and lesion development a mouse model of EM (C57/Bl6) was used. Mice were treated daily for 13 days using recombinant mouse LIF (300ng/100uL) and PBS (control) via intraperitoneal injection with blood samples collected on days 1, 7 and 14. Peritoneal fluid and spleen cells were harvested and processed with flow cytometry for myeloid and lymphoid markers. Plasma and peritoneal fluid were screened for inflammatory and angiogenic markers using multiplex analysis. Results: Preliminary results demonstrate a dysregulation of LIF across eutopic and ectopic sites. Significant downregulation (p<0.01) of LIF in eutopic EM tissues compared to controls was identified in the TMA. Transcript levels of LIF were significantly downregulated (p<0.05) while LIFR showed a two-fold upregulation of expression, when comparing ectopic and eutopic EM samples. At this time, remaining mouse and cell culture analysis are in progress. Our study for the first time provides evidence that EM lesions express LIF and that the dysregulation in LIF/LIFR in the lesion microenvironment might play a role in EM lesion sustainment. Mechanistic studies in our EM mouse model will shed light on the potential causal role of LIF and associated members within EM pathophysiology. In-depth investigation of normal and abnormal patterns has not been done due to lack of a non-invasive imaging modality. Electromyometrial imaging (EMMI) was developed to measure the magnitude, direction, and frequency of uterine peristalsis patterns quantitatively and objectively. Our goal was to image patients with endometriosis to determine if they have peristalsis patterns distinct from those with regular menstrual cycles. Methods: EMMI was performed using 128 body surface electrodes while incorporating MRI imaging of the uterus to image uterine peristalsis. We completed EMMI imaging during the four phases of the menstrual cycle. At each visit, electrodes are applied to the abdomen; simultaneously, a transvaginal ultrasound recorded direction of uterine peristalsis. Inclusion criteria included biologically female, age 18-40. Exclusion criteria included use of female birth control, pregnant or breastfeeding, BMI >40, or using medications which affect uterine contractility. Results: During this pilot study, we performed EMMI on two patients with clinically diagnosed endometriosis and compared results to four healthy controls (figure 1). UP waves during the menses phase in endometriosis patients had a shorter frequency and higher magnitude waves compared to controls (p<0.001). UP waves also predominately initiated in the cervix and propagated toward the fundus in endometriosis patients (mean 78%) but, in healthy controls, contractions were predominately fundus to cervix (mean 73.5%). UP waves in the peri-ovulatory phase were longer and weaker in endometriosis patients compared to controls (p<0.001). Conclusion: Our preliminary data suggest a difference in UP patterns in endometriosis patients compared to controls. Our data support Sampson's theory to explain endometriosis etiology due to cervix to fundus contractions during menstruation (retrograde menstruation). This novel tool can identify patterns to better understand pelvic pain and infertility related to endometriosis and potentially better diagnose or treat endometriosis non-invasively. Proteomics Analysis as a Tool to Understand Racial Disparities in the Development of Uterine Fibroids. Maria Victoria Bariani †, 1 Nicholas Bateman * , 2 Thomas Conrads * , 2 George L Maxwells * , 2 Qiwei Yang * , 1 Ayman Al-Hendy * . 1 Introduction: Uterine fibroids (UF) are the most common benign gynecologic tumors presented in reproductive-age women. It is well established that Black (B) women are disproportionately impacted by UF, experiencing greater prevalence and severity than White (W) women. To date, research is lacking on how UF develops and grows differentially in racial groups. This work aimed to identify differentially altered proteins and pathways involved in race disparity in normal and in at-risk myometrium in the context of UF in B and W women. Methods: Myometrial tissue from uteri without UF (MyoN, n=7 B, n=8 W), myometrium adjacent to UF (MyoF), and UF (n=10 B, n=5 W, each group) from consented women were collected at the University of Illinois at Chicago Medical Center. Global proteomic analysis was performed for representative tissue sections collected using laser microdissection followed by analysis using a multiplexed, quantitative proteomics approach employing liquid chromatography and high-resolution Orbitrap mass spectrometry. Differential analysis was performed for conditions of interest using LIMMA (p<0.01) and pathway analysis was performed using Ingenuity Pathway Analysis (IPA). The global proteomic analysis resulted in the quantitation of 6,407 total proteins across samples. comparisons. Moreover, we observed that the top pathway predicted as putatively activated in B-MyoF vs. B-MyoN is the PD-1/PD-L1 pathway and that this is unique to B patient samples. This work demonstrates that the same type of tissue presents a unique proteomic signature that corresponds to distinct cellular pathways related to race, highlighting the importance of distinguishing the racial groups in the UF research area. In addition, this analysis shows the different signatures of at-risk myometrium that may explain the underlying increased prevalence and burden of UF in B women. Future studies will allow us to distinguish elements that are linked to the observed racial disparity in the UF phytopathology context. To observe crosstalk in vitro, endometriotic epithelial cells (12Z) were cultured with MC-conditioned media. Multiplex cytokine analysis was performed on supernatant. To observe impact of E2 on MC recruitment in EMS, we induced EMS in 12 C57BL/6 mice and treated 6 with subcutaneous E2 pellets. After 10 days, peritoneal cells were analyzed by flow cytometry using MC markers CD117, FCERIα and MC progenitor marker integrin-β7. Mouse EMS lesions were stained with alcian blue to analyze MC density. Results: We observed significantly higher MCs in EMS lesions compared to matched endometrium. Compared to endometrium, EMS lesions had significantly higher SCF concentration (p≤0.05). Our targeted qPCR array revealed EMS lesions harbour a microenvironment conducive to MC recruitment and differentiation (upregulated CPA3, VCAM1, CCL2, CMA1, CCR1, and SCF, p≤0.05). Cytokine analysis showed MC-conditioned media significantly increased 12Z cells' production of cytokines IL-6 and IL-8 (p≤0.05). Flow cytometry analysis of mouse peritoneal immune cells showed significantly higher frequency of MC progenitors in E2-treated mice compared to non-treated mice (p≤0.05). The EMS lesions from E2-treated mice had significantly higher density of alcian blue stained MCs compared to healthy control endometrium (p≤0.05). Conclusion: Collectively, these findings suggest that EMS lesions provide a microenvironment necessary for recruitment and differentiation of MCs. In turn, MCs potentially release pro-inflammatory mediators that contribute to EMS pathogenesis. Antibiotics may have other adverse effects including a shift in the gut microbiota from beneficial to pathogenic bacteria. We determined the change in infant gut microbiota composition induced by antibiotic treatment with amoxicillin/clavulanate (A/C) in the in vitro proximal colon model (TIM-2). The TIM-2 colon model was inoculated with a pooled microbiota of 14 healthy human infants (3 months) using feces collected (diaper samples) from volunteers to allow for a standardized microbiota. The TIM-2 was loaded with A/C (25 μg/ml, twice a day) for 48 hours. Samples from the lumen of the system were obtained at 0, 24, 48 and 72 hours and analyzed for human microbiota by sequencing the V3-V4 region of the 16S rRNA gene using Illumina sequencing). Machine Learning of MRI for Detection, Evaluation, and Diff erentiation of Uterine Sarcomas, Atypical Leiomyomas, Benign Leiomyomas, and Normal Myometrium. Hiba Siblini †, Hui Li * , Sandra Laveaux * , Ricardo Lastra * , Maryellen Giger * , Ayman Alhendy * . University of Chicago, Chicago, IL, United States. Introduction: Uterine fi broids, also known as uterine leiomyomas (LM), are the most common uterine tumors in women and a major cause of gynecological morbidity and mortality. They can cause heavy menstrual bleeding, severe pelvic pressure, pain, and infertility. On the other hand, uterine leiomyosarcomas (LMS) are rare malignant uterine tumors of mesenchymal origin representing 3%-7% of primary malignant uterine tumors. Current practice defi nes typical leiomyomas as well circumscribed, homogenous masses with low signal intensity on both T2-weighted images and diff usion weighted images (DWIs), although up to 65% of leiomyomas undergo changes and do not present with this typical appearance.Therefore, given that leiomyomas may currently be managed with a more conservative treatment, it is crucial to distinguish them preoperatively from malignant LMSs or atypical leiomyomas. It is also important to rule out a malignant LMS prior to conservative treatment to avoid dissemination of the disease. Advances in computer power and machine learning algorithms have the conversion of complex imaging data sets into a multi-dimensional feature space with extractable characteristics. Hence, we aim to develop a computer assisted diagnostic tool as a machine intelligence model based on features of MP-MRI to diff erentiate between LM, LMS, and atypical LM. Methods: Our study population included female subjects above the age of 18 with an MRI scan obtained pre-operatively and a histopathology diagnosis of the pathology as a leiomyoma, leiomyosarcoma or atypical leiomyoma. We analysed the MRIs of uterine fi broid lesions found in 20 patients distributed between sarcomas (SA) and traditional fi broids (TF). Results: Using radiomics and machine learning, we generated a 3D plot of three computer-extracted characteristic features showing an appreciable separation between sarcomas and traditional fi broids. We also assess performance via ROC analysis of a simple classifi er in the SA vs TF task based on a leave-one-case-out validation [AUC=0.91]. Our current small and preliminary pilot study shows promising results in regards to developing a computer assisted diagnostic tool, a machine intelligence model, based on features of MP-MRI to diff erentiate between LM, LMS, and atypical LM. Functional enrichment analysis (FDR<0.05) of these genes revealed biological processes corresponding to nervous system, migration, cellular response, and immune system. Interestingly, besides synapsis, neuron development, morphogenesis and differentiation were associated with 21 of 30 top enriched functions from upregulated genes, suggesting nervous system processes to be important in UL physiology. These functions revealed some genes previously linked to cancer, as well as axonogenesis, neurogenesis and pain processes such as DRD2, CHRM2, SOX11 and NTNG1. Conclusion: This study showed that functions related to neuron development and synapsis were enriched in UL, which could be involved in tumor initiation and progression, as it occurs in other solid tumors. The enrichment of these functions suggests an aberrant innervation in UL that could explain the excessive pain that patients with UL suffer. Therefore, nervous system related processes may be important regulators of UL pathogenesis, and its reversion could be a promising therapeutic approach to reduce UL growth and survival. The levonorgestrel-releasing intrauterine system (LNG-IUS) is a highly effective long-acting contraceptive, and it is being recommended as first line contraceptive for all women in several guidelines. LNG-IUS has also been approved for the treatment of heavy menstrual bleeding in more than 100 countries. Although LNG-IUS acts mostly via local effects, a minute amount of LNG is released in the bloodstream. The possible metabolic changes and large-scale biomarker profiles associated with LNG-IUS use have not been studied in detail. The objective of this study was to examine, through the metabolomics approach, the metabolic associations of LNG-IUS use, their relation with the duration of use, and reversibility after interruption of use. Methods: Data were retrieved from five cross-sectional population-based studies (FINRISK and FinHealth surveys) carried out in Finland from 1997 to 2017. The study population included all fertile aged (18-49 years) women who participated in the surveys, with available information on hormonal contraceptive use, and metabolomics data (n=5649). A total of 211 metabolic measures were compared between 1006 current LNG-IUS users vs. control women (n=4643) not using any hormonal contraception via linear regression models. Results: After adjustment for covariates, 167 metabolites were associated with LNG-IUS use (effect size of around 0.25 SD, p-value adjusted by false discovery rate): lower levels of particle concentration of all lipoprotein subclasses, triglycerides, cholesterol and derivatives, apolipoproteins A and B, glycoprotein acetyls, glucose, aromatic and branched-chain amino acids, increased ratios of polyunsaturated fatty acids, omega-6 and linoleic acid, and reduced ratio of saturated fatty acids. With a few exceptions, the metabolic pattern of LNG-IUS use did not change with the duration of use (less than one year vs. 2 to 5 years vs. more than 5 years). No significant metabolic differences emerged between previous users of LNG-IUS and never-users. The use of LNG-IUS was associated with multiple modest metabolic changes, mostly indicative of a reduced cardiometabolic risk. The metabolic associations were essentially independent of the duration of use, and similar to never-users after discontinuation of LNG-IUS use. Reschke †, Sadia Afrin, Mostafa Borahay * . Johns Hopkins, Baltimore, MD, United States. Introduction: Uterine leiomyomas or fibroids, are the most common benign smooth muscle tumors of the female reproductive system, and represent a significant medical and economic burden. 1 Recent evidence suggests a strong association between body mass index (BMI) and uterine leiomyomas, with obese woman at two-three times greater risk 2, 3 The hormone leptin, a product of the obese (ob) gene, is primarily produced by adipocytes, and exhibits a strong positive correlation with total body fat and BMI. Leptin has been proven to stimulate tumor growth in various cell models. 4 We hypothesized that leptin influences leiomyoma proliferation and extracellular matrix deposition (ECM) in vitro. Methods: Immortalized human uterine leiomyoma (HuLM) cells were cultured and serum starved for 24 hours, followed by treatment with leptin (100ng/mL). At 6, 24, and 48 hours, cell growth and proliferation were assessed using the colorimetric MTT assay. Western blotting was performed for determining cellular proliferation marker, PCNA. ECM deposition was determined by western blotting for collagen I. Student's t-test was used to determine statistically significant differences (p<0.05). Results: Leptin treatment stimulated a significant increase in cellular proliferation as estimated by the MTT assay at 24 hours (p=0.04), with a decrease at 48 hours compared to untreated controls at respective time [1] . However, how polymeric NPs biodistribute after vaginal injection in pregnant mouse models is not known, limiting the use of NPs in pregnancy. Here we vaginally inject poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) NPs at two different gestational ages and characterize NP biodistribution to maternal organs and examine metrics of embryo and placenta development. Methods: NPs were synthesized by mixing PLGA and PEG-PLGA in a 3:1 ratio. DiD was added to the mixture, then the solution was added dropwise to poly(vinyl alcohol). NPs were purified and size and charge were measured. NPs or saline were administered vaginally to pregnant mice on embryonic days E14.5 or E17.5. Animals were imaged via IVIS immediately post-injection and 24 hrs later. Mice were euthanized, the major maternal organs, placentas, and embryos were excised and imaged via IVIS and placentas and embryos were counted and weighed. Results: IVIS imaging showed NPs are retained in the vagina 24 hrs post-delivery ( Figure 1A , B) and NP distribution was localized to the vagina with no distribution to other maternal organs ( Figure 1B) . No differences were observed in embryo ( Figure 1C ) or placenta number or weight between saline and NP treated mice, indicating lack of short-term effects on embryo development. To date, we cannot predict which fibroids will grow, the rate at which they would grow and if they could lead to serious medical consequences. We plan to investigate the use of improved magnetic resonance (MR) methods to evaluate physiological factors (perfusion, oxygenation and structure) of UF that are known to influence their growth and behavior and thereby identify predictive factors of UF growth. Methods: This is a prospective pilot study. Participants include reproductive aged (>18 years), English speaking, non-pregnant, nonbreastfeeding patients with ultrasound or MR confirmed diagnosis of uterine fibroids with at least one >2cm. Each subject has two scheduled visits, initial visit (month 0) and second visit (month 12), in which an MRI scan is done and a UFS-QOL questionnaire is administered. The MR acquisitions will include Axial T2 FSE whole pelvis, Axial T1 FSE whole pelvis, 3D T2 for volume estimation, Axial Diffusion (b=0, 50, 800), Dynamic contrast enhanced Axial T1-weighted gradient echo (DCEMRI), ADC, T2 map, R2* map, ASL and R2*mapping. Results: Of 207 eligible subjects, 32 were enrolled and 29 completed baseline scans. Two of the enrolled did not have MRI scans due to scheduling conflicts. 1 subject withdrew due to anxiety. Baseline scans were obtained for 96.6% of participants. 10 of 23 subjects eligible for 12-month scans (44%) have completed the study. Two subjects are scheduled for their second scan. Two had hysterectomies prior to their 12-month scan. Six patients are not due for the 12-month scan. Nine subjects are delayed for their scans but have been contacted. We will be conducting an interim analysis by February 2022. In addition to the quantitative analysis relating to the MRI parameters, we will perform qualitative analysis to study the demographic distribution of disease burden, the correlation of disease burden with UF-QOL (Uterine Fibroids Quality of Life Questionnaire), and the correlation of change in fibroid size at time zero and at twelve months with change in UF-QOL. Conclusion: This protocol was highly feasible given the limited scan time and further expansion of its sample size could be helpful for identifying features that correlate with UF growth, symptom development and/or disease progression. We anticipate that by the time we complete our interim analysis we will be able to identify the quantitative and semiquantitative MP-MRI parameters that correlate with UF growth as well as test for correlations between disease burden and quality of life. The findings of the 63 peer-reviewed papers provide insight from the pathophysiology process to possible treatment option. Two studies found an increased expression of 1α-hydroxylase in women with endometriosis, but concluded the enzyme was not an ideal indicator of endometriosis. Two studies, including a randomized control trial, reported no correlation between serum 1α,25(OH)₂D₃ concentration and endometriosis, whereas one study found an elevation. Regarding serum 25(OH)D₃ concentration of women with endometriosis, one study found an increase, a second found a decrease, and a third found no difference when compared to women without endometriosis. Two studies reported a change in vitamin D-binding protein gene expression and endometriosis, whereas two studies found no difference. Conclusion: Data were conflicting regarding the association between vitamin D and endometriosis. Further research is needed to assess the potential association between vitamin D and endometriosis. Interval Laparoscopic Tubal Sterilization -Understanding Factors That Influence Short Notice Surgery Cancellation. Megan Masten †, 1 Nicole Larrea * , 2 Aaron Lazorwitz * , 1 Claire Schultz * . 2 1 University of Colorado, Aurora, CO, United States; 2 Denver Health, Denver, CO, United States. Introduction: Tubal sterilization is the preferred contraceptive method for 25% of women in the United States. Laparoscopic sterilization has an anecdotally high cancellation rate. Cancellations effect operating room utilization and may reflect barriers to care. We aimed to identify the short notice cancellation (≤ 7 days from scheduled procedure) and no-show rates of laparoscopic sterilization, reasons for cancellation, and post-cancellation outcomes. We performed a retrospective chart review of patients aged 18-50 who canceled or no-showed a scheduled laparoscopic sterilization between May, 2016 and May, 2019 at an academic tertiary care hospital, and academic county hospital in Denver, Colorado. We reviewed electronic health records to determine the time between cancellation and surgery date and documented reasons for cancellation. We evaluated contraceptive methods used and pregnancies within a year after the canceled procedure. Results: The surgery cancellation rate was 24.6%, with 123 patients cancelling their procedure. Seventy-one percent of patients canceled their procedures on short notice and 32.5% (40/123) canceled same day. The most common reason for cancellation was patient choice (72%) followed by financial/insurance issues (13%). In the year after their canceled procedure, 20% (25/123) of participants obtained permanent contraception and 7% (9/123) had a subsequent pregnancy. Conclusion: Over 70% of participants canceled their laparoscopic sterilization procedure a week or less from their scheduled date. These short notice cancellations may adversely affect both patients and the healthcare system. More research is needed on interventions to reduce this rate while maintaining equitable access to permanent contraception. In Introduction: Identification of molecular mechanisms underlying tumorigenesis is one of the main goals for our field. Uterine fibroids (UFs) have been categorized into at least four subgroups according to driver gene alterations: MED12 mutation, HMGA2 overexpression, biallelic FH inactivation, and COL4A5/A6 deletions. However, recent developments combining high-throughput sequencing technology with "in silico saturation mutagenesis" might enable to detect novel driver mutations. Using this technology we aim the identification of novel driver mutations, not only in UFs but also in nearby myometrium suggesting its predisposition to tumorigenesis. Methods: We analyzed UFs and matched myometrium (MM) from 41 symptomatic women undergoing myomectomy. Exome-wide somatic mutations and differential gene expression were comparatively assessed. BoostDM machine learning method was used to perform in silico saturation mutagenesis (Muiños et al., 2021) in the identification of driver mutations from the sequenced UFs and MM exomes. Finally, we used the STRING database to predict protein-protein interactions. Results: We detected a high proportion of mutations affecting genes BCLAF1, CHD4, CREBBP, FOXA1, HLA-A, HLA-B, KIT, KRAS, NF1, TP53 and ERBB2 in UFs and MM. Among them, we identified a total of 25 tumor-associated driver mutations arising from mutation probability bias and positive selection. Interestingly, the c.3508C>G mutation in the ERBB2 gene, which was previously reported in the breast cancer model, was found in the 75% of our samples. This oncogene is involved in transcriptional regulation and signal transduction in the PI3K-Akt pathway, extensively reported in UFs. While we did not find significant differences in the expression of this gene, we found that it may directly interact with specific differentially expressed genes involved in collagen catabolic process, extracellular matrix disassembly and response to progesterone. Our results allow to map new mutations that may be important for the personalized medicine of gynecological tumors and tumorigenesis predisposition. (6) 2016). We aimed to test, using a sheep model of pregnancy, whether the low-dose ANS regimen proposed by WHO (ACTION III Trial) would achieve preterm lung maturation equivalent to that of the existing WHO dexamethasone treatment regimen, but with reduced risk of adverse outcomes. Methods: Following ethical review and approval, date-mated ewes with single fetuses received intramuscular injections of either i) 4 x 6mg dexamethasone phosphate q12h (n=22); or ii) 4 x 2 mg betamethasone phosphate q12h (n=21) or iii) 4 x 2mL saline q12h (n=16). Forty-eight hours after first injection, (124±1 d), lambs were delivered, ventilated for 30 minutes, and euthanised. Arterial blood gas, respiratory, haematological and biochemical data were analysed for group differences with ANOVA according to distribution and variance, with p<0.05 taken as significant. Results: After 30 minutes ventilation, lambs from both steroid-treated groups had significant and equivalent improvements in lung function relative to saline control (p<0.05). There was no significant difference in arterial cord blood pO 2, pCO 2 , pH, CtO 2 , lung compliance, or ventilator efficiency index. The mRNA expression of surfactant protein (SP) A, SP-B, SP-C, SP-D was equivalent between both steroid groups. Maternal and fetal plasma neutrophil, glucose levels and c-peptide levels were significantly elevated in the dexamethasone group, relative to the betamethasone group. Fetal plasma IGF-1 was significantly reduced in the dexamethasone group compared to the betamethasone group (p<0.05). We report that, in preterm lambs, a low-dose treatment regimen of 8mg betamethasone achieves lung maturation equivalent to that of a 24mg dexamethasone-based regimen, but with smaller perturbations to the materno-fetal HPA axis. These data also suggest that, given steroid pharmacokinetic differences between sheep and humans, that the betamethasone dosing proposed for Action III is likely ~50% greater than necessary. In previous our studies, we selected eight possible metabolic markers associated with PTB using nuclear magnetic resonance spectroscopy (NMR). Using the results of increased metabolites in PTB group, we performed CVF metabolic profiling by mass spectrometry (MS). P values <0.05 were considered statistically significant. The eight metabolic markers related to PTB group were acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine N-oxide by using NMR spectrometry in our previous studies. In verification these metabolites by MS, acetone was significantly increased in PTB group than TB group (P<0.01) and the lower birth weight of the newborn (P<0.05). Cervical length was also shortened at high concentration of acetone (P=0.07). Our study suggests that concentration of acetone in CVF could be a potential biomarker related to PTB. In the environment of vaginal dysbiosis, acetone in CVF may be associated to metabolites of microbiota, and such changes in the vaginal environment may be related to maternal lifestyle. Infancy in a High-Risk Population. Laura Jorissen †, Eva Mulder, Emma Janssen, Salwan Al-Nasiry * , Chahinda Ghossein-Doha, Sander Van Kuijk, Marc Spaanderman * . Maastricht UMC+, Maastricht, Netherlands. Introduction: Maternal, offspring and societal impact of pregnancies complicated by impaired fetal growth are substantial. Models allowing pre-pregnant prediction of subsequent small for gestational age (SGA) neonates in high risk women are lacking. These models would allow appropriate counselling with individualized risk estimates but also detail variables accessible to possible intervention. The objective is to develop a prediction model for SGA infancy based on pre-pregnant factors. Methods: Primiparous women with a history of SGA birth or preeclampsia admitted to non-pregnant cardiovascular and metabolic risk assessment were enrolled. The prediction model was developed using multivariable logistic regression with backward stepwise selection. Calibration and discrimination were evaluated after adjusting for overfitting with bootstrapping techniques. Results: We included 723 primiparous women with a history of preeclampsia (n=459), delivery of a SGA neonate (n=72) or combination (n=192). In the subsequent pregnancy, 118 women (16%) gave birth to a SGA neonate. Variables contributing to subsequent SGA were: first pregnancy gestational age at delivery and birthweight centile, maternal birthweight, fasting -insulin, -glucose and -cholesterol, circulatory plasma volume, maternal BMI and the use of aspirin prophylaxis in second pregnancy. Individual risk estimates varied between 0% and 71%. The initial AUC was 0.76 (95% CI 0.71-0.82), with minor correction for optimism after bootstrapping (0.02), indicating accurate discriminative ability. The calibration curve for deciles based on predicted probability ( Figure 1 ) showed adequate agreement between predicted and observed all along the range of risk estimates. We developed a well performing prediction model for delivery of a SGA neonate in high-risk women before pregnancy. The model consists of unmodifi able but also modifi able predictors. With this specifi c insight, women may strive to optimize their cardiovascular and cardiometabolic functioning before trying to conceive again. Upon external validation, this model can be used in clinical practice to aid physicians in personalized counselling to parental couples in advance of their subsequent pregnancy. In a non-human primate model, we have shown that off spring exposed to maternal high fat diet (mHFD) display persistent anxiety and a persistently altered gut microbiome, even when weaned onto a control diet for 2.5 years. Since the gut and its microbes infl uence behavior/neuroactivity, it is possible that mHFD exposure infl uences off spring behavior years after birth. To investigate the relationship between maternal diet and gut microbes on the neurometabolites relevant to anxiety, we hypothesized that mHFD exposure will produce a persistently altered gut metabolome profi le in vivo compared to maternal control diet (mCTR) exposure. Methods: Japanese macaques were fed mHFD, mCTR, or a mHFD-to-mCTR reversal diet during gestation/lactation then weaned onto CTR or HFD. Serum and fresh stool were collected longitudinally. Collection of intestinal tissue was performed at necropsy (early third trimester, 15 months, or 3 years). Serum and polar metabolite extractions of stool and intestine were analyzed by quantitative liquid chromatography-mass spectrometry targeting 34 metabolites spanning neuroactive pathways (serotonin, GABA, dopamine) and short chain fatty acids. Results: We quantifi ed 34 neuroactive metabolite levels in n=304 serum, n=390 stool, and n=547 intestine from n=79 fetal and n=134 juvenile off spring. We observed signifi cant alterations of the GABA neuroactive pathway in mHFD exposed offspring (see Figure 1 ). In post-wean CTR animals, we observe elevated serum GABA (fetal, p=0.030) and decreased stool glutamate (juvenile, p=0.038). In stool from post-wean HFD juveniles, we observe decreased GABA (p=0.001) and glutamate (p=0.009), in addition to serotonergic decreases in serotonin (p=0.009), tryptophan (p=0.051), 5-hydroxyindoleacetic acid (p=0.052). Conclusion: In our large experimental cohort of primates, metabolomic profi les of off spring were persistently altered by mHFD exposure, even after 2.5 years of post-weaning CTR feeding. These data suggest a programmable feedback mechanism in the early life gut that may be critical to development of the gut-brain axis in primates. . Prophylactic negative pressure wound therapy (NPWT) has been shown in some studies to reduce SSI after CD, but cost-eff ectiveness of NPWT is still largely unknown. We sought to determine cost-eff ectiveness of prophylactic NPWT compared to usual care after CD for persons with obesity. Methods: A cost-eff ectiveness model (CEA) compared NPWT to usual care (e.g., wound dressing with removal after 1-2 days postoperatively) for 100,000 persons with obesity after CD. All persons were assumed to be appropriate candidates for either strategy. Probabilities, costs, and utilities were derived from the literature. The CEA was conducted from a healthcare system perspective and the analytic horizon was the fi rst postpartum year. Incremental cost-eff ectiveness ratios (ICERs) for each strategy were calculated and compared. An ICER of $50,000 per quality-adjusted life year (QALY) was used to defi ne the cost-eff ectiveness threshold. One-way sensitivity and Monte Carlo analyses (MCA) were performed. Results: Compared to usual care, NPWT resulted in 2,293 fewer episodes of SSI, 194 fewer unanticipated outpatient wound visits, 71 fewer rehospitalizations, and 69 fewer reoperations, however it did not meet our pre-specifi ed ICER threshold (Table 1) . On one-way sensitivity analysis, the model was most sensitive to changes in the relative risk of SSI; probabilities of hospitalization and reoperation; and costs of treating SSI and using NPWT ( Figure 1 ). On MCA, usual care was cost-eff ective in 71.2% of runs. In our theoretical cohort, usual care was the cost-effective strategy. However, in almost 30% of runs, NPWT was cost-effective and the model was particularly sensitive to several inputs, highlighting the need for further research on the utility of prophylactic NPWT after cesarean delivery for persons with obesity. Introduction: Mitochondrial function in adipose tissue is an important yet understudied aspect in metabolic homeostasis. Our previous findings demonstrated that circulating levels of adrenomedullin (ADM) and mRNA and protein for ADM in omental adipose tissue were increased in patients with gestational diabetes mellitus (GDM) and contributes to altered lipid metabolism. These findings prompted us to further investigate the role of ADM in the regulation of mitochondrial biogenesis-signaling factors and its function in human adipocyte cell line. Methods: Differentiated human adipocytes were treated with increasing doses of glucose (1.5mg/ml to 3.0mg/ml) and ADM (0.1nM to 10 nM) with or without ADM antagonist ADM22-52 (100 nM) for 48 h. The mRNA expression for mitochondrial DNA (mtDNA)-encoded subunits of the electron transport chain was assessed by real-time qPCR with specific primers. The mitochondrial contents of the adipocytes were observed using mitochondrial-specific fluorescence MitoTracker green, the reactive oxygen species (ROS) was determined by MitoSOX red probe, and the mitochondrial function was tested by measuring oxygen consumption rate (OCR) using Seahorse Biosciences XF-96 Analyzer. Results: (1) Both glucose and ADM dose-dependently inhibited human adipocyte mRNA expressions of mtDNA-encoded subunits of electron transport chain, including nicotinamide adenine dinucleotide dehydrogenase 1 and 2, cytochrome b, and ATPase 6; (2) ADM does not affect the mitochondrial contents of adipocytes, but significantly increases mitochondrial ROS generation, and this increase is reversed by ADM antagonist, ADM22-52; and (3) ADM suppresses adipocyte basal and maximal oxygen consumption rate in a dose-dependent manner, thus impairing mitochondrial respiratory function. We report that ADM altered adipocyte mitochondrial function, and this may be through inhibiting mtDNA-encoded subunits of electron transport chain in mitochondria and stimulating ROS resulting in impairment of adipocyte function ADM blockade may improve adipocyte mitochondrial functions with significant protection from systemic metabolic deficiency seen in GDM. We aimed to investigate whether exceeding 20 mU/min, a commonly used maximal rate, was associated with adverse obstetric and perinatal outcomes in patients undergoing labor augmentation. Methods: This is a secondary analysis of a double-blind, single center trial of nulliparas with singleton gestations >=36 weeks' gestation randomized 1:1 to a high-dose oxytocin (HDO) titration regimen (6 mU/min) or standard-dose titration regimen (2 mU/min). A maximum rate limit was not specifi ed in the study protocol, and fetal heart rate patterns were reviewed before any rate change. For this secondary analysis, outcomes of women who received oxytocin and exceeded a dose rate of 20 mU/ min were compared to those of women whose rate remained <=20 mu/ min. Obstetric and perinatal outcomes were compared using bivariable and multivariable analyses. Characteristics with p<0.05 on bivariable analyses were included in multivariable models as potential confounders. Results: Of 1,003 participants, 955 received oxytocin and were included in this secondary analysis, with 190 (20%) exceeding a dose rate of 20 mU/min. Those who exceeded 20 mU/min were older, and more likely to have spontaneous rupture of membranes as their trial entry indication, be assigned to HDO regimen, receive oxytocin for longer, and have hypertensive disorders of pregnancy. Those whose maximum rates exceeded 20 mU/min were more likely to have a cesarean delivery, although the majority (74%) still delivered vaginally. While in bivariable analyses, there was a higher frequency of some adverse outcomes associated with exceeding 20 mU/min, those diff erences did not persistent after adjustment for potential confounding factors (Table) . Conclusion: In multivariable analyses, there are no diff erences in maternal or perinatal outcomes based on exceeding 20 mU/min of oxytocin. These data suggest that the higher rates seen in univariate analyses are related to patient characteristics that warrant the need for a higher maximum dose rather than the dose rate itself. Cost-Eff ectiveness of a Introduction: Women at risk for preterm birth are identifi ed by historybased and cervical length screening, though a new biomarker assay has entered the market intended for preterm birth screening of the entire pregnant population. We have developed an alternate hybrid digital/ biomarker (two-stage) screening platform to identify women at risk for very preterm delivery. The goal of this study was to determine the cost eff ectiveness of our hybrid platform compared to a commercially available biomarker-only approach. Methods: We developed a model to determine the cost-eff ectiveness of a two-stage preterm birth screening platform that utilizes a digital-only screen with refl ex to a progesterone (P4) biomarker assay for women who screen positive compared to a commercially available biomarker-only approach. The digital test identifi es risk for delivery less than 37 weeks' while the P4 biomarker assay identifi es risk for delivery less than 32 weeks. The primary outcome was the six-month cost to care for infants born prior to 32 weeks' gestation. Assumptions regarding test performance as well as costs of intervention were estimated from the literature. Women were assigned to full or intermediate bundles of care based on whether they screened high-risk for delivery at less than 32 weeks or less than 37 weeks, respectively. The costs of the screening tests were assumed to be as follows: digital screen ($100), P4 biomarker assay ($500), and commercially available biomarker assay ($800). Analyses were performed in PrecisionTree version 8.2 and the expected value of each testing option was compared. Sensitivity analyses were conducted varying the cost of the biomarker-only assay to determine the point at which the costeff ectiveness changed from one testing strategy to the other. Results: When choosing the hybrid screening platform, 43% of women were found to screen positive for preterm birth less than 37 weeks using the digital screen and 15.7% of those were found to screen positive for preterm birth less than 32 weeks using the P4 biomarker assay. When using the commercially available biomarker assay alone, 31% of women screened positive for preterm birth less than 32 weeks. The expected cost for the hybrid screening approach was $775 less per woman screened compared to the commercially available biomarker test. For every 1,000 women screened, the two-stage test saves $775,000. Sensitivity analyses showed that the hybrid screening platform remained cost-eff ective compared to the biomarker-only approach for all prices of the commercially available biomarker assay more than $80. Conclusion: A novel two-stage screening platform that utilizes a digital-only screen with refl ex to a P4 biomarker assay is cost eff ective in predicting women at risk for very preterm birth compared to a commercially available biomarker assay. PEC is associated with inflammation and dysregulation of innate and adaptive immune responses at the placental and serologic level, factors underlying pathogenesis of cardiovascular disease outside of pregnancy. We hypothesize that pre-pregnancy inflammation will correlate with subclinical cardiovascular dysfunction. Methods: This is a secondary analysis of a larger prospective observational study. Healthy nulliparas (n = 145) had detailed metabolic and cardiovascular assessment including body composition analysis, continuous non-invasive tonometric digital artery blood pressure, cardiac output monitoring, and clinical measures. In a selection of subjects, vascular compliance was assessed by pulse-wave velocity (PWV) and volume challenge. Pro-(IFNγ, IL-1β, IL-6, IL-18, TNFα) and antiinflammatory (IL-10) serum cytokines and chemokines (MCP-1) were measured by multiplex array (Mesoscale Discovery, Rockville MD). Relationship between pre-gestational serum cytokines with metabolic and cardiovascular markers were evaluated by Spearman correlations. IL-18, an inducer of IFNγ and Th1 responses, was also correlated with brachial beta stiffness (r = 0.25, p = 0.015). IL-10 negatively correlated with aorto-popliteal PWV (r = -0.19, p = 0.04), but not with cardiac ejection time. Aorto-popliteal PWV was no longer significant once corrected for cardiac ejection time, suggesting significance driven by vascular as opposed to cardiac stiffness. Conclusion: Pre-pregnancy subclinical metabolic and cardiovascular dysfunction are associated with a unique inflammatory environment. Markers of adaptive and innate immunity, TNFα and IL-18, were significantly correlated with endothelial adhesion molecules, which have been shown to be elevated pre-pregnancy in women who ultimately develop PEC, while anti-inflammatory IL-10 was inversely correlated with measures of vascular stiffness. Inflammation and dysregulation of adaptive and innate immunity may be the link to endothelial dysfunction that ultimately shapes maternal hypertension and the clinical phenotype. Introduction: Calculated blood loss (CBL), a formula estimating blood loss from body weight and hematocrit, has been proposed as a means to diagnose postpartum hemorrhage (PPH), but its utility remains to be determined. We compared CBL to provider estimated blood loss (EBL), hypothesizing that the CBL formula leads to higher rates of PPH. Methods: Cohort study of patients with nulliparous, term, singleton, vertex pregnancies, delivered 2016-2017. Exclusions were missing postdelivery hematocrit, blood transfusion, or diabetes. Provider-determined EBL was compared to CBL [percent change in hematocrit from pre-to post-delivery multiplied by estimated blood volume (body weight in kg x 85)]. The primary outcome was difference in rate of PPH (blood loss ≥1000mL) between CBL and EBL. Secondary outcomes included uterotonic use and percent of patients upgraded from normal blood loss to PPH using the CBL formula. Statistics by χ 2 , t-test, and Wilcoxon test; α=0.05. Results: There were 600 patients included; baseline characteristics are shown in Table 1 . Mean CBL was higher than provider-determined EBL (1096 ± 602 mL vs. 386 ± 245, p<0.001). Similarly, PPH rates were higher when using the CBL formula versus provider EBL (52% vs. 11%, p<0.001). In patients with a provider EBL<1000 mL, 49% (262/534) were upgraded to a diagnosis of PPH when using the CBL formula, but there was no difference in uterotonic use compared to those who were not upgraded (8% vs. 7%, p=0.57). Conclusion: CBL leads to significantly higher estimates of blood loss at delivery compared with provider estimates, leading to markedly higher rates of PPH. Given that uterotonic use was not higher in patients upgraded to PPH using the CBL formula, we suspect that CBL is overestimating blood loss at delivery. Thus, CBL may not be a reliable method of blood loss estimation in an obstetric population. Introduction: Obesity has been described as an infl ammatory condition with resulting systemic eff ects. In combination with pregnancy, obesity is associated with several comorbidities and pregnancy complications, but specifi c eff ects of obesity on the placenta and the potential downstream eff ects on fetal growth have not been well-studied. We sought to explore the relationship between obesity, placental pathology, and fetal growth. Methods: A cohort of 1467 women with singleton pregnancies from 2011-2020 was identifi ed from an ongoing obstetric registry, with available placental pathology data, as well as demographic, obstetric, and neonatal variables. Maternal weight categories were defi ned as: normal weight (BMI <25) and obese (BMI >=30) based on earliest weight recoded during gestation; the overweight category (BMI 25-29.9) was excluded for the purposes of this analysis. Associations between obesity, placental pathology, and fetal growth were assessed. Results: Pregnancies from 573 normal weight and 536 obese women were included. Maternal obesity was associated with large-for-gestation (LGA) placental disc (12% vs. 6%), increased incidence of decidual vasculopathy (DV; 37% vs. 11%), accelerated villous maturation (AVM; 19% vs. 14%), intervillous thrombi (IVT; 15% vs. 7%), chronic villitis (CV; 19% vs. 13%), and normoblastemia (40% vs. 25%). Of these, AVM, DV, and normoblastemia were associated with small for gestational age (SGA) infants, irrespective of obesity. In the setting of obesity, however, AVM appeared to have a more signifi cant eff ect on fetal growth, decreasing mean birthweight percentiles from 37.6 to 27.0 in normal weight mothers, but from 54.3 to 31.9 in obese mothers (p=0.01). Conclusion: Maternal obesity is associated not only with placental infl ammation (CV), but also with evidence of maternal vascular disease (DV), abnormal placental development (AVM), abnormal maternal blood flow in the placenta (IVT), and intrauterine hypoxic stress (normoblastemia). Our analysis suggests that while obese patients are more likely to have LGA babies, in the presence of specifi c patterns of placental injury (namely AVM), there could be a more severe blunting of fetal growth. Future analyses will focus on the eff ects of placental pathology on neonatal outcomes, beyond birthweight. In multivariable time-to-event models and in the Kaplan-Meier survival analysis, trends were again seen towards later delivery with all 3 rx, though not statistically signifi cant ( Figure 1 ). Conclusion: These data suggest a possible benefi t to rx with all 3 PTB treatments -cerclage, 17P, and vagP in high-risk patients with a TVCL <25mm, though fi ndings were limited by the sample size and should be confi rmed in a larger cohort. Metabolomic Methods: We performed metabolomic profi ling using maternal serum from the Pregnancy Outcome Prediction (POP) study at 12 and 20 wkGA and identifi ed candidate predictive metabolites using a mixed linear model fi tted to 185 cases of GDM and a random sample of 314 women. Candidates were validated at 28wkGA and predictive metabolites were defi ned sequentially by their ability to increase the bootstrapped area under the receiver operating characteristic curve (AUC) by >0.01 over maternal age, BMI, and any previously added metabolites. A predictive ratio of positively and negatively associated metabolites was used to validate fi ndings in two subgroups of the Born in Bradford (BiB) cohort with data from fasting plasma metabolites at 24-28wkGA. The predictive ability of a model including age, BMI and the four metabolites was compared with BMI>30 in the POP study. Results: 47 out of 829 metabolites were identifi ed as associated with GDM using the 12 and 20wkGA samples. Forward stepwise regression demonstrated an independent association for eight of them. Among these, seven were validated using the 28wkGA serum sample and four increased the AUC by >0.01. Two were positively associated (mannose and 4-hydroxyglutamate) and two negatively associated (1,5-anhydroglucitol and lactosyl-N-palmitoyl-sphingosine (d18:1/16:0)) with GDM. A predictive ratio discriminated GDM almost as well in the BiB subgroups (AUC=0.71 [1-sided P=8x10 -11 ] in BiB 1 and AUC=0.73 [1-sided P=7x10 -33 ] in BiB 2) as the POP study (AUC=0.75 [2-sided P=3x10 -15 ]) (Figure) . Introduction: Social deprivation affects the (mental) health of an individual and during pregnancy that of both the mother and the fetus. One of the most important pathways through which social deprivation aff ects health is through (chronic) stress exposure. Many studies have shown the negative eff ects of stress on the growth and development of a fetus, often resulting in adverse perinatal health. The other way around, adverse perinatal health also infl uences the perceived stress levels of the mother. The objective of this study was to examine whether perceived stress changes diff erently over time in social vulnerable women with or without a BIG2 outcome (i.e. presence of preterm birth and/or small for gestational age). Methods: As part of a pragmatic cohort study in vulnerable women, the Depression Anxiety and Stress scale (DASS-21) was used to assess the amount of perceived stress. The DASS-21 was fi lled out twice during pregnancy (at baseline and six weeks after start care) and six weeks postpartum. Data on a BIG2 outcome was obtained from the obstetric records of the participants. The median stress scores and interquartile ranges (IQR) were calculated for women with and without a BIG 2 outcome. Subsequently, the stress trajectories over time were categorized into no diff erence in stress levels (change -1 to 1 point), decreased stress levels (change -2 points or more) and increased stress levels (change 2 points or more). Results: Stress scores at baseline and six weeks postpartum were known for 191 women, and for 135 of these women also six weeks after start of care ( Table 1 ). The median stress scores did not really diff er for women with and without BIG2 at baseline and six weeks postpartum (Table 1 and Figure 1 ). However, women with BIG2 showed a larger improvement over time compared to women without BIG2 (6.50 -3.00 vs. 6.00 -4.00, respectively). Figure 2 shows the categorized stress trajectories. The stress scores were overall higher in women with a BIG2 outcome. Overall, stress scores increased most from baseline to six weeks after start care, irrespective of a BIG2 outcome. However, the biggest decrease in stress scores was seen in women with BIG 2 between six weeks after start care and six weeks postpartum. In this group of vulnerable women minimal eff ects of BIG2 was seen on the perceived stress levels over time. This might partly be explained by adaptive preferences, which is often observed in individuals living in social deprivation. BIG2 might however still infl uence the perceived stress levels in (pregnant) women not living in social deprivation. studies, methodological quality was addressed using the ErasmusAGE score (range: 0-10). The protocol was designed a priori and registered in PROSPERO (registration number: CRD42021273120). Results: We included 44 of the 5,873 unique records (median quality score: 6). Maternal circulating Trp concentrations were positively associated with pregnancy-induced hypertension (PIH), and negatively with antepartum depression, gestational diabetes, spontaneous abortion, and labor dystocia. Placental Trp concentrations were increased in early-onset preeclampsia (PE), and decreased in late-onset PE. In addition, maternal circulating kynurenine (Kyn) concentrations were elevated in spontaneous abortion, whereas reduced in antepartum depression. In preterm premature rupture of membranes (PPROM), circulating Kyn concentrations in umbilical cord blood were decreased. Higher maternal circulating kynurenic acid concentrations were associated with a greater risk of PE. In PE, circulating N-formylkynurenine, Kyn, anthranilic acid, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid, quinolinic acid and picolinic acid concentrations were unaltered in maternal blood and umbilical cord blood. Further details regarding differences in metabolite concentrations and reference intervals will follow. Conclusion: KP metabolite concentrations, in particular Trp, Kyn and kynurenic acid, are altered in maternal and fetal circulations in antepartum depression, gestational diabetes, PIH, PE, PPROM, spontaneous abortion, and labor dystocia, affecting pregnancy course and outcome in mother and child. Introduction: Due to the worldwide burden of obesity in women of reproductive age, bariatric surgery is increasingly practiced. Although bariatric surgery is an effective treatment to quickly and sustainably lose excess weight, the resulting lifelong iatrogenic malnourished state can interfere with pregnancy course and outcome. This study aims to evaluate the consequences of prepregnancy gastric bypass surgery on these factors. We performed a retrospective cohort study of 185 singleton pregnancies after gastric bypass surgery (due date between January 2009 and December 2019) at a bariatric surgery expertise center in Rotterdam, The Netherlands. Data from medical charts were evaluated, including maternal characteristics, birth weight, pregnancy and neonatal outcomes, and were compared with the Dutch Perinatal Registry data and morbidly obese women without bariatric surgery. Second and third trimester fetal growth parameters and estimated fetal weight (EFW) were analyzed using linear mixed models in 98 pregnancies with available ultrasound data and were compared with reference curves. Results: In postbariatric women compared to morbidly obese women, the risk for pregnancy complications such as gestational diabetes, preeclampsia and cesarean section was decreased (p<0.001), while the overall risk for pregnancy complications still remained higher than in the general Dutch population (p<0.001). Several individual fetal parameters (head circumference and biparietal diameter) and EFW were significantly decreased (p<0.001) in the second trimester, while EFW and abdominal circumference curves progressively worsened from 34 weeks of gestation onwards. In postbariatric pregnancies, birth weight was significantly reduced (p<0.001) and the risk for small for gestational age was increased (18.5 vs 10.4%, p=0.006), whereas the risk for macrosomia was significantly reduced (2.8 vs 10.9%, p=0.007). Conclusion: Pregnancies after gastric bypass should be regarded as highrisk pregnancies. Compared to women with morbid obesity, gastric bypass results in a maternal-fetal trade-off, favoring the mother. Fetal growth after gastric bypass surgery is most likely already impaired in the first trimester, which becomes worse during the third trimester, resulting in a signifi cantly lower birth weight. It is crucial to preconceptionally prepare postbariatric women contemplating pregnancy and to monitor longitudinal fetal growth for timely detection of growth restriction. Disparities in Clinical Management of Postpartum Hemorrhage Requiring Transfusion. Carolyn S Guan †, Theresa Boyer, Kristin C Darwin, Shari Lawson, Arthur Vaught * . Johns Hopkins University School of Medicine, Baltimore, MD, United States. Introduction: Postpartum hemorrhage (PPH) is a leading cause of maternal morbidity and mortality in the US and disproportionately aff ects pregnant persons of color. The objective of this study was to identify the demographic and obstetric characteristics of women who receive higher levels of antihemorrhagic intervention (uterotonics, procedures, and hysterectomy) in the setting of severe PPH requiring blood transfusion. We conducted a retrospective cohort study of patients with documented PPH (estimated blood loss (EBL) of ≥1000mL) and blood product transfusion. We defi ned 3 levels of antihemorrhagic intervention: (1) administration of uterotonics only, (2) performance of procedure (including B-Lynch suture, O'Leary stitch, Bakri balloon, dilation and curettage, laceration repair, or embolization), and (3) performance of hysterectomy. Maternal demographics, obstetric characteristics, and comorbidities were extracted from electronic health records. Ordinal logistic regression was used to estimate the odds of higher intervention level adjusting for maternal demographic and obstetric characteristics. Results: 368 patients were included in this study. 59.0% (n=217) received level 1 intervention, 31.8% (n=117) received level 2 intervention, and 9.2% (n=34) received level 3 intervention. Women receiving higher levels of intervention were more likely to have greater EBL (p<0.001), more transfusions (p<0.001), and be advanced maternal age (p=0.004) ( Table 1 ). Compared to White women, Black and Latino women were less likely to have received higher levels of intervention (p=0.029). After adjusting for advanced maternal age and placenta accreta spectrum, Black women remained signifi cantly less likely to receive higher levels of intervention (aOR 0.59, 95% CI 0.36-0.99) ( Figure 1 ). After adding an adjustment for EBL≥2000mL the diff erence was no longer signifi cant. Conclusion: Among women experiencing PPH and receiving transfusion, Black women are less likely to receive higher levels of antihemorrhagic intervention. This disparity is particularly concerning in this high-risk population. Methods: This is a secondary analysis of data from an observational cohort and randomized clinical trial (RCT). The control population recruited women at low risk for pre-eclampsia. In the RCT, patients defi ned as high risk for pre-eclampsia by USPSTF criteria were randomized to 81mg vs. 162mg of aspirin prior to 16 weeks' gestation. Serum was obtained at 11-13 weeks' gestation (prior to aspirin), at 28-32 weeks', and at delivery in cord blood. Serum CRP and sICAM concentrations were related to BMI > 30 at enrollment, log-transformed, and correlated with BMI by linear regression. Results: 190 women were studied. 65 controls were followed (25 obese, 38.4%). 60 patients were assigned 81mg aspirin (31 obese, 51.7%) vs. 65 to 162mg (38 obese, 58.5%). A higher proportion of patients in the obese subgroup had a history of chronic hypertension and prior pre-eclampsia compared to the non-obese subgroup. A signifi cant relationship was noted between BMI and both CRP and sICAM prior to aspirin exposure and in the third trimester, Introduction: Intrahepatic cholestasis of pregnancy (ICP) is a poorly understood pathology of pregnancy with potential adverse perinatal outcome. The incidence of ICP appears to vary by ethnicity, but it remains unclear whether disease severity and poor outcomes also do. We aimed to assess the association of ICP severity with ethnicity in a diverse, urban population. Methods: This is a case-control study of patients with ICP at a university hospital from January 1, 2013 -July 31, 2019. Patients were identifi ed by by ICD-10 code and grouped by total bile acid (BA) level (umol/L) at diagnosis: normal (BA <10), mild (BA 10-40), severe (BA 41-100), and extreme disease (BA>100). Normal BA were removed from the analysis. Patient race/ethnicity was grouped into non-Hispanic white, non-Hispanic Black, Hispanic, East Asian, South Asian and other to represent the major groups. We compared demographic and clinical characteristics using Chisquare, t-test, and ANOVA (signifi cance at p < 0.05). Table 1 , the following reference groups were used: singleton births, no previous PTB, no gestational or pre-pregnancy HTN, 30-34 years old, non-Hispanic White race, nonsmoker, no gestational diabetes, associate's degree or higher, and 13+ months since the last live birth. Conclusion: Black race is a signifi cant independent risk factor for PTB. However, the database does not provide some crucial parameters of socioeconomic conditions, such as household income, that may impact mothers' likelihood of PTB. Therefore, further research is needed to determine if Black mothers have a biological predisposition to PTB or not. to preferentially diff erentiate to orexigenic vs anorexigenic neurons. As mitochondrial function is critical for neurodiff erentiation, we hypothesized that mitochondrial dysfunction of hypothalamic NPCs from OB/HF off spring may be the underlying mechanism for hyperphagia. Methods: Female mice were fed either a control (10% kcal; N=4) or high fat (45% kcal; N=4) diet prior to mating and throughout pregnancy. Control and OB/HF off spring were born spontaneously. Hypothalamic tissue was obtained from 1 day old male off spring and NPCs cultured in complete media. Using the Seahorse assay, we determined mitochondrial oxygen consumption rate (OCR), including basal (basal metabolic rate), ATP-linked (capacity to meet energetic demands), maximal respiration (energy demand to meet metabolic challenge) and reserve capacity (extra ATP that can be produced by oxidative phosphorylation in response to increased energy demand). Data was analyzed by unpaired t-test. Results: Hypothalamic NPCs from OB/HF newborns exhibited reduced basal and ATP-linked OCR (~50%) and maximal respiration and reserve capacity (~75%) (Figure; Values are Mean±SE; *P <0.001). Conclusion: OB/HF newborns have impaired hypothalamic NPC mitochondrial OCR, suggesting that mitochondrial dysfunction potentially contributes to altered neurogenesis and hyperphagia evident in these off spring. Lipase Inhibitor Prevents Palmitate-Induced "Milk" Triglyceride Production in 3-D Mammary Culture. Mina Desai * , 1 Ken Kobayashi * , 2 Guang Han, 1 Michael G Ross * . 1 1 The Lundquist Institute, Torrance, CA, United States; 2 Hokkaido University, Sapporo, Japan. Introduction: Maternal obesity and excess infant weight gain increases the risk of childhood and adult obesity. Milk of obese/high fat diet women and dams have an increased fatty acid and caloric content, suggesting that milk composition refl ects maternal diet and serum composition. Notably, elevated plasma levels of free fatty acids and particularly saturated long-chain fatty acids such as palmitic acid are evident in women with obesity during pregnancy. Palmitate is the major saturated fatty acid in human milk representing 20-25% of fatty acids. We utilized a novel 3-D mammary epithelial cell (MEC) primary culture model to recreate the milk production pathway in vitro and examined the eff ects of exogenous palmitate on milk triglyceride content. We further tested whether a lipase inhibitor can prevent palmitate-induced milk production in MEC culture. Methods: Mammary glands from non-pregnant, non-lactating mice were collected and MECs cultured using cell inserts which separate the basolateral chamber containing lactogenic medium (nutrients) and the apical chamber containing secreted MEC "milk". Palmitate-albumin conjugate (50, 100 µmol/L) or a lipase inhibitor Orlistat (100 µmol/L) together with palmitate (100 µmol/L) were added to the basolateral chamber. After 48h, apical medium was collected and analyzed for β-casein (marker of milk) and triglyceride levels. MEC cells were analyzed for protein expression of β-casein, fatty acid synthase, the transcription factor SREBP1, and activated pSTAT5 (promotes milk synthesis). Data was analyzed by ANOVA with Dunnett's post-hoc. (Mean±SE; *P<0.01). Results: Following treatment with palmitate, MEC "milk" showed increased β-casein and triglyceride concentrations ( Fig A) and MEC cells showed increased β-casein, fatty acid synthase, SREBP1 and pSTAT5 ( Fig B) . Orlistat treatment normalized "milk" triglyceride concentrations to that of untreated MECs (Fig C) . Conclusion: Palmitate increases MEC synthesis and production of "milk" triglycerides, which is prevented by a lipase inhibitor. These fi ndings suggest that human milk production and composition may be modifi ed by maternal diet or pharmacologic intervention to normalize infant nutrition and weight gain. In Ctrl offspring, T cells increased (Fig.M, p<0 .05) by rIL-1b treatment, indicating that prenatal-inflammation-exposed offspring exhibited dysregulated T-cell immune responses to post-weaning challenges. Conclusion: Prenatal inflammation-exposed offspring exhibited dysregulation T-cell immune response to the postnatal challenge, which also revealed postnatal age-dependent differences. Given our results, we speculate that MI-exposed offspring are at increased risk for developing future immune-mediated diseases and hyper/hypo reactiveness in response to immunizations. Analysis of cfDNA Methylation Changes in Genes Associated with Hypertensive Disorders in Pregnancy. Jarmila Anna Zdanowicz †, Marialuigia Spinelli, Daniel Surbek, Martin Mueller * . University Hospital Bern, Bern, Switzerland. Introduction: Hypertensive disorders in pregnancy (HDP) are among the most serious complications of pregnancy and contribute substantially to perinatal morbidity and mortality of both mother and infant. The majority of methylation studies conducted so far have focused on the placental compartment and mostly on selected candidate genes, emphasizing outcomes and co-morbidities of pregnancy. Reports on cell-free (cf)DNA genome-wide differential methylation regions (DMRs), especially in early pregnancy, are absent. Using cfDNA between 11+0 to 13+6 weeks of gestation, we analyzed methylation profiles and gene methylation changes. We postulate that hypertensive disorders in pregnancy including preeclampsia (PE) have a shared cardiovascular predisposition. With previously selected samples from the first trimester, we matched patients according to known PE risk factors based on the Fetal Medicine Foundation (FMF) algorithm and identified three groups (n=5 each): PE and no chronic hypertension (PE), chronic hypertension and no PE (HT), and control (Ctr). CfDNA was isolated from all serum samples as previously described and used for further analysis. Consequently, we tested for differential methylation between pairs of experimental groups at the level of individual CpGs to generate methylation sequencing libraries and looked at methylation changes in genes. We detected pairwise changes in DMRs in the maternal compartment. To test the theory of shared vascular predisposition, we analyzed genes methylation changes. We summarized the significant DMRs and annotated genes in each pairwise comparison (the adjusted p-value is < 0.059 between all three groups, n=5 for each group). We detected a lower number of DMRs (86) and genes (36) in PE/HT compared to PE/Ctr (139 DMRs and 75 genes) and HT/Ctr (140 DMRs and 74 genes). This lower number of DMRs and genes in PE/HT comparison and the relatively large number of DMRs overlapping between PE/Ctr and HT/Ctr comparisons ( Figure 1 , compare overlapping boxes between PE/Ctr in red and HT/Ctr in green) point towards a shared background and/or predisposition of PE and HT groups. Intriguingly, we found that many of the annotated genes to be associated with cardiovascular disorders (highlighted in red in Figure 1 ). Conclusion: Analysis of specific genes point towards an association with cardiovascular disorders using cfDNA methylation from first trimester samples. Our data further supports the importance of imprinted genes but also allows a risk free assessment in early pregnancy. Introduction: Despite the well-recognized benefi ts of human milk, only 20% of infants are exclusively breastfeed through 6 months of age. Thus, a majority of infants are provided with formula-based nutrition at some point in early life. Among the components of human milk, the HMO composition partly modulates childhood growth as these complex sugars serve as prebiotics. Colonic bacteria digest HMOs with bifi dobacterium species especially well-suited to the digestion, in contrast to other "pathogenic" bacterium. In view of the importance of the microbiome, we assessed the impact of infant formula with and without supplemental lacto-N-neotetraose (LNnT) in an in vitro colon model. The TIM-2 colon model was inoculated with a pooled microbiota of 9 healthy human infants (1-2 months) using feces collected (diaper samples) from volunteers to allow for a standardized microbiota. The TIM-2 was loaded with infant formula with and without LNnT (200 and 1000 mg/L) for 72 hours. Samples from both the lumen and dialysate of the system were obtained at 0, 24, 48 and 72 hours and analyzed for fermentation products (eg, lactate). Results: Analysis of microbial metabolites (D-and L-lactate) demonstrated increased L-lactate with high dose LNnT, likely due to the stimulation of bifi dobacteria species that primarily produce L-lactate (Fig) . Conclusion: These fi ndings suggest that LNnT supplementation may be of benefi t for formula fed infants supporting the growth of benefi cial microbiota. The Effect of Maternal Metabolic Dysfunction on the Adipose Progenitor Population and Function in the Off spring. Taylor Scheidl †, Anna Mikolajczak, Radha D Singh, Jennifer A Thompson * . University of Calgary, Calgary, AB, Canada. Introduction: Childhood obesity is a burgeoning public health crisis, evident by a nearly 300% increase in obesity in Canadian youth over the last 30 years (Canada.ca). This has led to a declining age-of-onset for cardiovascular disease and type 2 diabetes, which historically presented in the aged population (Sahoo, 2015) . While sedentary lifestyle and poor nutrition undoubtedly contribute to the declining age-of-onset of cardiometabolic disease, contributions of the in utero environment remain underappreciated. The adipose tissue (AT) is implicated in developmentally programmed cardiometabolic disease risk. To expand, the AT depends upon a robust population and diff erentiation capacity of the adipose progenitor cells (APC) and the committed preadipocytes, which arise when APC commit to the adipocyte lineage. These progenitors are suggested to be both fi nite (Tang, 2011) and established in utero (Jiang, 2014) . Reduced progenitor availability for AT expansion accelerates the rate at which terminally diff erentiated adipocytes become lipidoverloaded and dysfunctional. Published data from our lab suggest that a metabolically adverse gestational environment leads to increased adiposity and accelerated adipocyte maturation in subcutaneous AT (SAT) of neonatal off spring, resulting in later life AT tissue dysfunction and lipid spillover, increasing cardiometabolic disease risk (Mikolajczak, 2021) . We postulate that the adverse intrauterine environment leads to AT dysfunction in adulthood by depleting the preadipocyte population and impairing the diff erentiation capacity of the APC and preadipocytes. We employ a murine model of maternal metabolic dysfunction by crossing females with heterozygous leptin receptor deletion (Het DB ) with wild-type (Wt) males. Wt off spring of Het DB pregnancies or of Wt pregnancies are stratifi ed to control-(CD) or high-fat diet (HFD). APC (CD36-;CD24+) and preadipocytes (CD36+;CD24-) isolated from the SAT are stained for fl ow cytometric analysis and identifi ed within the Lin-;CD34+;CD140a+ population. In vivo differentiation capacity of adipose progenitors will be determined by FACS isolation of APC from offspring ubiquitously expressing tdTomato. Isolated APC will be transplanted into the SAT of Wt recipient mice subjected to the adipogenic agent Rosiglitazone for 1 week. The number of tdTomato expressing preadipocytes will be determined by flow cytometry, and immunofluorescent imaging of LipidTox green stained SAT will be used to determine the number of newly formed adipocytes which express tdTomato. Results: Preliminary flow cytometric data shows that the proportion of committed preadipocytes is reduced in the offspring of Het DB pregnancies, while the proportion of APC is increased. Conclusion: Our preliminary data suggest that the commitment of APC to the preadipocyte lineage is impaired, reducing the preadipocyte population. We found increased TG-LCPUFAs in FGR cord plasma which correlated with US markers of compromised fetal growth. LPC-DHA (vital for brain development) is believed to be the lipid form for transport across the blood brain barrier, but our data show that LCPUFA packaging into TG (a storage form) may occur at the expense of LPC formation. We speculate that the increased TG-LCPUFA in FGR represents the fetal response to an adverse in utero environment, ie. oxidative stress, and may have detrimental consequences for ultimate LCPUFA transport to the brain for development. Hypoxia Increases P-glycoprotein (P-gp) Activity in Developing Human Brain Endothelial Cells: Implications for Fetal Brain Protection. Hafsah Mughis †, 1,2 Phetcharawan Lye, 1,2 Guinever Imperio, 1 Enrrico Bloise, 3 Stephen Matthews * . Introduction: P-glycoprotein (P-gp, encoded by ABCB1) is one of the primary multidrug resistance transporters expressed in the developing blood-brain barrier (BBB). It confers protection against the entry of harmful molecules (including drugs, pesticides and herbicides) into the fetal brain. During normal development, the fetus is exposed to relatively low oxygen concentrations (~ 5% O 2 ). However, pregnancy complications including pre-eclampsia may lead to even lower intrauterine O 2 levels. There is very limited information about the effect of hypoxia on P-gp at the fetal BBB. Our objective was to investigate whether hypoxia can alter P-gp levels in human fetal brain endothelial cells (BECs). We hypothesized that hypoxia will increase P-gp expression and function in human fetal BECs derived in 2 nd trimester. Methods: Human fetal BECs isolated from 2 nd trimester fetuses (N=6) were cultured to assess function and expression of P-gp under different levels of oxygen (1%, 5% and 20%) at 3 time points: 6h, 24h, and 48h. Media was equilibrated with 1% O 2 and 5% O 2 prior to adding to the cells. An oxygen sensor placed in the chambers was used to confirm oxygen levels. Changes in P-gp function were analyzed through accumulation of the fluorescent substrate calcein-AM. Protein and gene expression of P-gp/ABCB1 was assessed using Western Blot and RT-qPCR, respectively. Data was analyzed by 2-way ANOVA followed by Tukey's multiple comparisons test. Results: P-gp activity significantly increased under 1% hypoxia, compared to 20% O 2 , at all 3 time points investigated: 6h, 24h and 48h (P<0.05). No significant differences were observed in P-gp function at 5% O 2 , relative to 20% O 2 , after 6h and 24h, but an increase in function was observed after 48h. No significant changes in P-gp protein expression were observed, however ABCB1 mRNA expression level was significantly decreased under 1% O 2 (P<0.05) at 24h and 48h. Conclusion: This is the first study to show that severe hypoxia can lead to rapid and sustained increases in P-gp function in human fetal BECs. As has been previously reported in other model systems, including the placenta, hypoxia-induced changes in P-gp function are uncoupled from changes in ABCB1 mRNA and P-gp protein. The hypoxia-induced increase in P-gp function will potentially decrease transfer of xenobiotics across the fetal BBB, conferring protection of the developing brain. Given that hypoxia can occur in a number of pregnancy complications, our findings in a unique model system are highly clinically relevant. Further understanding of the regulation of P-gp at the developing human BBB, will potentially lead to the development of strategies to enhance fetal brain protection, particularly during vulnerable pregnancies. First Longitudinal measurements of crown-rump length (CRL), embryonic volume (EV), head circumference (HC), transcerebellar diameter (TCD), head volume (HV) in the 1 st trimester of pregnancy, and biparietal diameter (BPD), HC, TCD, abdominal circumference (AC), femur length (FL) and estimated fetal weight (EFW) in the 2 nd and 3 rd trimesters of pregnancy, were performed using 3D ultrasound scans and Virtual Reality techniques. Growth rates of body and head measurements were calculated between 9-11 weeks of gestation. Birth weight was collected from medical records. Associations between tHcy and embryonic/fetal growth parameters were investigated by linear regression and linear mixed models with confounder adjustment. Results: Maternal tHcy was negatively associated with levels of vitamin B 12 (B -0.21, 95% CI -0.25--0.17, p< 0.01) and folate (B -0.09, 95%CI -0.12--0.05, p< 0.01). tHcy was associated with reduced longitudinal CRL measurements (B -0.02, 95%CI -0.04--0.001, p= 0.04) and EV (B -0.01, 95%CI -0.02--0.003, p< 0.01). CRL growth rates (mm/day) reduced signifi cantly with increasing maternal tHcy levels (B -0.02, 95%CI -0.03--0.005, p<0.01), whereas this did not apply for growth rates of the HV, HC and TCD. In the 2 nd trimester, no signifi cant associations were found between tHcy and longitudinal BPD, HC, TCD, AC, FL and EFW measurements. Higher tHcy levels was associated with reduced neonatal birth weight (B -26,13, 95%CI -47.09--4.4, p=0.02). Maternal tHcy was not associated with preterm birth, small or large for gestational age neonates. Conclusion: High maternal tHcy levels are associated with delayed growth during the 1 st trimester of pregnancy and reduced birth weight, but do not associate with fetal growth parameters during the 2 nd and 3 rd trimester. Maternal tHcy levels do not associate with prenatal brain development. First Introduction: In-utero exposure to an adverse environment is associated with cardiovascular remodeling, aff ecting fetal cardiac function and causing possible long-term eff ects on cardiovascular health (CVH). Fetal cardiac function can be estimated by stroke volume (SV) measurements based on cardiac ventricle volumes. We aim to study feasibility and reproducibility of fi rst-trimester fetal cardiac ventricle volume (FCVV) measurements using Spatio-Temporal Image Correlation (STIC) and virtual reality (VR) techniques. Methods: First-trimester transvaginal STIC ultrasound datasets were recorded using regular B-mode (BM) and color Doppler (CD) mode in 20 pregnant women between 11 +0 -13 +6 weeks' gestation. FCVV measurements were performed in the end-diastolic (ED) and end-systolic (ES) cardiac phase using VOCAL, a validated method based on manual segmentation, and an innovative semi-automated volume measuring method using VR ( Figure 1 ) by two independent observers. SV was calculated by subtracting EDV from ESV. The inter-, intra-observer and inter-system agreement were assessed by the intraclass correlation coeffi cient (ICC) and Bland-Altman analysis. Results: Using VR, the mean SV was 31.60mm 3 and all measurements showed good to excellent intra-and inter-observer ICCs. The ICCs for BM were 0.86 and 0.74 and for CD 0.89 and 0.84 respectively. The inter-and intra-observer ICCs using VOCAL (BM) were 0.89 and 0.37, respectively. The inter-system agreement, comparing VR and VOCAL, showed an ICC of 0.36. Conclusion: Our study demonstrates that first-trimester FCVV measurements using STIC and VR are feasible and reproducible to assess cardiac function. Further, when compared to VOCAL as a golden standard, FCVV measurements using VR, show an increased feasibility and reliability. In the future, a fi rst-trimester fetal cardiac function assessment may provide insight into cardiac development during pregnancy and CVH throughout the life course. Metabolomic (n=10) were infused for 90 minutes with either IGF-1 LR3 (IGF-1) or vehicle control (CON). Each animal received both infusions spaced 2-3 days apart. Fetal plasma insulin and glucose were measured. At minute 90 of infusion, GSIS was measured utilizing a hyperglycemic clamp with frequent glucose and insulin measurements. At the end of the second infusion (IGF-1, n=5; CON, n=5), fetal islets were isolated and incubated with 1.1, 2.7, or 11 mmol/L glucose or 30 mmol/L KCl and then pelleted. Insulin concentrations of the media and cell pellet were measured by ELISA. Insulin secretion was calculated as the fraction of total islet insulin secreted into the media. Results: At the end of infusion, fetal plasma glucose concentrations were similar, but insulin concentrations were 48% lower in IGF-1 compared to CON (P<0.05). During the GSIS study, glucose concentrations were similar, but insulin concentrations were 66% lower with IGF-1 infusion compared to CON (P<0.0001). Insulin secretion in isolated fetal islets was not different based on infusion just prior to necropsy. Conclusion: IGF-1 LR3 infusion for 90 minutes into late gestation fetal sheep lowers plasma insulin concentrations and attenuates fetal GSIS. However, reduced insulin secretion does not persist in isolated fetal islets exposed to IGF-1 in vivo at the time of necropsy. Therefore, while acute increases in fetal IGF-1 may directly suppress insulin secretion, the fetal beta cell in vitro retains the ability to recover GSIS. With more prolonged elevations in fetal IGF-1, beta cells become programmed to secrete less insulin in response to glucose. We speculate that chronically, but not acutely, elevated fetal IGF-1 concentrations may underlie the risk of beta cell failure and diabetes later in life. Introduction: Intrapartum uterine contractions trigger the response of the fetal autonomic nervous system (ANS) producing fetal heart rate (FHR) decelerations. Phase-Rectified Signal Averaging (PRSA) analysis extracts predictive information from FHR, and is more resistant to nonstationarities, signal loss and artefacts than conventional FHR analysis techniques (i.e., short-term variability). In a normoxic near-term sheep exposed to repetitive umbilical cord occlusions (UCOs) modelling uterine contractions, we found that the average cardiac acceleration (AC) and deceleration (DC) computed by PRSA progressively increase with the severity of the UCOs suggesting an activation of ANS in response to acute hypoxia. In this study, we hypothesized the different ANS regulation under chronic hypoxia leads to a change in the FHR deceleration morphology and that the adaptation times would be different from those of healthy fetuses. Methods: 7 normoxic and 5 chronically hypoxic sheep fetuses underwent a strict experimental protocol of three levels of UCOs (mild, moderate and severe) as published. Firstly, using a trapezoid, we quantified the average characteristics of the fetal inter-beat time interval (FRR) series to UCOs in terms of baseline level (b), deceleration amplitude (a), time to reach the steady condition during UCO (τu) and time to reach the baseline (b) after releasing the UCO (τr) in the normoxic and hypoxic fetuses. The parameters were compared between the two groups and correlated with biomarkers of acid-base balance during the severe phase of UCOs. Then, we quantified whether AC, DC and the deceleration reserve (DR=AC+DC; AC is a negative quantity on FHR) were correlated with the morphological parameters. Results: Comparing normoxic and hypoxic fetuses, we found a clear difference for τu (24.8 ± 9.4 vs 39.8 ± 9.7 s; p<0.05), a (268.1 ± 109.5 vs 373.0 ± 46.0 ms; p<0.1) and Δτ (τu -τr) (13.2 ± 6.9 vs 23.9 ± 7.5 s; p<0.05). DR and DC correlated with Δτ and τu for a wide range of scales (max Pearson's correlation Rho = 0.9, p<0.05, and Rho=0.6, p<0.1, respectively). No other significant correlations between biomarkers and morphological parameters were found. Conclusion: Our findings support the hypothesis that hypoxic fetuses have a longer response time and larger asymmetry in the adaptation time, as FHR response to UCOs. The same difference can be assessed through PRSA based parameters. Nowadays, assessing these morphological parameters during labor is challenging due to non-stationarity, phase desynchronization and noise, further motivating future investigations on the translational potential of PRSA methodology for risk stratification on human cardiotocograms. Deep The relationship between organs in the fetal-placenta-cardiac axis during development is poorly understood. Our objective was to characterize placenta pathology and the transcriptomic signature in the cord endothelial cells (EC) among cases of prenatally suspected congenital heart disease (CHD) in order to elucidate mechanistic underpinnings. Methods: Cohort of confirmed CHD at a single institution with placenta pathology were included. CHD was categorized as septal defects, right heart defects, left heart defects, conotruncal anomalies, or other anomalies. Univariate analyses were conducted for maternal and neonatal variables across the various heart defects. Primary EC were isolated from cords immediately after delivery and RNA was isolated. Library preparation and sequencing was conducted at 50bp single-end reads at a depth of 30,000 million reads/sample and mapped. Differential gene expression was conducted Results: 140 cases of neonatal CHD were analyzed. Median maternal age was 32yo with varied medical comorbidities, family history of CHD, and IVF history (all p>0.05). Neonates with CHD were 46% female sex, median gestation age of 39 weeks, 45% had a vaginal birth, median birthweight of 2920g, and 25% with abnormal genetic testing, with no significant difference by subgroups(p>0.05). Placental pathology in this cohort was diverse: 7% thrombosis, 20% vascular infarction, 6% chorangiosis, and 4% hypomature villus. Newborns with right-sided heart defects had the greatest overall degree of placental abnormality. After adjusting for clinical covariates maternal age, fetal sex, gestational age, race and BMI (Figure 1 ), bulk RNA sequencing of the cord EC demonstrated significant modulation in genes involved in chondrocyte development and extracellular matrix (ECM) organization. Genes essential in the matrisome and ECM are significantly altered in fetal CHD and include Col1a1, Vcan, Dcn, Fbln1, Igfbp3, Tgfb1, Zplnd1, among others (FDR-adjusted P <0.05, log2FC > 1). The fetal-placenta-cardiac axis can be interrogated to understand molecular mechanisms related to CHD. We demonstrated a distinct placental pathology phenotype in cases of right-sided heart disease and a modulation in genes involved in the matrisome and ECM organization offering insight to the EC dysfunction in CHD. Maternal In view of this, we hypothesized that maternal HFD independent of obesity can impact the fatty acids profile in fetal hypothalamus and in the circuitry that controls energy homeostasis. Methods: Female mice were fed control (10% fat; n=8) or HFD (45% fat; n=9) prior to and during pregnancy. Considering that HF diet has a higher percentage of saturated FAs (38%) and a lower percentage of PUFA (23%) than control diet (28% and 40%, respectively), we evaluated body weight gain, adiposity, energy intake, inflammatory cytokines (IL1-b, TNFa, IL-6 by ELISA), glycemia and serum fatty acid composition (CG-MS) in control and HFD dams at gestational day e20 and compared to fetuses serum and hypothalamic fatty acid composition (n=8). Data were compared using Student's unpaired t-test. Results: At gestational day e20, non-obese HFD dams had higher level of PUFA than saturated FA though with comparable serum inflammatory cytokines as the control dams. Notably, e20 fetuses showed increased levels of serum and hypothalamic PUFA than saturated fatty acid. The HF diet has the highest n-6:n-3 ratio, which seems to contribute to the level of PUFA in maternal serum (27.97±2.1 HFD dams vs 21.53±2.63 C dams, p<0.001), with a signifi cant increase in n-6 (18:2). Similarly, we also observed the same profi le of PUFA and n-6 (18:2) in serum ( Introduction: Abnormal fetal growth is highly predictive of perinatal morbidity and mortality. Measurements of fetal growth provide a unique opportunity to study the association of fetal growth with maternal, fetal, and environmental factors during pregnancy. The objective of this study is to develop a computerized process to extract biometric measurements from fetal diagnostic ultrasound (dUS) reports and assess the quality of these measurements within a large healthcare system. Methods: Singleton pregnancies delivered between 2009-2020 were identifi ed from the KPSC electronic health records. The corresponding dUS images during pregnancy were retrieved. An automated computerized process was then developed and refi ned by iterative manual examinations to extract fetal growth-related measurements: crown rump length (CRL), abdominal circumference (AC), biparietal diameter (BPD), head circumference (HC), and femur length (FL). To assess the accuracy of the automated extraction process, 500 image reports (300 from reports with identifi ed fetal biometric measurements and 200 from reports without fetal biometric measurements) were randomly selected for manual examination. Lastly, the mean and standard deviation (STD) in centimeter for fetal biometric measurements were summarized. Introduction: CPAM: Congenital pulmonary airway malformation is one of the conventional congenital lung cystic diseases. The prognosis of this disease is determined on CVR (CPAM volume ratio), and CPAM volume is estimated using the formula for a prolate ellipse (length X Height X Width X 0.52). If CVR is over 1.6, the occurrence of hydrops is significantly increased. The mortality of CPAM with hydrops was reported extremely high, about 95 to 97%. We performed TAS (thoracoamniotic shunting) for CPAM at 25w3d of gestation, and managed the fetus to measure CVR during perinatal period, and were finally able to rescue the neonate of CPAM. Methods: Case:The mother was 35 years old, 4 gravity and 3 parity. At 20 weeks of gestation, her fetus was diagnosed with CPAM on ultrasonography, with multiple sized cysts at the fetal left lung. Then we performed TAS at 25w3d of gestation, because of deterioration in expanding CPAM lesion, hydropic feature, and elevating levels of CVR. After successful shunting, CVR was decreased under 1.6. However, the outside edge of shunt was dislocated into the fetal thoracic cavity, with elevated CVR in 1.41, so we administered Dexamethasone for regression of CPAM at 26 weeks of gestation. After administration of Dexamethasone, the CPAM lesion decreased and CVR was not elevated and underwent the range between 0.78 to 1.39. At 38w0d of gestation, the shunt tube was dislocated into CPAM, and CVR was elevated again over 1.6, so we determined induction of labor. The neonate was born on vaginal delivery at 38w3d of gestation. His birth weight was 2,930g, and his Apgar Scores were 8 and 9 at 1 and 5 min, respectively. Results: He was intubated because of respiratory distress at birth. He could be extubated on day 6. On day 20, he was put on ventilator again due to respiratory acidosis, with air trapping, so we performed upper left lung lobectomy for CPAM lesion, and resection tissue was pathologically diagnosed to CPAM type 1. He was discharged on 38th day without any respiratory device. Conclusion: We performed TAS and were able to rescue the neonate of CPAM (CVR >1.6, pathologically type 1) to prevent hydrops. HGSOCs, significant clinical benefit has been observed in non-HRD HGSOCs. However, HGSOC patients are developing PARPi resistance, which is becoming a significant clinical hurdle. Therefore, there is an urgent clinical need to understand and overcome resistance mechanisms to extend HGSOC disease-free intervals. We established isogeneic olaparib-sensitive and -resistant cells in a panel of HGSOC cell lines with varying BRCA mutations. Using these cell lines, we performed and analyzed RNA-sequencing to determine key genes and pathways to interrogate as potential therapeutic targets. Results were confirmed using luciferase reporter assays, qPCR, and western blotting. We used both genetic and pharmacologic approaches to investigate the importance of ATF6-mediated AP-1 signaling in mediating PARPi resistance. DNA damage was measured using comet assays and gH2AX foci formation. Lastly, an in vivo model was utilized to determine the efficacy of p38/MAPK14 inhibition in combination with olaparib in PARPi resistant HGSOC. We previously reported elevated Wnt signaling reduces PARP inhibitor sensitivity. Prior reports discovered that elevated Wnt signaling can enhance AP-1 transcriptional activity. In olaparib-resistant cell lines compared to sensitive cells, there is a significant increase in AP-1 transcriptional activity and DNA damage repair capacity. In an unbiased screen of 14 AP-1 subunits, we identified Activating Transcription Factor 6 (ATF6), a transcription factor that can be co-opted to upregulated AP-1 transcriptional targets. Upon ATF6 knockdown in resistance cells, there is decreased AP-1 transcriptional activity, increased DNA damage, and increased sensitivity to olaparib. AP-1/ATF6 activity known to be enhanced following phosphorylation by Mitogen-Activated Protein Kinase 14 (MAPK14/p38). In addition to ATF6/AP-1 activity, P38 phosphorylation is increased in PARPi resistant cells. We discovered that inhibiting p38 significantly inhibited AP-1 activity and mimicked ATF6 knockdown. In an in vivo model of olaparib resistance, p38 inhibition and olaparib combination significantly reduced tumor growth rate and tumor burden. Conclusion: This study highlights that a novel p38-ATF6 mediated AP-1 signaling axis contributes to PARPi resistance in HGSOC and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors. Introduction: Approximately 20% of women suffer from perinatal stress-related mood disorders including depression and anxiety, making these disorders among the most common. Stress-related disorders are associated with pregnancy complications including preeclampsia, which prime women for poor cardiometabolic health throughout life. However, the relationship and mechanism(s) linking maternal stress, vascular physiology and pregnancy outcome are unknown. The focus of this project was to investigate an animal model of chronic unpredictable stress (CUS) to study these relationships. Wild-type BALB/c female mice were randomized to control or 3-week CUS conditions prior to breeding. All mice were evaluated for anxiety and depressive-like behavior via Open Field testing. Placenta morphology was determined. Blood pressure was measured during pregnancy, and vascular function assessed by wire myography. Placental perfusion was measured by micro-MRI at 14.5 and 17.5 days pregnancy. Data analyzed by JMP Pro 15, mean±SD reported, significance accepted at p<0.05. Results: Following CUS exposure, mice exhibited increased anxiety like behavior including less exploratory and more freezing behavior (p<0.02). CUS led to significantly more spontaneous pregnancy loss (p<0.02). Pre-pregnancy CUS led to significant thinning of the decidual layer in the placenta (p<0.01). Mice exposed to CUS before pregnancy had significantly elevated blood pressure in mid (121/86±12/13 vs. 137/101±8/7, p<0.01) and late pregnancy (126/93±9/14 vs. 138/106±12/12, p<0.03). Arteries from mice exposed to CUS before pregnancy exhibited significantly blunted endothelial-dependent and -independent relaxation (p<0.02) during pregnancy. Placental perfusion data from MRI is being analyzed. Conclusion: Maternal stress is an important factor contributing to adverse pregnancy outcomes, including pregnancy loss, development of the placenta, elevated blood pressure, and vascular dysfunction. We have found that a mouse model of CUS may be a valuable tool to investigate the mechanism(s) by which stress contributes to adverse vascular and pregnancy outcomes. Early Postpartum Mesenteric Artery Remodeling in CD8 Deficient Mice. Elizabeth A. Bonney, Rebecca I. Fairchild, Kirtika Prakash, Nga Ling Ko. University of Vermont, Burlington, VT, United States. Introduction: Abnormal pregnancy is associated with future cardiovascular diseases and may be related to dysregulated interaction between maternal immune and vascular systems. Activated CD8 T cells contribute to vascular dysfunction. However, pre-pregnancy adoptive transfer of CD8 T cells to mice genetically deficient in T cells enhances postpartum (PP) vascular structural remodeling while impairing endothelial cell function. We sought to determine the effect of CD8 deficiency on maternal resistance arterial structure and function in the early postpartum (2-wk PP). Methods: CD8 deficient adult females, which have a spleen T cell population that is over 90% CD4 cells, were left virgin or mated to same strain males and allowed to litter. Two weeks after delivery of a first litter, second order mesenteric arteries were isolated, cannulated and pressurized in an arteriograph at 37°C in physiologic salt solution (PSS). Passive lumen diameters and wall thickness were measured in response to stepwise elevation in intralumenal pressure in relaxing solution (PSS with the addition of diltiazem and papaverine) using a video dimension analyzer. To characterize vasoconstrictor function, we evaluated arterial diameter in response to an increasing concentrations of phenylephrine (PE, 0.1 -30 µM), a selective α1-adrenergic receptor activator. To test endothelial function, arteries were pre-constricted with PE to 50-60% of the initial diameter and given acetylcholine (ACh, 0.01 -10 µM) in a cumulative fashion. To assess the role of nitric oxide (NO) in the modulation of mesenteric vascular function, the responses of arteries to PE and ACh were evaluated after 20 minutes pretreatment and in the presence of L-NG-Nitroarginine (L-NNA), a nitric oxide synthase (NOS) inhibitor. Vessels from virgin, aged matched CD8 deficient females were used as controls. Values obtained in response to increasing perturbation were analyzed with repeated measure ANOVA. Other data was compared using the t-test and reported as mean±SEM. Results: As compared to the mesenteric arteries from virgin mice, 2-wk PP vessels were not more distensible. At the physiological pressure of 50mmHg, lumen diameter trended lower (PP, 57.5±2 vs 76.6±9mm, p=0.1) and wall thickness was significantly smaller (PP, 3.2±0.2 vs 4.6±0.5, p=0.007). The vasodilation in response to ACh of the PP vessels was less than that of the virgin vessels, but the response of both types of vessels was regulated by NO. While vasoconstriction in response to PE was less in PP versus virgin vessels, only the PP vessel response was significantly mediated by NO. There is growing evidence that these cardiovascular adaptations may be impaired with advancing age, however, little is known about maternal cardiac function with advanced maternal age. We hypothesized that cardiac adaptations to pregnancy are impaired with advanced maternal age. Methods: Pregnant young (4 months) and aged (9 months; ~35 years in humans) rats were studied on gestational day 19 (term=22 days) and compared to age-matched non-pregnant rats (n=8-10/group). Twodimensional echocardiographic images of anesthetized rats were obtained using ultrasound biomicroscopy (Vevo 2100, VisualSonics). Images of the left ventricle (LV) short-axis were used to assess structure and function. were recorded for all patients who attended their 6-week postpartum visit. Two-tailed t-test analyses were performed to compare the frequency of postpartum depression in patients who successfully completed vaginal birth after Cesarean section and those who required repeat Cesarean section. Results: Patients who underwent successful vaginal birth after Cesarean section and patients who failed a trial of labor after Cesarean section scored similarly on the Edinburgh postpartum depression scale at 6-weeks postpartum (Average EPDS = 4.9 vs 5.4 respectively, p-value = 0.383). Patients who failed a trial of labor were significantly more likely to be on antidepressant medication at the time of their 6-week postpartum visit when depression scores were evaluated (10.0% vs 4.3% respectively, p-value = 0.046). There was no correlation between gravidity, parity, and postpartum depression score. There was no significant difference in rates of postpartum depression between patients who delivered vaginally after Cesarean section and those who underwent repeat Cesarean section after a failed trial of labor. However, patients with unsuccessful trials of labor were more likely to be using antidepressant medication at their 6-week postpartum visit. All meals and snacks were provided, the diets were eucaloric, and fiber content did not differ. The fecal microbiome was sequenced using shotgun metagenomics from 20 CHOICE and 17 CONV mothers at 31and 36 wks. Mothers consumed a standardized breakfast at 31 and 36 wks where fasting and postprandial (hourly 5hrs) glucose, insulin, triglycerides, and FFA were measured; 5-hour area-under-the-curve (AUC) exposures were calculated. Microbiome taxonomy and protein functional annotation of the assembled reads were analyzed by diet and maternal blood measurements. Results: Maternal glucose was controlled to conventional targets while FFA AUC decreased on CHOICE. There were no differences in alpha or beta diversity of the microbiome between the two diets. Women on CHOICE had significantly higher levels of Bifidobacteriaceae after 6 wks compared to women on CONV. Bifidobacterium adolescentis was the main species driving the relationship between diet and bifidobacterial abundance. Functional gene annotation revealed the CHOICE diet altered 105 microbial gene abundances, while the CONV diet altered only 31 gene abundances from 31 to 36 wks. Within CHOICE, there was an increase in the abundance of microbial genes involved with glycolysis/ gluconeogenesis, whereas these genes had decreased abundances in women on the CONV diet. There was an overall positive relationship between insulin AUC and Bifidobacterial abundance at baseline, which was associated with higher insulin AUC in women on CHOICE at 36 wks. In summary, we found that a eucaloric diet higher in complex carbohydrates and lower in fat (CHOICE) maintained normoglycemia, increased insulin AUC, and suppressed postprandial FFAs, while also increasing the natural probiotic Bifidobacteriaceae. These results suggest CHOICE improved insulin action, which may assist in reducing FFA levels and help lower insulin resistance. Given the beneficial effects associated with Bifidobacteriaceae to gut health in both mothers and infants, a higher complex carbohydrate/lower fat diet may provide additional benefits to mothers with GDM. Characterizing (Fig 1) . Fetal immune cells (mT + /CD45 + ) migration were seen in maternal lungs, heart, kidneys, and brain with maximum number of cells in maternal lungs (Table 1) . A dose of 10 4 E. coli increased mT + macrophages and CD4+ T cells in maternal lungs and a decrease in neutrophils compared to uninfected mice. In maternal heart and kidneys, infection associated PTB was associated with a decrease in mT+ M2 macrophage and neutrophils, while maternal brain showed no fetal immune cells (Table 1 ). PCR data determined that cells of fetal origin are present in maternal organs. We report the migration of fetal immune cells to maternal organs is varies and tissue dependent in response to an ascending intrauterine E. coli infection followed by PTB. Plasminogen Introduction: Pregnancy results in altered hemostasis, characterized by increased clotting potential and decreased clot lysis. The substantial increase in PAI-1 and PAI-2 are frequently cited as the basis for decreased fi brinolysis in pregnancy. Using thromboelastometry to characterize clot lysis dynamics, we investigated correlations between PAI-1 and/or PAI-2 and clot lysis parameters longitudinally prior to conception and over the course of pregnancy. Methods: This is a retrospective sub-analysis of a prospective study, using citrated, frozen platelet-poor plasma samples (n=39) collected pre-conception (follicular phase), in early pregnancy (12-14wks), and in the 3rd trimester (28-34wks). In the presence of 5pM tissue Factor and 4nM tissue plasminogen activator, rotational thromboelastometry was used to analyze the Lysis Time (the time at which clot fi rmness is decreased to 10% of the maximum clot fi rmness, LT). PAI-1,2 were quantifi ed using commercially available immunochemical assays. Data are expressed as mean, range. Pearson correlations were used to investigate the relationship between PAI 1,2 levels and LT, as well as the relationship between longitudinal changes in PAI 1, 2 and LT from pre-conception to early and late pregnancy. The threshold for signifi cance was set at p<0.05. Results: Average PAI-1,2 levels were highest, and LT longest in late pregnancy, with marked individual variation (Table 1) . PAI-1 was associated with LT in V1 (r = 0.693, p <0.001) and V3 (r = 0.385, p<0.05), but not V2 (r= 0.057, p= 0.72). There was no correlation between PAI-2 and LT at any visit. Longitudinal changes (Δ) in PAI-1, 2 and LT across visits demonstrated a strong correlation between ΔPAI-1 and ΔLT from V1 to V2, which persisted to a lesser degree from V2-V3 and V1-V3. There was no correlation between ΔPAI-2 and ΔLT across any visits (Table 2) . Conclusion: Despite > 100-fold increase in PAI-2 levels in late pregnancy compared to pre-pregnancy, PAI-2 appears to have no association with the decreased fi brinolytic potential observed with this methodology. In contrast, changes in measured PAI-1 correlate with an observed impairment in fi brinolytic potential. The PAI-1 levels reported here are lower than in other previous publications and may therefore represent a fraction of in vivo activity. Taken together, these results suggest that PAI-2 levels do not contribute to decreased fi brinolysis in pregnancy, while PAI-1 levels may contribute signifi cantly. (sPTB), the principal cause of perinatal mortality and morbidity and the most common cause of death in children under 5 years old, usually results from premature rupture of fetal membrane. The primary source of matrix and collagen support for the fetal membrane is the mesenchymal cell layer. The elasticity, integrity and strength of fetal membranes is infl uenced by mesenchymal extracellular matrix and adhesion (ECM-ADH) proteins and plays an essential role in preventing in preterm premature rupture of fetal membranes. The presence of extracellular vesicles (EVs, exosomes) in maternal plasma has been detected as early as 6 weeks of gestational age and increased along with gestational age. During pregnancy, placenta-derived exosomes (pEXO) take participating in intercellular transferring and communication various mRNAs, ncRNAs, proteins, and lipids. Our early study has demonstrated diff erential expression of placental ECM-ADH in sPTB. We hypothesize that sPTB-associated ECM-ADH may be detected in maternal plasma and plasma exosomes. Methods: Total protein was isolated from maternal bloods (plasmas and pEXO), which were collected from the fi rst, second, and third trimesters of full-term birth and sPTB. ELISA-based assessment was performed to quantitatively measure sPTB-associated ECM-ADH proteins CADM1, CADM3, ICAM2, HLDPB1, CNTN2, CNTN1, FKHR, CD43, PKR, ISGF, c-FOS, GP130, and IL-33, with CD9 as internal control. We fi rst validated the previously demonstrated placental ECM-ADH proteins would be measured in maternal plasmas and pEXO. Our results showed that all 13 proteins may be detected in plasmas and in pEXO at diff erent gestational age of three time points. Four proteins, the ISGF, c-FOS, GP130, and IL-33, but not the other ECM-ADH proteins, were shown a signifi cant reduction in pEXO, as well as plasmas, in both normal and sPTB pregnancies. This reduction in pEXO can be detected during the entire pregnant period except that c-FOS in full-term and ISGF in sPTB were not signifi cantly reduced. We then analyzed each ECM-ADH proteins at the three individual timepoints between full-term and sPTB. There was no signifi cance can be detected for any ECM-ADH proteins in pEXO between full-term and sPTB. However, to our surprise, amount of CADM1, CADM3, HLDPB1, and CNTN2 at the fi rst trimester; as well as CADM1, CADM3, and CNTN2 at the third trimester; were signifi cantly decreased in sPTB (p<0.05). Conclusion: Analysis of CADM1, CADM3, HLDPB1, and CNTN2 with maternal bloods demonstrated that measurement ECM-ADH proteins in the early stage of pregnancy may help clinical prediction of sPTB. (Fig, Panel A) , while CLT was faster at V1 and V3 (Fig, Panel B) . Depleted preparations exhibit minimal changes between the ± PTCI conditions, while the Intact preparations show a significant decrease in lysis times with the addition of PTCI (Fig) , resulting in lysis times approaching those of Depleted preparations across the Visits. Conclusion: Our findings demonstrate an increase in clot lysis potential from late pregnancy to the first 24 hours after birth. Decreases in clot lysis times observed in the Depleted preparations suggest that centrifugation removed sedimentible material with the capacity to impact fibrinolysis. These findings potentially implicate MVs as a mediator of the wellcharacterized decrease in fibrinolysis observed in late pregnancy. Postpartum Gut Microbiome Profile in Healthy Birthers: A Narrative Systematic Review. Wasana L Weerasuriya †, 1,2 Lilla Kis †, 2 Julia Saunders †, 2 Adetola Louis-Jacques * . 1 Introduction: There is emerging data on the importance of the maternal gut microbiome in pregnancy. However, there has been a limited focus on the maternal gut microbiome in the postpartum (PP). This study aims to systematically review and provide a narrative synthesis of the literature on PP gut microbiome composition and diversity in healthy birthers. Methods: Electronic search was conducted in November 2020 and updated in July 2021 using PubMed, CINAHL, Scopus and Cochrane Library databases. Keywords entered were mothers, maternal, patient, microbiome, postpartum, pregnancy and prepartum. OpenGrey.eu was also used and hand selection of conference proceedings, journals and references of relevant reviews was completed. Studies included healthy PP birthers of mainly term infants. Results: PRISMA flow diagram (see Fig. 1 ) depicts eligible studies (N=21). Ten studies assessed the early PP (after birth -6 weeks), 8 studies assessed the late PP (6 weeks -12 months) and only 3 studies assessed early to late PP longitudinally. In the early PP, Firmicutes was the predominant phyla, and Bacteriodes was the predominant genus. Firmicutes continued to be the predominant phyla in the late PP. Of the studies assessing the PP longitudinally, there were no changes in alpha or beta diversity over time. Conclusion: There is limited research on the PP maternal gut microbiome profile. Most studies assessed its influence on the infant gut microbiome. However, there is a need to understand the PP maternal gut microbiome for the birther's sake. We need to establish how it differs from nonpregnancy and pregnancy states and determine influential biological and environmental factors. Clinical studies suggest that pregnancy-related anxiety (PrA), which includes the woman's worries specific to the unborn baby's health and the pain of delivery, may be phenotypically distinct from general anxiety in the prenatal period, and may have differential prediction of child health outcomes. The current study tests the follow-up hypothesis that these measures of anxiety may be physiologically distinct. We focus on cortisol, a widely studied physiological marker of stress that is associated with poor maternal, perinatal, and child health outcomes. The central aim of this study was to test the hypothesis that pregnancyrelated and general anxiety have distinguishable associations with diurnal cortisol in a large diverse sample of women on whom psychological and physiological data were collected in each trimester. Methods: Data were collected from N=326 women enrolled in a longitudinal pregnancy cohort study. At each trimester, PrA was assessed using the Pregnancy Related Anxiety Questionnaire; general anxiety was measured using the Penn State Worry Questionnaire. Diurnal salivary cortisol was collected at each trimester using the MacArthur protocol (wake-up, 45' after wake-up, 2.5hrs after wake-up, 8hrs after wake-up, 12hrs after wake-up). Data were analyzed using multilevel mixed effect models to assess the diurnal, non-linear patterns of cortisol. Covariates considered for analyses included race/ethnicity, education, income, maternal age, parity, SSRI use, smoking, pre-pregnancy BMI, and sleep problems. Results: Diurnal cortisol data were available on n=217 participants. Multilevel model analyses included cortisol estimates for early morning level, cortisol awakening response, diurnal decline, and non-linear diurnal decline. Analyses indicated that PrA was reliably associated with diurnal cortisol patterns: for example, elevated PrA about labor was associated with a lower initial value (-.17 [.08]) and a fl atter diurnal slope (.01 [.007]); similar eff ects were found for PrA related to the baby's health. In contrast, no signifi cant eff ect was obtained for general anxiety. The fi ndings suggest that anxiety specifi c to both labor and the baby's health are signifi cantly associated with diurnal cortisol patterns across pregnancy. A stronger link between cortisol and PrA than with general anxiety may explain diff erential prediction to perinatal and child outcomes. Further analyses will include additional biological markers of prenatal maternal biology that may diff erentiate these phenotypes. Pregnancies Introduction: Up to fi ve years after preeclampsia, women frequently report mental and cognitive complaints. However, the effect of preeclampsia on the functional connections in the brain remains unclear. Therefore, we aimed to explore whether diff erences in functional brain organization, measured with MRI, are present in formerly preeclamptic women compared to parous control women with normotensive pregnancies, especially in regions that may explain these generally reported mental and cognitive complaints even years after the problematic pregnancy. In 55 formerly preeclamptic women (aged 37 ± 6 years, postpartum time 6 ± 4 years) and 17 parous control women (aged 39 ± 6 years, postpartum time 8 ± 4 years), structural and functional 7 Tesla MRI (Siemens Healthineers, Erlangen, Germany) scans were acquired. Using graph theoretical analysis, the efficiency and clustering coefficient of the functional brain network were investigated. Analyses were focused to the global (whole) brain, as well as to particular brain structures, i.e. regions of the limbic system and prefrontal cortex. Multivariable linear regression was used to investigate the relation between brain networkrelated graph measures and pregnancy condition (e.g. preeclamptic or normotensive pregnancy). Results: Significant differences in local functional organization were observed between the women with and without a history of preeclampsia. While a higher local efficiency was found in the prefrontal cortex (+5.5%, p = 0.048) and anterior cingulate cortex (+6,9%, p = 0.03), we found a lower local efficiency and local clustering coefficient in the amygdala (-11,7%, p = 0.004 and -16.1%, p = 0.02 respectively) and parahippocampal cortex (-9.5%, p = 0.007 and -14.5%, p = 0.008, respectively) in formerly preeclamptic women. No differences were found in global brain organization. Conclusion: Formerly preeclamptic women displayed a different local functional organization years after the pregnancy compared to the control women. These differences, especially in the limbic regions and prefrontal cortex, are in line with the often expressed psychological and cognitive complaints remaining after a preeclamptic pregnancy. Future studies should further explore the relationship between the functional cerebral network organization, psychological and cognitive complaints and targeted treatment strategies. Adaptations to Pregnancy in Advanced Maternal Age. Mazhar Pasha †, Raven Kirschenman, Amy Wooldridge, Roberto Villalobos Labra, Floor Spaans, Christy-Lynn Cooke, Sandra T Davidge. University of Alberta, Edmonton, AB, Canada. Introduction: Advanced maternal age (≥35 years) increases the risk of pregnancy complications, which may be due to impaired vascular adaptations to pregnancy. Aging is associated with endothelial dysfunction, attributed to increased oxidative stress via NADPH oxidases (NOX) and/or endoplasmic reticulum (ER) stress-mediated pathways. However, whether these pathways impact vascular adaptations in aged pregnancies, is not known. We hypothesize that vascular adaptations to pregnancy are impaired at advanced maternal age, due to increased oxidative and ER stress. Methods: Pregnant young (4 months) and aged (9.5 months of age; ~35 year in humans) rats were studied on gestational day 20 (term=22 days) and compared to age-matched non-pregnant rats (n=6-10/group). Mesenteric arteries were isolated and endothelium-dependent relaxation to methacholine (MCh; 0.003 to 3 µM) was assessed in the presence/ absence of a NOX inhibitor (apocynin; 100 µM) by wire myography. Given the potential role of oxidative/ER stress in modulating endothelial function, mesenteric artery NOX4 expression and levels of ER stress markers (GRP78, P-eIF2α, CHOP, and sXBP1) were measured (Western blotting Introduction: Inflammation has been posited to be a major mechanism in the aetiology and progression of many gut disorders (such as Inflammatory Bowel Disease, Necrotizing Enterocolitis), especially during early development of newborns. Gut cell lines can be used as viable experimental models to gain important knowledge of cytokine production and its regulation in the gut; both are crucial components of inflammation. We have therefore initiated studies of their production and its regulation in gut cell lines. Methods: Caco-2 and HT-29 cells were acquired from Sigma-Aldrich (Merck) and cultured as per the manufacturer's instructions. Cells were seeded in 24-well plates (50 000 cells per well) for 24 hours and treated with Interleukin (IL)-1β or Tumor necrosis factor (TNF)α at various concentrations for 24 hours, after which the media were collected. IL-6, IL-8 and IL-10 production (pg/mL/well/24h) were quantified using ELISA. Results: Overall, we have found that the Caco-2 cell line was highly responsive to cytokine stimulation whereas the HT-29 line was less so. Both IL-1β and TNFα caused concentration-dependent stimulation of IL-6 production by Caco-2 cells. Caco-2 cells produced an average of 0.25 ± 0.06 pg/ml/well/24h IL-6 at baseline, which was increased to 11.2 ± 0.31 pg/ml/well/24h with 50 ng/ml IL-1β stimulation. TNFα-stimulated (50 ng/ ml) cells had a maximal IL-6 response of 2.9 ± 0.16 pg/ml/well/24h. HT-29 cells did not produce IL-6 either basally or following stimulation by IL-1β or TNFα. We next determined the production of the anti-inflammatory cytokine IL-10 by the two cell types. Neither Caco-2 cells nor HT-29 cells secreted IL-10 constitutively or after stimulation with IL-1β or TNFα. Caco-2 cells produced IL-8 at prodigious rates and both IL-1β and TNFα treatment enhanced those rates of production significantly. Caco-2 cells produced an average of 7.2 ± 0.30 pg/ml/well/24h IL-8 at baseline, which was increased to 300 ± 8.4 pg/ml/well/24h with 50 ng/ ml IL-1β stimulation. TNFα-stimulated (50 ng/ml) cells had a maximal IL-8 response of 63 ± 1.46 pg/ml/well/24h. Surprisingly, the HT-29 cells also produced greater amounts of IL-8 and secretion rates were raised significantly in response to IL-1β or TNFα treatment in a concentrationdependent manner. HT-29 cells produced an average of 283 ± 6.36 pg/ml/ well/24h IL-8 at baseline, which was augmented to 3816 ± 85.8 pg/ml/ well/24h with 50 ng/ml IL-1β stimulation. TNFα-stimulated cells (50 ng/ ml) had a maximal IL-8 response of 6375 ± 89.43 pg/ml/well/24h. Data are mean ± SEM, representative of n=3. Conclusion: These results are a basis for a model that can provide greater insights into inflammatory mediator production and its regulation by gut cells. Immune Profile of Peripartum Anxiety. Morgan L. Sherer, Kristin Voegtline †, Gayane Yenokyan †, Sabra L. Klein, Lauren M. Osborne. Johns Hopkins, Baltimore, MD, United States. Introduction: Immune dysregulation has been linked to both psychiatric illness and pregnancy morbidity, including perinatal depression, but little is known about the immune phenotype of perinatal anxiety. Here, we sought to identify the unique "cytokine fingerprint" of antenatal anxiety. Methods: Pregnant women (n=74) were followed prospectively at 2 nd and 3 rd trimesters (T2, T3) and 6 weeks postpartum. Each visit included a blood draw and psychological evaluation, with clinical anxiety defined as a score > 21 on the Perinatal Anxiety Screening Scale. We examined the association of anxiety symptoms with 32 plasma-derived cytokines and chemokines (measured via multiplex assay). We derived a combined measure of proinflammatory and compensatory cytokine activity (PRO) from cluster analysis at T2. PRO (combining IFNy, IL6, CXCL-8, IL10, TNFa) explained 39% of the variance in anxiety symptomatology. Women with chronic stable anxiety had lower levels of PRO compared to women without anxiety symptoms at T3 (p=0.04). The slope of increase in PRO from T2 to W6 was significantly greater for women whose anxiety increased from below to above the clinical cutoff during pregnancy, compared to both chronically healthy (p=0.03) and chronically anxious women (p=0.02). The innate immune response throughout the antenatal period differs based on anxiety symptom profile, suggestive of a unique immune phenotype of perinatal anxiety. Adrenomedullin Results: Exp. 1: Immunofluorescent staining shows that ADM and its receptor components CRLR, RAMPs are expressed in mouse pancreatic islets and co-localized with insulin. Expression of ADM, CRLR and RAMP2 in islets from pregnant mice are reduced compared to that of nonpregnant mice. Exp. 2: Mouse NIT-1-β cells express mRNA for ADM and its receptor components, and these expressions are increased by glucose in a dose-dependent manner. Furthermore, ADM dose-dependently inhibits NIT-1-β cell proliferation, and this inhibition is blocked by ADM antagonist, ADM22-52. ADM inhibits glucose-induced insulin synthesis and secretion by NIT-1-β cells, and these inhibitory effects are associated with upregulation of endoplasmic reticulum (ER) stress biomarker genes. Exp. 3: ADM inhibited glucose stimulated insulin secretion in isolated islets from day 13.5 pregnant mice. Conclusion: This study unveils that reduced ADM and its receptors during pregnancy may play a role in stimulating β-cell mass and insulin production, while increased circulating ADM levels in GDM patients may contribute to the defective β-cells adaptation, and blockade of ADM actions with its antagonists may improve β-cell functions. exosomes are known to play an important role in immune function, act in intercellular communication, and have recently been explored as drug delivery vehicles. Gut cell lines can be used as viable experimental models to gain insight into cytokine production and it's regulation by proinflammatory cytokines (crucial components of inflammatory responses) and milk exosomes in the gut. Herein, we explore the role of exosomes sourced directly from cow's milk and from infant formula for their potential effects on intestinal inflammatory responses. Methods: Caco-2 and HT-29 cells were acquired from Sigma-Aldrich (Merck). Cells were seeded in 24-well plates (50 000 cells per well) for 24 hours and treated with IL-1β (1, 10 and 50 ng/ml) or TNFα (1, 10 and 50 ng/ml) for 24 hours, both with and without various exosomes (10 7 ,10 8 and 10 9 per ml) isolated from cow's milk or infant formula (Enfamil, Mead Johnson and A2, The A2 Milk Company). Exosome isolation was conducted using our published methods. IL-6 and IL-8 productions (pg/ mL/well/per 24 hours) were determined using measurements by ELISA. Results: Overall, we have found that neither the Caco-2 nor HT-29 cells basal production of IL-6 and IL-8 was altered significantly by cotreatment with any concentration of exosomes used. In the presence of stimulation by IL-1β and TNFα the production of IL-6 and IL-8 was again not significantly different from controls (see Figure) . There was no significant trend on cytokine production rates by cotreatment with increasing concentrations of exosomes from any source. Our results show that short term cotreatment of exosomes derived from cow's milk or infant formula does not alter cytokine production by gut cells. Legend: Interleukin (IL)-6 (A) and IL-8 (B) production (pg/ml per well 24h) from Caco2 cells stimulated with IL-1β. Within each stimulation group cells were treated with varying concentrations of stimuli (1, 10 or 50 ng/ml). As indicated in the graphs, stimulation was coupled with simultaneous exposure to cow's milk (GM) exosomes. Results are representative of n = 3. Introduction: Affective disorders, including mood and anxiety disorders, are associated with adverse pregnancy outcomes. The incidence of any mood or anxiety disorder during pregnancy is surprisingly high at approximately 18% overall. Uric acid is an inflammatory biomarker, and prior research has found impaired hippocampal response to psychosocial stress associated with elevated uric acid. The focus of this study was to quantify the prevalence of mood and anxiety disorders in early pregnancy, and to investigate the relationship between affective disorders and maternal uric acid levels in mid-pregnancy and pregnancy outcomes. Methods: Validated measures of pessimism, anxiety and depression were collected from 1349 women in early pregnancy (average 15.9±5.0 weeks). Uric acid levels in mid-pregnancy (average 20.3±2.0 weeks) were measured on a subset of women (n=572). Race and smoking status were self-reported at enrollment. Blood pressure and body mass index were measured by trained research nurses. Pregnancy outcomes were abstracted from medical records. Data analyzed by JMP Pro 15, mean+/-SD reported, significance accepted at p<0.05. The incidence of affective disorders was notably high in early pregnancy: depression= 12%, anxiety= 26%, and pessimism= 23%. Measures of optimism and lower anxiety and depression in early pregnancy were associated with significantly lower levels of maternal uric acid in mid pregnancy; and this relationship was also associated with maternal race (p<0.005). In addition, higher uric acid levels in mid-pregnancy were associated with earlier gestational age at delivery (38.5±3.3 vs. 39.2±2.3, p<0.05), and elevated maternal blood pressure in mid-pregnancy (p<0.01) and at labor & delivery (p<0.01). Conclusion: Measures of pessimism, anxiety and depression are notably high in early pregnancy. Lower uric acid levels are associated with being more optimistic, and lower anxiety and depression. Affective disorders and elevated uric acid are associated with earlier gestational age at delivery and elevated maternal blood pressure. Proteobacteria has been studied to be pro-inflammatory and play a role in gut dysbiosis in adults but also plays a large role in priming the infant immune system and decreasing their risk of obtaining various neonatal illnesses. Limitations of this study include non-standardized milk collection and storage. Further studies are needed to understand how maternal SDH can influence the gut health of neonates. Leukocyte Introduction: Short leukocyte telomere length (LTL) is a marker of cellular aging in non-pregnant adults, but little is known about its predictive utility in pregnancy. To address this gap in our knowledge, we evaluated associations between first trimester LTL and adverse perinatal outcomes. Methods: This was a prospective cohort study of pregnant people who presented for care at a single academic institution between January and October 2020. Nulliparous patients 18 to 50 with a viable pregnancy between 10w0d and 14w0d were eligible. Blood samples were collected on the day of enrollment. LTL was measured using quantitative PCR and reported as a telomere ("T") to single copy gene (human beta globin, "S") ratio, then converted to basepairs (bp) using a standard calculation. In the primary analysis, the exposure was first trimester LTL and the outcomes were perinatal complications such as hypertensive disorders of pregnancy and preterm birth that were abstracted from medical records. Secondary outcomes included responses to Patient Health Questionnaire-9 (PHQ-9) depression surveys. Given the normal distribution of LTL, we used t-tests to compare LTL between people with and without each categorical outcome, and computed Pearson correlations between LTL and gestational age at delivery. Results: 46 pregnant people were included. Mean ± SD maternal age at enrollment was 31.2 ± 3.4 years. Shorter first-trimester LTL was significantly correlated with earlier gestational age at delivery (coefficient 0.33, p=0.03, Figure 1 ). Although limited by small numbers, we found non-significant differences for shorter first-trimester LTL in people who developed preeclampsia (5511.4 ± 429.7 versus 5756.0 ± 249.7 basepairs, p=0.28) and spontaneous preterm birth (5488.8 ±262.9 versus 5745.3 ± 278.2 basepairs, p=0.23, Table 1 ). We identified significant correlations between shorter firsttrimester LTL and earlier gestational age at delivery. Our data also suggest possible associations between LTL and preeclampsia and spontaneous preterm birth that warrant further investigation in larger cohorts. Psychological Well-Being After Preeclampsia. Robert-Jan Alers †, 1 Introduction: Formerly preeclamptic women express reduced psychological well-being in the fi rst years after childbirth. However, the long-term prognosis is unclear. We assessed perceived psychopathology in a large cohort of women with a preeclamptic history and parous women without hypertensive gestational disorders. Methods: 980 formerly preeclamptic women (aged 39±8 years) and 502 normotensive gestation parous controls (aged 44±8 years) were included. Participants gave birth between 0.5 and 30 years ago. Symptomatic psychological distress was assessed using the Symptom Checklist-90-Revised. Higher scores refl ect more perceived psychological diffi culties. Obstetric history, educational attainment, height, and weight were recorded. A multivariable logistic regression model was run to examine possible relationships between psychological distress and a history of preeclampsia. The absolute and relative risks of a preeclamptic history on elevated scoring were calculated over postpartum time. Women with a preeclamptic history reported significantly more psychopathology compared to controls (fi gure). Although the impact on experienced mental well-being of formerly preeclamptic women lessened over time, a signifi cant eff ect lasted up to at least 18 years postpartum. In controls, psychological well-being was independent of postpartum time up to 30 years after childbirth. No association between reported psychopathology and early onset of preeclampsia, concurrent HELLP syndrome, preterm delivery, or perinatal mortality was observed. Formerly preeclamptic women with a high education were less likely to report psychological problems, while obesity negatively aff ected psychological well-being. Conclusion: Formerly preeclamptic women report impaired mental well-being for many years after the complicated pregnancy regardless of obstetric severity. A reduced psychological well-being may infl uence labor participation, eff ective social functioning, and overall quality of life. Potential underlying psychological or physical disorders remain to be identifi ed. The Impact of the Introduction: During pregnancy there is a high demand for micronutrients and B-vitamins to support one-carbon metabolism (1-CM), required for development of maternal and fetal tissues. A nutritional diet is important in the acquisition of these elements and for maintenance of a balanced maternal gut microbiome. Obesity is associated with a relative malnutrition, due to a low-nutrient diet. These defi ciencies derange 1-CM and gut dysbiosis, and are associated with adverse pregnancy outcomes. The microbiome and 1-CM interact with one another, but their interactions in obesity during pregnancy are poorly understood. This study investigates the impact of maternal obesity in pregnancy on the microbiome, 1-CM and off spring outcomes. Methods: A comprehensive literature search was conducted up to January 2021 resulting in 2,793 articles. Articles were included (n=70) if they met the selection criteria. Maternal obesity and 1-CM included 34 articles, maternal obesity and microbiome included 29 articles and maternal obesity and off spring outcomes included 7 articles. Results: 1-CM derangements are positively associated with obesity and increasing BMI, as well as metabolic disorders, including insulin resistance and glucose intolerance. During pregnancy, the microbiome seems to adapt in order to support the superior metabolic and immunesystem demands of pregnancy, resulting in a maternal microbiome with higher ratios of vitamin-producing phyla including Bacteriodetes. However, maternal-obesity microbiota show a trend of low ratios Bacteriodetes and dominance of Firmicutes, which contribute poorly to producing vitamins and supporting the demands in pregnancy. Maternal gut dysbiosis, due to obesity, seems to perturb 1-CM homeostasis and predispose to greater risks of obesity, hyperglycemia and dyslipidemia. Conclusion: Gut dysbiosis and derangements in 1-CM occur more often in pregnant obese compared to non-obese women. A better understanding of these interactions can be helpful to prevent adverse pregnancy complications and improve maternal and offspring health in the future by following the recommendations of weight reduction, diet adjustments, and lifestyle coaching. Especially the use of pre-and probiotics needs further investigation. Histone Lysine Demethylase KDM1A is a Regulator of Gene Expression and Mitochondrial Respiration Necessary for Placental Cell Proliferation. Jerry Bouma * . Colorado State University, Fort Collins, CO, United States. Introduction: Abnormal placental development and function lead to pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, with serious adverse health consequences for mom, the baby and future offspring. Placental development requires extensive placental (trophoblast) cell proliferation, migration, and differentiation into hormone-secreting cells, and in human placentation requires invasion and remodeling of uterine spiral arteries. The overall goal of our studies is to determine the role of histone lysine demethylase KDM1A in trophoblast cell proliferation and placentation. We hypothesize that trophoblast cell proliferation requires KDM1A regulation of mitochondrial respiration and genetic networks involving proliferation. To test this, we utilized in vitro approaches and an in vivo sheep model to study the effects of CRISPR-Cas9 mediated deletion of KDM1A in trophoblast cells. Methods: A CRISPR-Cas9 lentiviral mediated KDM1A knockout human trophoblast cell line was generated, and used to examine the effect of abolishing KDM1A on mRNA and protein levels of pluripotency and mRNA binding factor LIN28, nonhistone architectural transcription factor HMGA2, proto-oncogene cMYC, and angiogenic factor VEGFA. Furthermore, downstream changes in expression of let7 miRNAs were assessed. Regulation of mitochondrial function was examined by measuring mitochondrial respiration and ROS production in KDM1A knockout human trophoblast cells. Finally, using a placental-specific gene targeting approach we knocked-out KDM1A in the trophectoderm of hatched blastocysts and following embryo transfers examined the effect on trophoblast proliferation in pre-implantation sheep embryos. Results: Real-time PCR and western blot analysis revealed significant (P < 0.05) lower amounts of LIN28, HMGA2, cMYC and VEGFA in KDM1A knockout trophoblast cells. As expected, since LIN28 inhibits let7 miRNA, let7 levels were significantly (P < 0.05) increased. KDM1A knockout increases mitochondrial respiration and ROS production. Finally, knockout of KDM1A specifically in the trophectoderm leads to impaired trophoblast cell proliferation in vivo. Conclusion: Quantitative analysis suggests KDM1A is a key regulator of genetic networks controlling trophoblast cell proliferation. In addition, preliminary analysis indicate KDM1A regulates mitochondrial respiration in trophoblast cells, and is necessary for trophoblast proliferation in vivo. Introduction: During pregnancy, fetal trophoblasts detach from the placental anchoring villi and invade the maternal decidua. Failures in this process could be the underlying cause for various obstetrical syndromes, such as preeclampsia, fetal growth restriction, and preterm labor. However, key pathways driving extravillous trophoblast (EVT) differentiation, and particularly formation of decidual interstitial EVTs (iEVTs), remain largely elusive. Therefore, we aimed to describe transcriptional changes occurring between EVTs, found in placental anchoring villi, and EVTs that have deeply invaded into the decidua. Furthermore, trophoblast organoids were established from cytotrophoblast (CTB) progenitors of the same donors and differentiated into EVTs to investigate differences between in vivo and in vitro EVT populations. Methods: Donor-matched (n=3) placental EVTs (pEVTs) and iEVTs from decidua basalis were isolated from first trimester tissues and purified for HLA-G cell surface expression. Additionally, donor-matched placental CTB progenitors were used to establish trophoblast organoids. The latter were differentiated into EVTs. Purity was assessed by flow cytometry, using markers for decidual stromal cells, epithelial cells, and immune cell populations. Subsequently, bulk RNA sequencing (RNAseq) and comparative bioinformatical analyses were performed. Gene expression was further investigated using Immunofluorescence, Western Blotting, and qPCR. Results: While principal component analyses showed clear clustering of the trophoblast populations, iEVTs seem to be the most distinct population among all groups. A curated trophoblast-specific list of transcripts was created for gene set variation analysis, which indicated higher scores in iEVTs compared to pEVTs for secreted proteins. The iEVT-specific secretome showed increased mRNA and protein levels of pregnancyassociated factors, such as PAPPA2, AOC1, FN1 and, PRG2. Our results indicate that EVTs develop a distinct secretory phenotype upon invasion into the decidual tissue. This phenotype likely develops after detachment from the placental cell column and presumably peaks after trophoblasts have invaded into the uterine tissue. Many trophoblasts at that stage are in contact with maternal vessels, leading to the conclusion that upregulation of secreted proteins could have the purpose of preparing the maternal system for pregnancy-associated changes. A thorough analysis of the dynamics of these secreted proteins over the course of gestation could provide predictive markers for syndromes associated with defects in placentation in the future. derived lipids which may be protective in metabolic conditions. However, mechanistic studies characterizing the role of SPM in pregnancies complicated by obesity are lacking. We examined associations of placental SPM with pre-pregnancy body mass index (ppBMI), and ascertained their role in placental efficiency and fetal adiposity. We hypothesized that placenta from obese (BMI > 29.9 kg/m2) vs. lean (BMI 18.5-24.9 kg/m2) women will show reduced concentrations of SPM, which in turn will be associated with reduced placental efficiency, and increased neonatal adiposity. Methods: We assessed PUFA, prostaglandins (PGF2a), SPM and SPM precursor concentrations using UHPLC-ESI-MS in n=60 term c-section placentae. We assessed placental efficiency with the fetal/placental weight ratio, and body composition with air displacement plethysmography. We used Wilcoxon rank-sum for bivariate analyses, Spearman for correlations, and linear regression stratified by child sex and adjusted for maternal age, gestational weight gain, and gestational age to estimate associations of placental SPM and PGF2a with neonatal adiposity. Results: Median (IQR) maternal BMI was 27.9 (22.9, 38.6) kg/m 2 . Obese (vs. lean) placenta had higher concentrations of n-6 AA and n-3 DPA and DHA, though the downstream SPMs and their P450-, 12-LOX-and 15-LOX-enzyme derived precursors were significantly lower (Figure) . Placental SPMs positively correlated with placental efficiency in female (RvD1: r s = 0.50, p = 0.03; PD1: r s = 0.53, p = 0.01) and not male placenta. The ratio of placental pro-inflammatory PGF2a to anti-inflammatory SPM precursors was significantly associated with adiposity in females (PGF2a/17-HDoHE: β 1.5% (95% CI: 0.03, 3.04); PFG2a/14-HDoHE: 14.6% (95% CI: 4.6, 24.6) but not males. SPM and PGF2a/SPM ratios were not directly associated with adiposity. Conclusion: For the first time, we show that maternal obesity is associated with attenuated placental SPM, which may reflect inefficient SPM precursor synthesis, contributing to impaired placental efficiency and alterations in fetal adiposity accrual in a sex-dependent manner. The multinucleated syncytiotrophoblast (SynT), formed by fusion of proliferative cytotrophoblasts (CytT), serves a critical role in pregnancy maintenance by synthesis of steroid hormones and immune modulators, which protect the hemi-allogeneic fetus from maternal immune rejection. The placenta, has similarities to a tumor; it can grow under nutrient-restricted conditions, has reduced capacity to generate acetyl-CoA from citrate, and must protect itself from immune rejection. Thus, how do trophoblasts generate sufficient amounts of acetyl-CoA to fuel the TCA cycle, serve as a building block for synthesis of fatty acids and steroids and for permissive changes in chromatin structure? Our findings in cultured trophoblasts, below, have led to the novel hypothesis that, as in tumors, the nucleocytosolic enzyme, acetyl-CoA synthetase 2 (ACSS2), is critical for production of acetyl-CoA for SynT differentiation and function. Methods: Targeted metabolomics was used to assess temporal changes in levels of ~438 metabolites in human trophoblast stem cells (hTSCs; from Drs. Okae and Arima, Japan) cultured under conditions to promote CytT to SynT differentiation for up to 5 days. Metabolomics data were normalized by total ion content; statistical analyses were assessed by MetaboAnalyst. RT-qPCR was performed to analyze mRNA expression of enzymes responsible for synthesis of key metabolites. ChIP-seq and ChIP-qPCR were used to analyze histone acetylation of key genes. Results: Metabolomics data indicated that acetyl-CoA, which increased dramatically upon differentiation of hTSCs to SynT, was the 5 th most upregulated metabolite during SynT differentiation. This was accompanied by a 15-fold increase in ACSS2, which catalyzes acetyl-CoA production from acetate. Importantly, the NRF2/ACSS2 axis has been reported to contribute to metabolic reprogramming in cancer. Because we recently discovered that the redox-sensitive transcription factor NRF2 is a key mediator of the induction of the immune modulators, HMOX1, AhR, PD-L1, and GDF15, during SynT differentiation by direct binding to the gene promoters, we performed ChIP-seq to search for changes in histone acetylation of these immune modulators. Intriguingly, we observed that the active histone marks, H3K9Ac and H3K27Ac, were increased near the transcription start site of HMOX1 and GDF15. Conclusion: Our findings suggest that the marked increase in acetyl-CoA during SynT differentiation is mediated at least in part by the action of ACSS2. Acetyl-CoA serves as a key donor for generation of active histone marks at promoters of immune modulator genes. Thus, acetyl-CoA may serve a critical role in protection of the fetus from rejection by the maternal immune system. High-quality diffusion MRI and co-registered to conduct an automatic classification into three major placental compartments: intervillous space, placental tissue, and placental vessel. Diffusion basis spectrum imaging (DBSI) was applied to characterize the PV microstructure in intervillous space. Specifically, hindered water ratio, and free water ratio are derived to reflect PV's local membrane surface area. Results: T2W images are used to locate the placenta (Fig. 1a) . DBSI hindered water ratio (Fig. 1b) and free water ratio (Fig. 1c) can be computed reliably. The intervillous space, placental tissue, and the placental vessel can be accurately separated (Fig.1d) . Average DBSI hindered water ratio in the intervillous space increased over pregnancy ( Fig. 1g) , and average DBSI free water ratio in the intervillous space shown a significant decrease over pregnancy ( Fig. 1h) . The significantly increased DBSI hindered water ratio and decreased DBSI free water ratio suggested that more and more water molecules experienced the constrained diffusion (random walk), which reflected the increase of PV membrane surface area and the PV maturation during human pregnancy. In comparison, The average hindered water ratio and free water ratio in the entire placenta of the comparison group (17 patients) didn't show significant change over gestational age (Fig. 1e&f) . Conclusion: DBSI hindered water ratio and free water ratio in the intervillous space is promising imaging biomarkers capable of reflecting the placental villi maturation processing in human pregnancy specifically. Future studies aiming to detect the impaired and/or delayed placental villi maturation hold great promise to provide noninvasive biomarkers for early diagnosis. Vidya Dangudubiyyam †, Mishra S Jay †, Ruolin Song †, Sathish Kumar * . Introduction: Placental production of a range of hormones and cytokine factors play a multifaceted role in feto-placental development. The hemochorial type of placentation in rodents and humans renders them vulnerable to circulating endocrine-disrupting chemicals, such as perfl uooroctane sulfonic acid (PFOS). PFOS is positively associated with gestational hypertension, preeclampsia, and fetal growth restriction; however, the eff ect of PFOS on placental steroidogenesis is not known. In this study, we examined whether PFOS induces sex-specifi c changes in the expression of steroidogenic enzymes in the junctional zone (the major site of endocrine cross-talk) of the rat placenta. Methods: Pregnant Sprague-Dawley rats were administered with PFOS (50 μg/mL; n=7) through drinking water from gestational day (GD) 4 until term (GD20). Controls (n=7) received standard deionized waters with no detectable PFOS. On GD 20, pregnant dams were sacrifi ced. Placenta was isolated and sexed. The junctional zone was processed to assess mRNA expression of enzymes involved in steroid hormone biosynthesis. Plasma was collected to measure the levels of testosterone and estradiol using ELISA. Results: Placental weights (control: 0.54 ± 0.01 g; PFOS-exposed: 0.49 ± 0.01 g) were signifi cantly decreased in PFOS dams compared to controls; however, the junctional zone weight was not diff erent between the PFOS and control dams (control: 0.181± 0.01 g; PFOS-treated: 0.180 ± 0.01 g). In the junctional zone of male placentas, there was a signifi cant increase mRNA expression of Cyp11A1, 3β-HSD, Cyp17A1 in PFOS dams compared to controls. In the junctional zone of female placentas, there was a signifi cant increase in stAR, 17β-HSD3 in PFOS dams compared to controls. The mRNA expression of 17β-HSD2 was not altered in both male and female junctional zones. Plasma level of testosterone was signifi cantly increased, while estradiol was signifi cantly decreased in PFOS dams compared to controls. Conclusion: Our study demonstrates for the fi rst time that PFOS induced perturbations in placental steroidogenesis could contribute to adverse maternal and fetal outcomes. A better understanding of PFOS impacts on the placenta may provide critical information for assessing and ultimately controlling risks to pregnant women from PFOS exposure. Results: All but four cytokines (GM-CSF, IL-3, IL-4, MIP-2) were of signifi cance in relation to the increased risk of congenital ZIKV infection in the absence of bacteria (Fig. 1 ). Most of the tested cytokines including IL-1α, IL-2, and IL-13 showed signifi cantly increased concentrations in GF mock infected mice (p<0.0001), highlighting the profound impact of maternal microbiota on fetal immune responses in general. Interestingly, postnatal "rescue" of bacterial recolonization did not restore natural microbial anti-viral immune resistance (e.g., IL-13, p=0.0084) (Fig. 2) . Conclusion: Overall, our fi ndings suggest that developmental exposure to microbes is essential to later in life anti-viral immunity in pregnancy. cell-derived matrices (CDM), we previously showed impaired expression and topography of certain ECM components in FGRadv. Furthermore, these matrix diff erences resulted in diff erential eff ects on endothelial cell polarity and migratory properties. We hypothesized that there is aberrant placental ECM composition in FGRadv, which contributes to impaired angiogenesis in these placentas. We recruited subjects from three groups (N=6/group): (1) FGRadv, (2) Gestational age-matched, appropriately grown preterm controls (PTC), and (3) Uncomplicated, term controls (TC). Within one hour after delivery, 1 cm 3 of villous tissue was fl ash-frozen and stored at -80 o C. Samples were lyophilized and milled before 5 mg (dry wt.) of each sample was subjected to a 3-step, ECM-optimized extraction involving decellularization followed by ECM extraction via chaotrope and hydroxylamine hydrochloride chemical digestion. Samples were digested with trypsin and analyzed in 2 fractions using an Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoFisher). Data analysis was performed using MSFragger (version 3.3) and results were fi ltered to 1% FDR. Oneway ANOVA with multiple comparisons, where appropriate, was used for statistical comparison between groups. Results: Partial least squares discriminant analysis (PLS-DA) of the ECM fraction demonstrated signifi cant diff erences between FGRadv as compared to PTC and TC villous tissue. In contrast, there was some overlap between PTC and TC specimens, suggesting that gestational age has less of an eff ect on matrix composition than placental phenotype. A substantial proportion of proteins that were highly diff erentially expressed in FGRadv as compared to either TC or PTC but that did not signifi cantly differ between TC and PTC belonged to the basement membrane, including nidogen-1 (p=0.006), laminin α2 (p=0.0003), and laminin β1 (p=0.005), all of which were down-regulated in FGRadv tissue. Conclusion: Signifi cant diff erences in placental villous ECM composition exist between FGRadv when compared to either PTC or TC tissue, including down-regulation of key basement membrane proteins in FGRadv placentas. This may be one mechanism by which aberrant stromal matrix in FGRadv inhibits fetoplacental angiogenesis. Assessing Results: Basal uterine and umbilical glucose uptakes were suppressed (P ≤ 0.05) along with umbilical insulin concentrations in CSH RNAi pregnancies. Increasing the maternal-fetal glucose gradient via maternal hyperglycemic clamp normalized uterine glucose uptakes between treatments. Despite elevated maternal glucose concentrations, umbilical glucose and insulin concentrations in CSH RNAi fetuses tended (P ≤ 0.10) to remain lower, as did uterine and umbilical blood flows (P ≤ 0.10). The slope of the relationship between umbilical glucose uptake and the maternal:fetal arterial glucose gradient (CON RNAi, Y=29.54X +74.15; CSH RNAi, Y=19.05X + 52.40) was not statistically different (P = 0.34), but the y-intercepts and elevation were (P = 0.003), indicating reduced maximal glucose transport during maternal hyperglycemia. This may be explained in part by elevated (P ≤ 0.05) relative uteroplacental oxygen utilization (mmol/min/kg placenta) during the maternal hyperglycemic clamp, suggesting greater placental oxidation of substrates by the CSH RNAi placenta. Conclusion: Metabolic analyses suggest that perturbations in glucose transfer appear to be a combination of mechanistic defects along with reduced placental mass. Together, these data suggested that CSH likely plays a key role in modulating placental metabolism that ultimately promotes maximal placental glucose transfer. Supported by NIH HD093701. Introduction: COVID-19, caused by SARS-CoV-2, is linked to a higher rate of pregnancy complications, including premature birth and preeclampsia. Our work demonstrated that the increased incidence of preeclampsia in pregnant women infected with SARS-CoV-2 is related to dysregulation of the Renin Angiotensin system (RAS). SARS-CoV-2 pathogenesis is dependent on binding to the angiotensin-converting enzyme 2 (ACE2), a key enzyme in the RAS cascade. Autophagic activity is a crucial component of the placental syncytiotrophoblast barrier function and plays a key role in the vertical transmission of pathogens such as Zika virus. The SARS-CoV-2 genome encodes non-structural proteins (NSPs) shown to modulate autophagy in HeLa cells. Tissue-specific mechanistic links between the RAS and autophagy are not known. We tested the hypothesis that SARS-CoV-2 NSPs modulate the RAS via autophagy, leading to an increase in the proinflammatory arm of the RAS cascade in trophoblast cells. We probed SARS-CoV-2+ placental tissues for modulation of autophagic activity by immunofluorescence monitoring of autophagy flux markers LC3B and p62. The trophoblast cell line JEG-3 was transfected with expression plasmids encoding SARS-CoV-2 NSPs (NSP6, ORF3a, ORF3b, NSP8), critical elements of viral replication and transcription complex. The transfected cells were analyzed for autophagy flux markers and RAS cascade components (AT1R, AT2R, and downstream targets sFLt1 and VEGF) by western blots and immunofluorescence. We correlated expression levels of autophagy and RAS proteins with the area and number of lipid droplets. Lipid droplet biogenesis pathway genes were analyzed in JEG-3 cells transfected with SARS-CoV-2 geneencoding plasmids. Results: We found that SARS-CoV-2 activates the autophagy pathway via its ORF3a protein, but also inhibits autophagosome degradation. We observed significant lipid droplet accumulation in SARS-CoV-2+ placental tissues and JEG3 cells. We also found that viral NSP6 upregulates lipid droplet biogenesis, but ORF3b and NSP8 are not implicated in modulating these pathways. We observed that increased expression of AT1R and sFlt1, the pro-inflammatory arm of RAS cascade, is associated with increased autophagy but decreased autophagosomal degradation. In summary, we found that SARS-CoV-2 binding to ACE2 in placental trophoblasts leads to ACE2 degradation and dysregulation of the RAS cascade, and also promotes viral replication via activation of autophagy and lipid biogenesis mechanisms driven by the viral ORF3a and NSP proteins. Our findings also suggest that SARS-CoV-2 blocks autophagosome/lysosome fusion via ORF3a. Ongoing efforts are underway to further elucidate the specific role of SARS-CoV-2 NSPs and their related mechanisms in the placenta, and these may also provide attractive drug targets for intervention. ) and Ezh2(f/f):Cre-(flox-control) placentas (n = 8 per group) were generated and studied at dpc 17.5. RNA-seq revealed sex-specific effects of Ezh2 deletion in trophoblasts with a more significant shift in gene expression in female placentas (214 differentially regulated genes, ±2-fold, FDR p<0.05) compared to male (134 genes). GO analysis of the 117 genes uniquely increased in females with Ezh2 loss, revealed enrichment of immune response related pathways particularly systematic upregulation of pro-inflammatory chemokines/cytokines and complement genes. However, in male placenta, genes upregulated after Ezh2 loss were enriched for developmental pathways involving organ morphogenesis and skeletal development. Our studies point to a diverse set of mechanisms contributing to sex differences in placental gene expression which may contribute to offspring sex differences. Collectively, while these findings suggest that sex chromosomes and Ezh2 play critical roles in placental sex differences the precise mechanisms linking the two requires further study. We recently found that ferroptosis, a unique form of programmed cell death causes human trophoblast injury and related pregnancy complications. Modulatory ligand profiling has the potential to uncover new inhibitors of ferroptotic death mechanism. Whereas a single specific modulator marks the potential involvement of single molecule/protein, a cluster of modulators may reveal the involvement of cellular cascades/pathways. We hypothesized that coupling a robust screening approach with large-scale bioactive compound profiling, will yield a compendium of cluster regulators that define driving forces and regulators that underlie trophoblast sensitivity to ferroptosis. Methods: BeWo cells were seeded in 384-well culture plates employing Combi MultiDrop. After an overnight culture, RSL-3 (200 uM), a ferroptosis-specific inducer, was added along with 6520 individual bioactive compounds (1 uM each) from 3 different libraries, using a highthroughput platform. Vehicle (DMSO) or the specific ferroptosis inhibitor Ferrostatin-1 (0.5 uM) were used as controls. A luminescent cell viability assay was performed using CellTiter Glo. Results: Our screen revealed a large compendium of ferroptotic inhibitors (n=287). These compounds included many known ferroptosis inhibitors (serving to validate our approach), and dozens (n=72) of novel ferroptosis suppressors, including numerous compounds that appear to act via off-target iron-chelating activity or as antioxidants. Clustering analysis demonstrated that inhibitors of 5-LOX and FLP3, proteins that promote lipid peroxidation, inhibit ferroptosis. Inhibition of VEGF and EGF receptors and downstream cascades also inhibited ferroptosis, suggesting that these cascades play a key role in trophoblast sensitivity to ferroptosis. Finally, we confirm that esomeprazole and promethazine act as potent drugs that can be safely utilized to inhibit ferroptotic injury during pregnancy. Conclusion: Our study provides novel insights into the cellular mechanisms that may regulate trophoblast ferroptosis. Our approach suggests potential candidates for drug repurposing, aimed at inhibiting ferroptotic injury in human pregnancy. Effect The placenta anchors the fetus to maternal tissue and provides an interface for nutrient, gas, and waste exchange. Trophoblast stem cells differentiate into various lineages essential for executing the functions of the placenta. Trophoblast cell differentiation is orchestrated by the coordinated regulation of gene networks. A deeper understanding of the molecular basis underlying development of trophoblast cell lineages is a key to understanding the etiology of pregnancy-associated diseases. A suitable animal model can facilitate the discovery regulatory pathways controlling development of the uterine-placental interface. Unlike mouse, the rat exhibits deep intrauterine trophoblast cell invasion, a feature shared with human placentation. We have identified gene targets that play important roles in placental development. Leukemia inhibitory factor receptor (LIFR) and alkB homolog 1, histone H2A dioxygenase (ALKBH1) have been implicated in regulating placentation using mouse mutagenesis. In this study, we investigated the role of LIFR and ALKBH1 in the development of the trophoblast cell lineage in vivo using the rat. in placental development we generated in vivo rat models using CRISPR-Cas9 gene editing. Wild type and mutant placentation sites were examined histologically and through molecular and biochemical analyses. Results: Deletions were produced within LIFR and ALKBH1 loci yielding new null rat models. LIFR disruption resulted in placental and fetal growth restriction during midgestation and embryonic lethality in late gestation with no live pups at birth. On the other hand, ALKBH1 deficiency resulted in significant placental and fetal growth restriction beginning at midgestation but was compatible with postnatal survival. Histological assessment of both mutant models revealed placentas with structural abnormalities. LIFR null placentas possess an expanded junctional zone and a growth restricted labyrinth zone, whereas ALKBH1 null placentas exhibited growth restriction in both compartments. In summary, we have established two new models for the evaluation of genes important for development of hemochorial placentation. These efforts implicate these LIFR and ALKBH1 in the regulation of placental development and its importance in successful pregnancy outcomes. Alawadhi †, Narayana Kilarkaje * , Abdeslam Mouihate * , Maie Al-Bader * . Faculty of Medicine, Kuwait University, Jabryia, Kuwait. Introduction: Intrauterine growth restriction (IUGR) is due to the inability to achieve fetal growth potential for specific gestational age. IUGR is manifested by lower placental size and decreased placental vascularity indicated by reduced expression of vascular endothelial growth factor (VEGF). IUGR pregnancies also correlate with lower maternal progesterone levels. Previous studies showed progesterone-induced VEGF expression in different tissues. Therefore, the aim of the present study is to evaluate the effect of endogenous progesterone administration on pregnancy outcomes and placental VEGF expression as well as the modulation of nuclear and membranous progesterone receptors (PR) in DEX-induced IUGR model. Methods: Pregnant Sprague-Dawley rats were allocated into 4 groups based on daily intraperitoneal injection of: saline, dexamethasone (DEX 0.2 mg/kg), DEX and progesterone (0.2 mg/kg, 5 mg/kg, respectively) and progesterone (5 mg/kg). Injections started on gestation day (dg) 15 and lasted until the day of sacrifice (19 and 21 dg). ELISA was used to evaluate maternal progesterone levels. Real-time PCR and Western blotting were used to evaluate gene and protein expressions of VEGF, and nuclear and membrane progesterone receptors in labyrinth and basal placental zones. Immunohistochemistry was used to detect the localization of VEGF and the receptors in placental cells. Results: DEX had induced moderate IUGR with 16% reduction in fetal body weight and 27% reduction in placental weight. Progesterone co-administration with DEX was able to prevent the DEX-induced reduction in fetal body weight. DEX reduced the expression of VEGF in both placental zones and progesterone-supplementation prevented this reduction. Nuclear and membrane progesterone receptors showed tissue specific expression in different placental zones and responded differently to both DEX and progesterone. Conclusion: Our study proves that progesterone treatment improves the outcomes in IUGR pregnancy by increasing placental vascularity indicated by higher expression of placental VEGF. The enhancement in placental vascularity prevented the reduction in fetal body weight seen with dexamethasone treatment, at least partially, through improving nutrient delivery to the fetus and possibly through increasing steroidogenesis. The placenta is an indispensable organ for intrauterine growth and development in mammals. It establishes an exchange interface that is essential for nutrient and gas exchange between the mother and the developing fetus. A critically conserved step in the establishment of the maternal-fetal exchange surface in mammals is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast layer (SynT). Our lab discovered that the transcription factors, GATA2 and GATA3, are essential for trophoblast development and differentiation. In the early stages of mammalian development when GATA factors are selectively expressed in the trophoblast cells, simultaneous deletion of Gata2 and Gata3 genes in mice (GATA-DKO) severely impair placentation leading to embryonic death at embryonic day 8.0 (E8.0), a stage similar to first-trimester human pregnancy. The abnormal phenotype was accompanied by a reduction in the labyrinth layer that is composed mainly of syncytiotrophoblast cells. GATA2 and GATA3 expression is also conserved in the trophoblast cells of a human placenta, we observed a high expression of only GATA2 and GATA3 in the inner cytotrophoblast layer as well as the outer syncytiotrophoblast layer. However, the functional importance of these GATA factors in the context of human trophoblast development is yet to be defined. I, therefore, hypothesize that a conserved transcriptional program regulated by GATA2 and GATA3 is essential for the differentiation of human trophoblast progenitors towards SynT lineage at the maternal-fetal interface. Methods: To do this, we used a loss of function approach. Using lentivirus-mediated short hairpin RNA (shRNAs) we knockdown either GATA2 or GATA3 in undifferentiated hTSCs. The knocked down (KD) cell lines were then either cultured under the stem state or differentiated into the SynT lineage. The resulting phenotype was validated based on changes in the morphology as well as changes in expression of lineagespecific markers. Results: When these KD cells were cultured using proliferative media in a stem state condition, we observed that there was no effect of individual loss of function of either GATA2 or GATA3 on maintaining the stem state in the hTSCs. This implied that GATA2 and GATA3 have redundant functions in the stem state condition in the human TSCs and functional loss of one transcription factor can be compensated by the functional presence of the other. On the contrary, when these knockdown human TSCs were differentiated to the SynT lineage the loss of either GATA2 or GATA3 lead to impaired syncytialization. The cells did not fuse, nor did they express HCG but retained E-CADHERIN expression. Conclusion: Based on the observations we infer that GATA2 and GATA3 play an important role in the commitment of progenitor trophoblasts to the SynT lineage and are essential for establishing a functional maternal-fetal interface for exchange. Rey Diaz, Shuhan Ji, Emily J Su * . University of Colorado Anschutz Medical Campus, Aurora, CO, United States. Introduction: Cell-autonomous circadian rhythms (CR) are responsible for establishing temporal control of tissue function in all organ systems. CR are known to exist in the placenta, and circadian disturbances have been associated with adverse pregnancy outcomes including fetal growth restriction (FGR). Many factors associated with the FGR in utero environment can also regulate CR, including altered matrix mechanoproperties (i.e., increased placental fibrosis in FGR). In severe, early-onset FGR with abnormal umbilical artery end-diastolic velocities (FGRadv), fetoplacental angiogenesis is impaired, leading to diminished vasculature. We have previously shown that extracellular matrix (ECM) generated from placental fibroblasts regulates endothelial cell (EC) angiogenic properties through interactions with integrins αvβ3 and α5β1. Thus, our objective was to determine whether the extracellular microenvironment differentially regulates CR genes and rhythmicity of integrin expression in FGRadv. We hypothesized that not only does microenvironment influence periodicity but that rhythmicity is disrupted in FGRadv ECs regardless of the microenvironment. Methods: Human fetoplacental ECs were isolated from three groups (N=3/group): (1) FGRadv, (2) Gestational age-matched, appropriately grown preterm controls (PTCs), and (3) ECs is independent of BMAL1 and PER1. These findings suggest that periodicity is regulated by the microenvironment, and differences in circadian rhythms may regulate impaired angiogenesis associated with FGRadv. Oxidative Introduction: Fetal growth restriction (FGR) describes a fetus that fails to achieve its genetic growth potential and is a major risk factor for stillbirth. Placental uptake of glutamine and glutamate, important amino acids for placental metabolism and fetal growth, at initial rate (proxy of transporter activity), is reduced in FGR (1). Reasons for this remain elusive and cannot be accounted for by altered transporter expression. Oxidative stress, a common feature in FGR, can be induced experimentally by tert-Butyl hydroperoxide (tBOOH) which reduces amino acid uptake in BeWo cells (2). However, the effect of oxidative stress on glutamine and glutamate uptake by placental tissue has not been investigated. In this work we hypothesise that oxidative stress reduces steady-state uptake (intracellular levels) of glutamine and glutamate in placental tissue from normal pregnancy. Methods: Placental explants from normal pregnancy (individualised birthweight centiles 25-81; N=6) were cultured for 7 days and treated with 1 mM tBOOH on day 5 and 6 to induce oxidative stress. Uptake of 14 C glutamine and 14 C glutamate by placental explants was measured over 24h between day 6-7 (24h uptake was previously shown to represent steady-state intracellular accumulation) in the presence and absence of 1mM ouabain, a Na + /K + ATP-ase inhibitor, to account for non-transporter mediated diff usion of glutamine and glutamate into the explants. Culture medium was collected daily to measure human chorionic gonadotropin (hCG) secretion and lactate dehydrogenase (LDH) release, markers of endocrine function and tissue necrosis respectively. Results: 1mM tBOOH significantly reduced ouabain-sensitive (transporter-mediated) 24hr uptake of glutamine and glutamate to 5 (-1-34) % and 4 (0.9-60) % of control respectively (median and range: p=0.031; Wilcoxon signed rank vs 100%). tBOOH reduced hCG secretion (day 7 as % of day 5) to 44 (19-70) % of control (p=0.031; Wilcoxon signed rank vs 100%) but LDH release was not signifi cantly diff erent between groups (p=0.0625; Wilcoxon signed rank test). Conclusion: tBOOH reduced placental 14 C glutamine and 14 C glutamate uptake over 24h consistent with the hypothesis that oxidative stress reduces intracellular level of these amino acids. tBOOH inhibited hCG secretion but did not induce LDH release suggesting that tissue integrity was maintained. Oxidative stress could contribute to the reduction in placental amino acid uptake in FGR. Future studies will explore the mechanisms of tBOOH-induced inhibition of amino acid uptake, including suppression of the mTOR pathway which regulates glutamine and glutamate transport. (1) Over 15 million babies are born preterm and one million of them die every year worldwide. To date, the trigger and mechanism of spontaneous PTB remain largely unknown. There is limited effectiveness of therapies to prevent the occurrence of PTB, and a paucity of reliable predictive biomarkers. There is therefore the crucial need to improve our understanding of the molecular mechanisms of spontaneous PTB as this may inform improved diagnostics and therapeutics. Methods: To address this need, we conducted RNA-seq transcriptomic and qRT-PCR analysis on placental villous tissue from 20 idiopathic spontaneous preterm and 20 spontaneous term placentas to identify key genes and signalling pathways that are involved in the pathogenesis of PTB. Results: Our differential gene expression (DEG), gene ontology and pathway analysis revealed several dysregulated genes including OCLN, ARF6, KRT7, SLC6A13, SLC6A17, SLC26A8, KLF8 and FOXA1 which are associated with altered trophoblast functions characterised by aberrant cell motility, differentiation and micronutrients transport. We also detected significant upregulation of pivotal Wnt ligands WNT7A and RSPO4 suggesting an augmentation of the canonical Wnt/β-catenin signalling that may have detrimental effects on trophoblast function. By conducting bioinformatics analysis on DEGs we have identified four key miRNAs, including miR-524-5p, miR-520d-5p, miR-15a-5p and miR-424-5p which were significantly downregulated in the preterm placenta as confirmed by qRT-PCR. These miRNAs may have regulatory roles on the aberrant gene expressions that we have observed. Furthermore, we have identified two potential biomarker candidates, RSPO4 and SLC26A8 that need further exploration. Conclusion: Together, we provide new molecular insight for better understanding of the pathogenesis of spontaneous preterm birth. Further studies based on these observations may enable the development of new predictive biomarkers and therapies. Gestational Hyperuricemia Leads to Hypertension and Neonatal Growth Restriction in Mice. Benjamin P Luescher, Andreina Schoeberlein, Daniel V Surbek, Marc U Baumann * . Women's Health University Hospital, Bern, Switzerland. Introduction: Hyperuricemia is a common feature in pregnancies affected by preeclampsia. The role of uric acid in the pathogenesis of preeclampsia remains unclear. Glucose transporter-9 (GLUT9) is the major regulator of the placental uric acid transport system. We used two different GLUT9 knock-out (KO) mice models, i.e. a systemic GLUT9 KO to mimic fetal hyperuricemia and a liver-specific GLUT9 KO (LG9KO) to induce maternal hyperuricemia, to study the effect of elevated uric acid serum levels during pregnancy. Methods: Systemic GLUT9 KO mice were generated and their postnatal growth pattern were assessed by daily weight measurements. Histologic examinations were performed in fetuses and in animals sacrificed at postnatal day 70. In LGKO mice telemetric devices were implanted to measure blood pressure levels during pregnancy. Results: Neonates, exposed to hyperuricemia during pregnancy, developed severe renal defects and were significantly smaller than controls. Upon maternal hyperuricemia blood pressure levels were significantly increased in the second half of pregnancy compared to controls. Conclusion: Fetal hyperuricemia may cause impaired renal development and altered growth pattern. Maternal hyperuricemia during pregnancy may lead to hypertension. These findings suggest that uric acid plays an important role in the pathogenesis of hypertensive pregnancy disorders such as preeclampsia. These novel insights may offer the development of potential preventive strategies. Introduction: Senescence is the physiological process for removal of damaged cells in the body. Disruption of senescence is associated with several diseases such as Alzheimer's, osteoarthritis, hypertension and Type 2 Diabetes Mellitus, however its role in human placental function has not been explored. Placental syncytiotrophoblast (ST) display several characteristics of senescent cells in being multinucleate, terminally differentiated cells with an irreversible replication arrest. Furthermore, stressors such as increased oxidative stress, inflammation, metabolic imbalance, and insulin resistance observed in GDM pregnancies fuel senescence pathways in non-pregnant individuals. We hypothesized that senescence pathways play a role in ST differentiation and their disruption (possibly related to these stressors) could contribute to the placental dysfunction in GDM. Methods: Primary human cytotrophoblast cells (CT) isolated at term from lean (LN, pre-pregnancy BMI=18.5-24.9, n=5) and obese with type A2GDM (GDM; BMI=30-45, n=5) pregnancies were allowed to differentiate to ST. Expression of differentiation marker (β-hCG), senescence initiators (p21, p16) and autophagy mediators (LC3II, Beclin1) were assessed at 24, 48 and 72hrs by ELISA and western blotting. The number of senescent cells was assessed by staining for the marker Senescence associated β-Gal (SA-βGal). Results: Secretion of β-hCG increased over 72hrs in both LN and GDM confirming differentiation to ST. As CT differentiated to ST, p21 protein expression significantly declined at 48 and 72hrs relative to 24hrs in both LN (p<0.0001) and GDM cells (p<0.05) with a similar trend also observed for p16 (LN p=0.02 at 72hrs; GDM p<0.04 at 48, 72hrs). This pattern is consistent with cells undergoing senescence. However, the absolute levels of p21 were significantly lower (p<0.05 at 24, 48, 72hrs) and p16 higher (p=0.04 at 72hrs) in GDM compared to LN, suggesting differential activation of senescence pathways. Cells from GDM subjects also showed a higher proportion of senescence (SA-βGal + cells) compared to LN (p<0.04 at 24, 48hrs) which remained constant over time suggesting higher basal senescence. The proportion in LN however increased over time reaching significance at 72hrs (p=0.04). Unlike LN, GDM cells had higher levels of LC3II at 72hrs relative to 24hrs (p<0.005), suggesting an increase in autophagy as CT differentiated to ST. Compared to LN, GDM cells also had a lower ratio of LC3II:Beclin1 at 48hrs (p=0.006) and 72hrs (p=0.033) suggesting a progressive decline in their survival capacity. Conclusion: We show that ST differentiation involves senescence activation via both p21 and p16. Altered levels of p21, p16 and a higher proportion of senescent cells with GDM suggests dysregulation of senescence. Combined with increased autophagy and reduced cell survival capacity this could contribute to the placental dysfunction in GDM. Interface. Caitlin M Little †, Alicia S Liu †, Christina J Megli * , Malinda Schafer †. University of Pittsburgh School of Medicine, Pittsburgh, PA, United States. Introduction: S. agalactiae (GBS) remains a leading cause of maternal and neonatal infections. GBS colonization is associated with adverse perinatal outcomes, but a direct comparison to other ascending pathogens has not been well-described. Since the implementation of universal prophylaxis for GBS, maternal and neonatal invasive infections have declined. Yet, contemporary rates of infectious morbidity of GBS versus other pathogens is not well-described. Methods: Placental culture results from, a single center, were reviewed from 2014-2018 and pathogenic bacteria were selected for additional detailed review. Antimicrobial susceptibility and comprehensive maternal and neonatal characteristics were collected. Characteristics were compared by bacterial species (GBS in comparison to all other pathogens). Students t test was used for continuous variables and Fishers Exact test was used for dichotomous variables. p<0.05 was used as a cutoff for statistical signifi cance. Results: GBS was the major placental pathogen composing 43% of pathogenic placental isolates in this contemporary cohort. GBS was less likely to be found in a polymicrobial culture than other pathogens (13.0% v. 24.6%, p=0.006). Indications for placental culture, including IUFD, previable delivery, PPROM and preterm birth, were not diff erent between groups. Chorioamnionitis occurred more frequently with a GBS (44.16% vs 32.51%, p=0.0275) positive culture despite equivalent use of beta-lactam antibiotics between groups. Composite adverse maternal and neonatal outcomes occurred more frequently with a GBS positive culture (Table 1) . Conclusion: Despite implementation of universal screening and antibiotics with appropriate coverage, GBS remains a major pathogen associated with adverse maternal and neonatal outcomes. Future comparative studies on GBS and other pathogens may provide additional insight into unique mechanisms of pathogenesis. Race, Low-Dose Aspirin, and the Development of Preeclampsia in Low-Risk Nulliparous Patients. Kelsey Pinson †, Douglas Woelkers, Cynthia Gyamfi -Bannerman * . UC San Diego, San Diego, CA, United States. Introduction: Preeclampsia is a leading cause of maternal morbidity, with racial and ethnic minorities disproportionately aff ected. Low dose aspirin (LDA) has been associated with reduced risk of preeclampsia, however the relationship between race and eff ect of LDA for preeclampsia prevention is unknown. Our objective was to examine the relationship between LDA, preeclampsia, and race. We hypothesized that LDA would decrease risk of preeclampsia in all groups, without signifi cant diff erences by race/ethnicity. Methods: Secondary analysis of an RCT that examined LDA for preeclampsia prevention in nulliparous, low-risk women, defi ned as the absence of pre-existing hypertension, proteinuria, or other medical comorbidities. Participants were randomized to 60mg LDA or placebo between 13-25 weeks' gestation. We excluded subjects with terminations or spontaneous losses less than 20 weeks gestation. Our primary exposure, race/ethnicity, was self-reported and categorized into four groups: non-Hispanic White (White), Hispanic, non-Hispanic Black (Black), and Other. The primary outcome was preeclampsia. We fi t multivariable logistic regression models to adjust for diff erences present at baseline. Figure] . After adjusting for confounders, there was no diff erential eff ect of LDA by race. LDA was independently associated with a reduction in preeclampsia (OR 0.72, 95% CI 0.52-0.99, p<0.05). Conclusion: LDA was associated with decreased rates of preeclampsia in all race groups except for the small "other" group. The magnitude of the decrease was most pronounced in White women. Further study, including investigation of dosing, is needed to understand this observation. Preeclampsia Mouse Model of Placenta Specific Complement Activation. Manu Banadakoppa * , Kathleen Pennington, Ancizar Betancourt, Simone Hernandez Ruano, Chandrasekhar Yallampalli. Introduction: Semi allogenic nature of placenta may trigger maternal C activation at the feto-maternal interface. Correlative and genetic studies in preeclampsia (PE) patients have suggested that C activation may play an important role in PE pathology. Animal model to experimentally demonstrate role of C activation in PE is not available. In mice, C activation during pregnancy by the ablation of complement receptor 1 related protein y (Crry) is embryonically lethal limiting the use of gene knock out technology to study the role of C activation in PE. We employed a conditional and inducible gene knockdown method to develop a novel mouse model of PE. Methods: We used a tet-inducible promoter (TRE) to drive the expression of a miRE-based shRNA targeting Crry, in the presence of a reverse tet-transactivator (rtTA3). Using a recombination mediated approach, a tet-inducible GFP-coupled shRNA was inserted at the "safe-harbor" Col1a1 locus. To avoid leakiness of the TRE promoter a loxP-Stop-loxP cassette was used between TRE promoter and GFP-miRE expression. Mouse embryonic stem cells (ESC) were targeted for integration of these vectors by Flp/FRT recombinase-mediated cassette exchange (RMCE). Male Cr1l.634shRNA mice were then crossed with female B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae/J to obtain Cr1l +/+ RosartTA +/+ mice. To obtain placenta specifi c shRNA induction, we used Cyp19-Cre mice in which Cre is under Cyp19 promoter and hence expressed only in placenta. The Crry shRNA was induced using doxycycline (75µg/mL) in drinking water from 10.5dpc to 17.5dpc. Results: Doxycycline treatment reduced the expression levels of Crry in placentas by 30% resulting in increased C activation on placentas. In mice with placental C activation, systolic (147 v/s 117), diastolic (117 v/s 84), and mean (127 v/s 95) blood pressure were signifi cantly elevated compared to controls. Placental weight (96g v/s 87g), and fetal weight (973g v/s 898g) were also signifi cantly reduced in mice with C activation. These mice also had proteinuria as indicated by a signifi cant increase in urine albumin/creatinine ratio (264 v/s138), and reduced size of the placental junction zone. Consistent with blood pressure changes, mesenteric artery relaxation responses to acetylcholine are decreased and contractile responses to angiotensin II are increased, in these mice. Conclusion: Placental C activation exhibited hallmarks of PE such as elevated BP, reduced fetal growth and proteinuria. Similar to PE, placental and vascular pathology were also observed in these mice. This novel mouse model is highly suitable to study the mechanistic relation between C activation and PE pathology. were assessed by qRT-PCR and Western blotting and expressed relative to that of GAPDH and beta actin. Respirometry of OASMC was set up with Agilent Seahorse XF respirometer and oxygen consumption rates (OCR) were assessed. In brief, measurement was sequentially performed starting from 3 cycles of 2.5 μM oligomycin, 3 cycles of 4 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and 3 cycles of 0.5 μM rotenone + 0.5μM antimycin A. Production of ATP was defined as the difference between highest OCR during glucose phase and OCR at the end of oligomycin phase. Data were analyzed with Prism GraphPad Software using 1-way ANOVA or unpaired t test. P ≤ 0.05 was considered significant. Results: 1) CGRP dose dependently increased the mRNA encoding mitochondrial complex-1 proteins, ND1 and ND2, Cytochrome b (CYTB) (complex 3), Cytochrome c oxidase subunit I (CO1) (complex 4) and ATPase V mRNA (complex 5) (P<0.05); 2) sFLT-1 decreased OCR in response to maximal respiration and ATP production which is inhibited by CGRP; 3) sFLT-1 decreased mRNA of ND1, CYTB, CO1 and ATPase and these decreases were inhibited by CGRP (P<0.05); 4) sFLT-1 treatment decreased levels of mitochondrial protein complexes I, II, III and IV and sFLT-1 induced decreases in complex I and II were inhibited by CGRP and 5) sFLT-1 decreased mRNA for mitochondrial fusion protein mitofusin-1which was inhibited by CGRP. Conclusion: CGRP reverses sFLT-1-mediated decreases in OCR and MCPs in OASMCs, suggesting possible involvement of CGRP in mitochondrial biogenesis. This study supports a role for CGRP in protecting against sFLT-1 mediated mitochondrial dysfunction in vasculature during pregnancy. However, a less strict false discovery rate cut-off value (FDR<0.25) combined with a high fold change cut-off (log2FC>0.5) increased the number of DE proteins to 4 and 2 proteins at time-points 1 and 2 respectively, and reduced the number to 13 at time-point 3. In total, 66 proteins passed either of the two cut-off filters and were evaluated as potential diagnostic and/or predictive biomarkers. Among these were Soluble fms-like tyrosine kinase 1 (s-FLT1) and Fatty-acid amide hydrolase 2 (FAAH2), which both had 1.2-2 fold elevated expression levels in preeclamptic patients at all three time-points. Several studies have associated high levels of s-FLT1 with preeclampsia, while FAAH2 has been linked to implantation of the early embryo. Current studies by our group has also shown that FAAH2 is secreted from the placenta to the maternal and fetal circulation systems. Conclusion: Out of 5000 proteins, we identified 66 as potential clinical biomarkers for preeclampsia. Increased number of DE proteins between healthy and preeclamptic women towards birth indicate that there is a specific proteomic fingerprint of preeclampsia and that more distinct and deviating protein patterns develop towards the end of gestation. Reconsidering Aryl hydrocarbon receptor (AhR; a transcription factor) participates in many biological processes. However, the role of AhR in vasculature growth and function during pregnancy is poorly defi ned. Methods: Timed pregnant rats were treated with DMSO (vehicle) or an endogenous AhR ligand (ITE, [2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester]; 5 mg/kg BW/day) daily via tail vein injection on gestation days (GD) 10-19. Maternal blood pressure was recorded daily on GD 16-20 using the tail-cuff method, and uteroplacental blood fl ow was determined on GD20. Major maternal organs, fetuses, placentas, and maternal urine samples were collected on GD 20. Placental tissues were processed for histology, Western blotting, RNA-seq, and RT-qPCR analyses. Results: Compared with DMSO, ITE elevated maternal blood pressure by ~ 20% on GD16-17 and proteinuria by 50%, while it decreased uteroplacental blood fl ow by 20%. ITE did not aff ect maternal body weight on GD10-20. ITE reduced fetal and placental weights by 40% and 23%, respectively. Histochemical staining showed that ITE caused placental tissue lesions, cell death, immune-cellular infi ltration, and tissue edemas. Imaging analysis of placental tissue sections stained for CD31 revealed that ITE decreased placental vascular density (18%) and CD31 staining intensity (26%), which were accompanied by a robust decrease in placental CD31 protein levels in Western blotting. RNA-seq analyses indicated that ITE up-and down-regulated 411 and 907 genes, respectively, in female placentas, while ITE up-and down-regulated 571 and 1449 genes, respectively, in male placentas. RT-qPCR analysis on 17 selected genes confi rmed RNA-seq data (r>0.98). Biological processes enrichment analysis on ITE-dysregulated genes revealed that many biological processes (e.g., Cancer, Hypertension, Angiogenesis, Vasculogenesis, and Infl ammatory response) were commonly enriched in rat female and male placentas. Canonical pathways analysis indicated that 38 and 90 pathways were enriched in ITE-dysregulated genes in female and male placentas, respectively. These included those commonly (Cardiac Hypertrophy Signaling [Enhanced] , and Tight Junction Signaling), female specifi cally (Notch Signaling, Coronavirus Replication Pathway, and Caveolar-mediated Endocytosis Signaling), and male specifi cally (Role of Macrophages and IL-17 Signaling) dysregulated pathways. Diseases and bio-function enrichment analysis showed that ITE-dysregulated genes were associated with liver cancer, cardiovascular diseases, and kidney disease. Conclusion: These data indicate that excess endogenous AhR ligands can induce preeclampsia-like phenotypes (e.g., elevated blood pressure and proteinuria) in association with fetal sex-specifi c dysregulation of biological functions in rats. Methods: Data were obtained from a population of prospectivelyobserved pregnancies with SLE, APS, or antiphospholipid antibodies (aPL) alone (PROMISSE study; NCT00198068). Serum aD1 was assayed using the QUANTA Flash® system; 99th percentile for positivity was determined using sera from 125 healthy, non-pregnant females. aD1 levels were quantifi ed for 65 PROMISSE cases using serum obtained in early pregnancy. The primary outcome was APO defi ned as preeclampsia or placental insuffi ciency requiring delivery <34 wks GA. We compared sensitivity, specifi city, positive predictive value (PPV), negative predictive value (NPV), and c-statistic of positive aD1 and/or LA for APO. Results: Of 65 cases, the rate of APO was 51%, but 78% in those with positive aD1 and LA. Signifi cantly more APO cases tested positive for aD1 compared to cases without APO (52% v 25%, p=0.028, Table 1 ). Diagnostic performance of aD1 positivity for APO was superior to that of LA, with a PPV of 0.68 and AUC of 0.63 (Table 2 ). The intersection of LA and aD1 performed best for specifi city, and the union best for sensitivity; however, aD1 performed best in terms of NPV and AUC. In subjects with SLE, APS, or aPL, diagnostic performance of aD1 positivity for APO was superior to that of LA. Combined measures of LA and/or aD1 improves sensitivity and specifi city, aD1 alone outperformed all combinations as conveyed by the C-statistic. In this high-risk population, nearly 80% of subjects positive for both aD1 and LA suff ered an APO. New We hypothesized that hypertension during pregnancy will be associated with gestational age-specifi c dysregulation of mitochondrial genes. Methods: Publicly available high throughput transcriptomic data from maternal and fetal tissues were re-analyzed by diff erential gene expression analysis, time course gene set analysis, and functional enrichment analysis to detect diff erentially expressed mitochondrial genes (mtDEGs) and mtDEG-interacting genes throughout pregnancy and at delivery. Fetal data originated from placentas at delivery (20 hypertensive pregnancies, 21 controls). Longitudinal maternal data were from peripheral maternal blood collected at the 1 st , 2 nd , and 3 rd trimester and at delivery (9 hypertensive pregnancies, 8 controls). Presence of mtDEGs was used as a proxy for mitochondrial dysregulation. Associated expression of mtDEG-interacting genes was used to understand putative downstream consequences of mitochondrial dysregulation. Hypertensive pregnancies were represented by gestational hypertension and subtypes of preeclampsia. Results: Thirty diff erentially expressed mitochondrial genes (mtDEGs) were identifi ed in mothers with hypertension, while nine mtDEGs were detected in fetal placentas from hypertensive pregnancies. All identifi ed mtDEGs were nuclear-encoded. Only two mtDEGs were shared across maternal gestational ages, and no mtDEGs were shared between maternal and fetal tissues. Five maternal mtDEGs displayed changes in the expression of associated mtDEG-interacting genes that were diff erentially aff ected by time and condition. Maternal mitochondrial dysregulation was associated with increased infl ammatory pathway activity in early pregnancy and increased cell death/survival pathway activity in late pregnancy. This was also associated with dynamic dysregulation of placental development pathways. In fetal placentas from hypertensive pregnancies, mitochondrial dysregulation was almost exclusively associated with increased cellular secretion and extracellular vesicle release. We identify mitochondria-mediated maternal-fetal interactions during hypertensive pregnancy that indicate a novel mechanism for the delivery of pro-infl ammatory placental factors to maternal circulation. Our data also suggest generalized dysregulation of mitochondrial function in mothers and fetuses during hypertensive pregnancy. Overall, we link mitochondrial function to diverse processes characteristic of hypertensive pregnancy pathology. Sakuragi * , Eiji Shibata * , Emi Kodoh * , Yasuyuki Kinjo * , Kiyoshi Yoshino * . University of Occupational and Environmental Health, Japan, Fukuoka, Japan. Introduction: Pre-eclampsia (PE) are mainly caused by uterine placental perfusion disturbance due to uterine spiral artery remodeling disorder, but it is diffi cult to accurately detect blood remodeling disorder from the spiral artery to the intervillous space. Recently, measurement of vascularization index (VI), fl ow index (FI), and vascularization fl ow index (VFI) by 3-Dimensional power Doppler placental volume scanning via Virtual Organ Computer-aided Analysis (VOCAL) technique has been attracting attention as an evaluation of placental blood fl ow. We investigated whether VI, FI, and VFI correlate with the pathological structure of placenta and whether there was any diff erence in VI, FI, VFI and placental pathological structure between Normal group and PE group. Methods: 55 pregnant women (Normal group: n=27, PE group: n=28) underwent for VI, FI, and VFI at four locations on the placenta in the second and third trimesters. Two HE-stained specimens of post-partum placenta were prepared. We randomly selected two of these locations and used Image J, the open source image package, to quantify intervillous blood vessels (IBV), intervillous spaces (IS) and intervillous blood vessels + intervillous spaces (IBV+IS) per unit placenta and analyze their correlation with VI, FI, and VFI. Results: There was no positive correlation between VI, FI, or VFI and IBV, IS, IBV+IS. There were no signifi cant diff erences in VI, FI, and VFI between the normal and PE groups, but there were signifi cant diff erences in IBV, IS, IBV+IS in the PE group compared to the normal group. Conclusion: Placental hemodynamics measured by VI, FI, and VFI did not positively correlate with placental morphology at the third trimester. VI, FI, and VFI at the third trimester did not diff er between the normal and PE groups, suggesting that they may refl ect placental circulatory insuffi ciency. Cells. Maja Gajic. Medical University of Graz, Graz, Austria. Introduction: Preeclampsia (PE) is one of the leading causes of maternal and/or fetal morbidity and mortality. The exact mechanism of the disease is still unknown. However, increase cytokine production on maternal and fetal side, especially IL-8, is correlated with severity of the disease. Because, the only available treatment for PE is delivery, a need for new option is high. Due to its anti-inflammatory and immunomodulatory properties hydroxychloroquine (HCQ) represent good candidate. The hypothesis of this work is that there is higher expression of IL-8 mRNA in PE primary feto-placental arterial endothelial cells ( Introduction: Rich in polyphenols and oleic acid, EVOO has antiinflammatory and immunomodulatory effects in cardiovascular disease and hyperlipidemia. Subclinical pro-inflammatory state has been associated with preterm preeclampsia (PEC). We hypothesize EVOO will have an anti-inflammatory effect on cytokine profiles in nulliparas. Methods: This is a secondary analysis of a double-blinded randomized control trial. Healthy nulliparas were randomized to 8 weeks of daily 40 g of EVOO (n = 12) or identical dose of control sunflower seed oil (SSO) (n = 15). Subjects had detailed assessments at baseline and after intervention: continuous non-invasive arterial blood pressure (BP) monitoring, and vascular compliance assessment by pulse-wave velocity (PWV) and volume challenge. Metabolic measures included lipid profiles and measures of fat mass and insulin resistance. Pro-(IFNγ, IL-6, IL-18, TNFα) and anti-inflammatory (IL-10) cytokines, and chemokines (MCP-1) were measured by multiplex array (Mesoscale Discovery, Rockville MD). Cytokine levels were compared between groups with Student's t-test or Wilcoxon rank sum. Mean cytokine changes after treatment were compared with repeated measures analysis. Spearman correlations were used to examine the correlation of cytokine levels and their mean change over time with metabolic and cardiovascular variables. Results: Baseline cytokine levels did not differ between groups. Baseline serum IL-10 negatively correlated with age (r = -0.47, p = 0.014) and pulse (r = -0.44, p = 0.02). IL-6 was positively correlated with visceral fat mass (r = 0.57, p = 0.002) and triglycerides (r = 0.49, p = 0.01). HOMA-IR, a measure of insulin resistance, positively correlated with IL-6 (r=0.46, p = 0.017) and IFNγ (r = 0.54, p = 0.005) . Baseline MCP-1 positively correlated with systolic BP (r = 0.47, p = 0.014) and MAP (r = 0.38, p = 0.048). Uniquely, a reduction in MCP-1 from pre-to post-EVOO positively correlated to reduced systolic BP (r = 0.59, p = 0.042) and MAP (r = 0.59, p = 0.042), while rising MCP-1 in SSO controls inversely correlated with decreasing HDL (r = 0.56, p = 0.03) and rising AUC pulse in response to volume challenge (r = -0.82, p = 0.004). Group*time analysis showed a significant difference in IL-10 change between groups, with the SSO group increasing from baseline to post-treatment (+0.07 ± 0.03) while the EVOO group had no change (-.05 ± 0.03) (p = 0.01). Conclusion: MCP-1, which plays an important role in endothelial and vascular damage, is positively correlated with pre-gestational BP and decreases with EVOO supplementation. Surprisingly, EVOO did not increase serum levels of the anti-inflammatory IL-10 in our study. EVOO may be a useful part of pre-pregnancy metabolic and cardiovascular health, and aid in reducing adverse maternal and fetal outcomes associated with inflammation and insulin resistance. Use To focus on the male factor with self-oocytes, only couples whose women had normal ovarian reserve and didn´t have uterine defects were selected. Telomere length was measured by quantitative fluorescent in situ hybridization. Seminal and IVF ś parameters were assessed according to IVIRMA standard protocols. Results: A group of 18 NZ (43.4 ± 3.8 years) and 17 OAZ (44.2 ± 4.6 years) participated. Using donor oocytes, the fertilization (FR) and blastocyst development rates (BDR) were similar in NZ and OAZ groups. With self-oocytes, the FR was higher in NZ group (0.81 ± 0.12 (NZ), 0.65 ± 0.16 (OAZ), p =0.04), but the BDR was similar in both groups. The sperm telomere length (STL) was higher and statistically significantly different in the NZ group (121.0 ± 22.89 (NZ), 119.3 ± 50.14 (OAZ), p = <0.0001). Regarding correlations, a positive trend was observed between STL and FR in the OAZ group (r=0.49) and in the percentage of accumulation long telomeres in sperm and FR (r=0.31) using self-oocytes. In the case of NZ group, there is a positive trend between STL and FR only with donor oocytes (r= 0.41). A positive correlation between BDR and STL was found in NZ group with donor oocytes (r=0.776, p=0.01). In addition, a positive trend between BDR and the percentage of long telomeres was observed with donor oocytes (r=0.53). No correlation or trend was found in OAZ between BDR and STL, either with donor or self-oocytes. Conclusion: Longer telomeres in sperm ensure a better BDR with donor oocytes in NZ group. The OAZ group had shorter telomeres and worse fertility outcomes compared to NZ group, using the couple´s oocytes. Interestingly, the longer the STL is, the better the fertilization rates are in OAZ group using their own oocytes.This remarks the importance of long telomeres for embryo development, suggesting that as men ages longer telomeres are required for high-quality embryo formation. However, STL in OAZ group does not seem to be the only requirement for BDR, perhaps due to other genome alterations which may interfere with the development of good embryos. OTC counseling was delivered exclusively by REI subspecialists. The association between race and FP counseling also highlights disparities within FP counseling that should be addressed. Our study highlights the importance of comprehensive FP counseling and the need for further investigation into barriers to FP counseling. System-wide measures should be implemented to standardize the content and delivery of FP counseling, with consideration for involvement of REI providers whenever possible. (3) control medium without embryos, and compared using a Mann-Whitney test. Results: No significant differences were noted in the ORP of control medium between the arrested and blastocyst groups. Spent medium from blastocysts showed significantly higher levels of ORP than that of arrested embryos, but no difference compared to the control medium. Spent medium from arrested embryos showed a significant decrease in ORP from the control medium. Most time points were similar between the arrested embryos and blastocysts up to the 8-cell stage except at the time of 2pn fusion which was slightly slower in the arrested embryos (24.72±3.42 hr vs 23.22±2.68 hr; p=0.03). Conclusion: ORP in spent medium from blastocysts did not change compared to the control medium. This could indicate that the blastocysts have the capacity to maintain oxidative homeostasis, whereas poor quality embryos do not. Alternatively, the decrease in ORP for arrested embryos could be due to an increase in reductive stress, which may possibly be due to apoptosis. The control media between these groups showed no difference in ORP indicating that the differences seen were due to the embryos themselves and not the medium. Future studies need to be directed to ORP in the spent medium on Day 3 to provide more information about why some embryos arrest and others continue to develop. If the ORP levels shows similar trends on day 3 as in day 6, it could be a potential predictor for poor embryo quality and subsequent developmental competence. Introduction: Obesity in women is associated with decreased fertility, adverse pregnancy outcomes and relative hypogonadotropic hypogonadism, which we termed reprometabolic syndrome. We previously demonstrated that acute hyperlipidemia and hyperinsulinemia recapitulates this phenotype in normal weight women and exerts differential effects on the hypothalamic-pituitary-gonadal, adrenal and thyroid axes. We hypothesized that a eucaloric high-fat diet (HFD) designed to elevate insulin and circulating free fatty acids would also impact anterior pituitary trophic hormones and adipocytokines. Methods: 17 normal weight (BMI 18-24.9) healthy, cycling women (mean age 29±8) underwent frequent blood sampling (q10 min) in the early follicular phase (days 2-5) for 4 hours starting at 7 am, during a pre-diet cycle. They were subsequently provided a prescribed, eucaloric HFD (48% calories from fat) for the duration of their next menstrual cycle and the frequent blood sampling was repeated in their post-HFD cycle. Serum TSH, free T4 (fT4), total T3 (tT3), cortisol, GH, prolactin (PRL), IGF-1, HMW adiponectin, and leptin were measured by immunoassay. Wilcoxon signed-rank tests were used to compare hormone levels before and after the HFD intervention. Results: There was a small but significant decrease in tT3 (p=0.01) and cortisol (p=0.02) after the HFD. No changes in TSH, fT4, PRL, GH or IGF-1 were observed in response to the HFD. Similarly, leptin and adiponectin levels were not significantly different (Table) . A one-month HFD, designed to induce the reprometabolic syndrome of obesity, resulted in differential effects on the hypothalamicpituitary-gonadal, adrenal, and thyroid axes, implicated in reproductive function. The observed decrease in morning cortisol after the HFD is novel. Studies have not addressed the impact of eucaloric HFD on thyroid hormones and the clinical significance of the small reduction in tT3 is unclear. For example, previous studies have looked at understanding of fertility treatment in residents and MBA students, given that students in these professions are more likely to delay childbearing and in turn more likely to require fertility treatment as a result.. To date, there have not been documented studies on perceived barriers in medical students and their thoughts on fertility care. In this study, we aimed to examine the perceived barriers to fertility treatment in medical students and determine if there were differences in perceptions based on the participant's race. Methods: A 10-minute survey was sent to medical students at a single academic institution in the Midwest. This survey asked questions regarding perceptions of infertility treatment and perceived barriers to fertility treatment such as time, finances, awareness of options embarrassment in seeking care. T-tests were used to compare differences based on whether or not patients identified as White or Caucasian or as non-white on their survey Results: 139 students of the 750 students responded to this survey (19% response rate). Non-white participants were significantly more likely to report embarrassment with seeking fertility treatment (p=0.04). Additionally, non-white medical students were more likely to report three or more barriers than white medical students in this study. (p=0.048). Given that all medical students in this study indicated barriers to accessing fertility care, medical programs should plan to discuss fertility treatment with their students as part of student wellness. Additionally, given race-based differences in perceived barriers, special attention should be paid to explore etiologies of their perceived barriers and correct these for this population. Additionally, specific education should go into providing more transparency in fertility care to this population. Follicular Immune Signature of Polycystic Ovarian Syndrome in Women Undergoing Infertility Treatment. Soma Banerjee †, Fernanda B Levya Jaimes, Abigail A Zettel, Eryne T Jenkins, Jason Austin, Laura G Cooney * , Aleksander K Stanic * . University of Wisconsin,Madison, Madison, WI, United States. Introduction: PCOS is the most common cause of infertility associated with anovulation presenting at infertility clinics. Ovulation induction and IVF treatment results in only 20-50% of patients achieving pregnancy after the first IVF cycle. We are investigating whether PCOS is associated with an inflammatory functional bias and dysregulation of tissue-resident (follicular) versus systemic immune networks compared to non-PCOS control women undergoing infertility treatment. Methods: We prospectively enrolled a cohort of control and PCOS patients planning IVF treatment at an academic reproductive endocrinology and infertility practice and collected: plasma pre-treatment (visit 1) and at transvaginal oocyte retrieval (TVOR, visit 2), as well as follicular fluid (FF). A total of 30 patients had matched visit 1, 2 and FF samples (10 control and 20 PCOS), which were used for these analysis. Levels of adaptive helper cytokines (IL-2, IL-4, IL-5, IL-6, IL-9,IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFNy, TNFa) and innate response cytokines (TSLP,TGF-B1, IL-1α and β ,IL-11,IL-12, IL-15, GM-CSF, IL-18, IL-23, IL-33, IL-17, IL-1B) were determined employing multiplexed, custom cytometric bead arrays (LEGENDPlex, Biolegend). The immune signature of women with PCOS and non-PCOS controls were compared using student's t tests or Mann-Whitney U tests as appropriate for single comparisons. We examine the relationships between the serum and follicular fluid cytokines levels and clinical parameters, including age, BMI, and the number of stimulation days with univariate and multivariate linear regression to determine cytokine correlation with PCOS. Results: We find increased level of IL-18, IL-21 in follicular fluid of PCOS patients (p<0.05) and a trend towards increased IL-9, TNFa, IFNγ (0.0530) regularly cycling women, 24-48hrs before and after treatment with the GnRH antagonist cetrotide (3.25mg), followed by administration of hourly intravenous boluses of FSH (30IU) for 10h, in the early follicular phase. DNA was extracted from stool and bacterial profiles determined by analysis of 16S rRNA genes. Differences in fecal microbiota were analyzed in terms of microbial diversity (alphadiversity), overall community composition (beta-diversity), and relative abundance of individual taxa. Alpha diversity was compared by linear regression and differences in individual taxa determined using negative binomials adjusting for baseline. All analyses were stratified by weight category and used the Benjamini-Hochberg false discovery rate (FDR) to adjust for multiple comparisons. Results: Suppression of gonadotropins followed by reconstitution of pulsatile FSH was associated with significant differences in fecal microbiota that were modified by weight category. Higher Shannon diversity, richness, and evenness of intestinal bacteria were observed in pre-treatment samples compared to post-intervention. Women with obesity exhibited a larger effect size for those metrics. Among individual taxa that changed in relative abundance with treatment, 14 were significantly modified by weight category (FDR corrected p<0.05). These included Akkermansia, Bacteroides, Bifidobacterium, Enterobacteriaceae, Lachnospiraceae and Ruminococcaceae, alterations in which have been previously linked to obesity, infertility, hormonal contraception and PCOS. Conclusion: Modulation of FSH and LH and consequent alteration of estrogen levels are associated with changes in the intestinal microbiome, characteristic of reproductive endocrine dysfunction, that were modified in the context of obesity. Functional interactions between the hypothalamicpituitary-ovarian axis and gut microbiota, which impact circulating estrogen, may contribute to the detrimental effects on fertility and pregnancy in women with obesity, and thus provide novel avenues for therapeutic intervention. Nicola Tempest † * , 1,2,3 Evie Oliver †, 1 Hannan Al-Lamee †, 1,3 Emily Thomas †, 1 Andrew J Drakeley * , 3 Victoria Sprung * , 4 Dharani Hapangama * . Introduction: Infertility affects ~1 in 7 couples with many and varied causes that include lifestyle factors. Lifestyle changes are cost effective, non-invasive options for routine fertility care. Presently, information on habitual activity is not enquired from women and no consensus regarding optimum exercise exists for fertility patients. Therefore, we aimed to determine exercise habits of women attending the one stop infertility (OSI) clinic and assess enthusiasm to participate in further research examining exercise and fertility. We hypothesise that different exercise (type, dose and intensity) may affect fertility, despite even normal BMI. Methods: 250 women (mean age 34 years and BMI 29Kg/m 2 (range18-55.1kg/m 2 )) were recruited to this study from a tertiary OSI clinic over a period of 9 months. All participants completed a voluntary, self-reported, anonymous questionnaire, demographics were collected, and exercise habits captured utilising the International Physical Activity Questionnaire (IPAQ). Results: 60% of women reported no vigorous exercise and 71.6% no form of structured exercise. Interestingly, most vigorous/formal structured exercise was undertaken by women with a BMI <32. Out of the participants 90.4% were non-smokers, 58% consumed alcohol and 73.6% had a regular menstrual cycle. 31.2% of women were interested in taking part in future research relating to exercise and fertility. Conclusion: These novel data suggest that women attending OSI clinics perform minimal exercise, particularly obese women, implying that low activity levels and elevated BMI negatively influence fertility. A sub set of women, with normal BMIs, did a very high level of exercise, again potentially negatively influencing their fertility. Further evidence examining the effect of exercise as a non-pharmacological tool in fertility medicine are warranted as a first step in developing consensus guidelines for these patients and personalised plans of their fertility care. Conclusion: This novel study presents an improvement in embryo euploidy rates following intra-ovarian PRP application in infertile women with prior failed IVF cycles. The growth factors present in PRP may exhibit a local paracrine effect that could improve meiotic aberrations in human oocytes, thus improving euploidy rates. Whether PRP improves live birth rates and lowers miscarriage rates remains to be determined in large trials. Introduction: During menstrual cycle, uterine endometrium undergoes profound morphological and functional rearrangements, fundamental for successful embryo implantation. In response to the rise of progesterone levels in post-ovulatory phase, all the components of the endometrial tissue (stromal cells, luminal and glandular epithelium, vasculature, residing immune cells, and extracellular matrix) undergo a synchronized remodeling process, called decidualization .In decidualization, the role of endometrial stromal cells (EnSC) is particularly relevant: EnSC undergo complete functional and morphological remodeling, differentiating from fibroblast-like cells into an epithelioid, fully secretory phenotype. Decidualized EnSC secrete a wide array of protein cargoes (matrix proteins, hormones, cytokines, and growth factors) that reshape the local environment, modulate the crosstalk among resident cells and more in general allow the correct implantation of the embryo. As we recently published, decidualization of EnSC entails a complete readjustment of cytoarchitecture, both in terms of morphology and organelle volume: cells undergo a significant size increase, paralleled by a proportional increase of all the secretory organelles. Methods: In our experiments, we compared mitochondria before and after in vitro decidualization of EnSC. We analyzed mitochondria in high-resolution microscopy, followed by morphometric analyses. We also performed unbiased proteomic analyses of EnSC, further analyzing selected mitochondrial proteins by Western Blot. Results: In microscopy, we observed that mitochondria extend from the perinuclear area to the very periphery of the cells, only partially associating with the endoplasmic reticulum. Interestingly the mitochondrial network was completely reorganized in decidualization: mitochondria acquired a marked radial distribution and became more elongated, coherently with the post-mitotic nature of decidualized EnSC.When quantified by morphometry, mitochondrial volume was significantly increased in decidualized cells, in terms of both absolute value and relative occupancy of the cell, something that was not observed for secretory organelles.Moreover, in line with IF, both WB and unbiased proteomic analyses indicate that the mitochondrial proteins increase during decidualization relative to the total protein content. Conclusion: Taken together, these results suggest an important role for mitochondria in decidualized EnSC: the expansion of mitochondrial network and the morphological changes observed likely underlie the increased need for energy and biosynthetic intermediates that these cells require for their complex secretory task. The comparison of EnSC decidualization with another unrelated model of differentiation to protein factory (B lymphocyte to plasma cell) will be discussed, to better underline the special behavior of EnSC. Henarejos Castillo †, 1,2 Patricia Sebastian-Leon, 3 Alejandro Aleman, 1 Jose Remohi * , 4,2 Gorka Alkorta-Aranburu * , 5 Patricia Diaz-Gimeno * . 3 Conclusion: This is the first-time thermogenesis associated with ovarian failure, a process interwoven with menopause. Moreover, for pathways previously associated with OF, an increment of activity in the synthesis and secretion of aldosterone is suspected to be linked to impaired fertility; likewise with the parathyroid hormone; finally, an increment of autophagy would involve primordial follicle atresia. While further studies are needed for validation, this is the first study on ovarian failure to find accumulative DNA variants differences at the systemic level. This study deepens the molecular causes of OF for better precision medicine. Oocyte Oocyte stage-specific conditional ERβ depletion was carried out using conventional Cre-lox system in mutant mouse models. Changes in the primordial follicle activation were assessed by follicle counting in serial sections of whole ovaries. We detected an abundant expression and nuclear localization of ERβ in both granulosa cells and oocytes of dormant primordial as well as activated primary follicles. The nuclear localization of pERβ (Ser105) suggests that ERβ is transcriptionally active in oocytes and granulosa cells of both the dormant and the activated follicles. We observed that targeted depletion of ERβ in the oocytes of Erβ fl/fl :Gdf9-Cre mice increases primordial follicle activation to a similar extent of complete Erβ KO mice that lack ERβ in all cell-types. This novel finding emphasizes the essential role of ERβ in the oocytes for regulating primordial follicle activation. As Gdf9-Cre depletes the expression of ERβ in oocytes starting from the primordial stage, we examined the effect of ERβ depletion selectively in the activated follicles using a Zp3-Cre mouse line (Erβ fl/fl :Zp3-Cre). Both Zp3-Cre and Gdf9-Cre resulted in deletion of the floxed Erβ alleles in the pups verifying their oocyte-specific Cre efficiency. However, we did not observe any increased primordial follicle activation in Erβ fl/ fl :Zp3-Cre mice. Conclusion: These findings suggest that the presence of ERβ is essential in the primordial oocytes, but not in the activated oocytes for the regulation of primordial follicle activation. Ovarian Cellular Senescence as a Regulator of Fertility in Mice. Jesus R Lopez, Meirav Sela, Amanda N Kallen * . Yale School of Medicine, New Haven, CT, United States. Introduction: Cellular senescence, a response to cellular insult defined by loss of proliferative capacity and cell cycle arrest, is a key hallmark of aging in non-reproductive tissues. Senescent cells exhibit increased expression of the cell cycle inhibitor p16, a cyclin-dependent kinase inhibitor that enforces growth arrest. Age-dependent accumulation of p16-expressing cells impairs organ function in tissues such as kidney and heart and shortens healthy lifespan. Increasingly, however, senescence is also viewed as a beneficial response in young tissues which can promote damaged cell clearance. However, the contribution of senescent cells to reproductive aging is unknown. Because p16 is present in mouse primordial follicles and decreases with the onset of follicular recruitment, we hypothesize that senescent cells function as a protective mechanism to promote ovarian function in young mice, and that their clearance in young mice is detrimental to fertility. Methods: We utilized INK-ATTAC mice, which express a caspase-p16 fusion protein (permitting ablation of senescent cells with AP20187, a dimerizer that activates the caspase fusion protein) and a green fl uorescent protein (GFP) for visualization of senescent cells. 8-week mice were injected with 2ug/kg of AP20187 (AP) or vehicle (n=10/group) for 1 month, mated, and euthanized 13.5 days post conception. Uteri were collected for implantation site counting. Ovarian qRT-PCR was performed for p16 and Gfp (to confi rm senescent cell clearance) and Amh (to evaluate ovarian reserve). Results were compared using Student's t-test. qRT-PCR showed partial ovarian clearance of p16 and Gfp after one month of AP injections (a). After partial senescent cell clearance, AP-treated mice exhibited a strong trend toward reduced fertility, with (b) fewer pregnancies and (c) fewer implantation sites per mouse than controls. Nearly half (4/12) of AP-treated mice were infertile after clearance of senescent cells (c). Decreased ovarian Amh mRNA was observed after AP compared to vehicle (d). Conclusion: Our novel data demonstrate that partial elimination of senescent cells results in decreased ovarian reserve, fewer pregnancies, and complete loss of fertility in half of mice treated, supporting our hypothesis that senescent cells protect reproductive competence in young mice. Future studies will investigate the eff ect of improved senescent cell clearance on fertility and the age-specifi c eff ects of ovarian senescent cell clearance, providing a detailed understanding of the specifi c roles of senescence in the ovary across the lifespan. Endometrial were isolated from decidua basalis from term births and the expression of receptors (IL-20R1, IL-22R1 and TGF-βR1-3) was determined by fl ow cytometry. ILC3 were stimulated in vitro with cytokines from DSCs (IL-6, IL-7, IL-24, SCF, TGF-β1, TGF-β2) in the presence of blocking antibodies (IL-6bAb and IL-7bAb) and ESC-and DSC-conditioned (ESC-CM/DSC-CM) medium for 48 h. Changes of proportion within the cell population of NCR + and NCR -ILC3s were determined by fl ow cytometry. The data were analyzed by t-test and one-way ANOVA (Dunnett) and a p-value < 0.05 was considered as statistically signifi cant. Results: Stimulation of ILC3 with ESC-and DSC-CM, led to a decrease in the ILC3 cell fraction and the expression of CD69. The expression of ILC3-relevant cytokines IL-6, IL-7, SCF, TGF-β1 and TGF-β2 was detected in the CM of both ESCs and DSCs. DSCs secreted higher levels of IL-7 and lower levels of IL-6 than ESCs. The stimulation of ILC3s with IL-6 and IL-7 led to signifi cant increase of NCR -ILC3, while IL-6 also induced a decrease of CD69. The blocking of IL-6 and IL-7 on CM had no eff ect on ILC3 phenotype. The treatment with TGF-β1 lead to decreased mRNA expression of TGF-βR3 on in vitro generated ILC3s.Decidual ILC3s expressed higher levels of the IL-24-receptor monomer IL-22R1 than the IL-20R1 monomer, and the levels were higher in NCR + ILC3s than in NCR -ILC3s. However, in contrast to previous reports, IL-24 was not detected in ESC-and DSC-CM. Our results indicate that both ESCs and DSCs can infl uence uterine ILC3 biology by the release of soluble immune mediators. Sanchez-Reyes †, 1,2 Antonio Parraga-Leo †, 1,2 Patricia Sebastian-Leon, 1 Katharina Spath, 3 Antonio Pellicer * , 2,4 Jose Remohi * , 2,5 Dagan Wells * , 3, 6 Patricia Diaz-Gimeno * . Introduction: Oocyte quality plays a role in fertilization success and preand post-implantation embryo development. So far, only a morphological evaluation of the developmental stage and quality of the oocyte is assessed prior to fertilization with a limited predictive value. Therefore, we hypothesize that oocyte morphology assessment can improve fertilization success rate and preimplantation embryo developmental potential. Here we investigated whether oolemma surface area is associated with preimplantation embryo morphokinetic parameters and post-implantation clinical outcomes. Methods: 378 couples with an indication for in vitro fertilization (IVF) treatment or intracytoplasmic sperm injection (ICSI) were included in the VIRTUAL EmbryoScope study, part of the Rotterdam Periconceptional Cohort. 1,265 embryos were cultured in a time-lapse incubator. From embryos that were selected for transfer or cryopreservation, we recorded the time of fertilization (T0), pronuclear appearance (tPNa) and fading (tPNf), as well as the timing of reaching the 2, 3, 4, 5, 6, 7-and 8-cell stage (t2-t8), the start of blastulation (tSB) and reaching the full (tB) and expanded blastocyst stage (tEB). Oolemma area was measured at T0, tPNa and tPNf following ICSI (n=793), and only at tPNf following IVF, as IVF oocytes were transferred only after tPNa to an EmbryoSlide (n=472). Adjusted linear mixed models and logistic regression were used to analyze oolemma area and embryo morphokinetics and post-transfer outcomes, respectively. Subgroup analyses for IVF and ICSI treatment were performed. Results: Oolemma area significantly decreased from 10512 µm 2 at T0 to 9882 µm 2 at tPNf (p<0.001), with a mean shrinking rate of 28.36 µm 2 /h. In the total group, after adjustment for maternal age, type of ovarian stimulation and sperm retrieval method, oolemma area at tPNf showed a negative association with the moment of reaching t2 till t7 (e.g. t2 β= -8.7 hours, 95% CI -12.4, -5.0, p<0.001). Oolemma area at tPNf was not predictive of implantation (OR 23.9, 95% CI 0.7, 910.9, p= 0.08) or live birth (OR 16.6, 95% CI 0.4, 708.2, p= 0.13). Oolemma area at tPNf did not differ between IVF and ICSI, but oocytes reached tPNf earlier following ICSI (p<0.001). Subgroup analysis for IVF and ICSI demonstrated comparable results for embryo morphokinetics and clinical outcomes. Conclusion: Human oolemma area shrinks after fertilization and is independent of fertilization method. Larger oolemma area is associated with faster cleavage divisions to the 8-cell stage, but not with post-transfer outcomes. These data suggests that oocyte morphology is predictive of embryo developmental potential prior to zygotic genome activation and needs further investigation. Introduction: Regeneration of the endometrial stromal compartment in premenopausal women is likely maintained by the perivascular endometrial mesenchymal stem/stromal cells (eMSC) expressing Sushi domain containing 2 (SUSD2). The fate of SUSD2 eMSC during pregnancy and their role in decidualization is not fully known. The aim of our study was to determine the effect of progesterone on the stemness of the SUSD2+ eMSC isolated from non-pregnant uterine samples. Secondary objectives were to assess the mesenchymal to epithelial transition (MET) of the SUSD2+ eMSC on the publicly available singlecell RNAseq dataset (scRNAseq) from first trimester decidua. Finally, we aimed to characterize the functional capacity including differentiation into the mesenchymal cell lineage and CFU assays of SUSD2+ eMSC isolated from decidua at full term and compare it to the capacity of those isolated from non-pregnant uterine samples. The study included three groups of samples: non-pregnant uterine biopsy of endometrium (n=15), 1 st trimester deciduas (n=9) and uterine biopsies from healthy pregnant women (n=15). SUSD2+ eMSC isolated from non-pregnant and pregnant samples, included in the differentiation assay, colony forming unit assay and progesterone treatment. The decidual samples from the scRNAseq dataset were analysed using the Cell Ranger pipeline and the R package Seurat. Results: Progesterone treatment of the SUSD2+ eMSC from nonpregnant uterine samples resulted in significant changes in the gene expression profile; in particular genes involved in decidual vascularization, differentiation and decidualisation . However, the major MSC membrane surface markers, including SUSD2 remained unchanged. Histological analysis revealed a significantly lower abundance of SUSD2+ eMSC in 1 st trimester and full term samples compared to non-pregnant samples, p=0.0296 and 0.005, respectively. scRNAseq analysis confirmed the presence of SUSD2 in the pericytes but not in the decidual stromal cells. The differences in the MET gene expression remain yet to be investigated. The differentiation and the colony forming capacity did not differ significantly between the cells isolated from non-pregnant and pregnant uterine samples. Conclusion: Pregnancy reduces the abundance of SUSD2+ eMSC, however, eMSCs function remained intact. Our results suggest that SUSD2+ eMSC undergo decidualization in vitro, while retaining the MSC membrane surface phenotype. Therefore, eMSCs likely play an important role in the course of endometrial decidualization and embryo implantation. Analysis of the scRNAseq dataset will provide more information about MET during decidualization. year-old), and old women (>45 year-old), respectively. Animals underwent ovarian stimulation and mated. Mice were sacrificed 36h after to recover Metaphase-II (MII) oocytes and 2-cell embryos. Then, ovulation rate, oocyte quality, and embryo development were assessed. Ovaries were also collected to analyze follicular growth, stroma, and mitochondrial function. Results: A reduced number of follicles at different developmental stages was observed in old mice when compared to young and AMA (Fig. 1A) . The number of corpus luteum and MII oocytes were diminished in AMA, although these differences were higher in old mice ( Fig. 1B-C) . Oocyte quality analysis showed a decreased spindle area (Young: 270±50, AMA: 210±40, Old: 207±14 µm 2 ) and abnormal assembly (Young: 40, AMA: 84, Old: 83%) in AMA (p<0.01) and old (p<0.05) groups compared to young. Indeed, the 67.5% of oocytes from the old group were fragmented. The number of 2-cell embryos was reduced in AMA compared to young and no viable embryos found in the old mice (Fig. 1D ). Further in vitro embryo culture revealed impaired blastocyst formation (Young: 86%, AMA: 67%) and hatching rates (Young: 69, AMA: 47%). The age-related deficiencies were also observed in ovarian stroma, as both cell proliferation (Young: 13±1, AMA: 5±1, Old: 3±0%; AMA and Old vs. Young p<0.01) and microvessel density (Young: 15±5, AMA: 11±3, Old: 11±3%; p=N.S) decreased in the AMA and old. Moreover, a decreasing mtDNA copy number was also noticed in these models . Pearson correlation matrices on centred log-ratio transformed abundance raw counts for network analysis were generated using the Scipy Stats package (version 1.4.1) and visualised with Networkx (version 2.4). Considering taxa with significant Pearson correlation coefficient, a weight for each edge was computed as: abs(corr) -pval, where abs(corr) was the absolute correlation value between each pair of taxa and pval was the corresponding p-value. Results: The co-occurring EM bacterial networks showed a significant negative association between Lactobacillus and reproductive tract pathogens as Gardnerella, Atopobium, Streptococcus, Staphylococcus and Bifidobacterium in EF and EB. In EF, the EM networks in LB was denser and had a higher node degree distribution than co-occurrence networks of unsuccessful reproductive outcomes, with Lactobacillus being negatively related to pathogenic bacteria (Staphylococcus, p=0.03; Haemophilus, p=0.01) and positively correlated to a dense community of commensal bacteria (Streptomyces node, p=0). In NP, Lactobacillus negatively associated to Streptococcus, p=0.004; Gardnerella, p=0.001; Atopobium, p=0; Staphylococcus, p=0. In patients with BP and CM, these interactions disappeared, and the resulting networks were disconnected and formed sparse communities, although still showed significant co-occurrences. The analysis of EM co-occurrence networks for each ART reproductive outcome showed that LB networks had a higher node degree distribution than the networks of failed outcomes, with potential interactions that only occurred in patients with LB, suggesting a role of ecological relationships between taxa in EM stability and successful pregnancy. IM & DP-V contributed equally. knockout mice) and a GalT knockout immortalized mouse granulosa cell line (GalTKO:GC). We are determining how GalT deficiency impacts GC, focusing on the integrated stress response (ISR) 2,3 , its regulator eukaryotic initiation factor 2 alpha subunit (eIF2α), and the inactivating kinases HRI, PKR, PERK, and GCN2. 4 Methods: GalTKO:GC and parent OV3121 cells were used to measure expression at baseline, or, after treatment with the GC growth inhibitor anti-Müllerian hormone (Amh), the growth stimulator Tumor necrosis factor alpha (Tnfα), or both agents. eIF2α, inactivated Phosphoserine 51-eIF2α (P-eIF2α), and Casp3 expression were determined by western blot after treatment with vehicle, Amh, Tnfα (each at 10 ng/ml), or both agents, after 6 hours. Baseline eIF kinase expression was determined using quantitative RT-PCR. Our hypothesis was that ISR regulator and Casp3 protein levels would differ in cells deficient for GalT. Results: Significant differences (p<0.05, Welch's t test) were as follows. GalTKO:GC had higher levels of P-eIF2α than parent cells at baseline, and mutant cells also differed in their response to Tnfα. Parent cells had a lower ratio of P-eIF2α:eIF2α when untreated, and a higher ratio when treated with Tnfα. Conversely, GalTKO:GC had a lower ratio when treated with Tnfα. Amh treatment abrogated the stimulatory effect of Tnfα in both mutant and parent cells, but suppressed P-eIF2α below baseline only in Blake Vessa †, 1 Nischelle Kalakota †, 2 Qingshi Zhao * , 2 Sara Morelli * , 2 Nataki Douglas * , 2 Andy Babwah * . 3 1 Rutgers New Jersey Medical School, Newark, NJ, United States; 2 Rutgers New Jersey Medical School, Bloomfield, NJ, United States; New Brunswick, NJ, United States. Introduction: Adhesion G protein-coupled receptors (ADGRs) regulate male fertility but little is known about their roles in female reproduction. Recent studies suggest that in mice ADGRD1 facilitates embryo transit from the oviduct while ADGRG2 is required for human endometrial stromal cell decidualization. In a molecular screen comparing endometrial gene expression in the secretory phase of natural and ovarian stimulation cycles, 4 out of 22 ADGR genes, ADGRL1, L2, G1 and G2, were found to be differentially expressed. We sought to identify the endometrial cell type expressing these ADGRs and determine if ADGR expression was regulated by exposure to E2 and/or P4. Methods: Gene expression was determined in the estrogen and progesterone receptor-expressing human T-HESC (stromal) and Ishikawa (epithelial) endometrial cell lines. Cells were cultured in their respective medium lacking E2 or P4 (basal conditions) or in medium supplemented with 0.1% EtOH (vehicle), E2 (10 nM) or P4 (100 nM) for 48 hours (hormone-stimulated conditions). Gene expression was also determined in T-HESCs cultured under decidualizing conditions (10 nM E2, 1 µM P4 and 0.5 nM cAMP). After treatment, changes in gene expression were determined by RT-qPCR performed in triplicate using gene specific primers. Gene expression was calculated as relative expression to the housekeeping gene (18SN5), using 2 -∆∆Ct . Means were compared using unpaired t-tests and p<0.05 was considered statistically significant. Results: Under basal conditions, expression of ADGRL1 and ADGRG1 was higher in T-HESC as compared to Ishikawa [1.3-fold (p=0.02) and 1.4-fold (p=0.03), respectively], while expression of ADGRL2 and ADGRG2 was similar. Neither exposure to E2 nor P4 changed expression of ADGRs in T-HESC or Ishikawa cells. Exposure of T-HESC to E2, P4 and cAMP increased mRNA expression of IGFBP1, a marker of stromal cell decidualization (117-fold, p<0.01), ADGRL1 (9-fold, p=0.02) and ADGRL2 (2-fold, p=0.01). Conclusion: ADGRL1 and ADGRL2 mRNA expression increases with in vitro decidualizing conditions. However, they do not appear to be responsive to E2 or P4 treatment. These findings of increased expression with decidualization are similar to previous studies with ADGRG2. Further investigation is warranted to illicit the mechanism of increased expression with decidualization, as its regulation seems to be independent of E2 and P4 levels alone. Prioritized The human endometrium is a structurally complex organ and the site of embryo implantation. Whilst multicellular in vitro models of embryo-endometrial interaction have been established, there is a paucity of systems that replicate the histoarchitecture of the endometrium due to the absence of glandular elements. Epithelial glands and their secretions are essential for both decidualisation and blastocyst implantation. Therefore, we aimed to establish a three-dimensional model of embryo implantation incorporating primary epithelial organoids as glandular components to accurately recapitulate in vivo embryo-endometrial crosstalk. Methods: Biopsies of proliferative phase human endometrium were dissociated to allow separation of epithelial and stromal elements. Endometrial epithelial organoids were derived using an established protocol. Cells and organoids were embedded in hybrid type I collagenagarose scaffolds to mimic the cellular composition of the endometrium. Models were treated with oestrogen, medroxyprogesterone acetate and cyclic adenosine monophosphate to induce decidualisation. JEG-3 trophoblast spheroids were utilised as embryo surrogates and were seeded on top of the decidualised scaffolds. Markers of decidualisation and pregnancy establishment were assessed by immunostaining and enzyme-linked immunosorbent assays. Results: Addition of agarose to collagen scaffolds prevented contraction and degradation of the endometrial constructs over a culture period of 14 days. Epithelial cells seeded on the apical surface of scaffolds formed a monolayer reminiscent of the luminal epithelium and organoids retained a hollow interior containing secretions. Endometrial cells expressed phenotypic markers including CD10 and cytokeratin-18 for stroma and epithelia, respectively. In the presence of ovarian steroid hormones, the decidual marker IGFBP1 was significantly upregulated in treated constructs relative to controls, and epithelial organoids exhibited an increase in oestrogen and progesterone receptor expression. JEG-3 spheroids attached to the engineered luminal epithelium and remained viable for a 10-day culture period as evidenced by secretion of chorionic gonadotrophin β. In the presence of hormonal stimulation and an embryo surrogate, epithelial organoids demonstrated an increase in glycodelin expression. Here, we demonstrate a novel embryo implantation model by incorporating gland-like elements in the form of organoids. Both endometrial cell types are hormone responsive and display a decidual phenotype. The model supports trophoblast adhesion and facilitates embryo-endometrial crosstalk, thus providing a platform to study early pregnancy establishment. Such a platform may prove invaluable in the interrogation of reproductive disorders including recurrent implantation failure. Despite the insights about endocrinological and metabolic modifications, answers about the effect of menopausal transition in human myometrium are scarce. Here we aim to understand the effect of aging on the cellular and molecular heterogeneity of the human myometrium. Methods: Single cell RNA-seq (scRNA-seq) was performed on 27,322 single cells corresponding to myometrium from 4 menopausal women (age range 45-80 years), undergoing hysterectomy for uterine prolapse and patients from the organ donor program. Myometrial tissues were obtained from three different areas (anterior, posterior wall, and fundus). After tissue dissociation, single cell RNA-seq analysis were performed through the 10X Chromium system. Briefly, gel bead-in-emulsions containing the single cells, were used to generate barcoded cDNA libraries which were finally sequenced with Illumina NovaSeq 6000 system. Quality control and bioinformatic analyses were performed using standard Cell Ranger and Seurat protocols. Results: Dimensional reduction UMAP revealed the cellular heterogeneity of this tissue, which encompass 5 highly reproducible clusters across known cell types: fibroblasts, perivascular cells, endothelial, immune, and smooth muscle cells. Specifically, within the latter cluster, canonical marker genes allowed us to distinguish between the main smooth muscle cellular component (DES, ACTG2, CNN1) and the vascular smooth muscle cells (ACTA2, MYH11, MYL9). Also, RGS5 expressing cells denoted a broad population of perivascular cells, that could be separated into two different areas: the endothelium, and the contractile cells surrounding the vessels. Interestingly, within the endothelium, we have also identified an abundant population of cells expressing highly specific cell-cell adhesion molecules related to potential tight regulation of the interchange between circulating blood and smooth muscle. Conclusion: Our data reveal the first molecular and cellular cartography of the human menopausal myometrium through specific single cell transcriptomic signatures. This information is the basis for the of senescence, genomic instability, and changes in the immune and vasomotor system of human myometrium in menopause. Ovaries are reproductive organ in women, the degradation of this results to Primary Ovarian insufficiency (POI) and polycystic ovary syndrome (PCOS) including irregular menstrual cycle, various metabolic disorders and infertility. So far, although hormone replace therapy (HRT) is applied to patient for treatment, but it has restrictions on the appropriate treatment timing and patient age as well as side effects (e.g., breast tenderness, heart disease and carcinogenesis). Hence, Stem cells, which have been reported their potentials including higher proliferation, differentiation and cell to cell communication, are in the spotlight as a treatment to replace HRT. Several reports of ovarian function using various stem cells are increasing, but sill, their evidence is insufficient. Therefore, our aim in this study is to analyze the several factors secreted by stem cells play a major role in ovarian function. Methods: We used mesenchymal stem cells isolated from different organs (i.e., BM-MSCs, AD-MSCs, UC-MSCs) and cultivated up to passage 5. And we analyzed their cytokine factors in cell culture supernatants using by human cytokine array according to their manufacturer's protocol. In briefly, prepared all samples were concentrated by dialysis system and determined protein concentration by BCA assay. After samples labeled by biotin, it was incubated with each membrane at 4℃ for overnight. After each membrane were washed, it was incubated with labeled-streptavidin and detected by immunoblot system. The value of protein expression was measured by Image J with HRP-and Streptavidin positive control area. Also, we analyzed the human dermal fibroblast cells (HDF) and basal media without FBS as control. Results: We divided some factors according to categories of angiogenesis, inflammation, immune response, follicle development, metabolism and growth factor. As a result, nothing was detected in basal media and 3 factors (e.g., SPARC, EDA-A2 and IGFBP7) were detected in HDF. Co-detected factors of BM-MSCs, AD-MSCs and UC-MSCs are divided into angiogenesis (e.g., SPARC, Thrombospondin-1), inflammation, immune (e.g., EDA-A2, MIP1a), follicle development (e.g., Activin A and IGFBP7) and growth factor (e.g., HB-EGF Pregnancy. Lindsay K Doherty, Sean D Cleary, Angelo Elmi, Debra Bernat, Lorien Abroms. George Washington University, Washington, DC, United States. Introduction: Low-certainty evidence suggests that e-cigarettes could be an effective tool for smoking cessation; however, little information is known about the efficacy of e-cigarettes for smoking cessation in the pregnant population. This analysis examined the odds of cigarette smoking cessation and reduction attributed by e-cigarette use among pregnant smokers. Methods: This was a secondary analysis of a randomized trial of a text messaging program for smoking cessation during pregnancy (Quit4Baby). Data were collected in four surveys over six months. Participants with two or more surveys were included (n=463). The primary outcome was smoking cessation, defined as self-reported 30-day point prevalence abstinence from cigarettes. The secondary outcome was smoking reduction of at least 50% fewer cigarettes per day compared with baseline. Past 30day e-cigarette use was defined as self-reported use of e-cigarettes within the past 30 days. Past 7-day e-cigarette use and past 30-day e-cigarette use without past 7-day use were also examined. Multiple imputation was performed for intermittently missing data. Inverse probability weights estimated from logistic regression were applied to account for dropout and time-varying e-cigarette use in marginal structural models (MSMs). Odds ratios and 95% confidence intervals were estimated from both unweighted and marginal structural generalized estimating equations models with working independence correlation structures. The unweighted models indicated that past 30-day e-cigarette use significantly lowered the odds for smoking cessation compared with non-users. However, MSM results indicated that past 30-day e-cigarette users, as well as past 30-day users without past 7-day use, did not have significantly different odds of smoking cessation compared with past 30-day non-users (Table) . Among past 7-day e-cigarette users, odds of smoking cessation were 68% lower compared with past 7-day nonusers and 66% lower compared with past 30-day non-users. While the unweighted model indicated lower odds of smoking reduction in past 30day users, the MSMs found no significant associations between e-cigarette use and smoking reduction (Table) . Conclusion: Past 7-day e-cigarette use was associated with lower odds of smoking cessation, but not reduction, in this sample of pregnant smokers. Past 30-day e-cigarette use was not associated with smoking cessation or reduction. Smoking cessation and reduction by e-cigarette use Introduction: Pregnant women are ubiquitously exposed to phthalates through everyday consumer products. Phthalate exposure may be a risk factor for preterm birth, but most prior studies have had small numbers of preterm births. Our objective was to investigate the association between urinary biomarkers of phthalate exposure in pregnancy and subsequent preterm birth in the United States (US). Methods: Data were pooled from 16 pregnancy studies conducted across the US. A total of 6043 pregnant women, who delivered between 1983-2018 and provided ≥1 urine sample during pregnancy, were included. Preterm birth was defined as <37 weeks of gestation at delivery (n=538). Urinary phthalate metabolites were quantified as biomarkers of phthalate exposure. Concentrations of 11 phthalate metabolites were standardized for urine dilution and repeated measurements across pregnancy were averaged. Logistic regression was used to examine the association between a phthalate biomarker with odds of preterm birth. Quantile g-computation was used to assess the association between preterm birth and the overall phthalate mixture. Models pooled data using fixed-effects and adjusted for maternal age, race/ethnicity, education, and pre-pregnancy body mass index. Results: Phthalate biomarkers were largely detected in >95% of participants. Higher odds of preterm birth, ranging from 12-16%, were observed with an interquartile range (IQR) increase in concentrations of mono-n-butyl phthalate (OR = 1.12, 95% CI = 0.98, 1.28), mono-isobutyl phthalate (odds ratio [OR] = 1.16, 95% confidence interval [CI] = 1.00, 1.35), mono(2-ethyl-5-carboxypentyl) phthalate (OR = 1.16, 95% CI = 1.00, 1.35), and mono(3-carboxypropyl) phthalate (OR = 1.14, 95% CI = 1.01, 1.29). A joint IQR increase in the phthalate mixture was associated with 27% increased odds of preterm birth, but precision was lower relative to single phthalate analyses (OR = 1.27, 95% CI: 0.89, 1.79). Conclusion: Higher exposure to several common phthalates was associated with greater likelihood of preterm birth in a large US sample. The joint effect of exposure to phthalates was greater in magnitude than effects for any individual phthalate. Despite modest effect sizes, phthalate exposure is prevalent in the US and thus may have a substantial public health impact. The Effects of Placental Histopathology and Acute or Chronic Inflammation on Placental Efficiency in Term Newborns. Sadia Firoza Chowdhury †, 1,2,3 Ruchit Shah, 1,4 Mehrin Jan, 1,2 Serena Chen, 1,2 Jillamika Pongsachai, 1,2 Sylvia Dygulski, 1,2 Beata Dygulska, 2 Sanford Lederman, 2 Pramod Narula, 2 Carolyn Salafia * . 1, 2, 4 1 Placental Analytics, LLC, New Rochelle, NY, United States; 2 NYPBMH, Brooklyn, NY, United States; 3 University of Rochester School of Medicine and Dentistry, Rochester, NY, United States; 4 Institute for Basic Research, Staten Island, NY, United States. Introduction: Placental acute or chronic inflammation (AI, CI) is commonly identified as subclinical even in low-risk newborns. Yet, AI and CI and maternal and/or fetal vascular pathology (MVP, FVP) impact placental growth and, by extension, birthweight (BW) and placental weight (PW). We explore if the effects of AI, CI, MVP, and FVP impact placental efficiency in term newborns. Methods: A community hospital in an urban setting mandated a universal placental examination which led to gross and histologic examinations being performed with a standard protocol between January 2010 and March 2015. Inclusion criteria were limited to term singleton liveborns who had follow-up in resident or faculty pediatric practices to at least 2 years of age, and whose placentas had been fully evaluated. In 2226 children, maternal and fetal acute inflammation (MAIR, FAIR) was scored as 0-4 based on the number of affected sites (membranes, chorionic plate-maternal and umbilical cord and chorionic plate vessels-fetal). Chronic placental inflammation (CPI) was scored as "confined to basal plate and maternal-placental interface", and "villous infiltrates/chronic villitis". MVP considered villous fibrosis and lesions such as infarct and thrombosis; FVP was coded to distinguish terminal villous lesions and mural thrombi in the walls of muscular placental vessels. Analyses used nonparametric tests due to non-normal distribution of FPR and beta. Results: In this term population, strong sex-specific effects of both AI, CI impacted placental efficiency as measured by fetoplacental weight ratio (FPR), ponderal index (PI), and beta (ln(PW)/ln(BW)). Fetal and maternal AI impacts placental efficiency only in girls while fetal CI involving the placental villi impacts placental efficiency only in girls. Rates of histopathology lesions were low but their incidences vary by sex. Placentas of male and female newborns do not differ in size, while BW, BL and HC vary by sex. Conclusion: We have previously found associations with fetal AI and CI and conditions such as autism; prenatal inflammatory exposures have also been linked to increased risk of obesity. These sex-specific findings may guide us to understanding of fetal programming mechanisms associated with AI or CI exposure along with the vascular pathology. This project is an update from "Does Placental Acute or Chronic Inflammation Impact Placental Efficiency in Term Newborns in a Low Risk Community Setting" and "Does Placental Histopathology Impact Fetal Placental Weight Ratio in Term Well Newborns?" Placental Histology Patterns of Acute and Chronic Inflammation and Maternal and Fetal Vascular Pathology in ASD. Jillamika Pongsachai, 1,2 Mehrin Jan, 1,2 Sadia F Chowdhury, 1,2,3 Ruchit Shah, 1,4 Serena Chen, 1,2 Sylvia Dygulski, 1,2 Beata Dygulska, 2 Sanford Lederman, 2 Pramod Narula, 2 Carolyn Salafia * . 1, 2, 4 1 Placental Analytics, LLC, New Rochelle, NY, United States; 2 NYPBMH, Brooklyn, NY, United States; 3 University of Rochester School of Medicine and Dentistry, Rochester, NY, United States; 4 Institute for Basic Research, Staten Island, NY, United States. Introduction: A smaller study in this population with universal placental examination showed specific patterns of placental acute and chronic inflammation with significant odds of autism case status. We increased our autism sample size to confirm these findings. While we now know that oxidative stress pathways are identified as abnormal in children with autism spectrum disorder (ASD), vascular pathology in ASD is unknown; so we examined maternal and or fetal vascular pathology (MVP, FVP) in the placenta in ASD in a community-based sample. Methods: A community hospital-based sample with universal placental examination was searched for billing codes and medical records of developmental pediatrician visits for autism spectrum disorder (ASD) diagnoses among children. At least 2 ASD-related diagnoses as per Newschaffer et al were required to be an ASD case. Controls were selected as the next infant born of same sex, gestational age +/-2 weeks, and season of birth +/-2 weeks followed up to 2 years of age at our hospital with no diagnoses. Maternal and fetal acute inflammation (MAIR, FAIR) was scored as present/absent in the affected sites. Chronic placental inflammation (CPI) was scored as "confined to basal plate and maternalplacental interface", and "villous infiltrates/chronic villitis". Maternal uteroplacental vascular pathology (UVP) considered acceleration of villous maturation and lesions such as infarct and thrombosis; fetal vascular pathology (FVP) was coded to distinguish terminal villous lesions and mural thrombi in the walls of muscular placental vessels. Results: Placental coding was completed for 206 ASD cases and 583 controls. Only chronic anchoring villitis demonstrated a significant (but small) increased odds of ASD case status. CEM matched logistic regression shows only histology of abruption and reduced villous branching. Conclusion: There's an association of ASD case status with chronic inflammation of the placental villi that anchor into the maternal decidua, but not with chronic villitis from the basal plate. A lesion reflecting abnormal villous branching was significantly more common in ASD cases. Acceleration of maturation, shown by reduced villous branching, in a population of sporadic ASD suggests abnormal placental vascular branching at the capillary level as important to both genetic and sporadic ASD. This is an update for "Placental Histology of Acute and Chronic Inflammation in a Population Based Case Control Study of Autism" and "Roles of Maternal and Fetal Vascular Pathology in a Case-Control Study of Autism". Examining Weight Status and Weight Loss on Menstrual Cycle Length and Variability Among Reproductive Aged Women. Annie Caldwell, 1 Priyanka deSouza. 2 1 University of Colorado | Anschutz School of Medicine, Aurora, CO, United States; 2 University of Colorado, Denver, Denver, CO, United States. Introduction: Menstrual cycle disruptions have been observed when energy stores are low (underweight) or high (obesity), and when energy availability (energy in -energy out) is low in normal weight women during calorie restriction and/or increases in exercise. It is not currently known if these disruptions are reflected in menstrual cycle length or variability, or if disruptions occur when energy availability is low among women with obesity. Methods: This study included menstrual cycle length data from a large sample of reproductive aged women (18-40) who were regularly cycling (cycle length between 18-40 days), and not using hormonal birth control who were either using the CLUE app (underweight, normal weight, obese); and women participating in the Hormones during Weight Loss (HoWL; K01 HL 143039) study (obese with low energy availability). Women from CLUE had cycle length data over 12 months while those in HoWL had 4 months-the month prior to and the first 3 months of a behavioral weight loss intervention targeting a weekly energy deficit of 34% and a prescription to increase moderate intensity exercise (300 mins/week). The within-woman mean and coefficient of variation (CV) in cycle length over 12 months (CLUE) and 4 months (HoWL) was calculated, and we descriptively compare the mean of mean cycle length and variability across groups. Results: A total of n=1510 women were included from CLUE, (n=64 underweight; n=623 normal weight; n=621 with obesity), and n=19 from HoWL. Menstrual cycle length was notably similar across groups: underweight=28.49; normal weight=28. 11; obese=28.31; and HoWL=28.25 . Variability in cycle length among women in HoWL was lower (CV=7.2%) compared to the mean variability observed in CLUE (underweight=8.1%; normal weight=7.9%; obese=8%). However, the HoWL sample was much smaller and included fewer months of menstrual cycle data. Conclusion: These data suggest that among regularly cycling women, menstrual cycle length is remarkably preserved across weight status and during energy restriction, with rather high within-woman variability on average. Cycle length does not appear to be a good indicator of the underlying hormonal or ovulatory characteristics of the menstrual cycle that are disrupted by insufficient and surplus energy stores and low energy availability. Clinical Trials. Bhuchitra Singh †, 1 Ayman Al-Hendy, 2 Hugh Taylor, 3 Valerie Baker, 1 James Segars * . 1 1 Johns Hopkins School Of Medicine, Baltimore, MD, United States; 2 University of Chicago, Chicago, IL, United States; 3 Yale University, New Haven, CT, United States. Introduction: On January 25, 2018 the new Final Rule approved by the DHHS required all non-exempt, multi-site, domestic, human subject research protocols funded by the NIH to use a single IRB (sIRB). The aim of this study was to evaluate the overall functioning of the sIRB in the NICHD funded CONFIRM consortium of linked R01s and to identify the bottlenecks in the process of IRB approval for the multi-site studies. Methods: All funded studies in the consortium utilized the Johns Hopkins Medicine single IRB. All IRB applications for the linked R01s were prepared and monitored by the same team. The time required to accomplish an IRB approval and the number of modifications required to get the first approval was collected. Except for 2 sites, all of the other participating sites were SMART IRB platform signees. The time for participating sites to be onboarded was determined. Results: In total there were 16 participating sites. There were 7 for NatPro, 4 for Pre-FRIEND and 5 for the PREGnant study. On average, the initial approval required 3.96 months (2.00-6.21 months). The average number of issues raised by the sIRB prior to initial approval were 6, ranging between 3 to 8 queries. The onboarding time was defined as the duration between receipt of fully executed local context questionnaires and site-specific informed consent forms from the local site to final sIRB approval for that site. On average, the local sites took 2.89 months to return documents back to the sIRB. For the NatPro study participating sites, the average sIRB onboarding time was 1.90 months (1.67-2.13 months), for the Pre-FRIEND study it was 0.61 months (0.49-0.85 months). The sIRB onboarding process was noted to become more efficient over intervals of data collection. The greatest bottleneck was due to IND approvals, averaging 2.20 months. The sIRB reviewers' comments were favorable of the research strategy for all three studies. Conclusion: Even though the implementation of sIRBs was abrupt in response to changes in DHHS policy, the total time to approvals was similar to single-site IRB reviews for the initial review of the study protocols. Delays were introduced because most local IRBs insisted on reviewing the study protocol despite signing a Letter of Indemnification to the sIRB. Efficiency could be improved by addressing this bottleneck. The Impact of Race and Ethnicity on New Patient Conversion Rates. Tyler Soy. Vios Fertility Institute, Chicago, IL, United States. Introduction: In the attempt to create equity amongst patients of different racial identities, fertility clinics across the country have made efforts to expand their patient populations by implementing more effective methods of patient outreach. These tactics, along with an increase in treatment accessibility and provider representation have led to an increase in fertility patients from minority communities. The subsequent question is whether these patients of color are actually receiving the fertility treatment that they are in need of, once initiated as a new patient. Many start the process but do not move forward with treatment options. This study sought to investigate whether patients of color have lower rates of conversion from preliminary evaluation to treatment initiation. Methods: Retrospective cycle review of a private, multi-site fertility clinic. All patients who have self-reported race or ethnicity and also initiated a new patient evaluation from April 2016 to August 2021 were included. Patients were divided into self-reported ethnic/racial groups, White (non-Hispanic or Latino), Native American, Asian, Black, Hispanic/ Latino, Pacific Islander, and "other". Within each classification, patients were split into two further groups; those who continued with any form of fertility treatment and those who solely engaged in a new patient evaluation. Treatment cycles included in this study were IVF, IUI, OI, or FET. The differences in these two groups were quantified and converted into averages that this study defines as a "conversion rate". These rates were analyzed through sample t-tests to test for statistical significance using R. Results: A total of 6451 patient cycles consisting of 4649 White (non-Hispanic or Latino) (72%), 647 Black (10%) , 617 Asian (9%) , 412 Hispanic/Latino (6%), 15 Native American (>1%), 13 Pacific Islander (>1%), and 98 "Other" (2%) patient cycles were analyzed. The new patient conversion rates report as follows: 56% (White), 36% (Black), 54% (Asian), 52% (Hispanic), 60% (Native American) , 38% (Pacific Islander) and 44% ("other"). The difference between new patient conversion rates for white patients (56%) was found signifigant when compared to the average rate for non-white patients (47%, p =0.01) Conclusion: Our results suggest that inequities in fertility health goes further than improvements in diverse patient recruitment and expands to bridging the equity gap found in minority groups' usage of fertility treatment. With further evidence, this provides precedence for implementations that will lead to a higher retention of fertility patients of color. Introduction: Racial disparities in pre-existing diabetes (PDM) and gestational diabetes (GDM) remain largely unexplored despite associated adverse maternal and fetal outcomes. We examine national trends in PDM and GDM prevalence by race/ethnicity, and the association between these conditions and fetal death. Methods: A retrospective cross-sectional analysis was conducted using 2002 to 2017 pregnancy-related hospitalization records from the Nationwide Inpatient Sample. Exposures included PDM and GDM, and the outcome was stillbirth. Joinpoint regression was used to evaluate trends in prevalence over the study period. Survey logistic regression was used to evaluate the association between exposures and stillbirth. Reducing Preterm Birth and Racial Disparities: A Model of Stress-Induced Developmental Plasticity. Gabriella Mayne †, 1 Ayisha Buckley †, 2 Luwam Ghidei †. 3 1 University of Colorado, Denver, Denver, CO, United States; 2 Icahn School of Medicine at Mount Sinai Hospital, New York, NY, United States; 3 Baylor College of Medicine, Houston, TX, United States. Introduction: Preterm birth is a leading cause of neonatal mortality and is characterized by substantial racial disparities. Despite efforts to reduce preterm birth, rates continue to rise while racial disparities persist. Maternal stress is a risk-factor for preterm birth; however, often it is treated as a secondary variable rather than a key target for intervention. Stress is known to affect several biological processes leading to downstream sequelae. Methods: In this commentary, we present evidence of a model for stress-induced developmental plasticity where maternal stress is a key environmental cue impacting the length of gestation and therefore a primary target for intervention. We discuss what this model reveals about racial disparities in preterm birth and ultimately how to reduce preterm birth and the accompanying racial disparities. Results: Developmental plasticity is the biological capacity to alter developmental trajectories in response to environmental cues and results in phenotypic variation, including preterm birth. During pregnancy this plasticity is mediated through the maternal-fetal-placental neuroendocrine stress axis. Black women experience disproportionate and unique maternal stressors related to racism and discrimination. It is therefore not surprising that Black women have disproportionate rates of preterm birth. The downstream effects of racism on preterm birth pathophysiology reflect an appropriate response to stressors through this ancient, environmentally sensitive system. Fortunately, stress does not appear to be an all-ornone variable. Evidence suggests developmental plasticity is dynamic, functioning on a continuum. Conclusion: Therefore, simple, stress-reducing interventions that support pregnant women may tangibly reduce rates of preterm birth and improve birth outcomes for all women, particularly Black women. Introduction: COVID-19, caused by SARS-CoV-2, has significant impact pregnant women worldwide. Placenta is the structural and immunological barrier to limit pathogen transmission. SARS-CoV-2 can infect placenta, but little is known the response of placental innate immune system to maternal COVID-19 infection. Type I IFNs are considered potent antiviral cytokines. The objective of the study is to determine IFN induction pathway molecule expression in the placenta to test our hypothesis that innate antiviral immune system is activated at the placental maternal-fetal interface in response to COVID-19 infection. Methods: A total of 20 placentas were examined in this study, 12 from women infected with COVID-19 during pregnancy and 8 from women who were never infected with COVID-19 before and during pregnancy served as control. This study was approved by the Institutional Review Board (IRB) at Louisiana State University Health Sciences Center -Shreveport (LSUHSC-Sh). Patient demographic data and placental pathology report were assessed via medical record review. Placental formalin-fixed paraffin embedded tissue sections were used. In situ hybridization was performed to detect SARS-CoV-2 RNA expression and immunohistochemistry was used to evaluate expression of antiviral molecules STING, IRF3, MAVS, TLR7 and IFNβ in placental villous and fetal membrane tissue sections. Results: Among the 12 COVID placentas, SARS-CoV-2 RNA reactivity was detected in 6 placentas. The viral reactivity was mainly localized in syncytiotrophoblasts (STCs). Compared to non-COVID-19 control placentas, expression of STING, IRF3, MAVS, TLR7, and IFNβ were all upregulated with distinct cell-type specific distribution and pattern in placentas exposed to maternal COVID-19 infection, with STING in villous and decidual stromal mesenchymal cells; IRF3 in cytotrophoblasts (CTCs) and extravillous trophoblasts (EVTs); and MAVS, TLR7, and IFNβ in CTCs, EVTs, and STCs, and/or fetal vessel endothelial cells, respectively. Conclusion: STING, IRF3, MAVS, and TLR7 are key viral sensing molecules that regulate type I IFN production. Type I IFNs (IFNα/β) are potent antiviral cytokines to impair and eradicate viral replication in infected cells. This study is the first to demonstrate activation of type I IFN signaling pathway, with distinct cell-type specific distribution at the maternal-fetal interface in response to COVID-19 infection. Our findings also suggest that placental maternal-fetal interface has a welldefined antiviral defense system to restrict virus transmission and protect developing fetus from infection. Immunological Impact of SARS-CoV-2 and Pandemic Stress at the Maternal-Foetal Interface. Marie-Eve Brien †, 1 Elsa Bernier †, 2 Yasmine Kebiche †, 2 Josianne Clavel †, 1 Dorothée Dal Soglio †, 2 Isabelle Boucoiran †, 1,2 Sylvie Girard * . We recently published that over 80% of SARS-CoV-2+ pregnant women had placental abnormalities vs 25% in uncomplicated pregnancies. Interestingly, women exposed to pandemic-related stress solely but SARS-CoV-2-still had elevated rate of placental lesions (50%). Expanding on this work, we further assessed the inflammatory profiles at the maternal-fetal interface in relation to pandemic related stress +/-SARS-CoV-2 infection. Methods: We recruited 200 women at the CHUSJ between March 23 rd 2020 and July 14 th 2021. 80 had confirmed SARS-CoV-2 infection whilst 120 were negative and delivered during the same period (exposed to stress only). We also included historic controls (i.e. deliveries between April 14th 2016 and October 7th 2019, N=40). Placenta and fetal membranes samples were collected rapidly after delivery, snap frozen or fixed for protein and histological analysis. Maternal demographical data and obstetrical outcomes were evaluated through extensive review of the medical chart. Results: SARS-CoV-2+ women were predominantly from Black or Middle Eastern origins vs Caucasian in the other groups. Most women were positive in their 3 rd trimester (65%) and the rate of preterm birth was 16% in the SARS-CoV-2+ group vs 6% in the negative. In terms of inflammatory mediators, SARS-CoV-2+ women had elevated CRP and IL-1Ra levels in their placenta vs both negative and Ctrl (p<0.001). Surprisingly, placentas from pandemic stress-exposed women had increased level of IL-6 and MCP-1 (p=0.07 and p<0.05 respectively vs SARS-CoV-2+). Furthermore, anti-inflammatory cytokines IL-10 and IL-4 were elevated in both SARS-CoV-2+ and -groups (p<0.01) suggesting a potential protective mechanism within the placenta. Conclusion: The placental inflammatory profiles differed between SARS-CoV-2 status during pregnancy and was interestingly, also observed in placentas exposed to stress solely. This highlights the need to differentiate between the effects of pandemic-related stress and the added burden of SARS-CoV-2 infection on placental health and pregnancy outcomes. Current analysis are ongoing to assess the relationship between these inflammatory markers and placental lesions and relation to the trimester of infection. Mice. Patrick S Creisher †, 1 Ariana D Campbell †, 1 Ruifeng Zhou, 1 Jun Lei, 2 Kathleen R Mulka †, 2 Joseph L Mankowski, 2 Wayne Mitzner, 1 Andrew Pekosz, 1 Irina Burd, 2 Sabra L Klein * . 1 1 Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States; 2 Johns Hopkins School of Medicine, Baltimore, MD, United States. Introduction: Pregnant people are at an increased risk of severe complications of SARS-CoV2 infection and COVID19 compared to nonpregnant people. Pulmonary and immunological changes that occur during pregnancy, which can be investigated in animal models, may affect SARS-CoV-2 pathogenesis; animal models of SARS-CoV2 infection during pregnancy, however, are lacking. Methods: Pregnant outbred CD1 mice were used to model the genetic heterogeneity of humans, including maternal immune tolerance at the maternal-fetal interface during pregnancy. Pregnant CD1 mice were infected at embryonic day 10 with 10^5 TCID50 units of mouse adapted SARS-CoV2 (ma10 SARS-CoV2) or mock infected. Dams were followed for 7 days for morbidity (i.e. body mass change, rectal temperature, and signs of sickness) and mortality, with subsets euthanized throughout infection to measure pulmonary function, viral lung titers, and cytokines and inflammation in lung, spleen, and placenta tissue. Fetuses were weighed prior to flash freezing and pups were weighed at birth to evaluate intrauterine growth restriction. Results: Maternal SARS-CoV2 infection reduced the body mass gain throughout pregnancy compared to mock inoculated dams; although, there was no change in rectal temperature or significant signs of sickness. Fetuses collected from ma10 SARS-CoV2 infected dams were significantly smaller than those of mock inoculated dams at 7 days post infection. Assessment of viral, pulmonary, and immunological measures are ongoing. Conclusion: These preliminary results suggest that ma10 SARS-CoV2 infection of pregnant CD1 mice models the morbidity and adverse pregnancy outcomes associated with SARS-CoV2 infection of pregnant people. Future studies will investigate the role of inflammation versus viral dissemination in mediating morbidity and adverse pregnancy outcomes. Immunohistochemical Localization of ACE2 in the Male Reproductive Tract in the Rhesus Macaque: Implications for Nonhuman Primate Model Development for COVID-19. Hayly Hinkle, Sierra Block †, Ann Mitzey, Jenna Schmidt, Gregory Wiepz, Thaddeus G. Golos * . University of Wisconsin-Madison, Madison, WI, United States. Introduction: Angiotensin-converting enzyme 2 (ACE2) is a primary receptor for the SARS, MERS and SARS-CoV-2 coronaviruses. While its distribution has been described extensively in humans, protein expression has not been comprehensively reported in nonhuman primates (NHPs). NHPs provide critical animal models for understanding disease pathogenesis and establishing platforms for testing therapeutic interventions. With reports of SARS-CoV-2 detected in human semen, we conducted immunohistochemical staining for ACE2 in organs of the male rhesus macaque reproductive system. Methods: Testis, epididymis, ductus deferens, seminal vesicles, prostate, and bladder specimens were obtained at necropsy of 3 male rhesus monkeys, ranging in age from 5 to 8 years, and two fetal testis specimens. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned for immunostaining with anti-ACE2 antibodies. Kidney tissue sections were included in all staining experiments as positive controls. Semen was collected by electroejaculation from two additional males. Results: Intense expression of ACE2 was noted in the seminiferous tubules of all subjects examined. The pattern of staining suggested that Sertoli cells as well as spermatogenic cells of the testis highly expressed ACE2, however the densely packed nature of the tubules makes identification of individual cells complicated. Staining of fetal testis confirmed Sertoli cell ACE2 expression. There was, in general, diminished staining intensity in the downstream organs of the reproductive tract, with moderate expression in the caput epididymis, fainter expression in the caudal epididymis, and fainter/undetectable staining in the epithelia in the ductus deferens and seminal vesicles. Staining was modest and inconsistent in prostate secretory units, and faint in the transitional epithelium of the prostatic urethra and the bladder. Western blot of sperm cell pellets and seminal plasma indicated that ACE2 was associated with the cell pellet but not seminal plasma in samples from the two males evaluated. Conclusion: ACE2 is highly expressed in the testis, and western blot demonstration of ACE2 protein in the cell pellet of fresh semen samples suggests that cells of the spermatogenic lineage as well as Sertoli cells express ACE2 in seminiferous tubules. Immunostaining was not completely consistent with previous reports of ACE2 mRNA from scRNAseq analysis and may indicate that there is translational control of ACE2 expression, which is important to take into consideration when interpreting the pathophysiological outcomes of experimental infection in animal models. The presence of ACE2 in the testis may have implications for reproductive health, fertility, and sexual transmission. During the COVID-19 Pandemic. Danielle Mari Panelli, Mira Diwan, Giovanna I. Cruz, Stephanie A. Leonard, Jane Chueh, Ian H. Gotlib, Katherine Bianco * . Stanford University, Stanford, CA, United States. Introduction: Short leukocyte telomere length (LTL) is a marker of cellular aging, but little is known about it in pregnancy. Hypothesizing that pregnancy accelerates telomere shortening, we compared LTL between pregnant and non-pregnant people. Given the emergence of the coronavirus 2019 (COVID-19) pandemic and the association between LTL and stress, we also evaluated mental health measures. Methods: This was a prospective cohort of nulliparous pregnant and non-pregnant people between 18 and 50 who presented for obstetric or gynecologic care at a single institution from January to November 2020. Pregnant people were enrolled between 10w0d and 14w0d. All participants had two blood samples drawn for LTL; the first on the day of enrollment (Time 1) and the second approximately 7-8 months later (Time 2). LTL was measured using quantitative PCR and converted from telomere ("T") to single copy gene (human beta globin, "S") ratios to basepairs (bp). The primary outcome was LTL change rate between Time 1 and Time 2. Secondary outcomes included responses to the Patient Health Questionnaire-9 (PHQ-9) for depression and a survey about stress related to the COVID-19 pandemic. Differences in LTL were assessed using t-tests and linear regression models, crude and adjusted for age and comorbidities. Secondary outcomes were analyzed using Wilcoxon rank sum or Fisher's exact tests. Results: Of 76 people enrolled, 53 completed all assessments: 17 were non-pregnant and 36 were pregnant. There were no differences in the rate of telomere change between the groups (-6.5 ± 11.6 basepairs/30 days in non-pregnant versus -0.6 ± 20.5 in pregnant, p=0.19) . Similarly, the two groups did not differ in PHQ-9 depression scores. The majority of participants reported moderate or significant worsening of stress due to Table 2 ). Conclusion: In contrast to our hypothesis, our pregnant cohort did not have accelerated LTL shortening or worse mental health deterioration during the COVID-19. Whether this is due to our small sample size or a biologic effect related to resilience warrants further investigation. Immortalization and Characterization of Human Fetal Membrane and Maternal Decidua Cells. Enkhtuya Radnaa, Rheanna Urrabaz-Garza †, Ananth Kumar Kammala †, Ramkumar Menon * . University of Texas Medical Branch at Galveston, Galveston, TX, United States. Introduction: Human fetal membrane (FM) and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. The structural and mechanical integrity of this interface is essential for pregnancy maintenance and promotes parturition. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience diffi culties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. So, we report the creation of well characterized immortalized cells from primary isolates Methods: Primary cells were isolated from a normal healthy full-term, not in labor placenta. Primary fetal derived-AEC, AMC, CMC, and maternalderived DEC were immortalized using HPV16E6E7 retroviral system, and fetal-CTC were immortalized using SV40T lentiviral system. Infected cells were then sorted out with antibiotic selection (G418 and puromycin, respectively). The immortalized cells were characterized for the morphology (microscopy), cell type-specifi c markers (immunostaining), and cell signaling pathway activation (WB). Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats (STR) DNA analysis. Results: Immortalized AEC, AMC, CMC, CTC and DEC cells show their characteristic morphology (Fig.1) , and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated in AEC (R=0.857), AMC (R=0.812) CMC (R=0.798), chorion trophoblast cells (R=0.949) and in decidua (R=0.974) (Fig.2) . STR DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (1.5 (p value ≤0.05) between the TNL and TIL groups. Principal component and hierarchical clustering analyses grouped the samples according to their origin. Gene ontology (GO) analysis showed an over-representation of inflammatory and epithelial-mesenchymal transition (EMT) genes in TIL-cells. Differential accessibility analysis identified 15,378 openchromatin regions, of which 66 % were more accessible in TIL samples. The GO analysis shows again over-representation of genes linked to inflammation and EMT. We used Blood Transcription Modules to refine the analysis with a specific focus on immune cells: overexpressed genes in TIL were significantly linked to myeloid cells specific modules. FC analyses using a panel of 14 colors confirmed the presence of at least 20% of hematopoietic cells (CD45 + ). Several types of macrophages cells were found in different proportions between the two conditions, suggesting phenotypic myeloid heterogeneity. Conclusion: Our data confirmed that cells are keeping specific features of the tissue origin, both at the transcription and chromatin levels, even after one week of culture in vitro. Furthermore, myeloid subsets undergo changes in proportion and/or phenotype with labor that need to be deepen analyzed. The Role of Clarithromycin to Prevent Preterm Birth Induced by Sterile-Intra-Amniotic Inflammation. Jose Galaz †, 1 Roberto Romero * , 2 Marcia Arenas-Hernandez †, 1 Kenichiro Motomura †, 1 Nardhy Gomez-Lopez * . 1 1 Wayne State University, Detroit, MI, United States; 2 NICHD/ NIH/DHHS, Detroit, MI, United States. Introduction: Preterm birth, the leading cause of perinatal morbidity and mortality worldwide, is a syndrome of multiple etiologies. The only established causal link for this syndrome is intra-amniotic inflammation, which can be driven by microbes (intra-amniotic infection), or by alarmins, such as HMGB1 (sterile intra-amniotic inflammation, SIAI). Importantly, SIAI is more common than intra-amniotic infection in women with preterm labor and intact membranes. However, there is no clinically approved treatment for such a pathology. Therefore, we aimed to evaluate the potential utility and the mechanisms whereby clarithromycin (CLR), a macrolide with immunomodulatory properties that is approved for use during pregnancy, can serve as a treatment for sterile intra-amniotic inflammation. Methods: Pregnant mice received ultrasound-guided intra-amniotic injection of HMGB1 (n=10) as a model of SIAI or PBS (n=9) as control on 14.5 days post coitum (dpc). A second cohort of dams underwent intra-amniotic injection of HMGB1 (n=15) and were treated with CLR or vehicle control (n=15 each) after 6, 12, 24, 48, 72, and 96 hours. Pregnancy and neonatal outcomes were observed. Maternal and fetal tissues were collected on 18.5 dpc (before delivery) from a third cohort of dams intraamniotically injected with HMGB1 and treated with CLR or vehicle control (n=8 each) to evaluate inflammatory mediator concentrations in amniotic fluid and to assess gene expression in maternal and fetal tissues using high-throughput qPCR. Results: Intra-amniotic administration of HMGB1 shortened gestational length, increasing the rate of preterm birth compared to controls and validating our previously established model. Treatment with clarithromycin restored gestational length, decreasing the rate of preterm delivery. Importantly, a higher proportion of neonates born to dams treated with clarithromycin thrived until infancy compared to controls. Treatment with clarithromycin modestly reduced intra-amniotic inflammation prior to labor. Moreover, clarithromycin regulated the expression of key molecules involved in the process of parturition such as Oxtr, Il1α, and Il1β in the uterus as well as Gja1 and Mmp9 in the cervix. Notably, clarithromycin also downregulated the expression of multiple inflammatory transcripts in fetal tissues such as lungs, intestine, liver, and spleen. Conclusion: Intra-amniotic inflammation induced by HMGB1 induces preterm birth and neonatal mortality, which can be prevented by clarithromycin. The mechanisms whereby this macrolide prevents preterm deliveries are through the regulation of key molecules in the common pathway of labor as well as the modulation of inflammation in the fetus. This study provides evidence supporting the use of clarithromycin as a treatment for sterile intra-amniotic inflammation. Scott D Barnett † * , Hazik Asif †, Iain L.O. Buxton. University of Nevada, Reno School of Medicine, Reno, NV, United States. Introduction: Approximately 10-12% of US births are preterm. Tocolytics currently in use are ineffective at delaying preterm birth because most are 'generalized' smooth muscle modifiers not designed to target novel myometrial-specific pathways. Hypothesis: Mechanosensitive signaling in endothelial and myocyte compartments in pregnant uterus regulates quiescence. As pregnancy proceeds, increases in blood flow are sensed by micro-vascular endothelial cells (MEC). Gradual stretch as gestation proceeds is sensed by both MEC and uterine smooth muscle cells (USMC) resulting in relaxation in an NO-dependent fashion. Stretch-activation of Piezo1, an inward-rectifying cation channel preferential to Ca 2+ permeation, modulates Akt-dependent phosphorylation of endothelial nitric oxide synthase (eNOS) increasing NO production. NO acts via S-nitrosation of myometrial proteins rather than cGMP elevation. Here we seek to identify the expression and activity of Piezo1 in USMC and MECs, as well as the therapeutic potential of Piezo1 modulation as a tocolytic strategy. Methods: Organ Bath: Myometrial strips (n=3-5 TNL) were isometrically stretched in an organ bath then sequentially challenged with KCl (60 mM) and oxytocin (8 nM). Tissues were treated with either the Piezo1 agonist, Yoda1 (0.1-30 µM, cumulative -15 min intervals), or the Piezo1 antagonist Dooku1 (10µM). Some tissue were pre-treated with L-NNA (100µM) or the AkT/PKB inhibitor FPA-124 (10µM). Data were analyzed with LabChart®. Western Blot: Myometrial whole tissue lysate (n=5), or CD31+/-cell isolations (n=3) were analyzed for Piezo1 expression (Piezo1:GAPDH). Results: In myometrium, expression of Piezo1 remained consistent throughout pregnancy and parturition, with the exception of preterm (PTL), in which expression was decreased by 57.7% (±19.84) when compared to TL. Further, expression of Piezo1 in USMC (TNL, CD31-) was 59.5% (±9.2) of MEC (CD31+). Treatment of myometrial strips with a Piezo1 agonist (Yoda1) decreased contractions (AUC) in a dosedependent manner, resulting in an IC 50 of ~3µM, which was mitigated with the Piezo1 antagonist (Dooku1, 10µM). Additionally, pretreatment of tissue with the eNOS inhibitor, L-NNA (100µM), eliminated the effects of Piezo1, while pretreatment with FPA-124 (10µM), an Akt/PKB inhibitor, reduced the IC 50 Yoda response by 53.5% (±3.9), indicating a partial role of Akt in Piezo-1 induced relaxations. Conclusion: These data identify the mechanosensitive Piezo1 channel as a tocolytic target of interest. Not only is its expression down-regulated in PTL myometrium, potentially decreasing the availability of NO and protein S-nitrosation (quiescent modifiers) in uterine myocytes, but Piezo1 agonism serves as potent negative inotropic regulator of contractions. Taken together, these data highlight the importance of stretch-activated Ca 2+ regulation in myometrial tissue during pregnancy and parturition. Progesterone-Responsive Expression of ANKRD1 in Human Myometrial Cells. Emily Hamburg-Shields †, 1 Callista Smith, 2 Sam Mesiano * . 2 1 MetroHealth Medical Center, Cleveland, OH, United States; 2 Case Western Reserve University, Cleveland, OH, United States. Introduction: The molecular mechanisms by which progesterone maintains uterine quiescence remain incompletely understood. Elucidation of the uterine quiescent state is fundamental to the understanding of parturition and the pathophysiology of preterm birth. ANKRD1 is a gene that is expressed in human myometrium in the gravid state. It encodes ankyrin repeat domain 1, a protein that interacts with sarcomeric proteins, has DNA-binding function, and inhibits nuclear factor-kappa B (NF-kappa B) in cardiomyocytes. Its precise function in myometrial cells is unknown. We hypothesized that ANKRD1 is differentially expressed in response to progesterone signaling modulation and may act as a downstream mediator of progesterone signaling in this cellular context. Methods: To test our hypothesis, we examined the expression of ANKRD1 in immortalized human myometrial cells expressing progesterone receptor (PR) isoform A and isoform B (hTertA/B) in response to progesterone, mifepristone (a progesterone receptor antagonist), and promegestone (a selective progesterone receptor modulator). We measured expression of ANKRD1 relative to GAPDH using quantitative reverse-transcription polymerase chain reaction and results are pooled biological replicates from 3 independent experiments. Results: Human myometrial cells demonstrated constitutive (ligandindependent) expression of ANKRD1. Treatment with progesterone resulted in a 2-fold increase in expression of ANKRD1. Treatment with mifepristone resulted in a 0.6-fold change in relative ANKRD1 expression. This mifepristone-induced decrease in expression of ANKRD1 was normalized by the addition of progesterone. Addition of promegestone also normalized the relative expression of ANKRD1 in the presence of mifepristone. Conclusion: These results demonstrate that expression of ANKRD1 is modulated by manipulation of progesterone receptor activity via progesterone, mifepristone, and promegestone. The encoded protein has plausible functions in myocyte contractile behavior, regulation of NF-kappa B signaling, and regulation of gene expression, which will be explored in future studies. The Effect of Secretor Status and the Vaginal Microbiome on Birth Outcome. Samit Kundu * , Yun Lee, Lynne Sykes, Denise Chan, Holly Lewis, Richard Brown, Lindsay Kindinger, Anne Dell, Ten Feizi, Stuart Haslam, Yan Liu, Julian Marchesi, David MacIntyre, Phillip Bennett * . Imperial College London, London, United Kingdom. Introduction: Histo-blood group antigens (HBGA) are found on a range of cell types and in secretions such as cervical and vaginal. These antigens serve as attachment sites and energy substrates for microbes. The FUT2 gene encodes a fucosyltransferase enzyme, which fucosylates a glycan precursor forming the H antigen. Mutations in FUT2 have been identified that result in non-secretion of HBGA. We hypothesised that the composition of the vaginal microbiota in pregnancy might be influenced by maternal secretor status, with consequences for gestational length. Methods: We sequenced the second exon of the FUT2 gene to infer secretor status and undertook metataxonomic analysis of vaginal swab samples collected longitudinally from a cohort of 313 pregnant women. Results: We identified 28% of the cohort as non-secretors with the highest proportion in Afro-Caribbean women, 34.6%. Co-occurrence networks showed contrasting patterns of interactions in the microbiota of secretors and non-secretors: Lactobacillus species, in particular L.crispatus, were observed to have a greater proportion of negative edges in the nonsecretors. Regression modelling of gestational length showed that nonsecretors with Lactobacillus-depleted microbiota in early pregnancy had significantly shorter pregnancies compared with Lactobacillus-dominated non-secretors (p=0.045). This difference in gestational length was not observed in the secretors (p=0.28). Our results indicate that lactobacilli, particularly L. crispatus, are more refractory to co-colonisation in non-secretors and may offer a greater "protective" effect via competitive exclusion. Given this evidence of a more "protective" role of L.crispatus in non-secretors, this microbe represents a biotherapeutic candidate for these pregnancies. Dominance of the vaginal niche by lactobacilli is a hallmark of vaginal health, but our results demonstrate that this effect is nuanced by host secretor status. An enhanced inflammatory response, previously documented in non-secretors, may provide a mechanism linking gestational length to secretor status and the vaginal microbiota. Stratification by maternal secretor status offers a targeted intervention of "at-risk" pregnancies. Histomorphological Approach to Establish a Signature of Cervix Ripening in Women. Sandra E. Reznik, 1 Brigitte Vazquez, 2 Cynthia Janku †, 2 Sarah Chan †, 2 Gregory Dickinson, 1 Pe'er Dar * , 1 Steven M Yellon * . 2 1 Albert Einstein College of Medicine, Bronx, NY, United States; 2 Loma Linda University, Loma Linda, CA, United States. Introduction: Premature ripening of the cervix is associated with spontaneous preterm birth, the major cause of neonatal morbidity and mortality worldwide. In rodents, reduced cell nuclei density (CN), increased macrophage density (Mφ/CN) and degradation of cross-linked collagen in the prepartum cervix stroma are features of inflammation during ripening. Translating these endpoints to pregnant women is challenging due to sparsity of comparable peripartum reproductive tissues. Specimens from high-risk obstetrical patients at the Montefiore Health Center provided the opportunity to test the hypothesis that characteristics of inflammation are similar in the lower reproductive tract at delivery whether at term or preterm. Methods: With IRB approval, continuous full-depth tissue strips from the external cervical os to the lower uterine corpus were obtained from multipara women after post-delivery hysterectomy at 32-34 weeks (preterm not in labor, n=4) or 37-39 weeks (term, n=3, 2 post-induction, 1 not in labor). Fixed specimens were blocked, paraffin-embedded, and stained for Mφ (CD68) and CN (methyl green) or smooth muscle actinalpha (SMA-α). Approximately 10 photomicrographs/region from each whole section scan (Leica-Aperio) were subjected to a rigorous regionspecific analysis using the RandomSpot software plugin (Univ Leeds, UK) to assess Mφ and CN densities, as well as SMA-α area (NIH FIJI). Results: Mφ densities were higher at term than at preterm in the ectocervix and isthmus of the uterus (p<0.05, Student's t-test). However, Mφ densities were unchanged across reproductive tract regions of ectocervix, endocervix, internal os, and isthmus in both preterm and term specimens (p>0.05, ANOVA). As expected, SMA-α area appeared more abundant in the uterine corpus compared to the endocervix but did not differ between preterm and term. CN density was constant across anatomic regions and between term and preterm deliveries. Conclusion: Despite the small numbers of cases in this study, more Mφ/ CN in the ectocervix and isthmus in women at 37-39 weeks versus 32-34 weeks at delivery implies greater inflammation and ripening in these subregions as pregnancy nears term. Other morphologic features are unlikely to forecast preterm or term delivery. Importantly, histological confirmation of subregion anatomical boundaries rather than visual gross inspection could prove essential to understanding local inflammatory indices associated with parturition. Histomorphological analysis of post-partum hysterectomy strips could reveal region-specific molecular signatures to guide imaging studies and inspire novel therapeutic approaches to forestall or induce delivery. Maria Arianoglou †, 1,2 Sung H Kim * , 1,2 Phillip R Bennett * , 1,2 David A MacIntyre * , 1,2 Vasso Terzidou * . 1,2, 3 1 Imperial College London, London, United Kingdom; 2 March of Dimes Prematurity Research Centre at Imperial College London, London, United Kingdom; 3 Chelsea and Westminster Hospital, London, United Kingdom. Introduction: PTB is a complex multifactorial clinical syndrome, accounting for millions of deaths each year. A novel area of research has focused on epigenetics and the role of miRNAs in PTB. MiRNAs are 19-25 nucleotide-long, single stranded, non-coding molecules which regulate gene expression via degradation or silencing of the messenger RNA (mRNA). MiRNAs have tissue-specific physiological expression pattens and they shed into circulation. Increased expression of miR-150 has been found in plasma of pregnant women at 12-14 gestational weeks who subsequently had cervical shortening and/or PTB. We aim to elucidate the role of miR-150 in early pregnancy and the underlying molecular pathways leading to PTB. Methods: JEG-3 cell line was used as a trophoblast cell model, cultured at 0%, 3%, 8% and 21% O 2 to determine miR-150 endogenous expression levels. Transient transfections were performed at 8% O 2 with 0.5nM miR-150 mimics and 25nM miR-150 inhibitors for 24h using Lipofectamine 2000 (Invitrogen). RNA was extracted using Nucleospin miRNA extraction kit (Macharey-Nagel). Reverse transcription was performed for miRNA analysis using 500ng RNA and the miRCURY LNA RT miRNA cDNA synthesis kit (Qiagen) and for mRNA analysis using 2000ng/μl RNA and the M-MLV Reverse Transcriptase kit (Sigma-Aldrich), followed by quantitative polymerase chain reaction using miRCURY SYBR Green kit (Qiagen) and SYBR Green JumpStart TM Taq kit (Sigma-Aldrich), respectively. MiRNA and mRNA expression levels were determined using StepOne Software v2.3 and LinRegPCR v2017.1, while statistical analysis was performed using GraphPad Prism v9.0 (Graphpad). Results: The expression of miR-150 was significantly higher in hypoxic conditions (0%, 3% O 2 ) compared to normoxic (8%) and superoxic (21%) levels (0% -8% O 2 , p= 0.037, 3%-8% O 2 , p<0.0001, 3%-21% O 2 , p= 0.020, N=6, One way-ANOVA). MiRNA analysis showed that miR-150 was over-expressed and knocked-down using targeted mimics and inhibitors, respectively. The results showed a decreasing trend in the expression of MMP14 and ITGB3 in presence of mimics and increased expression of these genes with inhibitors (N=6, Mann Whitney or Unpaired t test). Conclusion: The level of oxygenation in trophoblast cells early in pregnancy may affect miR-150 expression, which is highly expressed in PTB cases. Subsequently, increased miR-150 expression could suppress ITGB3 and MMP14 expression, which regulate trophoblast invasion early in pregnancy. Impaired trophoblast invasion could lead to PTB-associated complications due to placental dysfunction. Intra-Amniotic Inflammation Induces Infiltration of Immune Cells into the Cervix, Leading to Cervical Shortening without Altering the Vaginal Microbiome. Jose Galaz †, 1 Roberto Romero * , 2 Andrew Winters †, 1 Kenichiro Motomura †, 1 Yi Xu * , 1 Tzu Ning Liu †, 1 Kevin Theis * , 1 Nardhy Gomez-Lopez * . 1 1 Wayne State University, Detroit, MI, United States; 2 NICHD/NIH/DHHS, Detroit, MI, United States. Introduction: The strongest predictor for preterm birth is a mid-trimester sonographic short cervix. There is an inverse correlation between cervical length and intra-amniotic inflammation driven by microbes (intra-amniotic infection) or alarmins [sterile intra-amniotic inflammation (SIAI)]. Moreover, there is an association between a short cervix and an altered vaginal microbiome. However, a causal link between these processes has not been demonstrated. Herein, we studied the relationship between intra-amniotic inflammation, cervical shortening, leukocyte infiltration, and effects on the vaginal microbiome. Methods: Pregnant mice received ultrasound-guided intra-amniotic injection of LPS on 16.5 days post coitum as a model of endotoxin-induced preterm birth (n = 6), IL-1α as a model of SIAI (n = 6), or PBS (n = 6). RU486 (n = 6) was utilized as a model of non-inflammatory preterm birth, or DMSO (n = 6). Cervical length was measured by ultrasound at time of injection and 6 h after, and was reported as percentage of cervical shortening. A second cohort of dams receiving the same treatments (n = 6 each) was used to collect cervical tissues 6 h after injection to perform multispectral imaging and identify leukocyte subsets. A third cohort of dams receiving the same treatments (n = 6 each) was used to evaluate vaginal microbiome using 16S rRNA gene sequencing. Results: Dams receiving intra-amniotic injection of LPS or IL-1α displayed greater cervical shortening compared to controls. In contrast, the administration of RU486 did not alter the cervical length. An increased infiltration of neutrophils and NK cells was observed in the cervices upon LPS administration compared to controls. Moreover, infiltration of neutrophils, macrophages, and NK cells was observed in the cervices from mice receiving intra-amniotic IL-1α compared to controls. There were no differences in the infiltration of immune cells in the cervices between mice receiving RU486 and DMSO. Microbiome analyses revealed no differences in the vaginal bacterial profile richness or heterogeneity for LPS vs. PBS, IL-1α vs. PBS, or RU486 vs. DMSO. Similarly, there were no differences in composition or structure of vaginal microbiomes, as well as in abundance of bacterial taxa in the vagina for any comparison. Conclusion: Alarmin-and endotoxin-induced intra-amniotic inflammation led to cervical shortening that was accompanied by immune cell infiltration. Such a process did not occur in a non-inflammatory model. Importantly, intra-amniotic inflammation-induced cervical shortening was not associated with an altered vaginal microbiome. This study provides a causal link between intra-amniotic inflammation and cervical shortening preceding preterm birth. Initial Validation of Uterine Microstructure Imaging Using Quantitative Histology. Wenjie Wu, 1 Zhexian Sun †, 1 Hansong Gao †, 1 Sicheng Wang †, 1 Zichao Wen, 1 Qing Wang, 1 Pamela K Woodard, 1 Hannah R Krigman, 1 Alison G Cahill, 2 Wang Yong * . 1 1 Washington University in St Louis, Saint Louis, MO, United States; 2 Dell Medical School, University of Texas, Saint Louis, MO, United States. Introduction: Microstructure of the uterus indicates the connectivity and contractility of myometrium cells allowing coordinated propagations of electrical signals in a laboring uterus. Uterine Microstructure Imaging (UMI) is a novel imaging tool to noninvasively quantify features of uterine microstructure by modeling their diffusion signatures using magnetic resonance imaging (MRI). We employed high-resolution ex vivo MRI of human uterus specimen to test the hypothesis that the UMIderived extracellular diffusion fraction and fiber density correlates with quantitative histology. Methods: A specimen of 10 × 20 × 3 mm 3 was dissected from the posterior of a total hysterectomy uterus after labor delivery due to placenta accreta. It was imaged with a Varian 11.7T MRI, using spin-echo sequence with 63-direction diffusion encoding (max b = 4500 s/mm 2 , voxel = 0.25 × 0.25 × 1 mm). After MRI, it was fixed in 10% neutral-buffered formalin, embedded in paraffine, and sectioned at 5 µm in the coronal plane and stained with H&E and Masson's trichrome. Whole slide images were digitized at 20× magnification. Maps of extracellular space density and uterine muscle fiber density were generated by segmenting the extracellular space in eosin stain and red muscle fiber stain in trichrome respectively. The histology quantification maps were registered to the corresponding MR images. We used in-house UMI software to generate a extracellular water diffusion fraction map from MR images by computing the fraction of hindered isotropic diffusion (diffusivity 0.2 -1.7 ×10 -3 mm 2 /s) in each image voxel and a UMI-fiber density map by computing the fraction of anisotropic diffusion (radial diffusivity ≤ 1 ×10 -4 mm 2 /s), which models the water diffusion within uterine muscle fibers. Results: 110 evenly spaced regions of interest (ROIs, 2 × 2 mm) were drawn in the H&E-, Trichrome-, and UMI-derived maps. The mean UMIderived extracellular water diffusion fraction correlates with histological extracellular space density (R = 0.46, P < 0.001), and the mean UMIderived fiber density correlates with histological uterine muscle density (R = 0.55, P < 0.001) in each ROI. Conclusion: The UMI-derived extracellular diffusion fraction and fiber density correlate with histological quantifications. Preterm Labor with and without Chorioamnionitis is Associated with Activation of Myometrial Inflammatory Networks: A Comprehensive Transcriptomic Analysis. Jason Phung †, 1 Carol A Wang, 1 Jocelyn Reeders, 2 Tamas Zakar, 1 Jonathan W Paul, 1 Craig E Pennell * , 1 Sonika Tyagi, 3 Smith Roger * . 1 1 University of Newcastle, New Lambton Heights, Australia; 2 John Hunter Hospital, New Lambton Heights, Australia; 3 Monash University, Clayton, Australia. Introduction: Term labor has been associated with increased myometrial expression of inflammatory genes. In preterm labor using quantitative PCR of a limited cohort of genes we reported only patients with chorioamnionitis showed an increase in inflammatory gene expression. Given the small number of genes assessed, this study aimed to use RNAsequencing and bioinformatics to assess the myometrial transcriptome during preterm labor with and without chorioamnionitis. Methods: Myometrium was collected at cesarean section from preterm patients not-in-labor (PTNIL, n=16) and preterm patients in labor with chorioamnionitis (PTL-C, n=8) or without chorioamnionitis (PTL-NC, n=6). Whole RNA sequencing was performed using Illumina NovaSeq. Reads were mapped to the human genome (hg38) and GENCODE gene annotation to generate gene level quantification. Differential gene expression (DGE) analysis, gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) were used to comprehensively interrogate transcriptomic differences and their associated biology. Results: DGE analysis revealed PTL-C compared to PTNIL was associated with 931 differentially expressed genes (P.adj <0.05), whilst PTL-NC compared to PTNIL was associated with no significant gene changes confirming our earlier results. In contrast, GSEA and WGCNA both demonstrated that PTL with and without chorioamnionitis were associated with enrichment of pathways involved in activation of the innate immune system and inflammation; as well as activation of G-protein coupled receptors. There was marked overlap in the pathways enriched in both PTL subtypes. Conclusion: Conventional DGE analysis demonstrate myometrium from PTL-NC and PTNIL groups are transcriptionally similar whilst the presence of chorioamnionitis is associated with marked DGE changes. In contrast advanced bioinformatic analysis supported the idea that PTL in general is associated with innate immune activation. Use of advanced transcriptomic analysis beyond single gene analysis provided additional insights and suggests that anti-inflammatory therapy may be relevant to the management of preterm labor of all etiologies. Hope M Wolf †, 1 Shawn J Latendresse, 2 Jerome F Strauss, 1 Roberto Romero, 3 Adi L Tarca, 4 Sonia S Hassan, 4 Nardhy Gomez-Lopez, 4 York P Timothy * . 1 1 Virginia Commonwealth University, Richmond, VA, United States; 2 Baylor University, Waco, TX, United States; 3 NICHD/NIH/DHHS, Detroit, MI, United States; 4 Wayne State University, Detroit, MI, United States. Introduction: The length of the cervix in the mid-trimester of pregnancy is a powerful predictor of maternal risk for spontaneous preterm delivery. We have previously reported that a customized assessment of cervical length (CL) accounting for maternal characteristics improves prediction of spontaneous preterm birth after mid-gestation compared to raw CL measurements. An unanswered question is whether patient specific changes in CL can provide additional information about the relationship between other maternal risk factors and spontaneous preterm birth. Methods: Non-linear latent growth curves were developed in Mplus to model within-patient change in CL across pregnancy in a cohort of 4,474 Black women carrying 5,111 singleton pregnancies. Pregnancies treated with progesterone or cerclage were excluded from the analysis. CL was measured using transvaginal ultrasound between 8 and 40 weeks of gestation. The median (interquartile range) of longitudinal observations per patient was 5 (3) (4) (5) (6) (7) (8) . Gestational age was determined based on LMP and confirmed by obstetrical ultrasound. Relevant medical history and birth outcome data were collected for each pregnancy. Mediation models were developed in Mplus to test whether the association between common maternal risk factors and gestational age at delivery is mediated by change in CL during pregnancy. Results: The median (interquartile range) gestational age at delivery in the cohort was 39.1 (38.1-40.0) weeks. A more rapid decrease in CL during pregnancy (steeper slope) is associated with a shorter pregnancy duration (p < 0.001). A higher maternal age, higher pre-pregnancy BMI, and a higher number of previous preterm births are significantly associated with a shorter pregnancy duration (p < 0.01). Change in CL was found to partially mediate the relationships between these maternal covariates and gestational age at delivery (mediation of BMI (p < 0.001); mediation of Maternal Age (p = 0.033); and mediation of previous preterm birth (p < 0.001)). A multiple mediation model provides evidence for the mediation of additional maternal risk factors, including gravidity, parity, previous abortions, and substance use during pregnancy. Conclusion: Serial measurements of CL across pregnancy were used to provide insight into how common maternal risk factors may mechanistically influence spontaneous preterm birth. Maternal risk factors may increase risk for preterm delivery through a cervical pathway, by contributing to a more rapid rate of cervical shortening during pregnancy. These results can generate new approaches to therapeutic interventions and lead to improved prediction of spontaneous preterm birth. Sunwha Park, 1 Young-Ah You, 1 Jeongsup Moon †, 2,2 Nayeon Kang †, 2,2 Young Min Hur †, 1 Soo Min Kim †, 1 Gain Lee †, 1 Young-Han Kim, 3 Yun Ji Jung, 3 Eunjin Kwon, 1 AbuZar Ansari, 1 Taesung Park, 2,2 Young Ju Kim. 1 1 Ewha Womans University Medical Center, Seoul, Korea, Republic of; 2 Seoul National University, Seoul, Korea, Republic of; 3 Yonsei University College of Medicine, Seoul, Korea, Republic of. Introduction: Preterm birth is a major cause of high morbidity and mortality rates in newborns. The main cause of spontaneous preterm birth is the activation of an inflammatory response derived from ascending genital tract infection. Though various studies seeking a correlation between vaginal microbiome and preterm birth have been conducted, it is still not easy to predict preterm birth based on microbiome using 16s metagenome sequencing techniques. Therefore, our study aimed to predict preterm birth through machine learning analysis of vaginal microbiome from pregnant women. Methods: In this matched case-control study, the subjects were pregnant women who visited Ewha Womans University Mokdong Hospital and Yonsei Severance Hospital from 2018 to 2020. Cervicovaginal fluid was collected from pregnant women in mid-pregnancy. 150 South Korean pregnant women were recruited in this research with 54 women giving preterm birth and 96 with term birth. Their demographic profiles and white blood cell count at mid-pregnancy were recorded, and the microbiome profiles of vagina were also analyzed with 16S rRNA gene amplification of the V3-4 region. Amplicon Sequence Variants analysis, grouping, and classification were conducted using DADA2. After univariate analysis with Wilcoxon rank-sum test, EdgeR, DESeq2, ZIBSeq, metagenomeSeq, ANCOM, CLR Permutation, and 10 markers were selected. Samples were randomly divided into a training set and a test set by the ratio of 2 to 1. The four machine learning models, including logistic regression, random forest, XGBoost, and support vector machine were used in this research. The model performance was compared with the area under the receiver operating characteristic curve. Results: When the forward variable selection was performed using the whole markers, the maximum validation AUC of the logistic regression model was 0.94. Ten markers, including Lactobacillus spp., Gardnerella vaginalis, Ureaplasma parvum, Atopobium vaginae, Peptoniphilus grossensis, Prevotella timonesis, were selected using univariate analysis and the validation AUC was 0.64 using logistic regression. As a result of developing several machine learning models, the performance of XGBoost was the best. The test AUC was 0.72 with 60% of sensitivity and 83% of specificity. When WBC which is one of the clinical information was added to the model, the test AUC was 0.71 with 55% of sensitivity and 93% of specificity. Conclusion: Our study demonstrates that several candidate bacteria could be used as a prospective predictor for preterm birth. We also confirmed that predictive rate can be improved as a model through machine learning techniques. Increasing Cervicovaginal CXCL10 Across Mid-Trimester Predicted Women Destined to Deliver Preterm. Emmanuel Amabebe †, Maria D Papageorgiou †, Dilly O Anumba * . University of Sheffield, Sheffield, United Kingdom. Introduction: The CXCR3 receptor chemokines (CXCL9, CXCL10 and CXCL11) are often implicated in chronic placental inflammation (CPI) associated with spontaneous preterm birth (sPTB). Overexpression of these and other chemocytokines in the cervicovaginal space may be indicative of chronic subclinical inflammation and risk of sPTB similar to their role in CPI. Hence, we measured the concentrations of CXCL9, CXCL10, CXCL11, TNF-α, Eotaxin and TNFR1 in cervicovaginal fluid (CVF) of asymptomatic pregnant women across mid-trimester and determined the ability of these chemocytokines to predict risk of sPTB before 37 weeks' gestation. Methods: CVF samples were collected from 51 asymptomatic high risk pregnant women at two gestational time points (GTPs): GTP1 -20 +0 -22 +6 weeks, n=51, preterm=17, term=34; and GTP2 -26 +0 -28 +6 weeks, n=45, preterm=14, term=31. Concentrations (pg/ml) of chemocytokines were quantified by multiplexed bead-based immunoassay (Cytometric Bead Array, CBA), while fetal fibronectin (fFN) concentration was determined by 10Q Rapid analysis. The GTP1/GTP2 ratios of cytokines and fFN concentrations were compared between preterm-and term-delivered women using area under the Receiver Operating Characteristics curve (AUC) to predict risk of sPTB. Results: Four (23%) of the preterm women experienced preterm prelabour rupture of membranes. None of the measured cytokines differed between the groups at GTP1. At GTP2, only TNFR1 was higher in the term compared to preterm women (70.3±69. 1 v 22.6±26.8, p=0.049, n=12) and distinguished both groups (AUC=0.88, p=0.005). However, when expressed as a ratio of GTP1/GTP2, only CXCL10 was higher in the preterm than term women and was predictive of sPTB (1.57±2.1 v 0.88±0.5, AUC=0.68, p=0.044, n=45). Combination of the ratios of CXCL10 and fFN improved sPTB prediction (AUC=0.74, p=0.007). The concentrations of CXCL11, TNF-α, Eotaxin (in all samples) and TNFR1 (in some samples) could not be determined as they were either below or above the detection limit of the CBA. Conclusion: These findings suggest increasing CVF CXCL10 with gestation in preterm women may indicate a chronic subclinical (noninfectious) inflammation of gestational tissues that increase the risk of sPTB. If confirmed in larger cohort studies, early estimation of CVF CXCL10 alone or in combination with fFN may identify the risk of inflammation-associated sPTB in asymptomatic women and guide preventive and/or therapeutic interventions. Physiopathology and Diagnostic Interests of Chemokines in the Preterm Premature Rupture of Membranes. Damien Bouvier * , 1 Salomé Lambert †, 1 Yves Giguère * , 2 Jean-Claude Forest * , 2 Bruno Pereira * , 1 Corinne Belville * , 3 Loïc Blanchon * , 3 Vincent Sapin * . France; 2 CHU de Québec, Québec, QC, Canada; 3 Université Clermont Auvergne, France. Introduction: Preterm premature rupture of membranes (pPROM), is responsible for 40% of spontaneous preterm births. Its complex pathophysiology involves inflammation. Thus, chemokines could be used as biomarkers. Methods: Serum concentrations of 4 previously selected chemokines (CX3CL1, CCL11, CCL26, CXCL9) during pregnancy (1st and 2nd trimester (T1, T2), delivery) were studied in a prospective cohort composed of three groups: i) pPROM (n=82), ii) spontaneous preterm birth (sPTB, n=64) iii) controls (n=292). Expression of 4 chemokines was studied in explants of fetal membranes at term (amnion, chorion). Results: Only CX3CL1 showed a predictive interest of pPROM at T1 (sensitivity 90,2%, specificity 38,4%) for a threshold of 180.6 pg/ mL, independently of the sPTB's group. Combination with known risk factors for pPROM improved diagnostic performance. Only CXCL9's RNA expression was detected in the chorion. Protein expression of the 4 chemokines was demonstrated in amnion and chorion with higher levels in the amnion for CX3CL1 and CCL11. Conclusion: Serum CX3CL1 assay at T1 is of interest in the detection of pPROM. Is Evening Primrose Oil Useful to Increase the Success Rate of VBAC? Daniel Roshan, John Migotsky * , Ariel Roshan †, Sarah Davis * , Boris Petrikovsky * . ROSH MFM, New York, NY, United States. Introduction: Evening primrose oil (EPO) has been used as a potentially effective cervical ripening agent for many years. Studies are controversial regarding its usefulness, we sought to investigate the effect of EPO as a slow-process cervical softening agent on vaginal birth after cesarean (VBAC) success rate when it's used starting from 35 weeks. Previous studies started it at 37 weeks or later. Evening primrose oil use through its polyunsaturated fatty acids leads to a gradual cervical softening through weeks therefore may leads to more favorable bishop score and more successful vaginal delivery. Methods: All our VBAC candidates with one or two previous low transverse cesareans were counseled about EPO 1,000 mg three times a day starting at 35 weeks. Patients with contraindication to VBAC, prior preterm deliveries, multiple gestations and seizure disorders were excluded. We conducted a retrospective chart review of patients who underwent VBAC trial at our private MFM practice from 2011 through 2018. all patients were managed and counseled by 4 obstetricians. Clinical information was extracted including peripartum hemorrhage and neonatal intensive care unit (NICU) admission. EPO use was coded as yes or no. The primary outcome was VBAC success. Descriptive statistics, including frequency counts for categorical variables and means, and standard deviations were calculated. Univariate/Multivariate analyses using logistic regression models were run to assess predictive factors of VBAC. Results: A total of 557 VBAC patients were identified Among 269 EPO users, 243 patients had successful VBAC (success rate 90%) as compared to 80% success rate among non-EPO users. No maternal or neonatal adverse effects from EPO were noticed. The rate of Preterm delivery and postpartum hemorrhage was not statistically different in the two groups Patients who took EPO has much softer and thinner cervices as compared to non EPO group.The univariate analyses revealed that the odds of having VBAC had significantly increased by twice with EPO (OR 2/17; 95% CI 1/31-3/57; p-value=0.003). In the subset of patients whose indication for past cesarean included "failure to dilate" (FTD) there was a threefold increase in the odds ratio for VBAC success rate. EPO use starting at 35 weeks was associated with much higher ripped cervices and progression in labor toward successful VBAC. Conclusion: EPO use appears to increase VBAC success rate without adverse side effects, especially in patients who had prior C/S for FTD. Based on our data its use starting at 35 weeks to gradually ripen and soften the cervix is highly beneficial to lower cesarean section rate. A large prospective randomized study is recommended to further confirm our findings. Lea Sarmiento, Theodore M. Nelson †, Erin Silva †, Tanya Glenn †, Clare Flannery, Shannon Whirledge * . Yale University, New Haven, CT, United States. Introduction: Uterine fibroids are benign tumors, highly prevalent in women of reproductive age, and associated with significant morbidity. There is an incomplete understanding of how these tumors grow and develop a profibrotic, extracellular matrix-enriched environment that limits the development of non-surgical, therapeutic options. We recently discovered that glucocorticoids significantly repress vitamin D receptor signaling in uterine fibroid cells, which is important due to the ability of vitamin D to negatively regulate fibroid cell proliferation. By repressing vitamin D action, we hypothesize that the glucocorticoid signaling pathway is a novel driver of uterine fibroid growth and development. The purpose of this study was to comprehensively identify all genes and pathways regulated by the endogenous glucocorticoid cortisol in uterine fibroid cells. Methods: RNA-sequencing was performed following 6 hour treatment with cortisol. Significantly regulated genes (p<0.05) were evaluated by Ingenuity Pathway Analysis (IPA) to identify highly enriched pathways. A bioinformatics analysis of the COREMINE Medical, MetaCore, IPA, AmiGo, and OMIM databases was performed to identify genes associated with proliferation and extracellular matrix. These genes were then cross-referenced to cortisol-regulated genes. Immortalized human uterine fibroid cells (UtLM) and primary fibroid cells were utilized to validate significantly regulated genes and evaluate whether cortisol enhances proliferation. Results: We identified 1,930 genes significantly regulated by cortisol in uterine fibroid cells. These genes are enriched for biological functions related to "cell death and survival" and "cellular growth and proliferation." "Molecular mechanisms of cancer" and "fibrosis signaling pathway" were among the top five enriched canonical pathways. Of the 1,930 cortisol-regulated genes, 743 were associated with the bioinformatics search term "proliferation" and 88 were associated with "extracellular matrix." Up-regulation of anti-apoptotic agents insulin like growth factor 1 receptor (IGF1R; 2.79 ± 0.33) and Bcl-2-like protein 1 (BCL2L1; 3.58 ± .15) were independently validated and significant with cortisol treatment (p=0.0005 and p<0.0001, respectively). Finally, we employed a cell metabolic assay to measure cell growth in the presence of cortisol. Compared to vehicle-treated cells, cortisol induced cell growth by 50% in UtLM cells (p<0.0001) after 48 hours. The effect of cortisol on cell growth was validated in primary human uterine fibroid cells (p<0.0001). Conclusion: These studies are the first to demonstrate that the endogenous glucocorticoid cortisol significantly regulates genes associated with proliferation and extracellular matrix in uterine fibroid cells, with functional consequences for fibroid cell growth. Introduction: Endometriosis is a common disease in women of reproductive age characterized by pain and infertility. Endometriosisassociated infertility is manifested at least in part due to attenuated response of the eutopic endometrium to progesterone (P4), but the full mechanism is poorly understood. RE1-Silencing Transcription factor (REST) is a transcriptional repressor of neuronal genes in nonneuronal tissue. REST may also play a role in mediating tissue steroid responsiveness, but a potential role in endometriosis pathophysiology is unknown. To fill this knowledge gap, we examined REST expression in eutopic endometrial tissue from women with endometriosis and functionally assessed its role on fertility and endometrial steroidresponsiveness using a novel mouse model. Methods: REST was localized by immunohistochemistry (IHC) in eutopic endometrium from women with and without endometriosis (N=10/study group with 5/proliferative and 5/secretory stages of the menstrual cycle in each group). Fertility was assessed in Rest conditional knockout female mice (Rest fl/fl ; Pgr Cre/+ , herein defined as Rest d/d ) and Rest fl/fl females (controls). A 6-month breeding trial was conducted (N=8 female mice/ genotype, 2 months of age). Number of pups born in each of 4 consecutive litters was recorded and uterine morphology of breeders determined at termination of study. Uterine steroid-responsiveness was assessed in ovariectomized, steroid reconstituted females (N=5/genotype/treatment). Uterine wet weight and uterine Foxo1a mRNA expression was assessed. Results: REST was localized to epithelial and stromal cell nuclei from control subjects and expression level did not differ between stages of menstrual cycle. In contrast, eutopic endometrium from subjects with endometriosis did not express detectable levels of REST protein by IHC. Fertility assessment of Rest d/d females revealed a significant reduction (P<0.01) in the number of pups born per litter from the first through fourth litter (4, 2, 0, 0) compared to Rest fl/fl females (8, 9, 9, 9) . Rest d/d females developed aberrant uterine morphology characterized by cystic glands, epithelial hyperplasia, and significantly (P>0.001) greater uterine wet weights. Ovariectomized, E2-treated mice revealed a hyper-estrogenic response, while P4 treatment confirmed a blunted P4 response on uterine wet weight in the Rest d/d mice as well as impaired P4 induction of uterine Foxo1a expression. Conclusion: REST is deficient in eutopic endometrium from women with endometriosis and its deletion from mouse uterine tissue recapitulates impaired fertility, aberrant steroid responsiveness and molecular signatures of endometrial tissue observed in human disease. Model. Ramanaiah Mamillapalli, Shutaro Habata, Hugh S Taylor * . Yale University, New Haven, CT, United States. Introduction: Endometriosis is a common gynecological chronic inflammatory disorder regulated by estrogen and characterized by the growth of endometrial tissue outside of the uterus. Endometriosis can cause debilitating pelvic pain and lead to infertility. Current treatments include surgical excision of endometriotic lesions and hormonal therapies that suppress the disease but are limited due to adverse side effects, loss of fertility and disease recurrence after discontinuation. Furthermore, no current treatment modalities are able to cure endometriosis completely. Here, we investigated the therapeutic role of a monoclonal antibody raised against Programmed Death-1 (PD-1; CD279) receptor in a murine model of endometriosis. Methods: Endometriosis was induced in 6-8 week old female C57BL/6 mice by suturing donor mouse endometrium into the peritoneal cavity of receptor mice. Sham surgeries were performed on control mice without implanting endometrial tissue. Recipient mice were allowed to develop endometriosis for 4 weeks and then treated with anti-PD-1 monoclonal antibody for four weeks (i.p, 10 mg/kg body weight, twice a week). Controls were treated with the respective isotype control (IgG2a). Mice were anesthetized and endometriotic lesions were collected and fixed in 4% paraformaldehyde for immunohistochemical and immunofluorescence studies. RNA was extracted from lesions and evaluated for differential gene expression using RTPCR. Histomorphology of the lesions was studied by H&E staining. Results: A total of 24 mice were included in the study, divided equally among the treatment and control groups. Endometriotic lesions from mice treated with anti-PD-1 antibody showed a significant reduction in lesion size and volume. Markers of endometriosis, including ER-α, ER-ß, Cyp19A (aromatase), KRAS 4A, KRAS 4B and IL-6 in anti-PD-1 antibody treated mice lesions were analyzed and compared to the untreated mice. In addition, differential expression of PD-1 target genes including SHP-2, TCR, CD28, IL-2, IL-21, BATF, ERK1/2, Caspase-7, STAT3, NFAT and NF-kB was examined, with most showing differential expression. Conclusion: Treatment with an anti-PD-1 monoclonal antibody decreased the size and volume of endometriotic lesions in a murine model. These data suggest a role for PD-1 in the pathophysiology of endometriosis. Moreover, treatment of endometriosis with anti-PD-1 antibody is a promising novel treatment option for endometriosis. Relationships with Cancer Genes and Pathways. Todd L Edwards, Jacqueline Piekos, Jing Wang, Sarah H Jones, Jacklyn N Hellwege, Digna R Velez Edwards * . Vanderbilt University Medical Center, Nashville, TN, United States. Introduction: Uterine fibroids (benign tumors of uterus) affect 77% of U.S. women by menopause and account for up to $34 billion in annual healthcare costs. Heritability estimates for fibroids range from 20-69%. Although studies have evaluated somatic mutations of fibroid tumor tissue or conducted genome-wide association studies (GWAS) of germline DNA, few studies have utilized RNA sequencing (RNAseq), despite successes from this approach in other complex traits. Methods: We conducted RNAseq on 35 fibroid tumors and 53 control myometrium (no-fibroids in uterus or history of fibroids) from women ages 18 to 50 undergoing hysterectomy with no cancer history, cancer treatment, or infectious diseases. Sequencing used paired-end 150 bp reads on an Illumina NovaSeq 6000. DESeq2 modeled differential expression between fibroid and myometrial tissue controlling for race. Functional enrichment analysis was performed using GSEA to identify biological pathways using all hallmark gene sets and gene ontology pathways. Results: There were 2,155 genes with significant differential expression (false discovery rate [FDR]-adjusted p [p adj ]<3x10 -6 ). The strongest association was observed at special AT-rich sequence-binding protein 2 (SATB2) (log 2-fold change = 4, p adj =4.68x10 -83 ). We observed significant associations with eight genes identified in the NHGRI GWAS catalog as associated with fibroids. From GSEA, 14 gene sets had p adj <0.05 and show evidence of upregulation, and eight show evidence of downregulation. Notable results were upregulated hallmark genes for MYC targets v1 (normalized enrichment score [NES] 2.8), oxidative phosphorylation (NES 2.6), and ESF targets (NES 2.5) and downregulated hallmark gene sets for estrogen response early (NES -1.6) that have been implicated in cancer, including ER+ breast cancer. Conclusion: Using RNAseq we identified multiple genes and pathways associated with fibroid risk that implicate cancer and tumorigenesis pathways. We also observed evidence of differential gene expression and several previously published GWAS loci, further supporting biological roles for these loci in fibroid risk. Novel Treatment of Endometriosis Using Adipose-Derived Stem Cells. S. J Huang * , 1 C-Y Huang, 2 Y-H Huang, 3 Ya-Chun Yu, 2 Shiu L-Y. 2 Introduction: Endometriosis is defined as the growth of endometrial glands and stromal cells in a heterotopic location under the cyclic influence of ovarian hormones with immune dysregulation. It is a common gynecological disorder manifested by chronic pelvic pain and infertility. Although various agents are available to treat endometriosis, the recurrence rate remains high. Recently, adipose tissue has been shown to be an abundant source of pluripotent adipose-derived stem cells (ADSCs), a mesenchymal stem cell. ADSCs display not only tissue regenerative effect, but also immune regulatory effect. Thus, the current study aims to test the effects of ADSCs on the growth of endometriosis. Methods: Adipose-derived stem cells (ADSCs) were isolated from adipose tissue obtained from lipoaspiration under Good Tissue Practice (GTP) and Good Manufacturing Practice (GMP) regulations. ADSCs and ADSC-derived conditioned medium (ADCM) were harvested at the third passage and subjected to quality validation, including karyotyping as well as growth promotion and sterility tests for microbial contamination. Autologous endometriosis mouse model was established by suturing 4 pieces of endometrial tissue 1 mm in diameter to the peritoneum followed by treating with ddH 2 O, ADCM, ADSCs or ADCM+ADSCs for 28 days. After sacrifice, the area of endometriotic cysts and the degree of pelvic adhesion were measured. The expression of ICAM-1, VEGF and caspase 3 was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Moreover, the numbers of macrophages and B-cells in the peritoneal lavage were determined by flow cytometric analysis. Results: Both ADCM and ADSCs passed quality validation. Compared with control, ADCM decreased the area of endometriotic cysts by 27.65 ± 0.03%. ADSCs alone did not show inhibition on endometriotic lesions. Moreover, the inhibitory effect of ADCM was obliterated by adding ADSCs. Although ADCM did not show improvement in peritoneal adhesion, the presence of ADSCs with or without ADCM increased the peritoneal adhesion. qRT-PCR demonstrated that ADCM inhibited ICAM-1 and VEGF mRNA expression by 39.15 ± 6.99% and 44.93 ± 4.46%, respectively, whereas the addition of ADSCs not only did not exhibit inhibition by itself, but also blocked the inhibition by ADCM. Consistent with qRT-PCR, IHC revealed that ADCM reduced ICAM-1 and VEGF expression by 43.74 ± 11.36% and 44.59 ± 16.84%, respectively, while the effect was obliterated by adding ADSCs. In peritoneal fluid, neither macrophages nor B-cells was regulated by the treatments. Conclusion: ADCM exhibits inhibitory effect on the development of endometriosis in a mouse model. This finding can potentially be translated to clinical treatment for human endometriosis. Endometriosis. Addie Luo, Ov Slayden * . Oregon Health and Science University, Beaverton, OR, United States. Introduction: Retrograde tubal transfer of endometrium during menstruation constitutes the popular theory for the etiology of endometriosis. However, the factors promoting cell adhesion and migration of the menstrual cells remain poorly understood. The extracellular matrix proteins, E-Cadherin (CDH1) and Beta Catenin (CTNNB1), are potential diagnostic markers in endometrial cancer. Both targets are downregulated in malignant tissue, where cell migration is favored. In this work, we assessed the expression of CDH1 and CTNNB1 in endometriosis-free endometrium, eutopic endometrium, and endometriotic lesion in menstruating rhesus macaques. Our premise is that downregulation of CDH1 and CTNNB1 facilitates the establishment of endometriotic lesions. Methods: Control endometrium (n=12) was obtained from endometriosis free monkeys that were treated sequentially with estradiol (E 2 ) and progesterone (P) to regulate the menstrual cycle. We collected the control samples during menstruation triggered by P withdrawal and confirmed by vaginal swab. Eutopic endometrium from monkeys with endometriosis (n=7) and endometriotic lesions (n=9) were obtained from menstruating, naturally cycling monkeys with advanced disease that were monitored for cycle stage by analysis of serum E 2 and P, and vaginal swabbing for menses. All samples were analyzed for CDH1 and CTNNB1 by immunohistochemistry (IHC), western blot and quantitative real-time PCR (qRT-PCR). Results: CDH1 and CTNNB1 expressions were significantly reduced in menstrual eutopic and ectopic endometrium compared to control endometrium (P<0.05). IHC staining of CDH1 and CTNNB1 localized to the glandular and luminal epithelial cells; CTNNB1 also localized to stromal cells in the superficial functionalis zone and to the endothelial cells. In the controls, CDH1 and CTNNB1 were expressed in the glands of non-bleeding basalis zone and in the luminal epithelium. In endometriosis, significantly weakened membrane staining of both targets was observed in both the eutopic and ectopic sites. Conclusion: Reduced extracellular matrix proteins CDH1 and CTNNB1 may lead to higher survival, proliferation, and migration of cells arising from menstrual debris. Therefore, retrograde menstruation may occur in many subjects, but expression of these proteins could contribute to the establishment of lesions. These results support the hypothesis that endometriosis exhibits a cancer-like cellular behavior in cell migration and adhesion. Supported by NIH HD098642 and OD 011092. The Role of PD1/PD-L1-Positive M2 Macrophages in Endometriosis Pathogenesis. Yuping Zhou †. Yale University, New Haven, CT, United States. Introduction: Endometriosis (EM) is an estrogen-dependent, immuneassociated disease that affects 10-15% of all reproductive age women and up to 50% of women with infertility and 80% of women with pelvic pain. The M1/M2 macrophages have distinct properties, with M2 macrophages creating an immune-privileged environment in some tumors. Similarly, PD1 and PD-L1 are immune modulators and important targets of cancer immunotherapy. Previous studies have established that PD-1/PD-L1 signaling inhibits numerous immune cell subsets in the tumor microenvironment, including macrophages. However, the role of macrophage types and function of PD1 and PD-L1 in endometriosis pathogenesis is not well characterized. We attempt to investigate the expression of PD-1/PD-L1 and the function of endometriosis-associated macrophages and endometriosis pathogenesis. Methods: Ectopic endometrial biopsies were collected from endometriosis patients and eutopic endometrial biopsies from patients both with and without endometriosis (n=10/group). Immunohistochemistry and immunofluorescence staining was performed for PD1, PD-L1, CD68, CD206. Human monocytes (THP-1) were differentiated into M0 macrophages with phorbol 12-myristate 13-acetate. M0 cells were polarized into M1 macrophages by IFN-γ and lipopolysaccharide, or M2 with interleukin (IL)-4 and -13. Endometriosis associated macrophages (EAMs) were generated from M0 in endometriotic cells-conditioned medium. M1, M2 or EAMs were co-cultured with endometriotic cells. Gene expression was quantitated by quantitative RT-PCR. Results: CD68 + macrophage number was significantly increased in ectopic endometrial tissue, compared to eutopic endometrium from either endometriosis patients or normal disease-free controls. PD-1 was detected only in ectopic endometrial tissues, but not in eutopic or normal endometrium. PD-L1 expression was dramatically higher in ectopic endometrial tissue than eutopic and normal endometrial tissue. PD-1 and PD-L1 were predominantly expressed by CD68 + macrophages. CD68 + and CD206 + double positive macrophages were dramatically increased in ectopic tissue (N=30, P<0.05). Endometriotic cell-conditioned medium induced M0 into EAMs with increased expression of the M2 marker CD163 and decreased expression of M1 markers IL-1β, IL-8 and TNF-a (N=3, P<0.05). When cocultured with EAMs, apoptosis of endometriotic cells was inhibited, while proliferation was enhanced, but not with M1 cells. Conclusion: Endometriosis is characterized by abundant M2-like CD68 + and CD206 + macrophages with high expression of PD-1 and PD-L1. Endometriotic cells may produce secreted factors that promote M2-like EAM polarization. EAMs are less cytotoxic and provide an immunoprotected environment, in contrast to M1 macrophages. Modulation of EAM/M1 polarization or the macrophage PD1/PD-L1 axis are potential targets for endometriosis immune therapy. Towards an Understanding of the Pathomechanisms of Uterine Fibroids and Associated Heavy Menstrual Bleeding Using Systems Biology Approaches. Chen-Yi Wang, 1 Darragh P O´Brien, 1 Anne Ndungu, 1 Jessica Malzahn, 1 Marina Maritati, 1 Beatriz Martinez-Burgo, 1 Kurtis Garbutt, 1 Krina T Zondervan, 1 Christian M Becker, 1 Adrian Harris, 1 Bianca De Leo, 2 Maik Obendorf, 2 Nicole Schmidt, 2 Joerg Mueller, 2 Thomas M Zollner, 2 Udo Oppermann, 1 Adam P Cribbs, 1 Benedikt M Kessler, 1 Martin Philpott * . 1 1 University of Oxford, Oxford, United Kingdom; 2 Bayer AG, Berlin, Germany. Introduction: Uterine fibroids (UFs) are benign tumours affecting up to 80% of women of reproductive age. Approximately 30% of fibroid patients suffer severe symptoms including painful heavy menstrual bleeding (HMB). Although mutations in MED12 or HMGA2 account for the majority of UF occurrence, the processes by which these lead to UFs and HMB remain poorly understood. Here we present a comprehensive systems biology study, merging clinical, genetic, transcriptomic and proteomic information to better understand the pathomechanisms underlying UF and associated HMB. Methods: UF, pseudocapsule, myometrium and endometrium tissues (where applicable) were collected from 137 donors undergoing hysterectomy, myomectomy or transcervical resection of fibroid (TCRF) at the John Radcliffe Hospital, Oxford, in accordance with guidelines under the ENDOX study (09/H0604/58). HMB status and use of hormone therapy was established from clinical notes and donor questionnaires. Menstrual cycle phase was determined by histopathology of the endometrium. UFs and myometrium of 91 donors were genotyped for MED12 mutations and profiled by genome wide SNP arrays. All tissues for these 91 donors were analysed by RNA-seq and proteomics. Systems level analysis was performed using Perseus software or the multiomics factor analysis (MOFA+) package for R. Results: Frequency of known UF MED12 mutations was 28%, which is substantially lower than other reports and may reflect a bias towards more complicated surgeries performed at the University Hospital. Our genotyping also revealed one novel exonic variant and multiple novel intronic variants in MED12. Systems level analysis of genotype, transcriptomic and proteomic data between myometrium and UF identified multiple interrelated gene sets involved in UF pathophysiology including extracellular matrix, protein glycosylation, sulphate biology and Wnt signalling. Similar analysis of endometrium stratified by donor HMB status revealed gene sets implicated in HMB, including vascular biology, clotting, cytokine signalling and inflammation. Conclusion: Utilising a larger cohort than previous studies, applying multiple 'omics approaches on individual tissue types and combining with clinical data has allowed a systems biology approach that expands our insight into possible pathomechanisms of UF and associated HMB. Resident Physician. Elham Neisani Samani †, Hamid Sanjaghsaz * . Garden City Hospital, Garden City, MI, United States. Introduction: Does the rate of miscarriage increase in the setting of adenomyosis? Background: Adenomyosis which is characterized by stromal and glandular endometrial tissue infiltration into the myometrium, is a frequent benign disease of women of reproductive age. There are studies questioning the association between adenomyosis and miscarriage. We conducted this study to determine whether the rate of miscarriage increases in the setting of adenomyosis or not. Methods: Literature was reviewed in PubMed, EMBASE, medRxiv, bioRxiv, and the Cochrane library on studies published from September 1990 until September 2021. The investigators included studies that evaluated miscarriage risk in pregnant women with adenomyosis by assisted reproductive technology, or with spontaneous conception. Data analyses were carried out by two authors. Results: 877 studies, which are comprised of 797,986 women, were included in the present study. Miscarriage risk increased in women with adenomyosis in spontaneous conception (OR: 1.71, 95% CI: 1.44-2.28) compared with those without adenomyosis. Compared with those without adenomyosis, women with adenomyosis had a higher rate of preterm birth, postpartum hemorrhage, and placenta previa (OR: 3.81, 95% CI: 1.44-5.47 ). Conclusion: Our finding suggested that the miscarriage rate was higher in women with adenomyosis compared with those without. Referral of women with adenomyosis and recurrent miscarriage to centers with a special interest in adenomyosis treatment may be necessary. Derek O'Neil, 1 Morgan Wasickanin, 2 Tiffany Katz, 1 Emily Wickersham, 2 Emilie Steed, 3 Laurel Gillette, 2 Christine Nadeau, 2 Novae Simper, 2 Sanam Bidadi, 1 Kjersti Aagaard, 1 Jan Sunde * . 1 Introduction: Implantation is fundamental to successful establishment of both the trophoblast (placenta) and embryo. Free-floating cells originating from the oviduct and uterus leads to benign ectopic lesions (endosalpingiosis, ES and endometriosis, EM respectively) and may also play a role in the pathogenesis of other ectopic implantations. We hypothesized that mechanisms driving ES will give crucial insight into early pregnancy implantations and failures, including recurrent pregnancy loss, placenta accrete spectrum, and ectopic pregnancies. To fully test this hypothesis, we developed a novel mouse model to interrogate the role of implantation signaling factors in promoting non-cancerous ectopic lesions. We developed an animal model of ES using auto-fluorescing (inherent in the donor mouse cells) oviductal tissue. To test how implantation factors affect ES and EM development donor mice were treated daily with progesterone from postnatal day 2-10 to induce infertility by blocking implantation (progesterone-induced uterine gland knock out, PUGKO). Oviductal and endometrial tissue from PUGKO and vehicle control (VC) mice was harvested. Recipient mice received either oviductal or endometrial tissue alone or co-injected with endometrial tissue (non-auto-fluorescing) to reintroduce implantation factors. After a month, recipient mice were euthanized to count auto-fluorescing legions. Results: PUGKO endometrium had significantly reduced implantation (2.14 lesions/mouse (L/M), p=0.042) compared to VC (4.71 L/M, figure 1 A-B). PUGKO endometrium with WT endometrium restored implantation. Additionally, oviduct implantation (0.92 L/M, p=0.028) was significantly increased when injected with WT endometrium (2.75 L/M, figure 1 C-D). Conclusion: Implantation factors are crucial to initiate and promote ectopic tissue growth. Our novel mouse model demonstrates that the removal of endometrial implantation factors reduces ectopic growth. Adding implantation factors increases oviductal tissue implantation. This model allows us to study the of implantation of these lesions, yielding the development of novel therapies. Progesterone Resistance and Altered Estradiol Responsiveness in Endometriosis. Valerie Flores †, Zhihao Wang, Hugh Taylor * . Yale School of Medicine, New Haven, CT, United States. Introduction: Endometriosis is a chronic gynecologic disease aff ecting 10% of reproductive-aged women. Progestin-based therapy is fi rstline, however 1/3 of women fail fi rst-line therapy. We have previously demonstrated that non-responders (NRs) to progestin-based therapy have decreased expression of the progesterone receptor (PR), while responders to progestin-based therapy have high PR. Interestingly, there was a subgroup of women with a moderate level of PR who also did not respond to progestin-based therapy, suggesting altered reliance on sex-steroids for endometriosis disease progression. As such, we aimed to determine if endometriotic lesions display unique proliferation patterns when exposed to various concentrations of estradiol (E2) and progesterone (P4). Methods: Primary eutopic endometrial cells from women with (n=6) and without (i.e., controls, n=3) endometriosis (ENDO) were cultured and plated at a concentration of 3x10^3 cells/well. Cells were incubated in DMEM media for 24hr, then in in serum-free media for 24hr. Cells were treated with one of the following treatments at time of BrdU labelling:E2 or P4: 10^-12, 10^-10, 10^-8, 10^-6, 10^-4; E2 at 10^-7 + varying P4: 10^-12, 10^-10, 10^-8, 10^-6, 10^-4. After 24 hr of treatment, BrdU cell proliferation assay was performed. Treatment groups were performed in triplicate, and cell samples was normalized to their untreated cells. An endometrial stromal cell (HESC) line was used as an internal control. Student's t test was used to compare proliferation patterns of ENDO to controls. Results: Both control subjects and HESCs demonstrated increased proliferation upon exposure to E2, which was blocked by addition of P4 (P4 alone and E+P treatment). In 3 ENDO subjects, increasing concentrations of P4 did not result in decreased proliferation when compared to control cells (p<0.05). Similarly, E2+P4 treatment did not attenuate proliferation (p<0.05). Three ENDO subjects demonstrated decreased proliferation at a P4 of 10^-6 (p<0.05), but continued to proliferate even at low E2 concentrations (10^-12) when compared to control cells (p<0.05). Conclusion: While control subjects and HESCs responded normally to hormonal treatment, we found two distinct phenotypes in cells from women with ENDO-those that grew as a result of progesterone resistance (i.e, continued proliferation despite increasing P4 concentration), and those that respond to P4 but were hyperresponsive to even low levels of estradiol. The two diff erent phenotypes of endometriosis we found highlight the heterogeneity of this debilitating disease, and the need for an individualized approach to treatment-estrogen deprivation agents for those who are progestin-resistant, and progestin-based therapy for those that have diminished requirements for estradiol for growth. Doing so can allow clinicians to avoid trialing futile treatments, and allow a precision medicine approach to treating endometriosis. A Single-Cell Atlas of the Leiomyoma Vascular Capsule Reveals Activation of Endothelial and Immune Cells. Martin Philpott, 1 Chen-Yi Wang, 1 Dylan Windell, 1 Jessica Malzahn, 1 Warren Baker, 1 Marina Maritati, 1 Salwa Lin, 1 Anna James-Bott, 1 Benedikt Kessler, 1 Mark Coles, 1 Christopher Buckley, 1 Krina T Zondervan, 1 Christian M Becker, 1 Bianca De Leo, 2 Joerg Mueller, 2 Nicole Schmidt, 2 Thomas M Zollner, 2 Adrian Harris, 1 Adam P Cribbs, 1 Udo Oppermann * . 1 1 University of Oxford, Oxford, United Kingdom; 2 Bayer AG, Berlin, Germany. Introduction: Uterine fi broids (UF) are benign smooth muscle-derived tumours and are the most common pelvic uterine tumours of women in reproductive age. Although women with UF are often asymptomatic, large numbers of patients present with symptoms of heavy or prolonged menstrual bleeding, dyspareunia, or pelvic pain and are aff ected by pregnancy loss or infertility. Whilst several genetic lesions in the HMGA2 and MED12 genes have been associated with uterine fi broid pathologies, the causes of clinical sequelae are far from being understood. We hypothesized that better understanding of the molecular pathology is needed to address the unmet clinical need of improved therapeutic strategies for UF. To progress towards this goal we here initiated a single cell atlas of human fi broids and in particular the pseudocapsule of the tumour, representing an anatomic entity that surrounds the myoma and separates it from normal myometrium Methods: UF were collected from patients undergoing laparoscopic surgery at the John Radcliff e Hospital, Oxford, in accordance with guidelines under the ENDOX study (09/H0604/58). Tissue samples were prepared for histology (paraffi n fi xed) or directly fl ash frozen for single cell analyses. Single cell analyses were carried out by nuclei preparation, followed by encapsulation using the 10X Genomics platform and Illumina short-read sequencing. Cycling immunofl uorescence (CellDive, Leica Microsystems) was carried out using multiple rounds of multiplexed immunofl uorescence staining using an in-house designed marker panel targeting immune, stromal, and endothelial cell populations. Results: Single cell transcriptomics identified a robust set of 16 distinct cell clusters within the uterine fi broid pseudocapsule. The data suggest substantial infi ltration of innate and adaptive immune cells and furthermore provide detailed transcriptomic maps of fi broblast/stromal and endothelial cell activation states. Spatial information was obtained by immunofl uorescence mapping of cell types identifying organisation and interactions of myeloid/dendritic cells with the vascular and lymphatic endothelium. The results constitute a fi rst step to deliver spatial and phenotypic cellular data to advance our understanding of molecular events underlying leiomyoma pathology and provide novel insights into aberrant immunological processes occurring in the tumour microenvironment. Hyperandrogenemia. Rachel C Wilson †, 1 Fangzhou Luo, 1 Tanner Grenz, 2 Ov D Slayden * . 1 1 Oregon National Primate Research Center, Oregon Health and Sciences University, Beaverton, OR, United States; 2 Knight Cancer Institute, Oregon Health and Sciences University, Portland, OR, United States. Introduction: Women affl icted with PCOS exhibit ovulatory dysfunction and subfertility, but the impact of PCOS on abnormal uterine bleeding remains unclear. We've recapitulated, in part, the PCOS phenotype in rhesus macaques (Macaca mulatta) by long-term treatment with slightly elevated testosterone (T) and/or a western-style diet (WSD). We reported previously that macaques treated with T and WSD displayed a high incidence of endometriosis (PMID: 33554152). Therefore, we examined changes in uterine bleeding associated with endometriosis, and in response to treatment with T and/or WSD. Methods: Female monkeys were treated with T and/or WSD for 7 years as part of a larger cohort. Weekly blood draws were performed to quantify serum progesterone (P), along with daily vaginal swabs to classify bleeding as spotting, light, or frank. Day 1 of the menstrual cycle was defined as the first day of frank bleeding after a drop in P and the start of the secretory phase coincided with a rise in P. We used analysis of covariance (ANCOVA) to determine how treatment uterine bleeding with a covariate as number of cycles. We performed separate repeated measures analysis of variance (ANOVA) to analyze if P levels differed with either T and/or WSD treatment or endometriosis status. Finally, t-tests were used to determine if bleeding differed with disease state. We also examined vascular and structural proteins in the endometrium and cervix. Results: Serum P measured in the third and fourth week of cycles were significantly higher (p = 0.01), but did not differ with treatment or endometriosis. The covariate of number of cycles was significant for proliferative phase bleeding (Spotting: p = 0.02; Light: p < 0.01; Frank: p = 0.01) and luteal light bleeding (p = 0.03). The individual and combined treatment of T and WSD increased the number of light bleeding days during the proliferative phase ( Fig. 1a ; T: p = 0.001; Diet: p = 0.02; T*WSD: p = 0.02). And females with endometriosis had significantly more spotting (Fig. 1b ; p = 0.04) and light bleeding ( Fig. 1b ; p = 0.05) in the secretory phase. Conclusion: The deferential bleeding phenotypes associated with endometriosis and PCOS risk factors support the notion that management of aberrant uterine bleeding could aid in addressing issues associated with infertility. Funded by HD071836 and P51 OD11092. Transcriptome Analysis Reveals BRD9 Inhibition-Induced Distinct Pathways in Uterine Fibroids. Qiwei Yang, Maria Victoria Bariani, Ayman Al-Hendy. University of Chicago, Chicago, IL, United States. Introduction: Uterine Fibroid (UF) is the most common benign tumor in women of reproductive age. As the "readers" of lysine acetylation, Bromodomains are responsible for transducing the signal carried by acetylated lysine residues and translating it into various normal phenotypes. Bromodomain-containing proteins (BRDs) can have a wide variety of functions via multiple gene regulation mechanisms. However, the role and mechanism of BRDs in the pathogenesis of uterine fibroids are lacking. The present study aimed to determine the mechanism underlying the inhibitory effect of UF cell proliferation via BRD9 inhibition. Methods: To determine the role of BRD9, we assessed the effect of two BRD9 inhibitors (IBRD9 and TP472) on human UF cell (HuLM) proliferation and transcriptome alterations. Cell proliferation was performed by trypan blue exclusion test. To determine whether the altered transcriptomic profiles of UF cells in response to two BRD9 inhibitors, we performed RNA-seq analysis of vehicle-treated UF cells (n = 4), IBRD9-treated UF cells (n = 4), and TP472 -treated UF cells (n=3). Bioinformatic statistics were performed using the packages in R. Differentially expressed genes (DEGs) were identified as those having a false discovery rate (FDR) corrected p-value < 0.05. The levels of genes showing differential expression by RNA-seq were validated by qPCR (p<0.05, n=4). Results: Proliferation assessment demonstrated that both IBRD9 and TP472 significantly inhibited the HuLM proliferation in a dose-dependent manner (p<0.05). PCA plot of control vs. BRD9 inhibitor treatments showed that BRD9 inhibitor treatments were clustered separately from the controls. A total of 2372 and 2321 genes were differentially expressed in the IBRD9 and TP472 groups compared with controls, respectively (FDR<0.05). Venn diagram analysis revealed 853 and 610 common up and down DEGs in both BRD9 inhibitor groups respectively. To determine if the DEGs affect the biological functions implicated in the process of cell proliferation inhibition, gene set enrichment analysis was performed and showed that nine common gene sets, including E 2 F targets, G 2 M checkpoint, and MYC targets were significantly enriched in IBRD9and TP472-vs. vehicle-treated groups (FDR<0.05). In addition, gene ontology analysis of DEGs revealed that the biological process plays a dominant role in the IBRD9-and TP472-induced inhibitory effect of UF cell proliferation. By validation analysis using q-PCR, both IBRD9 and TP472 treatments significantly downregulated the RNA levels of cell cycle-related genes, including cyclinD1/3, CDK2, and PCNA (p<0.05). Conclusion: Abnormal expression of BRD9 correlates to uterine fibroid disease. Targeting BRD9 suppresses UF phenotypes via altering multiple pathways, including E2F, G2M checkpoint, and MYC targets. These novel mechanisms might provide a non-hormone, epigenetic therapy option for patients with UFs. Cell-Penetrating Nanogel Loaded with Antibiotic Reduced Uropathogenic E. coli (UPEC) Infection and Strengthen Urothelial Cell Survival. Humberto Escobedo †, Nicholas Zawadzki, James Till, Andres Vazquez-Torres, Marsha Guess, Guankui Wang, Dmitri Simberg, Devatha Nair, Michael Schurr. University of Colorado Anschutz Medical Campus, Aurora, CO, United States. Introduction: Urinary tract infections (UTIs) are prevalent bacterial infections globally, leading to healthcare costs over $1 billion annually. Uropathogenic bacteria form intracellular communities that enable them to avoid antibiotic destruction and immune clearance. We developed a cell-penetrating, nanogel (NG)-based system that can locally deliver an antibiotic to urothelial cells in a targeted manner. The objective of our study was to determine if our NG can reduce Uropathogenic Escherichia coli (UPEC), UTI89, in an in vitro and in vivo UTI infection model. Methods: Human bladder cells (HTB-9) were infected with UTI89 at a multiplicity of infection of one. After, a gentamicin (Gm)-loaded NG conjugated with a urothelial cell-penetrating peptide (NG-CPP+Gm), Gm alone or no treatment were delivered to the cells. Four hours after treatment, the number of colony-forming-units per milliliter (CFU/mL) and urothelial cell survivability were determined. Additionally, UTI89 (10 7 CFU) was delivered transurethrally to C57BL/6J mice. After 1 day post infection (dpi), mice were treated twice per day for 2 days with one of the following: a) 25µg of Gm-loaded NG-CPP delivered transurethrally (n = 16); b) 200µg of Gm delivered subcutaneously (n = 16); c) no treatment (n = 15). After 3 dpi, the mice were sacrificed, bladders were harvested, and the number of CFU/mL were determined. One-way ANOVA with post-hoc comparison analysis was performed. A p-value <0.05 was considered statistically significant. Results: HTB-9 cell survival was highest for NG-CPP+Gm (~87%), followed by Gm (~68%) and untreated (~64%), while both NG-CPP+Gm and Gm alone treatments significantly reduced the bacterial load compared to untreated HTB-9 cells at 4hr (p<0.05). Murine bladders treated with NG-CPP+Gm and Gm both significantly reduced bacterial load when compared to the untreated (p<0.01). Conclusion: We demonstrated that a targeted delivery of a cell-penetrating nanogel containing gentamicin improved urothelial cell survival significantly more than gentamicin alone and led to a comparable reduction in UPEC infection in vitro. Furthermore, treatment with NG-CPP+Gm using ¼ the dose of gentamicin led to a comparable reduction in bacterial load as gentamicin alone compared to untreated mice. Our findings suggest that delivery of an antibiotic using a targeted nanogel may serve as a novel, effective way of efficiently treating UTI with lower antibiotic concentrations that may help to minimize side effects. Characterizing Ion Channels in the Endocervix. Mackenzie Roberts †, Shan Yao, Jeffrey T Jensen, Leo Han * . Oregon Health and Science University, Beaverton, OR, United States. Introduction: The characteristics of cervical mucus change throughout the menstrual cycle and regulate fertility. Ion channels regulate mucin protein unfolding, hydration and ultimately the consistency of mucus. While essential channels have been described in other mucus secreting epithelia, prior studies have not determined which channels mediate mucus changes in the cervix. Here, we characterized ion channels in the endocervical epithelium under different hormonal conditions using a non-human primate (NHP) model. Methods: We produced a primary endocervical cell culture from adult female rhesus macaques (Macaca mulatta) using conditional reprogramming. We treated cells grown at an air-liquid interface using the following conditions: no hormone control, estradiol (E2) x 7 days, or E2 x 5 days followed by 2 days of progesterone (P4). We utilized the BioMark HD (Fluidigm) system to measure gene expression for the following ion channels: CFTR, ANO6, ATP2A3, CLC2, CLCA1, SCNN1, NKCC1, SLC26A2 as well as estrogen receptor (ER) and progesterone receptor (PGR). We performed immunohistochemistry (IHC) of select ion channels. Results: Both ER and PGR showed increased gene expression under E2 only conditions, and down regulation with the addition of P4. Compared to controls, we found that E2 increased gene expression for CFTR, CLCA1, NKCC1, and SLC26A2 (See Table 1 ). E2+P4 downregulated gene expression for ANO6, ATP2A3, and SCNN1 compared to E2 only. Using IHC we were able to confirm CFTR, SCNN1, SLC12A and KCNN4 in the cervix. Conclusion: Several ion channels in the endocervix demonstrate hormonal regulation; E2 up-regulates and P4 down-regulates ion channel gene expression. The direction of this effect is consistent with the clinical observation of decreased mucus viscosity and increased permissiveness to sperm movement seen under the influence of estrogen in the follicular phase. As ion trafficking is a major determinant of mucus hydration and consistency, channels that are E2 and P4 regulated may be potential targets for novel therapies in disease physiology, fertility management, and contraceptive development. Introduction: An association between phthalate esters, bisphenol A, organochlorinated environmental pollutants and endometriosis prevalence has been largely suggested (Sirohi et al. 2021) . However, there is scarce research focused on other environmental pollutants such as trace metals, which have been reported to interact with both the endocrine system and oxidative stress protection. We aimed to investigate the possible association between a set of trace metals found in blood and urine of women with and without endometriosis. Methods: Urine and blood samples were collected from 23 patients with endometriosis and 24 controls confirmed by laparoscopy. Fifteen trace elements were quantified in both matrices using inductively coupled plasma mass spectrometry. Logistic regression models were employed to examine the association between quartiles of trace metals and endometriosis prevalence after age adjustment. Results: After adjustment, the relative risk (95% CIs lowest vs. highest quartile) of endometriosis was 2.67 (95% CI: 0.39, 4.94; p = 0.018) for copper/zinc (Cu/Zn) ratio and 2.42 (95% CI: 0.16, 4.69; p = 0.030) for cesium measured in blood. Conversely, an inverse association was observed for selenium -3.05 (95% CI: -5.94, -0.15; p = 0.033), titanium -3.65 (95% CI: -6.55, -0.74; p = 0.011), lithium -2.10 (-4.18, -0.01; p = 0.042), and barium -2.50 (95% CI: -5.09, 0.08; p = 0.050) levels in blood. In urine, and inverse association was observed for selenium -2.65 (95% CI: -5.00, -0.30; p = 0.023) and rubidium -4.62 (95% CI: -8.12, -1.11; p = 0.008). We found that environmentally relevant concentrations of some understudied trace elements are associated with endometriosis prevalence. Among them, we highlight the alteration of some biomarkers of oxidative stress, such as an elevated blood Cu/Zn ratio associated with a higher incidence of endometriosis and the consistency of selenium, in both blood and urine, as a protective factor for the disease presence. Further studies with broader populations are needed to elucidate the potential role of selenium and zinc supplementation for endometriosis treatment and the biological significance of other trace elements observed associations. Support: PFIS (PI/00009), APOTIP/2020/013, Miguel Servet Contract (CPII18/00002) and ISCIII FIS project (PI17/00931). The therapeutic efficacy of curcumin against various human diseases has been documented. In uterine leiomyoma cells curcumin has demonstrated an anti-proliferative effect in-vitro possibly mediated by activation of peroxisome proliferator-activated receptor-γ, increased apoptosis and decreased production of extracellular matrix (ECM) components. Our main objective was to examine the overall impact of curcumin on in-vivo 3-dimensional (3D) human leiomyoma xenografts. Methods: Leiomyoma and myometrial cells derived from patient tissues were grown in 3D format before implantation into the flanks of NOD/ SCID immune-deficient ovariectomized mice. Hormone pellets containing estradiol and progesterone were implanted before xenograft implantation. The mice were divided into two groups; exposed to curcumin-fortified diet and control diet. Blood was collected at the end of the study; serum analyzed via LCMS for curcumin and its metabolites. Results: Curcumin and its metabolites, predominantly in the glucuronide and sulfate forms, were present in the serum of mice on curcumin-fortifi ed diet. The leiomyoma xenografts of the curcumin-fortifi ed diet demonstrated a signifi cant 67% decrease (p=.0004) in the tumor area. Myometrial xenografts were also aff ected demonstrating a 20% decrease (p=.09) in tumor area. To determine if the steady-state serum levels of curcumin provided by the diet was playing a preventive role we examined the tumors histologically. Microscopic analysis of hematoxylin and eosin stained sections of the xenograft demonstrated a change in the matrix morphology. Sections showed a breakdown of the ECM as decreased number of cells and structural compactness of the matrix, compared to the control sections. Immunohistochemically, curcumin exposed xenografts relative to the controls, demonstrated decreased intensity of staining for fi bronectin protein that plays a major role in extracellular matrix assembly. Conclusion: Curcumin, a natural compound demonstrates therapeutic properties for treatment/prevention of uterine leiomyomas in-vivo. The compound has been shown to be safe when consumed at high concentrations. Our data suggests high curcumin consumption may provide benefi cial eff ects on overall uterine fi broid burden. Fibroid Cells and is Inhibited by EGCG. Md Soriful Islam, Lena W Chen †, Kamaria C Cayton Vaught, Joshua T Brennan, James H Segars * . Johns Hopkins University School of Medicine, Baltimore, MD, United States. Introduction: The excessive accumulation of a fi brotic extracellular matrix (ECM) is a distinguishing characteristic of uterine fi broids. However, the underlying mechanisms of fibrosis are incompletely understood. Fibroid treatments are limited, and eff ective non-hormonal medical treatments are urgently needed. Recently, we and others reported that Hippo/YAP signaling is involved in fi broid cell fi brosis. Epigallocatechin gallate (EGCG) is a natural compound found in green tea reported to reduce fi broid size. Previously, we reported that EGCG inhibited the fi brotic phenotype, at least in part, by altering YAP signaling in fi broid cells. Here, we tested the hypothesis that β-catenin signaling may be involved in fi brosis in fi broid cells and targeted by EGCG. Methods: Based on a viability test, we treated both human fi broid (P51F) and myometrial (P51M) cells with EGCG at 100 µM concentration for 24 h and measured protein levels of the active form of β-catenin. To explore the role of β-catenin in the fi brotic phenotype, we used ICG-001. ICG-001 disrupts β-catenin interaction with CBP (CREB-binding protein) via binding to CBP. Human fi broid cells (P51F and P57F) were treated with ICG-001 at 2.5 µM concentration for 24 h. Protein or mRNA levels of fi bronectin (FN1), versican (VCAN), and plasminogen activator inhibitor 1 (PAI-1), actin alpha 2, smooth muscle (ACTA2), Yes-associated protein 1 (YAP1), and TEA domain transcription factor 1 (TEAD1) were assessed. Diff erences were considered signifi cant at p<0.05. We found that protein levels of active β-catenin were higher (1.9-fold) in P51F fi broid compared to P51M myometrial cells. EGCG treatment decreased β-catenin (active) protein levels by 51% in fi broid cells (p<0.01). In P51M myometrial cells, the protein levels of β-catenin (active) were not signifi cantly aff ected by EGCG treatment. Treatment of P51F fi broid cells with ICG-001 resulted in a 49% reduction of fi bronectin protein levels (p<0.05), suggesting a role for β-catenin in fi brosis. This was further supported by a 48% reduction of PAI-1 protein expression (p<0.05) and a 73% reduction of ACTA2 mRNA expression (p<0.01) with ICG-001. In contrast, transcript levels of FN1, PAI-1, and VCAN were increased in fi broid cells by ICG-001 treatment. Interestingly, we found that ICG-001 treatment reduced protein levels of transcriptional eff ectors of Hippo signaling YAP (active) by 37% (p<0.05) and TEAD1 by 24% in P51F fi broid cells, suggesting a possible interaction of β-catenin signaling with YAP/TEAD to induce the fi brotic phenotype. Conclusion: EGCG treatment reduced β-catenin (active) in fi broid cells. The downregulation of critical proteins of ECM components and Hippo signaling by ICG-001 suggests that β-catenin signaling in cooperation with YAP/TEAD may promote fi brosis, which can be eff ectively inhibited by EGCG. Introduction: Pelvic organ prolapse (POP) results when supportive tissues of the pelvic fl oor become compromised. In a blinded histological analysis of uterosacral ligament (USL) biopsies from POP patients (n = 195) and matched controls (CTRLs; n = 52), we identifi ed 3 histologic POP phenotypes of USLs. 1 One phenotype (POP-I) scored signifi cantly higher for neutrophil infi ltration compared to CTRLs. Hypothesizing that stable neutrophilia might correspond to infl ammatory tissue degeneration, genome-wide transcriptomic and DNA methylome studies were performed to determine whether chemoattractant factors and their receptors might be dysregulated in POP-I USLs. Methods: RNAseq was performed on POP and matched CTRL USL specimens (n=10 per group). Expression levels were validated separately by quantitative RT-PCR, For methylome analysis, DNA was extracted from 16 USL biopsy samples (8 POP, 8 healthy controls), bisulfi te converted, and assayed on 2 Ilumina Human Methylation EPIC BeadArrays. Results: Bioinformatic analysis of datasets revealed that several immune response pathway gene sets, including immune chemotactic factors are aberrantly expressed. Notably, the receptor CXCR4 and its ligand CXCL12, and homing receptor Selectin L (SELL), whose combined action has been shown to favor neutrophil homing and survival. 2,3 Transcript fold change in expression and DNA methylation at sites of interest are summarized in the Table. Conclusion: There is a strong correspondence of signifi cantly elevated CXCR4 transcripts and reduced methylation within its promoter in the POP-I USL. This was not matched by elevated CXCL12 ligand expression. SELL elevation also favors increased neutrophil attraction and survival. We interpret these data as showing that POP-I stable neutrophilia occurs due to aberrant reduction in CXCR4 promoter methylation. Targeting CXCR4 and SELL upregulation in women may improve pathological infl ammatory-type pelvic fl oor support compromise. Oregon HS University, Portland, OR, United States. Introduction: Endometriosis is common, occurring in approximately 10% of reproductive age women. It is associated with infertility, pelvic pain, progression, and a slightly increased risk of cancer. Genetic variants of WNT4 (eg. rs2235529) have been identifi ed that are signifi cantly more common in women with endometriosis. The WNT4-beta catenin pathway is also implicated in endometrial and ovarian endometrioid adenocarcinomas. We hypothesized that the WNT4 intronic variant associated with endometriosis may be associated with high stage [severe] endometriosis and an increased risk of endometrioid adenocarcinoma. ), documented history of infertility (yes or no), and at least 5 years of documented clinical outcomes to evaluate for disease persistence and de novo presentation of adenocarcinoma in the follow-up period. Selection criteria yielded 399 cases for chart review and 173 with suffi cient tissue biopsies comprised predominantly of endometriosis to provide adequate DNA for WNT4 (rs2235529) allelic discrimination by Taqman. We tested for associations between severe disease and infertility, overall cancer risk in follow-up, and a specifi c relationship between the minor T-allele and endometrial cancer. Results: All study subjects had self-selected as "white, non-hispanic." Patient age at the time of index biopsy diagnosis in severe (median 38 yrs) and mild (median 39 yrs) was not diff erent between groups. Similarly patient age was not signifi cantly diff erent between women with WNT4 minor allele (t) and without (cc). Median clinical follow-up was 12 years (7-18). As expected, infertility was more common in cases of severe endometriosis (32/132, 24%) than mild low-stage disease (17/267, 06%) [p<0.0001]. In the subset of 173 endometriosis cases with suffi cient DNA for testing, the Tmaf was 0.31 (expected 0.15) and signifi cantly greater in severe disease (0.37) compared with mild disease (0.29) [p<0.05]. Overall there was no association with reported cancer in follow-up, but the Tmaf in women who later developed endometrial cancer (6/7, c/t; maf 0.43) may be increased compared with other types of cancer (eg. breast, ovary, colon) (9/18, ct; maf 0.25) [Fisher's p-value =0.18). The WNT4 intron variant loosely associated with endometriosis is signifi cantly more common in high-stage severe disease and may also imply an increased risk of endometrial adenocarcinoma. If confi rmed in larger ongoing studies, identifi cation of cancer driver mutations in index biopsies of endometriosis may better classify long-term cancer risk in these women. The cystic fi brosis transmembrane conductance regulator (CFTR) is a cAMP-regulated ion channel found in epithelial cells including those in the airway, intestine, kidney and cervix. Chloride (Cl-) and bicarbonate (HCO3-) transport through CFTR plays a critical role in mucin unfolding, hydration, and ultimately the consistency of mucus secretions. CFTR is counter regulated by the sodium epithelial channel (ENaC) which promotes sodium and water reabsorption. Mutations in the CFTR gene are the cause of cystic fi brosis, an inheritable disease where dysfunctional mucus secretion leads to thickened mucus secretions, obstruction of mucosal passageways and multi-organ dysfunction. Functional studies of CFTR allows for both mechanistic experiments as well as drug-discovery testing of mucus producing epithelia. However, functional testing of CFTR has never been performed in mucus producing endocervical cells. We sought to assess the feasibility of evaluating CFTR function through epithelial monolayers electrical resistance measurements and short circuit current using conditionally reprogrammed endocervical cells from adult female Rhesus Macaques (Macaca Mulatta). Methods: We differentiated conditionally reprogrammed primary macaque endocervical cells at an air-liquid interface and measured transepithelial electrical resistance (TEERS) using a voltohmmeter (WPI). We then treated diff erentiated cells with CFTR-inhibitors Inh-172 (10 uM) and GlyH-101 (20 uM) and compared TEERS to vehicle-only controls over 48 hours. We also mounted diff erentiated cells on an Ussing chamber apparatus and measured activation and inhibition of CFTR as well as the inhibition of ENaC using amiloride. Results: Diff erentiated cells formed a mono-layer with electrically tight cellular junctions (TEERS>400 ohms/cm2). Compared to no-vehicle controls, both CFTR specifi c inhibitors increased epithelial resistance in all cells at 2, 24 and 48 hours after treatment. Endocervical cells mounted on an Ussing chamber demonstrated signifi cant loss of short circuit current with amiloride inhibition. Forskolin (CFTR activator) increased the short circuit current and addition of INH-172 (CFTR inhibitor) decreased short circuit current. We demonstrate that inhibition of CFTR in conditionally reprogrammed endocervical cells results in higher epithelial electrical resistance and decrease in the forskolin activated short circuit current. These results support using functional testing of CFTR in primary endocervical cells as a method for understanding tissue specifi c physiology of the channel as well as a platform for testing channel specifi c inhibitors on endocervical cells. Features Associated with Leiomyosarcoma in Premenopausal Women. Sarah Lauren Cohen Rassier, Sepideh Yadollahi, Ramyar Ghandriz, Timothy L. Kline, Bohyum Kim, Shannon K. Laughlin-Tommaso. Mayo Clinic, Rochester, MN, United States. Introduction: Leiomyosarcoma (LMS) is a rare but aggressive uterine cancer that can be diffi cult to distinguish from benign uterine fi broids (UF). Although the incidence of LMS increases with age, it has also been reported in premenopausal women. The objective of this study is to identify features that may be associated with higher risk of LMS particularly in the premenopausal population. Methods: This retrospective cohort study was approved by the local IRB. Cases were identifi ed from a pre-existing institutional database of pelvic imaging performed at our academic referral center. Medical records were reviewed to obtain demographic information, medical history, imaging details, symptoms and lab values. All premenopausal LMS cases were included, and representative control patients with symptomatic UF were selected as well. Univariate analyses were performed to compare diff erence between baseline features and reported symptoms in the two groups. Results: LMS patients were found to have a lower baseline hemoglobin prior to diagnosis (10.7+1.6 vs 12.3+2.3 g/dL, p=0.021), larger mean tumor dimension (13.5+4.7 vs 7.4+4.2 cm, p<0.001) and more often had a solitary uterine mass (82.4% vs 20.7% of cases, p<0.001). Regarding symptoms at presentation, UF patients were more likely to report menstrual irregularity (57.1% vs 17.6% of cases, p=0.009), excessive duration of bleeding (44.8% vs 11.8%, p=0.021) or constipation (48.3% vs 11.8%, p=0.012) compared to LMS patients. Conclusion: Several diff erences in baseline features and presenting symptoms were noted among women with LMS and UF, including significantly more abnormal bleeding in women with UF. These fi ndings can be used as part of counseling and treatment planning for premenopausal women with symptomatic uterine masses. The Efficacy of Nano-Colloidal Silver in Treatment of Postpartum Endometritis in Dairy Cows. Gamal El-Amrawi * , Dina Gad El-Karim. Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt. Introduction: Postpartum endometritis due to bacterial infection is one of the most common postpartum diseases affecting dairy cows, and antibiotics are widely utilized for its treatment. Recently, bacterial resistance to antibiotics became the most important problem facing the scientific community. so, our study hypothesized that intrauterine administration of nano-colloidal silver, would help in elimination of bacterial infection causing endometritis, due to its wide spectrum of bacteriostatic and bactericidal activity against enormous range of bacterial strains at low concentration, and such recovery could be detected early through estimation of peripheral level of some chemokines and acute phase proteins and later through detection of pregnancy rate. Methods: In a private farm, twenty one Holstein dairy cows were diagnosed with postpartum endometritis at 35-40 days postpartum, relying on clinical and ultrasonography examination. The treatment protocol depends on intrauterine administration of 50 ml of nano-colloidal silver solution (25 ppm/ml, particles diameter of about 15-25nm), for three consecutive day, and one dose of PGF-2α analogue (cloprestenol) for animals with CL on their ovaries. Blood samples were drawn just before the treatment and on 7th day from start of the treatment. Serum level of ceruloplasmin, CRP were assessed (immuno-turbidmetric), in addition to serum amyloid -A (SAA), haptoglobin, TNF-α and IL-6 using ELISA Kits. Also, the uterine state was monitored after the treatment using ultrasonography. The animals were re-examined at 49-54 days postpartum to detect the recovery rate. Then, the cured animals were artificially inseminated on their observed estrus; the pregnancy was diagnosed on 40th day post-service. Data were analyzed by t-test using SPSS software. Results: Treatment with nano-colloidal silver, significantly decreased the serum level of SAA, CRP, TNF-α and IL-6 (P<0.001). The level of haptoglobin and ceruloplasmin did not changed significantly (P<0.001) after treatment. Also, the uterine secretions was decreased or disappeared, and endometrial thickness was decreased upon ultrasongraphy examination on 7th day post-treatment. At re-examination, 14 animals out of 21 animals (66.6%) were recovered successfully from endometritis. The pregnancy rate was 71.4 % (10 out of 14 animals were found pregnant). The study indicated the effectiveness of nano-colloidal silver in treatment of postpartum endometritis in dairy cows. So, it could substitute the treatment with antibiotic in such cases, which may share in alleviation of the antibiotics resistance problems, due to uncontrolled usages of antibiotics. Further studies are needed to investigate the effect of different treatment period and solution concentration of nano-colloidal silver on treatment of endometritis in dairy cows. Defining which are believed to have distinct biological functions. However, the expression and functional profile of each CBX protein as it relates to uterine fibroidogenesis has been limited. This work aimed at identifying the expression profile of these proteins in uterine fibroids and to assess their downstream functional role in the pathogenesis of uterine fibroids. Methods: We mined published RNA-seq data (GEOdataset: GSE120854; 9 myometrium and 12 fibroids). Real-Time PCR and western blot analysis were performed on a separate cohort of uterine fibroids and patientmatched myometrial tissues (n=18) to determine expression profile of CBX proteins. Individual CBX family genes (CBX2/4/8) and control NLS constructs were overexpressed in a previously characterized UT-TERT myometrial cell line, and RNA-seq was performed. Differential analysis was performed by DESeq2, and enriched pathway analyzed using Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA). Results: RNA-seq analysis identified significant overexpression of CBX2/4/8 and downregulation of CBX7 in uterine fibroids compared to myometrium. In congruence with RNA-seq, RT-qPCR analysis on a separate cohort, further confirmed overexpression of CBX2 (P= 0.0002), CBX4 (P= 0.0001), CBX8 (P= 0.0001), and decrease in CBX7 (P= 0.0025) transcripts. Immunoblot analysis identified an average 2-fold increase of CBX2 in uterine fibroids. IPA analysis of CBX2 overexpressing UT-TERT cells revealed significant positive enrichment of signaling networks of "Cell Cycle Control" (P = 6X10 -18 ) and "Cyclins and Cell Cycle regulation" (P = 8X10 -9 ), and a negative enrichment of "G1/S checkpoint" (P = 1X10 -10 ) and "G2/M DNA damage" (P = 1X10 -10 ). GSEA analysis identified "Cell Cycle checkpoints" and "DNA replication" to be enriched, indicating that CBX2 could act as a driver of transcriptional alterations to promote tumor growth. Similarly, RNA-seq analysis of CBX8 overexpressing cells mimicked pathways identified in response to CBX2 overexpression. In contrast, CBX4 overexpression identified positive enrichment of "RHOGD1 signaling" (P = 9X10 -5 ), and "P53 signaling" (P = 1X10 -11 ). Conclusion: In uterine fibroids, CBX family proteins are differentially expressed compared to matched myometrium and may contribute uniquely to the pathogenesis of uterine fibroids. Further studies will explore the combined consequence of CBX2/4/8 overexpression with simultaneous CBX7 knockdown to determine dysregulated differentiation program as it relates to fibroidogenesis. Maternal Height as a Risk Factor for C-Section: Analysis of US Birth Data. Nicole Yao †, 1 Isabella Gomez †, 1 Narvella Sefah †, 1 Maria Teresa Benedetto-Anzai * , 2 Yuzuru Anzai * . 2 1 University of Chicago, Chicago, IL, United States; 2 Lenox Hill Hospital, New York City, NY, United States. Introduction: Despite the abundance of data on maternal BMI and Cesarean Delivery (CD) risk, there have been few reports regarding maternal height and CD. Previous reports, which showed an inverse correlation between maternal height and CD, were based on homogenous populations. It is not clear if this holds true across different ethnic and racial groups. Therefore, we investigated if maternal height affects CD in the US, which has a diverse patient population. The Centers for Disease Control and Prevention (CDC) provides US birth data. Using 2016-2019 data, we examined how maternal height relates to the likelihood of CD. Our analysis focused on full-term, nulliparous, singleton births constituting a total of 5,239,407 births. In addition to maternal height, we examined the following factors: maternal race, age, BMI, birth weight, and paternal race. SPSS was used to perform descriptive analysis, followed by logistic regression with multiple imputed data to calculate odds ratios and corresponding 95% confidence intervals. The following factors showed significant associations with CD: maternal height, race, BMI, age, and fetal birth weight. The rate of CD decreased as maternal height increased. This correlation remained significant after controlling for other factors and regardless of maternal race or ethnicity. Compared to 64-inch-tall mothers, women 60 inches in height have an aOR of 2.1 of CD, whereas women taller than 71 inches have an aOR of 0.58 (Fig. 1) . Based on aOR, maternal height had a stronger influence on CD than BMI>30, maternal age up to 39 years, and fetal birth weight up to 4499g, making it one of the most significant factors to affect CD rates. Conclusion: In this analysis of national birth data, maternal height is an independent risk factor for CD, with the odds of CD increasing as height decreases. Along with established factors such as age and BMI, height should be considered to be one of the prognostic indicators of CD. Even though maternal height is not a modifiable factor, this information is valuable for practicing physicians when assessing and counseling pregnant patients. Neonates born to mothers with gestational diabetes mellitus (GDM) have 3.7 times greater risk of GBS sepsis but mechanistic insight is lacking. We hypothesize that gestational diabetes renders women and their neonates more susceptible to GBS by perturbing immune defenses and enhancing GBS virulence. Methods: To induce GDM, C57BL6/J mice were placed on either a high fat, high sucrose diet or a low fat, no sucrose control diet one week prior to mating and maintained throughout pregnancy. Mice were vaginally inoculated with GBS on days 14.5 and 15.5 of pregnancy. On day 17.5, mice were euthanized, and tissues were collected to quantify GBS burden in maternal vaginal, cervical, and uterine tissues as well as placentae and fetal livers by plating on GBS selective agar. Cytokine levels (IL-1β and MIP2) in maternal vaginal and uterine tissues were quantifi ed by ELISA. In a second cohort of animals, dams gave birth and we assessed pup survival, weight, and GBS liver and intestinal burdens during the fi rst week of life. P of <0.05 was considered signifi cant. Results: Day 17.5 fetuses from GDM dams were signifi cantly more likely to exhibit GBS dissemination and had signifi cantly greater GBS placental and liver burdens compared to fetuses from control dams. Additionally, GBS vaginal burdens were signifi cantly higher in GDM dams compared to controls. Vaginal and uterine cytokine levels were similar between GBS-infected GDM and control groups. Pups born to GBS challenged GDM dams had signifi cantly worse survival and decreased weights in the fi rst week of life compared to pups from GBS challenged control dams. Conclusion: Our model recapitulates clinical observations of increased GBS susceptibility in GDM both in terms of in utero GBS dissemination and worse pup outcomes. Ongoing work seeks to profi le maternal and fetal immunity and determine how the diabetic environment alters GBS gene expression. In summary, this model provides a unique platform for mechanistic and therapeutic insight into GBS dissemination in pregnancy. Introduction: Fetal surgery can treat birth defects in utero, improving long-term outcomes for children; however, preterm labor remains a common complication of these surgeries. The immune system plays an important role in the success or failure of a pregnancy, but changes in immunity following possible trauma induce by fetal surgery are weakly understood. We hypothesize that fetal intervention will lead to increased infi ltration of immune cells into the placenta due to surgery-induced infl ammatory responses. We undertook a retrospective cohort study of pregnancies seen at a single, tertiary center in the Midwest. Three groups were compared: 1) fetal surgery (n=25), 2) indication but no surgery (n=14) and, 3) normal controls matched by gestational age at delivery (n=40). Additionally, a subset of our cohort having residual tissue available were selected for staining (n=15 surgery; n=5 controls). Immunohistochemical (IHC) analysis was used to identify the following immune cells in the villi: CD4, CD8, CD56, CD138 and CD163. Comparison of continuous variables between groups was performed using Kruskal-Wallis testing or Pearson's chi square test for nominal variables. Results: There were no signifi cant maternal diff erences in age, race, gravidity, and body mass index between all three groups. Most of the cases undergoing fetal intervention were for diagnoses of congenital diaphragmatic hernia (34%), spina bifi da (20%), hypoplastic left heart syndrome (20%), or twin-to-twin transfusion syndrome (14%). Fetuses who underwent surgery were more likely to be born preterm (p=0.01) and have a lower birth weight (p=0.05). The diagnosis of common placental pathologies, such as chronic villitis, acute chorioamnionitis, fetal vascular malperfusion or chronic deciduitis, were not found to be increased in fetal surgery cases compared to those who did not have surgery or gestational age-matched controls. We did observe a signifi cant increase in avascular villi in the placentae of the fetal surgery group compared to control (p=0.025). Additionally, IHC showed no increase in the infi ltration of CD4+ T helper cells (p=0.15), CD8+ cytotoxic T cells (p=0.15), CD56+ NK cells (p=0.23) or CD138+ plasma cells (p=0.28) between surgery and control groups, respectively. Lastly, CD163+ fetal Hofbauer cells showed no diff erences in control and surgical cases (p=0.68). Conclusion: Our data suggests that fetal surgery does not lead to increased pathological fi ndings or immune cell infi ltration in the placenta. This provides reassurance, both to fetal surgeons and patients considering in utero intervention, that surgery may not lead to obvious changes in placental histology associated with an infl ammatory response. The likelihood of successful pregnancy in subjects with unexplained recurrent early pregnancy loss <10 weeks gestation (uERPL) is generally favorable. However, some have found that even one unexplained fetal death (uFD) between 10-20 weeks gestation is associated with lower rate of subsequent successful pregnancy. We investigated pregnancy outcomes associated with a history of solely uERPL compared to those with any uFD. Methods: Ours was a prospective study of 828 individuals seeking consult for a history of ERPL, defi ned by >2 losses <10 weeks gestation, or >1 FD. Subsequent pregnancy data after recruitment was obtained via electronic survey, phone-call interviews, and/or review of medical records. We excluded those with a known/suspected cause of pregnancy loss, those who did not become pregnant, and those with unavailable pregnancy outcome. 373 subjects were included. The primary exposure was history of uFD or uERPL (without a history of FD). The primary outcome was the pregnancy outcome (livebirth or loss) in the first pregnancy after consult. Results analyzed with chi-squared tests. We included 184 people with uFD and 189 with uERPL. The overall rates of loss in the subsequent pregnancy were similar: 113 (39%) among people with uFD, 68 (36%) among people with uERPL (p=0.6) ( Table 1) . When more granularly comparing the timing of loss, groups differed significantly in 3-category outcome (p<0.001), with fetal demise occurring more frequently (16% vs 3%) and early loss occurring less frequently (23% vs 33%) among people with uFD than those with uERPL. Of pregnancies that progressed >10 weeks, FD occurred in 20% of people with a history of uFD vs. 5% of people with a history of uERPL (p<0.001). Conclusion: People with a history of uFD have a similar rate of live birth in their next pregnancy compared to those with a history of uERPL. However, those with a history of uFD are more likely to have a subsequent FD. At 10 weeks gestation, 1 in 5 people with a history of uFD had another FD vs 1 in 20 with a history of uERPL. Lindsay K Doherty, Sean D. Cleary, Angelo Elmi, Debra Bernat, Lorien Abroms. George Washington University, Washington, DC, United States. Introduction: The harmful effects of cigarettes to the fetus are wellestablished, but little knowledge exists about e-cigarettes and fetal health. Many pregnant people who smoke cigarettes believe that e-cigarettes are a safer alternative and and can help their efforts to quit smoking. This analysis evaluates risks of adverse neonatal outcomes in association with combined e-cigarette and cigarette use compared with cigarette use only. Methods: This was a secondary analysis of a randomized trial of a text messaging program for smoking cessation during pregnancy (Quit4Baby). Data on e-cigarette and cigarette use were collected at four surveys throughout the study period. Pregnant smokers enrolled in the trial with available data on cigarette and e-cigarette use during pregnancy and birth outcomes were included in this analysis (n=295). The primary exposure was any past 30-day dual use, defined as self-reported use of e-cigarettes and cigarettes within the last 30 days, in a survey during pregnancy. Past 7-day dual use was also examined. The comparison group used cigarettes only. The primary outcome was low birth weight (< 2500 grams); secondary outcomes included preterm birth (< 37 weeks gestation), admission to neonatal intensive care unit (NICU), prolonged neonatal hospital stay, and perinatal death. Relative risks and 95% confidence intervals were estimated from multivariable log binomial regression models. Results: Overall, 141 (48%) reported past 30-day dual use in at least one survey during pregnancy, and 154 (52%) reported cigarette-only use. The prevalence of adverse neonatal outcomes was similar among babies born to dual users compared with cigarette-only users (Table) . In multivariable analysis, no associations were found between low birth weight and past 30day dual use or past 7-day dual use. Risk for preterm birth, however, was 50% lower among past 30-day dual users compared with cigarette-only users. No other neonatal outcomes were associated with dual use (Table) . Past 30-day dual use was associated with lower risk of preterm birth compared with cigarettes only. Past 30-day and past 7-day dual use during pregnancy did not affect birth weight in the offspring of smokers in this sample. Data presented as n (%) or aRR (95% CI) *Data missing for birth weight (n=20), preterm birth (n=20), NICU admission (n=24), and length of neonatal hospital stay (n=24). †Adjusted for maternal age, pre-pregnancy BMI, trimester at enrollment, employment status, and metropolitan area residence. ‡Multivariable regression analysis not performed for perinatal death due to the small number of events. The Association Between Low Diabetic Screen and Neonatal Morbidity. Jia Jennifer Ding †, Lisbet Lundsberg, Jennifer Culhane, Caitlin Partridge, Moeun Son * . Yale University, New Haven, CT, United States. Introduction: While deleterious effects of hyperglycemia on pregnancy outcomes are well studied, whether low diabetic screen (DS), a proxy for hypoglycemia, is associated with adverse perinatal outcomes remains unclear. Several international studies report an association between low DS and small for gestational age (SGA) birthweight, but this remains unexamined in a U.S. population. Methods: This is a retrospective cohort study of pregnant women with a 1-hour 50-gram glucose challenge test (GCT) result who delivered at a single tertiary-care academic center from 2013-2021. Women with pregestational diabetes and multifetal gestations were excluded. The exposure of interest was low DS, defined as cohort-specific GCT <10 th percentile (<79 mg/dL). Women with a low DS were compared to women who had a cohort-specific GCT >10 th percentile (>80 mg/dL) and passed DS, and to women diagnosed with gestational diabetes (GDM) with GCT >200 mg/dL or who met Carpenter-Coustan criteria. These three groups were compared using bivariate analyses. The primary outcome was a composite neonatal morbidity measure. Secondary outcomes included SGA and large for gestational age (LGA) birthweight. Using multivariable logistic regression, the association of low DS (compared to those with GCT >80 mg/dL and passed DS) and morbidity outcomes was tested, adjusting for baseline differences with p<0.05. Results: Of 36,300 eligible women, 3432(9.5%) had a low DS (GCT <79 mg/dL), 29047(80.0%) had GCT >80 mg/dL and passed DS, and 3821(10.5%) had GDM. A low DS was significantly associated with SGA, and largely drove the increased risk of the neonatal morbidity composite, even after multivariate adjustment. Conclusion: A low DS is signifi cantly associated with increased neonatal morbidity and SGA. Women with low DS may be prone to hypoglycemia or other glycemic dysregulation and benefi t from enhanced antenatal surveillance given the risk of SGA and its comorbidities. Direct National University of Singapore, Singapore, Singapore. Introduction: Antenatal steroids (ANS) are standard of care for women at imminent risk of preterm delivery. ANS accelerate maturation of the preterm fetal lung. Current dosing regimens see the mother exposed to unnecessarily high levels of steroids which disrupt the maternal HPA axis and glucose regulation, altering placental function and fetal growth. Using a sheep model of pregnancy, we aimed to demonstrate that maternal antenatal steroid administration was redundant in driving preterm fetal lung maturation. Methods: Ewes and fetuses at 120d gestation underwent recovery surgery to install a fetal jugular catheter. Animals were then immediately randomized to either: i) fetal intravenous betamethasone phosphate infusion to maintain fetal plasma betamethasone levels at 2ng/ml for 26 hours (fetal low-dose group; n=16); ii) fetal intravenous saline infusion for 26 hours and two maternal intramuscular injections of 0.25mg/kg betamethasone phosphate + betamethasone acetate (maternal clinical treatment group; n=12); or iii) fetal intravenous saline infusion for 26 hours (negative control group; n=10). Fetuses were delivered 48 hours after treatment was initiated, ventilated for 30mins to allow collection of lung function and physiological data, and euthanized. Quantitative PCR was used to measure changes in mRNA transcript for markers of lung maturation-surfactant proteins (SPA-D), aquaporins (AQ-1,5) and sodium channel subunits (ENAC-B). The average betamethasone dose for the fetal low-dose group was 1% (0.3mg) of that used in the maternal clinical treatment group (30mg). At 30 minutes of ventilation, arterial paCO2, pH, heart rate and VEI were signifi cantly (p<0.05) and equivalently improved in both the fetal lowdose and maternal clinical treatment group relative to negative control. Relative to saline control, SP-A, SP-C and AQ-5 mRNA expression was signifi cantly higher in both the fetal low dose group and maternal clinical treatment group. Conclusion: Maternal steroid administration was not required to elicit fetal lung maturation. Targeted fetal ANS treatments may allow the use of materially reduced antenatal steroid exposures, signifi cantly reducing disruption to the materno-fetal HPA axis and the risk of adverse outcomes. Darios Getahun * , 1,2 Morgan R Peltier, 3 Vicki Y Chiu, 1 Michael J Fassett * . This study aimed to determine if pregnancy after bariatric surgery is associated with autism spectrum disorder (ASD) risk in the off spring, and how this association is infl uenced by race/ethnicity, sex, timing of pregnancy post-surgery, and bariatric surgery type. Methods: A retrospective cohort study of children born from singleton pregnancies at Kaiser Permanente Southern California hospitals (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) in women who received bariatrics surgery (n=1,711) or were eligible but did not receive the surgery (n=16,010). All data were extracted from the electronic health records (EHR). Bariatric surgery registry records were used to ascertain the surgery timing and procedure type. Specialistconfi rmed diagnosis was used to ascertain ASD cases. Adjusted hazard ratio (aHR) and 95% confi dence interval (CI) were used to quantify the associations. Results: 10% of eligible women had the surgery. Compared with children of women without BS, children of women with BS were more likely to be of African-American race/ethnicity. Children born to women that had BS were more likely to be diagnosed with ASD We hypothesize that coaching on healthy lifestyle, here with focus on diet, using the eHealth coaching program www.SmarterPregnancy.co.uk (SP) improves maternal CV disease risk factors during the fi rst trimester of pregnancy. Methods: We examined the associations between SP intervention and maternal blood pressure (BP), anthropometrics, physical activity and smoking behaviour during the fi rst trimester in 132 pregnant women (intervention) compared to 1,093 pregnant controls in the Rotterdam Periconception Cohort. The intervention period was considered as a minimum of 6 weeks program usage up to a maximum of 24 months after the full program completion (that is 24 weeks). Outcomes were compared using regression models adjusted for maternal covariates including age, BMI, parity, gestational age, pregnancy status, smoking, alcohol use, geographic origin, education level and conception mode. Generalized estimating equations were used to track changes in dietary behaviour among the intervention group at 12 and 24 weeks after program activation, by means of a dietary risk score for vegetable, fruit, fast food, savory and sweet snack intake. The intervention group showed lower systolic BP (β adj -2.312; p<0.05), diastolic BP (β adj -1.879; p<0.01) and mean arterial pressure (βadj -2.023; p<0.01), smaller hip circumference (β adj -1.259; p<0.05) and higher waist-to-hip ratio (WHR) (β adj 0.015; p<0.05) compared to controls. We found no differences between the groups for BMI, waist circumference, smoking and physical activity. Stratifying for conception mode (besides WHR) associations remained significant for women conceiving following assisted reproduction. SP intervention improved dietary behaviour at 12 (β -0.834; p<0.001) and 24 weeks (β -1.073; p<0.001). Conclusion: Promoting healthy lifestyle behaviours with a focus on diet and using SP improved maternal cardiovascular health, importantly as a mean of lower blood pressure measurements, during the first trimester of pregnancy. Oregon HS University, Portland, OR, United States. Introduction: Fetal growth restriction and developmental programming of adult onset disease have a multifactorial etiology. Maternal environment, including diet, have been well-studied, but the synergistic effects of maternal diet and genetic predisposition require further exploration. We hypothesized that the combination of dietary risk and genetic risk would have a greater affect than either factor alone. Methods: Prospective case:control animal study employing a wellcharacterized transgenic (TG) mouse model of fetal growth restriction compared with wild-type (WT) controls. Genetic risk (AGTdup in C57Bl/6J background) was based on relatively elevated maternal angiotensinogen (AGT) expression/angiotensin II. Dietary risk evaluated low-sodium (0.15%), high-sodium (4%), and normal chow (1.3%) beginning 1 month prior to mating with WT males. Food intake was monitored. Progeny weights were recorded at birth and then weekly. Fetal sex was recorded Results: Progeny from TG dams were born smaller (1.3 grams) than WT controls (1.4 grams) when mothers were fed a normal chow diet. As expected, low-sodium diet led to fetal growth restriction in WT dams (1.3 grams; p<0.01). Despite returning maternal diet to normal after delivery, their progeny did not catch up in weight by 6 weeks' (~20 grams vs ~29 grams). TG dams repeatedly miscarried with no viable litters when fed low-sodium diet. High-sodium diet lead to larger litters in WT dams, but only ¼ TG dams became pregnant, which ended in stillbirth. Conclusion: Maternal low-sodium diet negatively affected both TG>WT dams, but high-sodium diet only adversely affected TG dams. This is intriguing provided human AGT-related genetic risk appears to arise out of more "protective" variants, which are seen at lower frequencies in high-risk populations. Introduction: Gestational diabetes (GDM) is a common pregnancy complication characterized by second trimester maternal hyperglycaemia. Untreated, GDM relates to increased risk on adverse maternal and neonatal outcome. Either beta cell dysfunction or insulin resistance are thought to underlie impaired gestational glucose tolerance. Understanding the dominant underlying mechanism predisposing to GDM may be important to provide timely and effective treatment, improving perinatal outcome. In this study we tested the hypothesis that insulin resistance rather beta cell dysfunction predisposes to GDM. Methods: A 75g oral glucose tolerance test (OGTT) was performed in 2112 second-trimester pregnant women to determine the relationship between insulin resistance (HOMA-IR), beta cell function (HOMA-β) and prevalence of abnormal glucose handling. Results: As compared to women with normal insulin resistance and normal beta cell function, high insulin resistance raised the risk on GDM (RR 6. 1, ). Beta cell dysfunction raised the risk on GDM (RR 3.8, 95%CI [2.7-5.4]). High insulin resistance but not beta cell function increased the necessity for additional glucose lowering medication on top of low carbohydrate diet in women diagnosed GDM. Conclusion: Both high insulin resistance and beta cell dysfunction increase the risk on GDM. As increased insulin resistance rather than beta cell function relates to insufficient response to low carbohydrate diet, we speculate that insulin sensitizers rather than insulin therapy may be the most suited and targeted therapeutic treatment modality in low carbohydrate diet-insensitive GDM in Northern European women. Metformin Inhibits Mitochondrial Metabolism and Increases Amino Acid Uptake in Primary Human Trophoblasts Cells. Amy Kelly †, Lana Madi, Anita Kramer, Theresa Powell, Thomas Jansson * . University of Colorado Anschutz, Aurora, CO, United States. Introduction: Metformin supplementation improves glycemic control in pregnant women with diabetes and reduces infant birth weights through largely unknown mechanisms. To explore the effect of metformin on human placental function, we tested the hypothesis that clinically relevant doses of metformin inhibits mitochondrial function and amino acid transport in primary human trophoblast cells. Methods: Placentas were collected from healthy women with normal BMI and Primary Human Trophoblast (PHT) cells were isolated and cultured. At 66 h, a physiological (1000 ng/mL), and supraphysiological dose of metformin (5000 ng/mL) or vehicle (cell culture media) with or without insulin (1nM) were added (n=4). At 90 h, the rate of PHT cell respiration was determined using Seahorse MitoStress protocol. System A and L amino acid uptake was measured by radiolabeled Na + -dependent uptake of [ 14 C]methylaminoisobutyric acid and 2-amino-2-norbornanecarboxylic acid (BCH)-inhibitable uptake of [ 3 H]leucine, respectively. PHT cell signaling (insulin, mTOR, AMPK, PGC1α) was assessed by Simple western. Statistical significance was tested by repeated measures ANOVA with Dunnett's post hoc test. Results: Metformin in physiological concentrations reduced respiration at basal (-66±15%, P=0.01) and at uncoupled, maximal (46±2%; P=0.02). Metformin did not change non-mitochondrial respiration. Physiological concentrations of metformin increased System A uptake (28.1 pmol/mg/ min) compared to control (8.1 pmol/mg/min; P<0.01) in the presence but not in the absence of insulin (n=4). Metformin increased AMPKphosphorylation and inhibited mTOR signaling. Physiological doses of metformin increased phospho (473)-AKT 40% as compared to control (n=7; P < 0.05). In agreement with effects in other tissues, metformin activates AMPK, inhibits mTOR signaling, and inhibits trophoblast oxidative phosphorylation. This may decrease placental ATP production in vivo, inhibiting energy requiring processes such as active nutrient transport and protein synthesis, contributing to reduction in fetal growth. We speculate that the stimulatory effect of metformin on trophoblast system A amino acid transport represents an acute effect on amino acid transport, possibly through increased insulin sensitivity. In vivo this would be counteracted by decreasing placental ATP levels following chronic treatment with metformin. There are not enough studies to clarify the role of MVF in prognosis. Due to this panorama full of uncertainties, the present study aims to establish whether the histological alterations of MVF has an impact on the severity of the clinical outcome. Methods: Observational, descriptive, cross-sectional study was performed. Maternal and newborn outcome information, as well as the gross and microscopic description of the placentas were obtained. The information collected was recorded in a RedCap web-based format. The Institutional Research and Ethics Committee approved this study. Diagnoses of newborns were taken into account. For mothers, diagnosed pre-pregnancy maternal conditions and pregnancy-related diseases and complications were collected. UC alterations including: length, insertion, coiled, direction of the coiling; number vessels, entanglements and true UC knots. Gross and microscopic alterations of the placenta were registered. Gross description and sampling of the placentas followed institutional protocols. Microscopic criteria followed de Amsterdam consensus. The association between FVM and interest clinical outcomes was evaluated by calculating raw odds ratio (OR) analysis with 95% confidence intervals (CI). Statistical analysis was performed using Stata 14.2. Only variables with a P-value <0.05 were retained in the final statistical models. Results: A total of 387 had any criteria of FVM and 345 newborns had any complication. A positive association were seen for respiratory distress syndrome(p:0.04; OR1.6(CI 1. 2-5.8)) non-physiological jaundice(p:0.004; OR1.87(CI 1.21-2.89)), IUGR(p:0.007; OR2.55(CI 1.27-5.12)) and patients with congenital heart defects (p:0.019; OR2.35(CI 1.13-4.89)). Association with maternal pregestational conditions also were significant: chronic hypertension(p:0.03; OR4.35(CI 1.01-18.56)) and obesity/ overweight(p:0.009; OR1.98(CI 1.17-3.32)). Finally, maternal pregestational conditions such as HDP also shown statistical significance associated with FMV(p:0.02; OR1.72(CI 1.06-2.79)). The association between FVM and adverse neonatal prognoses is known, so achieving an early and pertinent characterization of the most relevant diagnostic criteria will allow, in the long term and in future studies, the development of early interventions by clinicians in these newborns which in turn could decrease their morbidity and mortality. Introduction: Complications from preterm birth (PTB) account for the leading cause of death and disability in those under five years. Whilst the role of omega-3 supplementation in reducing PTB is well-established, growing evidence suggests supplementation use in those replete has no benefit and may increase the risk of early PTB. Current methods for assessment of omega-3 serum levels are often expensive and invasive. Therefore, we aimed to develop an affordable and non-invasive clinical tool to identify women who were replete in omega-3 fatty acids (total omega-3 serum levels above 4.3%) in early pregnancy. We conducted a prospective observational study recruiting 250 participants from three clinical sites in Newcastle, Australia. Participants were eligible if they had a singleton pregnancy between 8-and 20-weeks' gestation. Estimated intake of omega-3 fatty acids and sociodemographic data was collected using an electronic questionnaire. Total omega-3 serum levels were determined from a venous blood sample via gas chromatography. The optimal cut-point of estimated omega-3 intake that predicted individuals at risk of total omega-3 serum levels above 4.3% was developed using multivariate logistic regression, adjusting for maternal age, body mass index, socioeconomic status, and use of omega-3 supplementation. Models were evaluated using sensitivity, specificity, area under receiver operator characteristic (AUROC) curve, true positive rate (TPR) at 10% false positive rate (FPR), the Youden Index, Closest to (0,1) Criteria, Concordance Probability, and Index of Union. Model internal validation was performed via bootstrapping to generate 95% confidence intervals for the assessment of the model's performance in the study cohort. Results: Of the 227 participants included in model development, 59.0% had total omega-3 serum levels above 4.3% of total fatty acids. Predictors in the optimal model included dietary intake of omega-3 with adjustment for maternal age, body mass index, socioeconomic status, and omega-3 supplementation use. The optimal model had a moderate discriminative ability (AUROC 0.75, 95% CI 0.69-0.82) with 82.1% sensitivity, 59.1% specificity and 38.8% TPR at 10% FPR. Conclusion: Our clinical tool was a modest predictor of pregnant women with total omega-3 serum levels above 4.3% based on their dietary intake and sociodemographic characteristics. Its current performance is not yet adequate for clinical use but may be improved with adjustment for total energy intake. Overall, our study demonstrates potential for the prediction of omega-3 serum levels using non-invasive techniques. Abnormal Glucose Tolerance in the Third Trimester After Early Normal Screen and Associated Pregnancy Outcomes. Emily Lee †, Lisbet S Lundsberg, Jennifer Culhane, Caitlin Partridge, Moeun Son * . Yale University, New Haven, CT, United States. Introduction: Early glucose tolerance testing (GTT) for gestational diabetes (GDM) is recommended in patients with risk factors. It is unknown whether patients with a normal early GTT who fail a repeat third trimester GTT have different risk profiles and different rates of adverse maternal and neonatal outcomes compared to women who pass repeat screening. Methods: This is a retrospective cohort study of women with singleton pregnancies delivering between 2013-2021 who underwent early GTT (<20 weeks' gestation) and passed (<140mg/dL), and subsequently had a repeat GTT >=24 weeks' gestation. Women were classified as pass/ pass if both early and repeat GTT values were <140mg/dL. Conversely, women were classified as pass/fail if their second GTT was ≥140mg/dL. The composite neonatal morbidity outcome included birth injury, large for gestational age birth weight, respiratory distress, hypoglycemia and/or hyperbilirubinemia requiring NICU admission, and perinatal death. The maternal composite morbidity outcome included hypertensive diseases of pregnancy, cesarean delivery, obstetric anal sphincter injuries (OASIS), and postpartum hemorrhage. Bivariable and multivariable analyses were performed. Median values of early GTT were compared between both groups using Wilcoxon test. Results: Of 45,345 deliveries, 4577 (8.9%) women had early and repeat GTT. 3499 (76.4%) passed both GTT tests and 1078 (23.5%) failed repeat screening. Women who failed repeat GTT were significantly more likely to be >35 years old, married or partnered, multiparous, have commercial insurance, and use metformin before pregnancy. Women who failed repeat testing were more likely to be non-obese and identify as Caucasian. Compared to women who passed both GTT, women who failed their second screen were at increased risk for the neonatal composite outcome (34.7% vs. 25.0%, OR 1.60 (95% CI 1.38-1.85)), which persisted with multivariable analysis (aOR 1.58 (95% CI 1.36-1.83). Similarly, they were at increased risk for the maternal morbidity outcome (57.8% vs. 54.0%, OR 1.17 (1.02-1.34); aOR 1.16 (1.01-1.34)). Median value of early GTT was higher in women who failed the second screen (115mg/ dL, IQR 102-127) compared to women who passed both screens (99 mg/ dL, IQR 85-114); p<0.0001. Women with normal early GTT who failed their second screen had fewer risk factors than expected, but were more likely to experience both adverse neonatal and maternal pregnancy outcomes. This group also had significantly higher median early GTT value. Therefore, women who underwent early screening and passed still remain at risk for complications and should undergo timely repeat GTT in the third trimester. Further research to investigate alternative early GTT thresholds in women with risk factors may be warranted. Fetal Acidemia at Time of Cesarean Delivery: The Effect of Abdominal Wall Thickness. Rebecca Rimsza †, Fan Zhang, Katherine Bligard, Ebony Carter, Jeannie Kelly, Anthony Odibo, Methodius Tuuli, Nandini Raghuraman * . Washington University School of Medicine, Saint Louis, MO, United States. Introduction: Maternal obesity is a risk factor for fetal acidemia at time of cesarean delivery (CD). One hypothesis is that increased abdominal wall thickness leads to elevated intraabdominal pressure and decreased uterine perfusion. We investigated the relationship between subcutaneous depth and umbilical artery (UA) pH among patients with obesity. Methods: This is a secondary analysis of a multicenter randomized trial of negative pressure wound therapy among patients with obesity undergoing planned or unplanned CD. Subcutaneous tissue depth was measured intraoperatively. The primary outcome was UA pH. The secondary outcomes were UA pH <7.1, <7.0, other UA gas parameters, NICU admission, and APGAR scores. Outcomes were compared between patients with subcutaneous depth ≤2cm and >2cm. Multivariable logistic regression and the Zhang method were used to estimate adjusted relative risks. Results: Of the 1624 randomized in the parent trial, 1312 had subcutaneous measurements and UA gases. 415 (31.6%) had subcutaneous depth ≤2cm and 897 (68.3%) had depth >2cm. Patients with depth >2cm were more likely to be White, have private insurance and have a higher body mass index. There was no difference in rates of scheduled or unscheduled CD between groups. Indication for CD was significantly different between groups, with more CD for labor arrest or non-reassuring fetal status in the group with >2cm depth. UA pH was not significantly different between groups (7. Conclusion: Cesarean birth was more common among NTSV people with epilepsy, and was associated with severe pre-eclampsia/eclampsia, gestational diabetes, and gestational age >41 weeks at birth. Hospital and regional differences were noted. Further investigation into whether this increased cesarean rate is related to SMM among people with epilepsy is warranted. Clinical factors associated with NSTV cesarean births among people with epilepsy. The greatest risk is found in women who are obese before conceiving and gain significant weight during pregnancy. In addition, obesity is associated with placental lipo-toxicity, oxidative stress, and inflammation. Here we aim to investigate how high-fat diet (HFD)-induced obesity affects placental gene expression and development. Methods: To induce obesity, adult female FVB mice were fed a HFD (60 kcal% fat) while control mice were given a low-fat diet (LFD, 10 kcal% fat). Two feeding schedules were applied: (1) a HFD during mating and throughout pregnancy (HFD pregnancy); and (2) a HFD initiated 4 weeks prior to mating and during pregnancy (HFD pre-conception). Maternal weights were monitored daily. Glucose levels in blood collected from tails or by cardiac puncture were measured using a glucometer. At different gestational ages (E7.5, 8.5, 9.5, 11.5, and 16.5) , placentas were collected for total RNA isolation. Gene expression levels were then quantified using custom-designed PCR arrays and compared between all groups. Results: Significant maternal weight gain was found in mice on both feeding schedules compared with pregnant LFD controls. However, glucose levels in the HFD and LFD groups were not significantly different. Expression levels of several placental genes were significantly different in both HFD groups compared with LFD controls. Specifically, HIF1A, HO-1, SOD-1, VEGFA, Angpt1, IL-6, AP2, Nrf2 were downregulated and Gpx1 was upregulated. Although this trend was similar for both HFD groups, it was more dramatic in the HFD preconception group. Conclusion: A HFD can induce gestational obesity in mice, and subsequently affect the expression of placental genes that are associated with oxygen sensing, oxidative stress, angiogenesis, and inflammation. Preconceptional obesity can aggravate these alterations and therefore may heighten the risk for pregnancy complications. Placental Pathogenic Cultures Associated with Infectious Etiologies of IUFD. Alicia S Liu, Caitlin Little, Christina Megli * . University of Pittsburgh School of Medicine, Pittsburgh, PA, United States. Introduction: Intrauterine fetal demise (IUFD) is a devastating complication of pregnancy that can be infectious in etiology. Although ascending infection can cause IUFD, the organisms that are associated with IUFD are not well characterized. Moreover, the utility of placental culture in the work-up of stillbirth is not well defined. At our institution, placental cultures are routinely sent as part of the work-up of an IUFD. We sought to evaluate the utility of placental culture in defining an infectious etiology for IUFD as well as elucidate the organisms isolated from infectious causes of IUFD. Methods: From 2014-2018, 910 placentas had bacterial cultures sent. 422 cultures resulted in pathogenic bacterial isolates. The maternal chart was reviewed for diagnosis of IUFD and then maternal and pregnancy characteristics were collected. Histopathology placental results were reviewed and categorized into infectious, vascular and indeterminate etiologies of stillbirth. Infectious etiology was defined by being positive for chorioamnionitis on histopathology or diagnosis of PPROM preceding IUFD diagnosis. A two-way ANOVA was performed comparing probable, possible and non-infectious etiology. Results: Our cohort included 79 stillbirths. In this large cohort, 61 cases were identified as having probable or possible infectious etiology. All had positive placental cultures. In the probable cohort, 64.5% (n=20) were GBS, 25% (n=8) were other pathogens. A minority, 6% (n=2) had E. coli. In the possible infectious etiology group, 41.9% were positive for GBS (n=13), 6.5% had E. coli (n=2), and 19.3% grew E. faecalis (n=6). Other pathogens made up the rest, 32% (n=10). Of note, in a controlled cohort with a clear vascular etiology of the IUFD, pathogenic bacteria were also detected. The proportion of pathogenic bacteria approached significance p=0.07 when comparing between the 3 categorized etiologies of IUFD. Conclusion: In this contemporary cohort, pregnancies that resulted in stillbirths of probable or possible infectious etiology were not significantly associated with pathogens that are known causes on intrauterine infection at increased rates compared to stillbirths of non-infectious etiology. This suggests that more research is necessary to determine the utility of placental culture in context of determining IUFD causes. Identification of the Role of RNA Binding Proteins in the Selective Enrichment of microRNAs in Small Extracellular Vesicles in Gestational Diabetes Mellitus. Soumyalekshmi S Nair, 1 Nanthini Jayabalan, 1 Andrew Lai, 1 Dominic Guanzon, 1 Martha Lappas, 2,3 Carlos Salomon * . Introduction: Gestational Diabetes Mellitus (GDM) is the glucose intolerance first diagnosed in pregnancy. It is the most common medical complication of pregnancy with short-term and long-term consequences to mothers and offspring. Small extracellular vesicles (sEVs) are nanosized vesicles and miRNAs in sEVs are involved in regulation of insulin sensitivity in target cells. The aim of the present study is to identify the molecular interaction between sEV miRNAs and proteins that leads to recruitment of miRNAs in sEVs. We identified the repertoire of miRNAs highly enriched in sEVs in Normal Glucose Tolerance (NGT) and GDM primary trophoblast cells using small RNA library synthesis and next generation sequencing. Using Motif Discovery based on Short Nucleotide Sequences (MDS2) we performed the miRNA motif discovery and enrichment analysis and the miRNA protein interaction was analysed using pull down assay. Using RBPmap, a computational tool that enables accurate prediction and mapping of RBPs binding sites on any RNA sequence we analysed the interaction of miRNA motifs and proteins. Results: This study identified that the miRNA content of sEVs correlated with that of the trophoblast cells of origin but identified a specific set of miRNAs which were differentially expressed in sEVs compared to cells. We identified that miR-1246 and miR-1285 were the most highly enriched miRNAs in sEVs in NGT and GDM respectively. We identified constitutive sequences 4-6 bps long with coverage greater than 75% with a statistically significant p-value and unique expression as the miRNA motifs. In addition, a repertoire of RNA binding proteins (RBPs) interacting specifically with the candidate miRNAs were identified. We identified the motifs UUCU and UCUCU are overrepresented in miR-1246 in NGT interact with proteins hnRNPH2 (heterogeneous nuclear Ribonucleoprotein), YBX3 (Y Box protein), EIF4B (Eukaryotic translation initiation factor 4B) and GRSF1 (G-rich sequence factor 1). In GDM, the motifs GUAG and GAGU overrepresented in miR-1285 were identified to interact with proteins HDLPB (High Density Lipoprotein Binding Protein), ACADVL (Very long-chain specific acyl-CoA dehydrogenase) and CWF19L1 (CWF19-like protein 1). Conclusion: Specific molecular interactions between specific miRNA motifs and RNA binding proteins might be involved in the recruitment of miRNAs in small extracellular vesicles Indication for Emergent Cesarean Delivery and Risk of Neonatal Metabolic Acidosis. Gabriela Dellapiana †, Savannah Gonzales †, Candace Levian †, Richard Burwick * . Cedars-Sinai Medical Center, Los Angeles, CA, United States. Introduction: Emergent cesarean delivery (CD) is associated with increased risk of neonatal metabolic acidosis; we hypothesize that risk varies by delivery indication. Methods: Cohort study of patients undergoing emergent CD from July 2012 to April 2020. Cases were stratified by delivery indication: nonreassuring fetal heart tracing (NRFHT), cord prolapse, malpresentation in labor, failed operative vaginal delivery (OVD), uterine rupture, or vaginal bleeding. The primary outcome, reflecting neonatal metabolic acidosis, was a composite of arterial cord pH ≤7.0, base excess ≥12, or 5-minute Apgar <7. Patient characteristics were evaluated as confounders. Statistics by χ 2 , t-test, test of medians, and logistic regression; alpha <0.05. Results: From 50,494 deliveries, 464 (0.9%) had emergent CD and were included in the analysis. The leading indication for emergent CD was NRFHT (n=328, 70%), followed by cord prolapse (n=47, 10%), failed OVD (n=35, 8%), malpresentation in labor (n=27, 6%), vaginal bleeding (n=16, 3%), and uterine rupture (n=11, 2%). The median (IQR) time from skin incision to delivery was 2 min (1-3) and did not vary by Friday Posters delivery indication. The primary composite outcome occurred in 26% (119/464) of patients, including 15% (n=61) with arterial cord pH ≤7.0, 12% (n=49) with base excess ≥12, and 16% (n=72) with 5-minute Apgar <7 (Table 1 ). The composite neonatal outcome occurred at a similar rate in all groups except uterine rupture. Compared to patients delivered for other indications, those with uterine rupture were more likely to have the composite outcome (64% vs. 25%, P=0.009; OR 5.2, CI 1.5-18); pH≤7.0 (44% vs. 13%, P=0.02; OR 5, ; and 5-minute Apgar <7 (45% vs. 15%, P=0.01; OR 4.6, CI 1.4-16); these fi ndings were unchanged after multivariable adjustment. Conclusion: After emergent CD, neonatal metabolic acidosis occurs at a high rate regardless of indication. However, uterine rupture carries a much greater risk compared to all other indications, reinforcing the need to prevent this adverse outcome. Introduction: Pre-pregnancy obesity in pregnancy is a known risk factor for cesarean birth. However, little is known about the risk of cesarean birth in patients that had BMI<30 pre-pregnancy but after gestational weight gain became obese. Therefore, the objective of this study was to compare the rates of cesarean births in pregnant patients who became obese during pregnancy to those who maintained their fi rst trimester body mass indices (BMIs). Methods: We conducted a retrospective cohort analysis of patients with known fi rst trimester and delivery BMIs who delivered between 2014 and 2017 at a single academic medical center. The primary outcome was the rate of cesarean birth. Analysis was stratifi ed by three cohorts: (Cohort 1) patients who maintained normal or overweight BMIs (<30 kg/m 2 ) throughout pregnancy; (Cohort 2) patients with pre-pregnancy BMI of <30 kg/m 2 who became obese (≥30 kg/m 2 ) during pregnancy; and (Cohort 3) patients with pre-pregnancy BMI ≥30 kg/m 2 who remained obese throughout pregnancy. Bivariate and multivariate analyses were conducted to assess independent association between BMI and cesarean birth. Results: The analysis included 515 patients. In unadjusted analysis, cohort 2 (42.7%) and cohort 3 (46.5%) had higher rates of cesarean birth compared to cohort 1 (32.5%) (p = 0.013). After adjusting for confounding factors, cohorts 2 and 3 were independently associated with a higher risk of cesarean birth, with cohort 2 having the highest risk (aOR 1.77, 95% CI 1.04 -3.00) (Table) . Conclusion: While all patients with BMI ≥30 kg/m 2 at the end of pregnancy had higher risk of cesarean compared to patient with BMI<30, patients who became obese during pregnancy had the highest odds ratio of cesarean birth. This fi nding adds additional evidence to the detrimental eff ect of excessive gestational weight gain on maternal health. Introduction: Blood transfusion is a contributor to severe maternal morbidity and iron defi ciency anemia (IDA) is a risk factor, with increased prevalence in those of low socioeconomic status. This study assesses the relationship between IDA and transfusion, and investigates if insurance type, as a proxy for socioeconomic status, modifi es that relationship. Methods: Retrospective cohort study of all deliveries > 20 weeks gestation from 10/2015 -09/2018 at a tertiary referral center. Demographic, obstetric, and socioeconomic factors and hemoglobin (Hgb) and mean corpuscular volume (MCV) on delivery admission were abstracted from medical records. Primary exposure was presumed IDA, defi ned as Hgb < 11 g/dL and MCV < 85 on delivery admission. Primary outcome was transfusion, identifi ed by ICD-10 codes or blood bank records. Secondary outcome included massive transfusion (> 4u product). Insurance was defi ned as public, private, and self-pay. Bivariate analyses compared individuals with IDA to those without. Multivariable logistic regression was used to evaluate factors associated with transfusion with an interaction between anemia and insurance type. Results: Of 11491 included deliveries, 790 (6.9%) had IDA at admission. There were 207 transfusion events. Table 1 shows baseline characteristics and outcomes by presence of anemia. In multivariable logistic models, anemia was associated with a 3-fold odds ratio (OR) of transfusion (aOR 3.6, 95% CI 1.9 -6.7). Native Hawaiian/Pacifi c Islander race, delivery type, and estimated blood loss were independently associated with transfusion (Table 2) . Though IDA is more common among the publicly insured, insurance type did not modify the relationship between anemia and transfusion. Conclusion: IDA is a modifi able risk factor associated with increased transfusion risk independent of insurance type. Identifying social determinants that increase risk for IDA on admission for delivery may help to target interventions. Methods: We performed a survey study of second stage experience among women with singleton, term, vaginal deliveries at a tertiary care center between August-November 2020. Patients undergoing cesarean delivery were excluded due to second stage biases associated with unplanned cesarean delivery All eligible patients were approached on randomly selected days of the week. Postpartum patients completed a 13-question survey on childbirth education, details of the second stage, and patients pushing experience. Bivariate analyses were used to compare survey responses between White and Black patients. Results: Of the 38 patients surveyed, 24(63%) were White and 14(37%) were Black. White women were older in age (30±5 vs 25±6 yrs), lower in body mass index (29.3±4.7 vs 31.9±9.3 kg/m2) and delivered babies with higher birthweights (3495g±413 vs 3222g±792) compared to their Black peers (p < .05 for all). The length of the second stage and rates of nulliparity, induction, and epidural use were similar between groups. 25% of White women reported receiving childbirth education via private or hospital-based classes whereas none of the Black women received formal education (p=0.04). Black patients were more likely to have knowledge about the second stage from observing other deliveries (29% vs 4%, p = .003). Black and White women were equally satisfied with provider support during the second stage (93% vs 96%, p=0.3). Both groups felt additional instruction would help with pushing (White: 80%, Black: 64%, p=0.45). White and Black patients agreed on what would be most helpful to prepare them for the second stage with "hearing others experiences" and an "instructional video" as most common (46% vs 21% and 29% vs 21%, respectively, p >.05 for both). Conclusion: Black women are significantly less likely to receive formal childbirth education and second stage guidance. This disparity highlights the need for innovative approaches to childbirth education that are accessible to all patients. Future work should examine the effect of such approaches on effective pushing and adverse outcomes related to prolonged second stage. Strategies for Successful VBAC. Daniel Roshan, Marjon Mobasseri * , Jasmine Roshan †, John Migotsky * . ROSH MFM, New York, NY, United States. Introduction: Vaginal birth after cesarean (VBAC) remains an important option for many women, especially those who desire multiparity. We thought to develop strategies to enhance successful VBAC and reduce complications. 1. Use of Evening primrose oil (EPO) which has been used by many as a slow process cervical softening agent from 35 weeks till delivery. 2. Maternal weight gain management and guidelines to prevent excessive weight gain and large fetuses. 3. Avoiding induction, waiting for spontaneous labor until 42 weeks if clinically possible. 4. Letting patients to be mobile during labor as much as possible and avoiding early admissions to labor suit. We applied the above strategies to our private practice group patients from 2011 till 2018. Through our computerized data base, we collected relevant data from all our patients who were a candidate for VBAC after one or two c section (C/S) without any contraindications. Maternal average age was 31, mean pregnancy weight was 147 lbs, and BMI 25.6. All patients were managed by the same team of 4 obstetricians and one MFM doctor. All patients were extensively counseled on VBAC and patients who has contraindication to VBAC were removed from the study group. Results: We identified 562 patients who underwent VBAC trial, 479 patients had successful VBAC, 85% success rate which deemed to be much higher than published reports by ACOG and our hospital control group(71%). Of the successful VBAC group 139 patients had two prior c/s, 33 had operative vaginal delivery (5 vacuums and 28 outlet forceps). Our complications rate was comparable to prior reports by ACOG. One case of uterine rupture which was diagnosed during labor and managed without fetomaternal harm, uterus was repaired. the rate of 3 rd and 4 th degree perineal tear was 1.9%, episiotomy rate 11% and blood transfusion rate of less than 2 units was 2.7%. There was no side effects reported from the use of EPO. Neonatal complications rate was similar to non VBAC group. Conclusion: The above strategies was extremely helpful to increase successful VBAC rate and lower complications of childbirth. Weight management preconception and throughout the pregnancy, use of natural slow progress cervical softening agent such as EPO, avoiding unnecessary inductions and awaiting spontaneous labor with less medical intervention, all are extremely useful strategies to achieve successful VABC. Many experienced clinicians and prior published abstracts attest to our findings however a large prospective studies are required to further confirm our proposed strategies. Mina Desai * , 1 Ken Kobayashi * , 2 Guang Han, 1 Michael G Ross * . 1 1 The Lundquist Institute, Torrance, CA, United States; 2 Hokkaido University, Sapporo, Japan. Introduction: Infants born to obese mothers are at markedly increased risk of childhood and adult obesity due to in utero exposure and excess postnatal infant weight gain. The increased fat and calorie content in milk of obese mothers suggest that maternal diet, metabolic milieu and mammary epithelial cell (MEC) fatty acid uptake and synthesis can alter milk composition. Importantly, milk long chain fatty acids (e.g palmitate) originate from maternal diet or from mobilized fat stores whereas short and medium chain fatty acids are from de novo lipogenesis in MECs. Increased plasma palmitate levels are seen in obese women during pregnancy and palmitate is the major saturated fatty acid in human milk representing 20-25% of fatty acids. We used a novel three-dimensional (3-D) human MEC culture model to determine the effects of exogenous palmitate on MEC cellular pathways and secreted mRNA. Methods: MEC cultures were established using mammary glands from non-pregnant, non-lactating mice. For 3-D MEC cultures, cell inserts were used to separate the basolateral chamber containing lactogenic medium (nutrients) and the apical chamber containing secreted MEC "milk". Palmitate-albumin conjugate (100 µmol/L) was added to the basolateral chamber. After 48h, apical medium and cellular MEC were collected separately, extracted for mRNA and analyzed expression levels of fatty acid uptake enzyme (lipoprotein lipase), fatty acid transport protein (FABP3), fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD1). Data was analyzed by ANOVA (Mean±SE; *P<0.01 treated vs. untreated MECs). Results: Following treatment with palmitate, MEC "milk" had increased mRNA expression of lipoprotein lipase and FAS with decreased expression of SCD1 and unchanged FABP3. An identical pattern of changes was noted in mRNA expression in MEC cells (i.e., increased lipoprotein lipase and FAS with decreased SCD1 (Fig) . Conclusion: Palmitate-mediated increased lipoprotein lipase and decreased SCD1 expression suggest increased palmitate uptake and suppression of fatty acid desaturation. Similar changes in mRNA profile of MEC "milk" and cellular MEC suggest that milk composition can be modulated by maternal diet with secreted milk mRNA reflecting MEC cellular responses. Mechanisms of Fetal Programming of Metabolic Dysfunction by Placental mTORC1 Nutrient Sensing. Megan Beetch †, Brian Akhaphong, Briana Clifton, Emilyn Alejandro * . University of Minnesota, Minneapolis, MN, United States. Introduction: Placental dysfunction is a leading cause of pregnancy complications, such as fetal growth restriction (FGR), which can impact offspring metabolic tissues and lead to obesity and type II diabetes (T2D) in adulthood. Mammalian target of rapamycin (mTOR) is a nutrient sensor kinase that integrates signals to promote growth. Human studies have shown decreased mTOR activity in the FGR placenta. Methods: Using murine models of direct mTOR manipulation in the placenta (loss or gain of mTOR activity by deletion of mTOR or TSC2, a negative regulator of mTORC1), we have demonstrated a role for placental mTOR signaling in modulating birthweight and adult offspring susceptibility to insulin resistance and obesity. Results: In female offspring, the loss of placental mTOR (mTORKO Pla ) increased susceptibility to insulin resistance and obesity under a high-fat diet (HFD) challenge, whereas enhanced placental mTORC1 activity (TSC2KO Pla ) conferred protection. To better understand the molecular mechanisms governing the offspring phenotypes, we hypothesized that placental mTOR regulates insulin sensitivity in the adult offspring through regulation of insulin genes, thereby insulin levels, and insulin signaling in peripheral tissues. We found that mTORKO Pla female offspring on HFD displayed hyperinsulinemia. In contrast, TSC2KO Pla female mice did not have heightened serum insulin levels in response to HFD and showed increased insulin sensitivity measured by HOMA-IR. Differentially programmed insulin sensitivity in the placental mTOR-manipulated offspring was further supported by alterations in Akt signaling (a proxy for insulin signaling) in the liver of mTORKO Pla and TSC2KO Pla mice in a sexually dimorphic manner. Furthermore, Ins1 expression was significantly increased in mTORKO Pla female islets and significantly decreased in TSC2KO Pla female islets, corresponding with lack of hyperinsulinemia observed in TSC2KO Pla female offspring under a HFD challenge. RNA sequencing of TSC2KO Pla and mTORKO Pla liver and islets revealed differences in lipid metabolism, inflammation, and phagocytosis pathways. For example, a liver candidate that encodes MUP1, a secreted protein implicated to regulate whole-body insulin sensitivity, was increased in mTORKO Pla and reduced in TSC2KO Pla females. In addition to delineating metabolic consequences of decreased/increased placental mTOR nutrient sensing in adulthood, we explored local alterations in the mTORKO placenta. Female-specific increase in mRNA and protein levels of amino acid transporters from Systems L and A were identified in the mTORKO placenta and may contribute to the differential programming of male and female fetuses. Conclusion: Understanding the landscape of fetal programming by placental mTOR and its impact on offspring metabolic outcomes will fill a knowledge gap with potential for new therapeutic strategies for obesity and T2D. Programming of NAFLD in Mice. Ashok Mandala †, Rachel C. Janssen, April M. Teague, Wanke Zhao, Jacob E. Friedman * , Karen R. Jonscher * . Center, Oklahoma City, OK, United States. Introduction: Predisposition to juvenile NAFLD is programmed in early life by maternal Western diet (WD), but mechanisms are poorly understood and nutritional interventions are a primary target. We found that offspring of dams receiving WD (WD-O) during gestation and lactation have reduced serum levels of the tryptophan metabolites indole (Ind) and indole-3-acetate (I3A), concomitant with hepatic steatosis and inflammation. We hypothesized that maternal supplementation with these metabolites blunts development of WD-induced NAFLD in the offspring. Methods: Dams were fed WD, with or without indole or I3A supplemented in drinking water, for 2 weeks prior to mating and through gestation and lactation. Male offspring were weaned onto chow diet and, at 12 weeks of age, challenged with WD for 4 weeks. Tissues were harvested at 16 wks. Hepatic lipid accumulation was evaluated by Oil Red O staining and measurement of liver triglyceride levels. Expression levels of genes involved in de novo lipogenesis, inflammation and fibrosis were measured using qPCR. In vitro, we treated RAW264.7 macrophages with lipopolysaccharide (LPS), and HepG2 hepatoma cells with free fatty acid (FFA), in the presence or absence of indole or I3A. We measured expression levels of genes involved in inflammation and lipid metabolism. Results: While body weight was significantly reduced in offspring from Ind (Ind-O) at 16 weeks of age vs. WD-O (P<0.01), I3A-O were unchanged. Both Ind and I3A significantly (P<0.05) reduced indices of NAFLD in offspring, including steatosis, hepatic triglyceride levels, and expression of genes involved in de novo lipogenesis and fibrogenesis. Expression levels of pro-inflammatory genes were reduced in Ind-O while anti-inflammatory genes increased in I3A-O, suggesting these molecules act via distinct mechanisms. Co-treatment of FFA-loaded hepatocytes and LPS-treated macrophages with Ind or I3A significantly reduced inflammation and expression of genes involved in de novo ceramide synthesis. Addition of exogenous ceramide to macrophages had no effect on the anti-inflammatory effect of Ind but blunted the effect of I3A on inflammatory gene expression, indicating a crucial role for ceramide in pharmacological actions of I3A but not Ind. Conclusion: Our findings suggest that maternal supplementation of endogenous microbiota-derived I3A and Ind targets liver inflammation and ameliorate NAFLD in vivo. Our in vitro studies suggest a potential mechanistic role for ceramide metabolism in this process. Ind and I3A are natural ligands for the aryl hydrocarbon receptor, a mediator of immune homeostasis, and future studies will probe the role of signaling through this receptor in the developmental programming of NAFLD. Maternal Obesity Programs Adverse Metabolic Phenotypes in F3 Neonatal Lambs. Jessica King †, Christopher L. Pankey * . West Virginia School of Osteopathic Medicine, Lewisburg, WV, United States. Introduction: Maternal obesity (MO) programs negative metabolic phenotypes in future generations. Obesity during gestation promotes hypercortisolemia, providing a mechanism for multigenerational programming as both the fetus (first generation (F1)) and its associated germ cells (future second generation (F2)) are directly exposed to the adverse maternal endocrinology. Whereas multigenerational programming encompasses phenotypic changes in F1 and F2 following a maternal exposure, transgenerational programming effects are not observed until the third generation (F3), as they are never directly exposed to the original insult. We hypothesized that MO would program adverse metabolic phenotypes in F3 neonates, demonstrating a transgenerational programming mechanism. Methods: Control (CON) and obese (OB) ewes (generation 0; F0) were bred to a single ram to produce the first generation of offspring (F1). From 60 d prior to conception through term, CONF0 ate 100% National Research Council recommendations (NRC), while OBF0 ewes ate 150% NRC. All F1, F2, and F3 ate 100% NRC after weaning. All mature F1 ewes were bred to a single ram to generate CONF2 (n = 4) and OBF2 (n = 5). Mature F2 ewes were bred to a single ram to produce CONF3 (n = 4) and OBF3 (n = 5). An intravenous glucose tolerance test (GTT) was performed in F2 ewes at 90% gestation. F3 Morphometrics were recorded immediately after birth. Dual-energy X-ray absorptiometry scans (DEXA) were performed in F3 on d1 of life to determine body composition. Results: OBF2 ewes exhibited markedly greater (P < 0.0001) plasma cortisol than CONF2 throughout gestation. GTT revealed OBF2 ewes had a higher (P < 0.05) insulin response with similar glucose, resulting in greater (P < 0.05) insulin resistance. DEXA revealed no diff erences between OBF3 and CONF3 neonates in mass, fat mass, or lean mass (p < 0.05). DEXA did show decreased BMD in OBF3 females vs CONF3 females, but no diff erence in OBF3 and CONF3 males. Morphological measurements reveal greater (p < 0.05) birthweight and abdominal girth, and a trend (p < 0.1) for greater skin fold thickness and thoracic girths in OBF3 relative to CONF3. No treatment diff erences were identifi ed in biparetial distance, crown rump length, or shoulder height. Conclusion: These data suggest a transgenerational, serial programming mechanism resulting from F0 obesity that lessens in severity across generations. Future studies should be directed at elucidating the phenotypes observed following metabolic stressors in OBF3. Introduction: Monochorionic-diamniotic twins are at risk for twintwin transfusion syndrome (TTTS) resulting from a circulatory imbalance between twins. Predicting disease progression and outcomes remains a challenge owing to an incomplete understanding of disease pathophysiology. We performed analyses of amniotic fl uid (AF) exosomes and metabolites to identify dysregulated pathways and identify molecular phenotypes of TTTS. Methods: We compared AF from the recipient collected prior to laser (cases) vs. singletons (controls). RNA was extracted from AF exosomes isolated by diff erential centrifugation. After library preparation, indexed libraries were pooled and sequenced (Nextseq 500). Diff erential gene expression and gene set enrichment analyses were performed (FDR < 0.1). To validate RNA-seq fi ndings, metabolite profi ling (LC/MS) was done for a separate group of AF samples. Principal component analysis was carried out using the most variable metabolites, both within all samples and within cases only. Diff erential metabolites were calculated based on an absolute fold change > 2 and adjusted p < 0.001. Results: RNA-seq analysis included 7 cases vs. 6 controls. TTTS patients tended to be younger and at a later gestational week (20.5 vs. 16.8); most were stage III. Gene set enrichment analysis identifi ed downregulation of genes involved in oxidative phosphorylation, TCA cycle, fatty acid, and amino acid metabolism. Metabolomics on 22 stage III cases and 10 controls revealed distinct metabolic patterns (Fig A-C) . When analyzing diff erential metabolites, 2 clusters emerged from the cases (Fig D) . Compared with Cluster 1, Cluster 2 had low urea and increased metabolites involved in amino acid metabolism and fatty acid oxidation (Fig E-F) . Cluster 2 fetuses appeared to have a more severe phenotype based on clustering of Cluster 1 with controls. Conclusion: TTTS-associated metabolic disturbances diff erentiate stage III cases into distinct clusters. Cluster 2 metabolite pattern may refl ect: 1) renal injury (cardiorenal physiology), or 2) metabolic derangement from oxidative stress and dysregulated fatty acid metabolism. Breast Fig 1) and average milk (75±9 vs 114±7 kcal/dl) was signifi cantly increased in OW/OB. Amongst all women, there were two distinct groups of foremilk: Low Fat (LF <2.0 mg/ dl; N=6) and High Fat ( > 2.0 mg/dl; N=15), with no relation to maternal BMI. The net increase in fat (6.3 vs 3.1 g/dL) and calories (55 vs 25 kcal/ dL) from fore-to hindmilk was signifi cantly greater in HF vs LF (Fig 2) , and infants of HF women had a trend towards increased milk ingested at the study feed (57 vs 41 g). Conclusion: Milk fat increased ~3-fold from foremilk to hindmilk, with milk of OW/OB women having markedly increased caloric content. As BMI did not correlate with foremilk fat content among LF and HF women, we speculate that maternal diet contributes to high milk fat and calorie content. Novel approaches to calibrate the caloric intake of infants of OW/ OB mothers and prevent childhood obesity are needed. signaling, which has been linked to several health conditions, including cardiovascular disease. These effects are sex dependent due to variations in estrogen levels. After regulatory agencies declared BPA to be a toxic substance in 2010, manufacturers turned to substitutes such as bisphenol S (BPS). BPS exposure is on the rise; a recent study revealed that BPS was detectable in 81% of adult urine samples in the United States . BPS can cross into the placenta, and it accumulates in the fetal compartment to a greater extent than BPA. BPS may also disrupt estrogen-dependent activation of nitric oxide (NO) production, a vasodilatory agent that plays an important role in maintaining vascular health. This project focuses on understanding the sex-dependent risks of developmental exposure to BPS on later-life vascular function. Our objective was thus to determine if prenatal BPS exposure will influence adult vascular health by modulating blood vessel regulation in a sex-dependent manner. Methods: Mesenteric arteries were excised from male and female C57BL/6 mice prenatally exposed to 250 nM BPS and mounted on a pressure myograph. After pre-contraction with phenylephrine, cumulative doses of acetylcholine were added to assess dilation. Vessel stiffness of the aorta was assessed with a wire myograph. Results: Male derived vessels did not exhibit changes in their dilatory or contractile response after prenatal BPS exposure. Increased dilation was observed in female BPS-exposed mice, and sensitivity to contractile agonists was increased. Female vessels but not male vessels exhibited increased myogenic reactivity. Mouse aortas and mesenteric arteries showed no difference in vessel elasticity after prenatal BPS exposure. Conclusion: Although BPA has been linked to negative health outcomes, little is known regarding the effects of its substitutes and their role in developmental programming of cardiovascular disease. Here, we demonstrate that prenatal BPS exposure leads to alterations of vascular function in a sex-specific manner via differential regulation of vascular development. Maternal At 130dGA, fetuses were euthanized, and cardiomyocytes isolated for fatty acid uptake (2µM BODIPY-C 12 and C 16 , 5%CO 2 , 39°C; BODIPY-C 12 represents long chain fatty acids, BODIPY-C 16 represents very long chain fatty acids). Z-stack images were collected at 60 minutes (Zeiss 880 LSM with Fast Airyscan, 488 laser, 90-130 slices, 0.2 µm thick per frame (18-26 µm thickness, 7-12 frames per animal)) and maximum intensity projections performed in ZEN Black (Zeiss). Images obtained for lipid droplet number and size were referenced to cell area and analyzed with Fiji software. Western blot and quantitative PCR analyses investigated lipid transport and processing enzymes (n=7 Vehicle, 8 T3). Data were analyzed using Student's t-test. Results: Cells from T3-infused fetuses had increased uptake of BODIPY-C 12 , resulting in more lipid droplets, although droplets were not larger than in the Vehicle group. BODIPY-C 16 lipid droplet size and number were not different between groups. Gene and protein expression of fatty acid transporter CD36 (+50%) and mitochondrial transporter carnitine palmitoyltransferase 1a (CPT1a: +50%) were elevated in T3 hearts. Beta oxidation (very long/long chain acyl-CoA dehydrogenase, VLCAD: +100%; LCAD: +200%), hydroxyacylcoenzyme A dehydrogenase (HADH: +100%) and tricarboxylic acid cycle genes (isocitrate dehydrogenase, IDH: +100%)) were also elevated in T3 hearts (p<0.05 vs Vehicle). Esterification genes were not different between groups. Conclusion: Increased transporter and enzyme expression, and increased lipid droplet number coupled with unchanged expression of esterification and lipid storage genes suggest that T3 promotes fetal cardiomyocyte uptake and utilization of free fatty acids. Offspring. Ruolin Song †, Jay S Mishra, Sri V Dangudubiyyam †, Jyoti Watters, Tracy Baker, Sathish Kumar * . University of Wisconsin-Madison, Madison, WI, United States. Introduction: Obstructive sleep apnea (OSA), characterized by repeated events of ischemia-reperfusion, is highly prevalent in pregnancy, negatively affecting the gestation process and particularly the fetus with development of hypertension in the adult male but not female offspring. It is unclear what contributes to this gender difference. Additionally, metabolic dysfunction has been reported in male offspring born to gestational intermittent hypoxia (GIH) exposed dams. It remains unknown whether the female offspring are also adversely affected. Since female rats reach their maximum estrogen levels at puberty, we hypothesize that estrogen may be involved in stabilizing blood pressure and glucose metabolism in adult females born to GIH exposed dams. Methods: Experimental subjects were Sprague Dawley rats (n=4-5/ group), born to dams that underwent GIH (21% FIO2 interspersed with 10.5% FIO2, 15 episodes/hour, 8 hours/day) or normoxia exposures (control, alternating room air) from gestation day 10 through 21. At five weeks of age, baseline blood pressures were non-invasively recorded in female control and GIH offspring using the CODA System. Following this, female offspring rats underwent ovariectomy (OVX) or left with intact ovaries. Blood pressures were monitored biweekly through 16 weeks of age. At 16 weeks of age, an intraperitoneal glucose tolerance test was done after overnight fasting, and then the rats were euthanized and tissues collected for western blotting. Results: In prepubertal female offspring at 5 weeks of age, the blood pressure was significantly higher in the GIH females compared to control females (overall effect of GIH, p=0.004). The GIH females became normotensive by 11 weeks of age, coinciding with increased levels of estradiol associated with puberty. However, OVX led to a significant increase in blood pressure in GIH females with no significant effect in controls (GIH-OVX vs. control-OVX, p<0.0001). GIH females exhibited glucose intolerance with elevated insulin levels following a glucose tolerance test. OVX induced glucose intolerance in controls and exacerbated this effect in the GIH females. Protein expression levels of insulin receptor-β (IR-β) and glucose transporter type 4 (GLUT4) were significantly decreased in GIH-OVX offspring (GIH-OVX > GIH-intact = control-OVX > control-intact). Conclusion: With functioning ovaries, GIH offspring could reverse the GIH-induced hypertensive effects observed before puberty, suggesting a protective role of estradiol. While GIH itself was able to impair insulin signaling, loss of ovary in the GIH offspring exacerbated the metabolic dysfunction due to alterations in IR-β and GLUT4 expression levels. These results suggest that estrogen contributes to normalization of blood pressure and control of metabolic function in adult GIH female offspring. Texture-Based Analysis of the Placenta and Fetal Liver: Application to Fetal Growth Restriction. Aya Mutaz Zeidan †, 1 Paula Ramirez-Gilliland †, 1 Ashay Patel, 1 Zhanchong Ou, 1 Dimitra Flouri, 1 Nada Mufti, 1, 2 Kasia Maksym, 2 Rosalind Aughwane, 2 Sebastien Ourselin, 1 Anna David * , 2 Andrew Melbourne * . 1 1 King's College London, London, United Kingdom; 2 University College London, London, United Kingdom. Introduction: Fetal growth restriction (FGR) affects approximately 10% of all pregnancies and is diagnosed by failure of the fetus to reach its genetically predetermined size. The multiple aetiologies, coupled with the risk of fetal complications -encompassing neurodevelopmental delay, neonatal morbidity, and stillbirth -motivate the need to improve holistic assessment of the FGR fetus using MRI. We hypothesised that the fetal liver and placenta would provide insights into FGR biomarkers, unattainable through conventional methods. Methods: Here we explore the application of model fitting techniques, linear regression machine learning models, and texture analysis (by measuring Haralick features from multi-contrast MRI modelling) for a multi-fetal organ analysis of FGR. We employed T2 relaxometry and diffusion MRI datasets for 12 normally grown and 12 FGR gestational-age (GA) matched pregnancies (Estimated Fetal Weight below 3rd centile, Median GA 28 +4 wks±3 +3 wks). We applied models based on the Intravoxel Incoherent Motion Model, which describes circulatory properties of the fetal organs, and analysed the resulting features distinguishing both cohorts. The placenta and fetal liver presented key differentiators between FGR and normal controls, with decreased perfusion, abnormal fetal blood motion and reduced fetal blood oxygenation (all p<0.05). This may be associated with the preferential shunting of the fetal blood towards the fetal brain, affecting supply to the liver. These features were further explored to determine their role in assessing FGR severity, by employing simple machine learning models to predict FGR diagnosis (100% accuracy in test data, n=5) and GA at delivery (RMSE=3.06 weeks). Image texture analysis of the fetal organs demonstrated prominent textural variations in the placental perfusion fractions maps between the groups (p<0.0009), and spatial differences in the incoherent fetal capillary blood motion in the liver (p<0.009). Conclusion: This research investigated the effect of FGR on fetal organs, measuring differences in perfusion and oxygenation within the placenta and fetal liver, and their prognostic importance in automated diagnosis using simple machine learning models. Results: Given the low frequency of proliferating myocytes, they were grouped as inactive (G 1 /G 0 ) and active (S/G 2 /M). There were no differences in the relative proportion of inactive (95.9±0.4% CON vs. 95.8±0.5% IUGR) vs. active (4.1±0.4% CON vs. 4.2±0.5% IUGR) myocytes. Since a shift in cell cycle could not explain differences in growth phenotype with IUGR, MRFs were plotted on a 3-dimensional axis to identify five distinct populations of myocytes: satellite cells (high Pax7, smallest cells), myoblasts (high Pax7), early (low Pax7, high MyoD), late (low Pax7, high myogenin), and fusing (low MyoD, high myogenin) myocytes (Fig. 1) . The rate at which myocytes progress through the cell cycle is normal in IUGR muscle. We speculate that a larger proportion of IUGR myocytes have exited the cell cycle (G 0 ) vs. reside in G 1 ; future studies will use markers to separate G 0 from G 1 . We developed a flow cytometry assay to test how differential regulation of myocyte differentiation contributes to reduced myogenesis in the IUGR fetus. Novel identification of the fetal satellite cell also may inform muscle regeneration potential in IUGR. per 1000 live term births. Infants that receive current treatments (e.g., hypothermia) may still develop adverse neurologic outcomes. Maternal gestational creatine supplementation has been proposed as a potential prophylactic neuroprotective therapy, with evidence of reduction in perinatal brain injury in rodent and sheep studies. The current study aims to characterize aspects of creatine synthesis, metabolism and transport in gestational and neonatal tissues following maternal oral creatine supplementation. Methods: Pregnant rhesus macaques were assigned to control (n=5) and creatine supplementation (0.6g/kg, Creatine monohydrate, PO, BID for ~30 days prior to delivery; n=5) groups. All neonates were exposed to 10 min umbilical cord occlusion at the time of C-section delivery (155±1 dGA; Term=167d). Maternal tissues were collected at delivery. Neonates were resuscitated and received neonatal intensive care for a period of four days until post-mortem neonatal tissue collection. Creatine concentrations were estimated in fluid and tissue samples. RT-qPCR was performed for genes involved in creatine metabolism. Statistical significance (p$It;0.05) was assessed by Student's t-test and F-test to compare variances, or Mann-Whitney U-test after testing for normal distribution (Shapiro-Wilk). Results: In response to creatine supplementation, a significant increase was observed for genes involved in creatine synthesis (Gatm, Gamt) in neonatal liver. Expression of the creatine transporter gene (Slc6a8) was also increased in neonatal kidney and quadriceps in the creatine group. There was a significant increase in creatine kinases (Ckmt1, Ckmt2, Ckb, Ckm) across multiple neonatal tissues. In contrast, Ckmt2 and Ckm were significantly downregulated in placenta. Across gestation, a higher expression of creatine metabolic genes was present during the 2 nd and reduced in the 3 rd trimester. Conclusion: Creatine synthetic gene expression across gestation likely signifies the high energy demand for placental and fetal growth and nutrient transfer. Increased creatine kinase and transporter gene expression may indicate increased creatine tissue storage demands with supplementation. Understanding the response of endogenous creatine metabolic pathways to gestational creatine supplementation is important for the safe use of creatine as a therapeutic agent during pregnancy. Identification of Novel Nutrient-Gene Interactions in Neural Tube Defects. Marina White †, Jayden Arif-Pardy †, Kristin L Connor * . Carleton University, Ottawa, ON, Canada. Introduction: Neural tube defects (NTDs) remain among the most common congenital anomalies. Contributing risk factors include genetics and nutrient deficiencies, however, a comprehensive assessment of nutrient-gene interactions in NTDs is lacking. We hypothesised that multiple nutrient-gene interactions would be evident in gene signatures associated with NTDs. We aimed to identify novel nutrient-sensitive gene regulatory networks using gene expression data from fetuses with spina bifida (cases) and fetuses with no congenital anomalies (controls). Methods: Amniocyte gene expression data for cases (n=3) and controls (n=5) were obtained from GEO (GSE4182) and re-analysed. Differentially expressed genes (DEG) were identified (eBayes; FDR q value<0.05, absolute fold change>2) and screened for having nutrient-cofactors (Scott-Boyer, Nature 2016). Transcription factors (TFs) with nutrient cofactors that regulated DEG, and nutrient-sensitive miRNAs that had a previous link to NTDs, were identified and used to construct DEG regulatory networks (iRegulon v1.3, miRWalk2.0). Results: Of the 880 DEG in cases (725 down-and 155 up-regulated) vs. controls, 10% (n=87) had at least one nutrient cofactor, including vitamins B2 (for n=5 genes), B3 (n=7), B5 (n=2), B6 (n=1), folate (n=1) and iron (n=6). Next, miRNA-targetome network analysis revealed that collectively, the 22 unique nutrient-sensitive miRNAs identified targeted 39% of DEG in cases (n=344; Fig 1A) . The miRNAs targeting the largest proportion of DEGs were miR-123-3p (n=87 DEG) and miR-17 (n=80 DEG), which are both sensitive to choline, inositol, iron and vitamin D activity. Last, we identified 16 TFs that had nutrient cofactors and target down-(n=5) and up-regulated (n=10) DEG, or both (n=1; Fig 1B) . Together, these 16 nutrient-sensitive TFs are known to regulate 52% of DEG (n=460). Zinc and vitamin B5 were cofactors for E1A binding protein p300, which, of the TFs identified, is known to regulate the highest number of DEG in cases (n=310 [35%], 301 down-and 9 up-regulated). Conclusion: We identified multiple novel nutrient-sensitive gene regulatory networks associated with spina bifida, which may relate to NTD pathogenesis, and indicate new targets to explore for NTD prevention or to optimise fetal development. The Evidence also demonstrated that pregnancies complicated by CHD had higher rates of placental disease, highlighting the significance of the fetal-placenta-cardiac axis in the context of CHD. Our objective was to analyze neonatal birthweight (BW) and placental weights (PW) based on type of CHD to investigate an association between placental insufficiency and CHD phenotypes. Methods: A retrospective cohort of all pregnancies that underwent a prenatal fetal echocardiography at a single institution from January 2011 -September 2021 with postnatal confirmation and placental pathology. Categories of CHD were assigned following review of postnatal echocardiography. CHD was classified as septal defects, right heart defects, left heart defects, conotruncal anomalies, or other anomalies. Descriptive analyses including frequencies and medians were conducted for all maternal and neonatal variables, including birthweight to placental weight ratios. Published sex-stratified nomograms for placental and birthweight were used. The Kruskal-Wallis test was used to analyze continuous variables in this data set. Results: Our final cohort included 140 liveborn singletons. Median BW:PW ratio was 6.96 for all CHD diagnoses, 6.92 for septal defects, 6.63 for right heart defects, 7.16 for left heart defects, 7.30 for conotruncal abnormalities, and 6.70 for other anomalies, with no significant difference between groups by type of CHD (p=0.30). Placenta weight-to-birth weight (PW:BW) ratios were <10 th percentile for 78% (n=109) and <3 rd percentile for 54% (n=75) of the cohort, with no significant difference between CHD categories (p=0.65 and p=0.49, respectively). Conclusion: More than half of cases of CHD had a PW:BW ratio <3 rd percentile. There was no significant difference in BW:PW ratios based on types of cardiac malformations in singletons with CHD. This suggests that placental insufficiency and an abnormal maternal-placenta-fetal axis of circulation may contribute to the development of CHD in singleton pregnancies. Introduction: Acute perinatal white matter injury (apWMI) is the most common form of brain injury in preterm infants, yet the mechanisms underlying neurologic dysmaturation in this disease are far from understood. Reactive astrocytes are a hallmark of apWMI, but the roles that astrocytes play in disease pathogenesis remain unclear. Studies in the mature brain highlight the formation of molecularly distinct reactive astrocyte subtypes in response to brain injury, some that are strongly neurotoxic and others that mediate brain repair. The current research project tests the hypothesis that neurotoxic reactive astrocytes (nrAs) form in apWMI and are drivers of disease outcomes. We tested the formation of neurotoxic reactive astrocytes across multiple rodent apWMI models. apWMI was confi rmed using immunohistochemistry for myelin basic protein at postnatal day 11. nrAs were identifi ed using in situ Hybridization (ISH) for nrA marker C3. Microfl uidic qRT-PCR of mRNA isolated from immunopanned and FACS purifi ed primary rodent astrocytes evaluated the expression of a panel of established reactive astrocyte subtype-specifi c genes. C1q -/-, Il1-a -/-, TNF -/mutant mice were used to investigate the necessity of nrAs for myelination defects. Results: We demonstrate the formation of neurotoxic reactive astrocytes following acute perinatal brain insult in rodent apWMI disease models. These cells form within the fi rst 24 hours of brain injury. C1q -/-, Il1-a -/-, TNF -/transgenic mice unable to form nrAs display a signifi cant reduction in myelination defects in periventricular white matter after injury compared with wildtype controls. Our experiments provide evidence that neurotoxic reactive astrocytes not only form following acute infl ammatory/hypoxic perinatal brain injury but also appear to play a causal role in the production of myelination defects associated with this disease. This fi nding opens the door to investigating nrAs as therapeutic targets in apWMI. Ongoing experiments aim to defi ne a therapeutic window for rescuing myelination through the modulation of nrAs and to confi rm formation of nrAs in human postmortem perinatal brain specimens aff ected by apWMI. Methods: This is a planned secondary analysis of a 17-center retrospective cohort study of twin pregnancies with fi rst trimester cfDNA screening from 12/2011 -2/2020, excluding those with chromosomal abnormalities and vanishing twin. CfDNA testing was performed by a single lab using a MPSS approach. Baseline characteristics and birth weight of pregnancies with normal FF were compared with those with low (<5%) and high (>95%) FF using descriptive statistics, including parametric and nonparametric tests as appropriate. Results: FF distribution is shown in Fig 1. Chronic hypertension, elevated BMI, and Black race were associated with low FF in the 1,052 pregnancies included (Table 1 ). There was no diff erence in the median FF in those with low (<10%) and normal birth weight (Fig 2, p=0.11 ). There was no association between FF and birth weight <10% or <=5% (Table 2) . Conclusion: There was no association between fi rst trimester cfDNA FF and low birth weight, suggesting that fi rst trimester FF is not a reliable predictor of fetal growth in twin gestation. Methods: A case study of two patients who presented to a private, multi-site fertility clinic with previously diagnosed autosomal genetic conditions. Treatment outcomes, emotional strain, and fi nancial burden were examined along with PGT-M guidelines from multiple reproductive and genetic organizations. Results: In both cases, care was established with knowledge of aff ected genetic status, but screening was off ered and denied in both cases. Patient 1 had congenital hearing loss. Patient 2 and her partner were carriers of the BRCA-2 mutation. For Patient 1, indecisiveness led to delayed fi nancial inquiry and subsequent lack of coverage. Eventually, coverage was off ered but declined and they proceeded with a frozen embryo transfer. After a successful birth, she returned to reestablish care and pursue PGT-M. Coverage for biopsies was denied and a transfer of an unaffected embryo ended in miscarriage. Later, PGT-M for another IVF cycle was denied for coverage again. Patient 1 discontinued care after the retrieval yielded no unaffected embryos. Overall, Patient 1 expressed severe distress during treatment. Patient 2 was advised to create a probe before starting treatment. The pre-authorization for biopsy was submitted promptly by the financial team but was denied since the couple declined genetic testing. Patient 2 had a failed transfer with her only unaffected embryo, testing paid for out of pocket. After another round of IVF and a transfer of a carrier embryo, she achieved a successful pregnancy. Conclusion: Although various organizations have general guidelines for PGT-M, there are no unified clinical care guidelines for testing in patients that present with known genetic conditions. Although both cases resulted in a successful pregnancy, they demonstrate the importance of efficient treatment in these unique patients intending to do PGT-M. We implore a need for timely communication with the patient about the clinical and financial process of PGT-M and the expected treatment timeline. By establishing unified practice guidelines, patients can achieve pregnancy faster, reduce overall treatment costs, and receive a more satisfactory treatment experience. Introduction: Chemotherapy is often ineffective in advanced stage and aggressive histologic subtypes of endometrial cancer. Overexpression of the receptor tyrosine kinase AXL has been found to be associated with therapeutic resistance, metastasis, and poor prognosis in endometrial cancer. Our objective was to identify whether inhibition of AXL through a selective targeted agent, AVB-500, would improve sensitivity to paclitaxel (pac). We also sought to determine whether the mechanism for this improved sensitivity was through metabolic regulation. Our study provides strong pre-clinical rationale for combining AVB-500 with pac in aggressive endometrial cancer models. Additionally, continued identification of viable biomarkers is critical to the success of targeted therapies such as AVB-500. Given AXL's role in glucose homeostasis, one or more glycolytic intermediates may prove to be a useful biomarker to help predict sensitivity in patients, thus tailoring who would benefit from this treatment combination. Impact of Obesity on the Endometrium. Mike R Wilson, Ronald L Chandler. Michigan State University, Grand Rapids, MI, United States. Introduction: Obesity is defined by a disproportionate body weight relative to height and an accumulation of adipose tissue. The global percentage of overweight or obese people has increased by 28% from 1980 to 2013, and as such, obesity has been described as a pandemic. In 2014, the World Health Organization reported 1.5 billion overweight and 640 million obese people globally. The continued rise of obesity is expected to negatively impact life expectancy in the United States. Obesity is associated with numerous chronic conditions, including type 2 diabetes, cardiovascular disease, and cancer. Obesity also has a negative impact on female fertility. Hyperinsulinema during obesity increases ovarian androgen production and promotes polycystic ovarian syndrome, negatively impacting fertility. While obesity is known to impact oocyte yield and embryonic health, evidence suggests that the endometrium is also impacted during obesity. Increases in BMI lead to reduced fecundability, even in eumenorrheic women. In additional to effects on fertility, obesity promotes endometrial cancer, for which incidence rates are steadily rising as a result of obesity. Methods: In this study, we sought to characterize the effects of obesity on the endometrial epithelium. We utilized a mouse model of obesity in combination with established methods for isolating uterine mouse cells. CD-1 mice were fed a high-fat diet or control diet for 18 weeks. Cells were isolated for RNA-seq, and statistical analysis was performed using DEseq2. Results: Obese mice displayed a reduced glucose tolerance, hyperinsulinemia and signs of fatty liver disease. By magnetic sorting, we collected endometrial cells from control and high-fat diet mice and performed RNA-seq. Among differentially expressed genes, we observed pathway enrichment for pathways related to protein transport, localization and secretion. We identified transcriptomic similarities between the obese uterus and the obese liver, including induction of the Lcn2 gene. We observed similarities to our previously published mutant endometrium models in the CD-1 background, such as upregulation of inflammatory response genes including Il6. We observed an increased abundance of macrophages localized to the obese mouse endometrium, which may be the result of increase inflammatory signaling. Conclusion: The results described herein suggest a role for obesity in driving a cancer-like inflammatory transcriptomic profile in the endometrial epithelium which results in differential macrophage recruitment. We treated three uterine serous cancer cell lines (ARK1, ARK2, PUC198) that expressed Her2/neu and AXL receptors with AVB500 (Aravive Biologics) in combination with Trastuzumab. Clonogenic proliferation assays were performed after 3 days of treatment, and relative cell viability was calculated using GraphPad Prism. Next, using a Matrigel invasion assay, we examined the invasive capacity of the cell lines after 24 hours of treatment with vehicle, AVB500 alone, Trastuzumab alone, or combination Trastuzumab and AVB500. We also performed a proximity ligation and IF colocalization assays to show that Her2/neu and AXL receptors form a complex that results in the transphosphorylation and activation of the AXL. We performed in vivo studies using nod-SCID mice injected with 1 x 10 7 ARK1 and ARK2 cells intraperitoneally, followed treatment with vehicle control, AVB alone, or Trastuzumab alone or in combination with AVB500 for 35 days and 21 days, respectively. Results: In uterine serous (ARK 2 and PUC 198) cells, there was a significant reduction in cell proliferation when treated with combination Trastuzumab and AVB500 compared to the Trastuzumab-alone group (0.16nm vs 0.20nm, p=0.015; 0.29 vs 0.015, p=0.0044) in ARK 2 and PUC 198 cell lines respectively. We also observed a reduction in the number of invading tumor cells when cells were treated with combination Trastuzumab and AVB500 versus Trastuzumab alone (3.9 vs 17.7 invading tumor cells/hpf, p=0.0001; 3.2 vs 17.8 invading tumor cells/hpf, p=0.0004) in ARK 1 and ARK 2 cell lines, respectively. Furthermore, the proximity ligation and colocalization assays demonstrated physical closeness of AXL and HER2 receptors in ARK2 cells that could account for the coregulation of these receptors. Finally, we found that mice treated with Trastuzumab plus AVB-500 developed significantly less tumor burden than mice treated with Trastuzumab alone (0.032 g vs 0.083 g, p= 0.024) in ARK1 xenograft model and (0.142g vs 0.443 g, p= 0.0015) in ARK2 xenograft model. The GAS6/AXL inhibitor AVB-500 potentiated the effect of Trastuzumab to decrease uterine serous cancer cell proliferation and invasion in vitro and tumor burden in vivo. There was co-localization of AXL and HER2 receptors seen in both uterine serous cancer cell lines. Our data suggest that the addition of AVB-S6-500 increased the therapeutic efficacy of Trastuzumab therapy in uterine serous cancers. Additional therapeutic and mechanistic experiments are ongoing. Examining Interactions Between Bone Marrow-Derived Adipocytes and Ovarian Cancer. Zachary L Watson * , Kathleen M Gavin, Bitler G Benjamin. University of Colorado, Aurora, CO, United States. Introduction: Hormonal shifts during aging and menopause lead to increased visceral abdominal fat and chronic inflammation typified by elevated secretion of inflammatory cytokines, resulting in a peritoneal adipose environment that is highly favorable for ovarian cancer progression. However, the ultimate source of elevated inflammation has not been described. Bone marrow-derived adipocytes (BMDAs) may be a key source of proinflammatory signaling. BMDAs begin as myeloid cells that traffic to adipose tissue and differentiate into adipocytes. While histologically similar to conventional mesenchymal adipocytes (CMAs), BMDAs are a highly inflammatory subtype. BMDAs accumulate within visceral adipose depots and their production increases with age. In mice, ovariectomy (model of pseudomenopause) results in greater BMDA production, suggesting that BMDAs are likely to extensively accrue in postmenopausal women. We posited that human BMDAs would exhibit greater inflammatory signaling than CMAs, and that ovarian cancer cells would respond to signaling from BMDAs by altering their own physiology and proliferation. Methods: We sorted and differentiated human adipocyte precursors into BMDAs and CMAs. We examined cytokine secretion in adipocyte conditioned media by multiplex ELISA. We incubated conditioned media on PEO1 ovarian cancer cells and analyzed the cells by reverse phase protein array (RPPA). We incubated ovarian cancer cells (PEO1, OVCAR4, and OVCAR8) in conditioned media and assayed proliferation using IncuCyte imaging. Results: ELISA showed that BMDA cells secreted elevated amounts of IL-6 and IL-8. By RPPA, we observed differential expression of proteins involved in signaling and metabolism (e.g., PI3K/AKT) between cells incubated in BMDA or CMA media. We found that cells incubated in BMDA media upregulated markers of epithelial-to-mesenchymal transition (EMT), including fibronectin (FN1) and tissue transglutaminase (TGM2), yet also downregulated programmed cell death protein 4 (PDCD4), suggesting alterations to survival pathways. Depending on the patient sample, cell lines showed differential growth rates in BMDA media versus CMA controls. Conclusion: ELISA data suggest that BMDAs may be a source of inflammatory signaling within the omentum and other visceral fat depots that are often colonized by ovarian cancer cells. RPPA and proliferation data indicate that ovarian cancer cells respond to signaling from BMDAs by altering their metabolism and survival. The finding of elevated EMT markers suggests that BMDAs may influence cancer cell migration and drug response. In summary, our findings suggest that BMDAs may contribute to the establishment of a pro-tumor niche within the omentum. Further studies are underway to examine the influence of BMDAs on the tumor microenvironment, including cancer cell migration, drug response, and immune cell infiltration and function. . Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p<0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p<0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Conclusion: Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer. Targeting the Class I Histone Deacetylases in Uterine Leiomyosarcoma. Qiwei Yang, Maria Victoria Bariani, Ana Corachan, Ayman Al-Hendy. University of Chicago, Chicago, IL, United States. Introduction: Uterine leiomyosarcoma (LMS) is a rare and aggressive uterine malignant tumor, representing 3-4% of all uterine malignancies. Irrespective of treatment, the LMS is characterized by a poor prognosis. The current therapy for LMS patients exhibits resistance to currently available therapies. Class I histone deacetylases (including HDAC1,2,3, and 8) is one of the major classes of the HDAC family, catalyze the removal of acetyl groups from lysine residues in histones and cellular proteins, Class I HDACs have distinct cellular and subcellular expression patterns and are involved in many biological processes and diseases through diverse signaling pathways. However, the link between class I HDAC and LMS is unknown. In this study, we assessed the role and mechanism of the class I HDACs in the pathogenesis of LMS. We performed Immunoblot analysis to measure the Class I HDACs protein levels in four cell lines covering the spectrum from a normal myometrial cell line (UTSM) to benign uterine tumor cell line (HuLM), and uterine malignant cell lines (MES-SA, SK-UT-1). Cell proliferation was performed using trypan blue excluding assay in the presence or absence of Tucidinostat (Class I HDAC inhibitor). RNA-seq was performed to determine the molecular mechanism underlying Class I HDACs inhibition in LMS cells (n=4 for each group). A variety of R packages was used for this analysis. The differentially expressed genes (DEGs) were identified as those having a false discovery rate (FDR) corrected p-value < 0.05. In addition, data were analyzed by a two-tailed unpaired Student's t-test between any two groups. Results: Immunoblot analysis demonstrated that the expression levels of HDAC 1, 2, and 3 exhibited a graded increase from normal and benign tumors to malignant uterine sarcoma cells ( Introduction: Hypertensive-complicated pregnancy predisposes to remote cardiovascular disease (CVD). Whether hypertensive-complicated pregnancy modifies the age-related decline in vascular function is unknown. Therefore, we investigate if the age-related impairment in endothelial-dependent and -independent function is more pronounced among women with a history of hypertensive-complicated pregnancy as compared to normotensive gestation. Methods: This cross-sectional cohort study included former pregnant women (≥ 18 years) with a history of preeclampsia (PE) or uncomplicated pregnancy. Brachial artery flow-mediated dilation (FMD; absolute, relative and allometric) and sublingually administered nitroglycerinemediated dilation (NGMD; absolute and relative) were measured using brachial ultrasound and calculated. Associations of age with FMD and NGMD were investigated by linear regression. Models were adjusted for body mass index, smoking, antihypertensive drug use, mean arterial pressure, fasting glucose, menopausal state, family history of CVD and stress stimulus during measurement. Models evaluating absolute FMD and absolute NGMD were additionally adjusted for baseline diameter. Potential effect modification of PE was studied by including an interaction term with age. Results: In total, 1217 former pregnant were included, of which 803 (66.0%) with and 414 (34.0%) without a history of PE (age 22-62 years). In formerly preeclamptic women as compared to controls, relative FMD (-0.57%/10yr (95% CI -0.69 --0.09) vs. -0.39%/10yr (95% CI -0.81 --0.33), respectively; p-value interaction 0.361) and relative NGMD (-1.08%/10yr (95% CI -1.59 --0.59) vs. -1.39%/10yr (95% CI -2.02 --0.77), respectively; p-value interaction 0.443) decreased comparably with age. Similar results were found for absolute FMD, allometric FMD, relative NGMD and after adjustment for confounders. A history of preeclampsia itself did not independently affect FMD and NGMD. In young-to middle-aged parous women, irrespective of obstetric history, there is an age-related decrease in FMD and NGMD, being similar in former preeclamptic women and those with normotensive gestation. Novel Role of Angiotensin Type 2 Receptor in Promoting Angiogenesis in Primary Human Uterine Artery Endothelial Cells. Jay S Mishra, 1 Sri V Dangudubiyyam †, 1 Ruolin Song †, 1 Dong-Bao Chen, 2 Sathish Kumar * . Madison, WI, United States; 2 University of California, Irvine, CA, United States. Introduction: Angiogenesis is vital for placental vascular development and uterine artery remodeling, leading to a low-resistance and a lowpressure system in pregnancy. The renin-angiotensin system (RAS) is upregulated in pregnancy with increased expression of angiotensin type 2 receptors (AT 2 R) and unchanged angiotensin type 1 receptors (AT 1 R) in uterine arteries (UA). AT 2 R blockade increased vasoconstriction and blood pressure and decreased UA blood flow, indicating that AT 2 R plays an important role in modulating vascular function during pregnancy. However, the role of AT 2 R in angiogenesis during pregnancy has never been studied. In the present study, we mechanistically evaluated the regulatory effect of AT 2 R on angiogenesis. Methods: Primary human uterine artery endothelial cells (hUAECs) established from fresh uterine artery tissues collected from pregnant women (n = 5) undergoing hysterectomy were cultured in complete ECM with 5% serum and 1% antibiotics and treated with AT2R agonist Compound (C21) at 1, 10, and 100 nM for 24 hours. Cell proliferation and chemotactic motility were assessed using the MTS cell proliferation and transwell migration assay. Capillary tube formation was determined in a growth factor-reduced matrigel system. To explore C21 regulated angiogenic pathways, membrane-based angiogenic protein and phosphorylation array were done. mRNA levels of the target genes were analyzed by qRT-PCR. Results: AT 2 R activation with C21 induced dose-dependent proliferation of hUAECs at 10 and 100 nM doses while 1 nM dose showed a similar effect as vehicle control. Compared to controls, C21 also dose-dependently induced chemotactic motility of hUAECs as determined by transwell migration assay. On matrigel matrix, C21 induced robust capillary-like tube formation. The semiquantitative angiogenesis antibody array showed that C21 induced increased expression of 12 of the 43 angiogenic factors, including EGF, bFGF, Leptin, PLGF, IGF-1, and Angiopoietins. qRT-PCR showed that C21 induced upregulation of these pro-angiogenic proteins at the mRNA level. Surprisingly, C21 did not significantly alter the phosphorylation of proteins related to MAPK, AKT, NFkB, and TGFb pathways. Conclusion: These results, for the first time, show that AT 2 R activation significantly increases proliferation, migration, and tube formation in in vitro treated hUAECs and that these effects are related to activation of pro-angiogenic proteins such as EGF, bFGF, Leptin, PLGF, IGF-1, and Angiopoietins. The proportional increase in mRNA transcripts suggests that C21 regulates these targets at the transcriptional level. These results identify C21 as a novel agent with therapeutic potential in pregnancy disorders associated with reduced angiogenesis, such as preeclampsia and fetal growth restriction. Like Symptoms in Mice. Kathleen Pennington, Yuanlin Dong, Simone Hernandez Ruano, Akansha Mishra, Chandrasekhar Yallampalli. Baylor College of Medicine, Houston, TX, United States. Introduction: Adrenomedullin (ADM), a member of the calcitonin peptide superfamily, is expressed by and secreted from a variety of cell types, and has known functions in cell growth, inflammation, hormone secretion, vascular adaptations to pregnancy and fetal growth. ADM is increased in Gestational Diabetes, a common obstetrical complication affecting up to ~10% of US pregnancies. Previously, utilizing in-vitro and ex-vivo cell culture, we have shown increased ADM results in adipose tissue and beta cell dysfunction. We have also shown that ADM and its receptors are decreased in pancreatic beta cells in normal pregnancy. Here, we hypothesize that increased ADM plays a central role in the pathophysiology of GDM. To test this we evaluated the in-vivo effects of ADM in pregnant mice. Methods: Eight weeks old C57bl/6 female mice were bred to proven breeder males. Day of observed copulatory plug was denoted as day 0.5. On day 6.5 of pregnancy osmotic mini pumps were implanted with either ADM (1.2 µg/g BW/day) or Saline (control). Glucose tolerance tests were performed at day 16.5 of pregnancy. One group of mice was euthanized at day 13.5 and a second group of mice was euthanized at day 17.5 of pregnancy. Blood was collected for serum insulin measurement, and visceral adipose tissue (VAT) for ex-vivo lipolysis analysis. Pancreatic islets were isolated for glucose stimulated insulin secretion (GSIS) assays. P-value of less than 0.05 was considered significant. Results: At day 13.5 of pregnancy, islets isolated from ADM infused dams had decreased GSIS and increased VAT lipolysis compared to control dams. At day 16.5 of pregnancy, ADM infused dams had decreased glucose tolerance compared to control dams. At day 17.5 of pregnancy, ADM infused dams had increased VAT lipolysis, however GSIS was not affected. Serum insulin levels were not different at day 13.5, however there was a trend (P=0.08) for serum insulin to be decreased at day 17.5 of pregnancy. Further analysis is underway to analyze the effects of in-vivo ADM infusion on VAT and pancreatic islet gene expression. Conclusion: ADM infusion results in glucose intolerance as well as adipose tissue and beta cell dysfunction in-vivo, further supporting the hypotheses that increased ADM plays a role in the pathophysiology of GDM. Further work focuses on the mechanisms by which ADM regulates the pathophysiology of GDM as well as the effects of ADM antagonist as treatment for GDM. Methods: A retrospective cohort study of pregnant persons with SUD receiving care through our PATHways treatment program were included in the analysis. Subset analysis completed on cesarean deliveries between 2017 and 2021. Pharmacovigilance program implemented on October 2019 with a 3-month run in period. The program featured a quantity prescribed based on utilization in the 24 hours preceding discharge. The quantity and dosage of prescribed narcotics were assessed pre-and postintervention. Fisher's exact test and linear regression were used in analysis. Results: 304 patients in the program delivered during the interval with 117 (38%) undergoing cesarean section. Narcotic dosing data was available 92 of these patients: 33 (36%) of these patients had a primary cesarean section versus 59 (64%) repeat cesarean sections. Demographics from 2017-2019 (n=59) were similar to 2020-Present (n=33). All but one patient was prescribed oxycodone 5/325 mg formulation (10/325 mg). Between 2017-2019, patients were prescribed 26.3 ± 8.22 pills versus 13.2 ± 9.17 between 2020-2021, P=0.0001 as seen in Table 1 . Figure 1 shows the average quantity of narcotics prescribed pre and post implementation of the pharmacovigilance program. The median buprenorphine dose at delivery was 12 mg (IQR 8-16) from 2017-2019 and 14 (IQR 8-16) from 2020-21. From 2020-21, the quantity of narcotics was not correlated with buprenorphine dose based, P=0.581. From 2020-21, the quantity of narcotics above the median buprenorphine dose at delivery was 14.3 versus 11.3 prescribed pills. The implementation of a pharmacovigilance program statistically decreased the quantity of narcotics prescribed. A 51% reduction in the number of prescribed narcotics were achieved. Diff The chronic hypoxia of high-altitude (HA) residence reduces uterine artery (UtA) blood fl ow during pregnancy, contributing to an increased frequency of preeclampsia and intrauterine growth restriction (IUGR), yet not all babies are growth-restricted at high altitude. A key contributor of these pregnancy complications is less pregnancy rise in UtA blood fl ow. Large-conductance Ca 2+ -activated (BK Ca ) and ATP-sensitive (K ATP ) K + channels regulate vascular function, and their activity is modulated by hypoxia. We hypothesized that increased K + channel-dependent myometrial artery (MyoA) vasodilation can serve as compensatory response to maintain UtA blood fl ow and fetal growth at HA. Methods: Myometrial biopsies were obtained from the lower uterine segment of non-laboring, healthy residents of HA (>2600 m) or low altitude (LA, <1700m) undergoing Cesarean section at term (38-40 weeks, singleton pregnancies). MyoA arteries were isolated and studied using wire myography. We measured vasodilator responses to the BK Ca or K ATP channel openers, NS11021 or pinacidil (1 nM-100 µM) with or without channel blockers, tetraethylammonium (TEA, 1 mM) or glibenclamide (Glib, 30 µM), respectively. 459±19, p<0.05) but not at LA (496±21 Glib vs. 479±40). Supporting reduced vasodilation by BK Ca in -e MyoA at HA, voltage-dependent K + currents tended to be reduced in MyoA smooth muscle cells from HA vs. LA (AUC: 3.5±0.5 and 6.6±1.7, respectively, p=0.0637). Conclusion: Our results suggest a differential regulation of MyoA K + channels in LA and HA pregnant women. Specifically, whereas BK Ca channel activity appears reduced in HA MyoA in a smooth muscledependent manner, endothelial K ATP channels are more active in HA MyoA. These observations indicate that K + channel-dependent vasodilation is a compensatory means for maintaining UtA blood flow in uncomplicated pregnancies at HA. Future studies will assess the mechanisms responsible, and whether K + activity differs in MyoA from healthy vs. IUGR pregnancies at HA. Lactogenesis Requires Cross- Talk Introduction: Breastfeeding protects both mother and infant against disease with increased duration of breastfeeding conferring increased protection. The AAP recommends exclusive breastfeeding for six months, however, the 2020 Breastfeeding Report Card from the CDC reports that in the U.S. only 25.6% of mothers adhere to these guidelines. Low milk supply is a primary driver of premature lactation cessation, and the underlying molecular mechanisms are poorly understood. We seek to define lactation at the molecular level to improve breastfeeding success. Based on previous findings that accumulation of lipid in the gland can trigger death of milk secreting cells and regression of the gland, we hypothesize the converse: that a signal originates from secretion of the milk fat globule to feed-forward overall milk secretion and drive lactation in the gland. Methods: We used genetically-modified mouse models to define the mechanisms regulating the initiation of milk secretion post-partum. Two proteins, Xdh and Btn1a1, previously shown to mediate milk fat secretion, were genetically deleted in milk secreting cells (n=5-7 animals/group). The growth of cross-fostered pups was measured. Glands were collected at day 3 post-partum and analyzed by histology, immunohistochemistry and transcriptomics. Mammary explants were stimulated with oxytocin to measure the response of the contractile cells. Results: Deletion of either Xdh or Btn1a1 results in retention of lipid within the milk secreting cells and delayed litter weight gain, indicating delayed lactogenesis. Transcriptomic analysis of genes upregulated in both strains (108 genes) at day 3 post-partum using PANTHER Overrepresentation Test showed an 81-fold enrichment of genes classified as Smooth Muscle Contraction (p-value: 1.5E-15) suggesting increased assembly of the contractile apparatus in the myoepithelial cells which mediate milk ejection. Immunostaining showed an increase in the levels of smooth muscle actin and non-muscle myosin in these contractile, myoepithelial cells. Mammary explants from wild-type and knockout dams, stimulated ex-vivo with oxytocin, were equally able to respond by contracting. Interfering with milk fat secretion at the molecular level delays onset of milk production. Despite the deletion of milk fat globule docking proteins in the milk secreting cells, a transcriptional response is seen in the contractile, myoepithelial cells of the mammary gland. This result suggests milk-secreting cells communicate secretory defects to nearby contractile cells creating compensatory responses in their contractile protein networks possibly to increase the strength/duration of contraction. Work to identify this signal is ongoing. This work was supported by National Institutes of Health: 2R01HD45965 (JLM & JM) and 5P01HD038129 (JLM). Introduction: Women with preeclampsia exhibit altered hemostatic features, which may be evident outside of pregnancy, and into subsequent pregnancies. Here, we investigate the hypothesis that women with a prior preeclamptic pregnancy demonstrate differences in hemostatic balance prior to a subsequent pregnancy, compared with nulliparous women. Methods: This is a retrospective sub-analysis of a prospective study. Plasma samples were collected from healthy, normotensive women with preeclampsia in a prior pregnancy (Prior PE, n= 12), and healthy, normotensive nulliparous women (P0, n=24) at the following Visits (V): V1: pre-conception (follicular phase), V2: early pregnancy (12-14 weeks), and V3: late pregnancy (28-34 weeks). Previous deliveries in the Prior PE group occurred an average of 2.3±1 years earlier. Platelet-poor plasma collected at the three timepoints was analyzed for thrombin generation (TG) and clot lysis (CL) potential. TG assays were tissue factor (TF)initiated (5pM), and analyzed peak level (Peak TG), time to peak level (tPeak), maximum rate, and endogenous thrombin potential (ETP). CL assays used rotational thromboelastometry (ROTEM) and were TF and tissue plasminogen activator (tPA)-initiated (5pM and 4nM, respectively). Unpaired Student t-tests were used to compare parameters between groups at each visit. Linear regression with generalized least squares analysis was used to analyze the change in parameters from V1 to V2, V1 to V3 and V2 to V3 between groups. Data are expressed as mean and standard error, with significance set at p < 0.05. There was no difference between P0 and Prior PreE in lysis parameters at any visit, nor in the change in lysis parameters between visits. Conclusion: Non-pregnant women with a history of a preeclamptic pregnancy demonstrate a slower time to peak TG compared to women with no prior pregnancy. In the transition from pre-conception to the late first trimester, both peak TG and ETP increase more in the women with prior preeclampsia, thereby exhibiting a comparatively procoagulant transition into early pregnancy. In contrast, there does not appear to be a difference in fibrinolytic potential between the two groups, either pre-pregnancy, or in the transition to early pregnancy. Future studies exploring the impact of parity on the non-pregnant hemostatic profile would allow a more extensive investigation into our findings. Pregnancy. Henrike Schroeder, Damián O. Muzzio, Jens Ehrhardt, Marek Zygmunt * . University Medicine Greifswald, Greifswald, Germany. Introduction: Infections are leading causes of neonatal death. Gender differences in susceptibility to bacterial and viral infections, affecting male newborns with higher incidences, are increasingly being studied. The immune defences of the newborn rely greatly on the passive immunity acquired during gestation. Immunoglobulins pass through the placenta via a transport process mediated by the neonatal Fc receptor (FcRn). Little is known about the placental regulation of receptor expression. We hypothesize that FcRn might be increasingly expressed or regulated differently in female neonates. Methods: Placental tissue was obtained from term placentae after informed consent (f=11, m=10). All participants delivered by C-Section. Isolated trophoblasts were stained for FcRn protein expression and binding capacity of IgG1 was determined by flow cytometry. Relative FCGRT mRNA expression was detected by qRT-PCR. Regulation of protein expression was assessed using an immortalized trophoblast cell line (HTR8/SVneo). Cells were exposed to hCG, TGF-β, Dexamethasone (DEX), Progesterone (P4), Estradiol (E2) for 48 h, 72 h and 5 days. Factors diff ering in cord blood of males and females were given exclusively to the cells for 48 h and 5 days (Testosterone (TT), TNF-ɑ, IFN-γ, IL-1β). After treatment, FcRn expression and IgG1 binding capacity were determined by fl ow cytometry. Data were analyzed by students t-test and one-way ANOVA. A p < 0.05 was considered statistically signifi cant. Results: No signifi cant diff erences were found in the FCGRT mRNA expression or the protein expression of FcRn between female and male newborns. In vitro, HTR8/SVneo cells showed a signifi cant increase of FCGRT mRNA expression after 48 h treatment with hCG, DEX, P4, E2, while there was a decreased expression after TGF-β treatment. In contrast, no changes on the protein expression levels were observed. IgG1-binding cells were reduced after 72 h and 5 days of TGF-β-treatment and increased after 5 days stimulation with P4, DEX and E2. A decreased protein expression of FcRn could be observed after 5 days of treatment with TNF-ɑ, IFN-γ, IL-1β and TT, which was refl ected in a reduced IgG1 binding capacity. Conclusion: This data suggest that while there is no diff erence in protein expression of FcRn between female and male placentae, there are diff erences in its regulation of function. DEX and P4, both increased at the end of pregnancy, lead to better binding capacities and higher mRNA expression. The male sex hormone and other substances correlating with TT in cord blood may as well play an important role in FcRn regulation and may be responsible for declined transport properties of the placental Ig-transporter. Introduction: Women with a history of preeclampsia report cognitive problems such as impaired concentration and memory in the early years after pregnancy, but remote prognosis is unclear. We evaluated perceived executive functioning, cognitive processes necessary for goal-oriented, effi cient, and socially adapted behaviour, in a large cohort of formerly preeclamptic women and normotensive gestation parous controls. Methods: We included 976 formerly preeclamptic women and 505 women with normotensive gestation in their history between 0.5 and 30 years after childbirth. Cognitive assessment was evaluated by measuring selfreported executive functioning using the behaviour rating inventory of executive function scale for adults (BRIEF-A). Higher scores refl ect more perceived diffi culties. Obstetric history, educational attainment, height, and weight were documented. Potential relationships between perceived executive functioning and a history of preeclampsia were examined through multivariable logistic regression. The relative and absolute risks of reporting elevated BRIEF-A scores after preeclampsia were calculated over postpartum time. As compared to controls, former preeclamptic women experienced signifi cantly more problems in executive functioning (fi gure). While controls had stable cognitive functioning in the three decades after gestation, in formerly preeclamptic women, the negative impact on cognitive functions diminished over time but lasted signifi cantly up to at least 16 years. Neither onset of preeclampsia, nor concurrent HELLP syndrome, preterm delivery, or perinatal mortality related to worse cognitive functioning. Obesity negatively aff ected cognitive performance while educational level positively aff ected functioning. Conclusion: Irrespective of clinical severity, women with a preeclamptic history report more cognitive diffi culties up to many years after the troublesome pregnancy. These cognitive complaints may substantially affect effective social functioning and labor participation. Possible underlying disorders, either psychological or physical, remain to be elucidated. We had previously shown that lactation results in long-term maternal cardiometabolic health benefi ts, which were associated with increased oxytocin (OXT) levels later in life. During lactation, oxytocin is produced in response to mechanical nipple stimulation and psycho-social maternal-off spring interaction. The objective of the current study was to diff erentiate between the roles of these two mechanisms in hypothalamic oxytocin production during lactation. Methods: Nonpregnant CD-1 mice were either thelectomized (had nipples removed surgically, TL group) or sham-thelectomized (surgical incision was made, but nipples were not removed, STL group). After 7-day recovery period mice were bred. At delivery pups were removed from TL mice. In STL mice pups were culled to 8 pups/mom. One TL and one STL with her pups stayed in the same cage together until weaning. Group of STL had their pups removed at delivery and were housed separately with no exposure to pups (CTR-STL). One month post-delivery (1 month in mice age is approximately 3.3 years in humans), dams were euthanized, and their tissues collected. The relative expression of OXT and oxytocin receptor (OXTR) were measured in hypothalamus by realtime quantitative PCR and analyzed using One-Way and Kruskal-Wallis ANOVA with appropriate post-hoc tests (statistical signifi cance: P<0.05). Results: Total body weight was not signifi cantly diff erent at 1 month post-delivery among the three groups of dams. Adiposity (the % of excised adipose tissue weight from total body weight) were signifi cantly higher in TL (P=0.001) and CTR-STL (P=0.04) mice when compared to STL group. OXT gene expression was signifi cantly lower in TL (P=0.003), and it was downregulated in CTR-STL, though did not reach statistical signifi cance (P=0.43) in comparison to STL mice. OXTR gene was signifi cantly downregulated in TL (P=0.03) and CTR-STL (P=0.02) groups as compared to STL mice. None of the parameters investigated were signifi cantly diff erent between TL and CTR-STL groups. Conclusion: Lack of lactation resulted in higher adiposity and lower levels of hypothalamic OXT and OXTR at 1-month post-partum in thelectomized mice, but exposed to pup interaction, and in mice, totally separated from off spring at delivery. Our current results suggest that both, mechanical nipple stimulation and maternal-off spring interaction, are equally important in OXT and OXTR production. The maternal pelvis has been traditionally classifi ed into 4 types: gynecoid, anthropoid, android, and platypelloid. While most women's pelvises may be intermediate as they do not fi t one defi nition perfectly, this classification helps physicians to better understand the mechanism of labor. As such, diff erent pelvic shapes should be incorporated into simulation models. However, at present, no model exists replicating these diff erent pelvis types, and most of the current birthing simulators do not even contain a bony pelvis as a component. We sought to create diff erent maternal pelvis models using 3D printing technology for educational and training purposes. Methods: The Cancer Imaging Archive provides access to a TCIA radiology portal. From this resource, a CT folder of a female patient was downloaded and converted into a 3D model with the program Slicer©, exposing only the pelvis. Then, using MeshMixer©, the original pelvic shape was manipulated and altered in the likeness of the four classifi cations of female pelvises. The four 3D-printed prototypes were also segmented into three sections to illustrate the anatomical joints. Two fetal skullsone made of elastic fi lament and the other out of hard plastic-were also constructed following the same protocol. Results: Four pelvic models representing classical female pelvis types were 3D-printed along with two fetal skulls. They replicate the characteristics of the four pelvic shapes and allow the examiners to appreciate their diff erences during clinical pelvimetry. They also demonstrate their eff ects on the birthing process. For example, the fetal head is sometimes unable to pass the android pelvis when it is presenting as an occiput posterior, and the platypelloid pelvis prevents internal rotation. Finally, the application of forceps is aff ected by the pelvic shapes, making it more diffi cult to insert and articulate the forceps in a non-gynecoid pelvis. Conclusion: These models allow physicians and trainees to have a more realistic learning experience in the clinical assessment of the maternal pelvis, understanding how the mechanism of labor changes in diff erent pelvis types, and becoming more profi cient in operative vaginal delivery. These newly constructed pelvis models should be incorporated into future birthing simulators. Previously we have shown that mice exposed to a brief high fat high sugar (HFHS) display complications similar to GDM including: glucose intolerance, decreased beta cell numbers and serum insulin levels as well as maternal dyslipidemia. The objective of the current study was to further characterize beta cell function and insulin resistance in this mouse model. Methods: Mice were randomly assigned to either control (CD) or HFHS (45% fat, 18% sugar) diet and mated one week later. Exp. 1: At day 0 (day of mating) mice were fasted and tested for intraperitoneal insulin tolerance. Mice were then sacrifi ced and pancreas were collected for histological analysis. Exp. 2: At day 8.5 of pregnancy surgery was performed to insert catheters for Euglycemic Hyperinsulinemic Clamp experiments, and mice were allowed to recover. Clamps were performed in conscious day 13.5 pregnant (P) or non-pregnant (NP) mice fed either CD or HFHS for 21 days (equivalent diet exposure time to d13.5 mice). Exp. 3: Pancreatic islets were isolated and glucose stimulated insulin secretion (GSIS) assays were performed on P CD and P HFHS dams at day 0, 13.5 (mid) and 17.5 (late) of pregnancy. P-value of less than 0.05 was considered signifi cant. Results: Exp. 1: At day 0, insulin tolerance and beta cell numbers were not diff erent between CD and HFHS mice. Exp. 2: At day 13.5, glucose infusion rate (GIR) was reduced in P HFHS dams compared to all other groups (NP CD, P CD, NP HFHS). P HFHS dams also had reduced glucose disposal rate compared to all other groups. Basal glucose production (after 4 hours fast) was not diff erent among groups. Under hyperinsulinemic conditions, all groups had suppression of hepatic glucose production. The percent suppression in glucose production from basal to clamped state decreased with diet alone as well as pregnancy status however interaction between pregnancy status and diet was detected. P HFHS dams had decreased glucose suppression compared to NP CD females. Fetal and placental glucose uptake were increased in HFHS compared to CD dams. Exp. 3: GSIS was not diff erent at day 0, however at day 13.5 and 17.5 GSIS was decreased in islets isolated from P-HFHS compared to P-CD dams. Conclusion: Brief HFHS diet results is beta cell dysfunction and insulin resistance specifi c to pregnancy. This data further supports the use of this brief HFHS diet model as a useful model to study the pathophysiology of GDM. Future work is planned to utilize this animal model to study the mechanisms regulating beta cell function and insulin resistance in normal and diabetic pregnancies. Use Introduction: Preeclampsia is associated with elevated thrombotic risk in pregnancy and postpartum, and is linked with increased risk for future cardiovascular morbidity. Here we report plasma coagulation factor levels and thrombin generation potential in women examined outside of pregnancy and grouped by parity and prior pregnancy outcome. Methods: This is a cross-sectional retrospective analysis of two approved studies. Citrated platelet poor plasma samples were obtained during the follicular phase from women classifi ed as nulliparous (P0, N=36), primiparous with an uncomplicated pregnancy history (P1Healthy, N=10), and primiparous with a history of preterm preeclampsia (P1PreE, N=10). The groups were predominantly non-Hispanic Whites, representative of the local population. Previous deliveries were 2-4 years prior (Figure) . Samples were analyzed for functional levels of coagulation factors (II, V, VII, VIII, IX, X, antithrombin (AT), and protein C (PC)). Thrombin generation (TG) potential was measured using the Calibrated Automated Thrombogram protocol and reagents. Briefl y, reactions were recalcifi ed and coagulation initiated with exogenous tissue factor (TF) or TF with 10 nM soluble thrombomodulin (TF+TM) to assess the inhibitory protein C pathway. Peak TG levels from each condition were compared, as was the ratio of TF+TM peak thrombin relative to the TF condition alone ("TF+TM"/TF). Means ± SD are reported. Welch's t-test was used to compare groups, signifi cance was set at p < 0.05. Results: P1 Healthy women were older than the P0 and P1 PreE groups. There were no statistically signifi cant diff erences in BMI (Figure) . Levels of factor II, V, and IX were not diff erent between P0 and P1Healthy, however prior preeclamptics had higher levels. Levels of factor VII, VIII, and X were not diff erent between the groups. PC levels were diff erent between groups, with P1PreE having the highest levels. While the peak TG was not different between any of the groups under either assay condition (TF or TF+TM), the P1PreE group had a higher APC sensitivity as represented by the peak ratio of the TF+TM to TF conditions. Conclusion: Over 2 years after an index preeclamptic delivery, women with prior preterm preeclampsia exhibit a slightly more procoagulant coagulation factor profile compared women with a prior normotensive delivery, which may be attenuated by higher PC levels and increased APC sensitivity. Introduction: Ubiquitin specific protease 19 (USP19) is a membrane bound deubiquitylase (DUB) involved in the misfolding-associated protein secretion (MAPS) pathway, removing cellular debris, which has recently been implicated in muscle atrophy and cachexia as well as glucocorticoid and insulin signalling. USP19 -/-mice show a lean phenotype and are less prone to obesity and insulin resistance. As the underlying molecular mechanisms are not yet understood, we explored USP19 as a novel target in adipogenesis, polycystic ovarian syndrome (PCOS), cachexia and human diseases that involve muscle atrophy. Methods: Five known USP19 inhibitor compounds were profiled by proteomics for direct cellular target engagement and selectivity. Descriptive target validation and novel USP19 substrate discovery experiments was performed in an MCF7 breast cancer cell line using advanced proteomics and ubiquitomics pipelines. To support pre-clinical studies, an in-house knockout mouse was also generated, and wildtype and USP19 -/-mice (n = 11 females per group) were exposed to a high fat diet (HFD) regime. Brown/white adipose and gonadal/inguinal muscle tissues were extracted from each mouse and subjected to an equivalent discovery proteomics and ubiquitomics procedure. Results: USP19 inhibitor capacity was determined for three compounds in vitro and in cells. Analysis of the body weight of the WT and USP19 ko mice revealed a clear reduction in overall body weight in the absence of USP19 when fed a HFD. From proteomics and ubiquitomics, we identified several pathways involved in adipogenesis, thermogenesis, and insulin and glucocorticoid signalling in both cell lines and the HFD rodent model. Furthermore, USP19 compounds attenuate adipocyte differentiation and, based on public studies, reduce the extent of muscle wasting. Conclusion: Our results support the therapeutic potential for USP19 inhibition, leading to reduced body weight and attenuated adipocyte differentiation and muscle wasting. We propose that USP19 inhibition may represent a novel option to treat obesity and female-specific cardiometabolic traits, in addition to cachexia and other muscle atrophies. aff ecting the endocrine, infl ammatory and 1-carbon (1-C) metabolic pathways. We hypothesize that identifying biomarkers affected by maternal obesity during the periconceptional period can unravel the pathophysiologic mechanisms and identify individuals at risk of adverse clinical outcomes. Therefore, a systematic review was conducted to identify periconceptional biomarkers of the endocrine, infl ammatory and 1-C metabolic pathways infl uenced by maternal obesity. Methods: The databases of Embase, Medline All Ovid, Web of Science Core Collection and Cochrane central register of trials were searched until December 31 st , 2020 to identify articles that studied biomarkers in relation to maternal obesity, overweight/obesity or BMI during the periconception period. The protocol was registered under the PROSPERO registry of systematic reviews and the ErasmusAGE score was used to assess the quality of the studies included. Results: Fifty-one articles were included in the review, which evaluated over 30 biomarkers. Endocrine biomarkers associated with obesity included leptin, insulin, thyroid stimulating hormone, adiponectin, progesterone, free T4 (FT4) and human chorionic gonadotropin (hCG) (Figure 1 ). C-reactive protein (CRP) was associated with obesity as part of the infl ammatory pathway, while the associated 1-C metabolism biomarkers were folate and vitamin B12 (Figure 1 ). BMI was positively associated with leptin and CRP, and negatively associated with FT4, progesterone and hCG. Obesity as well as BMI were associated with increased insulin resistance. Concerning the remaining studied biomarkers, conclusions could not be established defi nitely due to limited or contradictory data. Conclusion: Maternal obesity aff ects a variety of biomarkers of the endocrine, infl ammatory and 1-C metabolic pathways. Future research should focus on a composite determinant of clinically relevant biomarkers to identify individual risks for a timely prevention of adverse pregnancy outcomes in maternal obesity. Introduction: Women who experience hypertension in pregnancy have increased risk of both chronic hypertension and dementia. High blood pressure is associated with increased evidence of white matter hyperintensities (WMH) in brain imaging. WMH are disruptions of the white matter of the brain, are associated with vascular disease, occur more frequently in people with hypertension, and are associated with cognitive impairment. We evaluated the relationship between WMH and cardiovascular function in healthy young women. Methods: Sixty-two women were assessed during the follicular phase of the menstrual cycle after a 3-day sodium/potassium controlled diet. Cardiovascular assessments included tonometric blood pressure monitoring, dual echocardiography with Doppler ultrasound to assess cardiac ejection times and pulse wave velocity of the brachial artery (to assess vessel stiffness), volume loading with tonometric blood pressure monitoring to assess vascular compliance, flow mediated vasodilation of the brachial artery to assess endothelially mediated dilatory response, and MRI scan including T2 FLAIR imaging. Blood was drawn to assess inflammation. Two investigators reviewed each MRI scan for evidence of WMH. WMH were qualified using the Fazekas rating scale, an ordinal categorization of healthy, punctiform, early confluent and diffuse confluent WMH. In the event of a discrepancy in reported Fazekas score, a third adjudicator reviewed the images. All reviewers were blinded to cardiovascular assessments. Eighteen women had evidence of WMH, and 46 had normal white matter structure. Results: Mean age was 31 ± 5 years, with body mass index 26 ± 6 kg/m 2 . Women were 88% Caucasian, and included 50% prior preterm preeclamptics, 50% nulliparas. There were no differences in Fazekas score by pregnancy history. Women with evidence of a Fazakas score >0 had exaggerated cardiac output response to volume loading compared to women with a Fazakas score of 0, 15.7 ± 14.8 vs. 6.6 ± 11.7 L, p=0.016 suggesting reduced cardiovascular compliance. They had longer cardiac ejection times, 0.160 ± 0.021 vs. 0.147 ± 0.020 sec, p= 0.03. Fazekas scores of >0 trended towards decreased brachial compliance in response to flow-mediated vasodilation (4.6 ± 6.4 vs 8.3 ± 7.4%, p=0.085), and white blood count (6.3 ± 1.7 vs. 5.6 ± 1.3 x10 9 /L, p=0.065) compared to those with no evidence of WMH. Conclusion: Women with WMH have reduced cardiovascular compliance, and a trend towards decreased endothelial responsiveness compared to those without WMH. These data demonstrate that the relationship between cardiovascular and brain health is detectable in young, healthy, reproductive-aged women, and may play a role in later risk of clinical disease. These findings may help identify women tho are potentially at risk for cognitive decline and pathological aging. Methods: Systematic searches were performed electronically across PubMed, CINAHL, Scopus and Cochrane Library databases in November of 2020 and July of 2021. The following keywords were used: mothers, maternal, patient, microbiome, postpartum, pregnancy and prepartum. In addition, OpenGrey.eu was utilized and conference proceedings, journals and references of relevant reviews were hand selected. Results: A total of 8 studies were eligible out of 2512 studies screened as illustrated in the PRISMA flow diagram (Fig. 1 ). Conditions and clinical intermediate measures investigated for their association with the postpartum gut microbiome consisted of early-onset preeclampsia, gestational diabetes, inflammatory bowel disease, postpartum depression, excessive gestational weight gain, overweight/obese pre-pregnancy and postpartum body mass index, and other anthropometric measurements. Enrichment and/or depletion of various microbial taxa was observed in 7 studies. Lower alpha diversity was found in the gut microbiome of women with overweight/obese pre-pregnancy BMI and urolithin metabotype A, the latter of which was associated with significant reduction in waist-tohip ratio postpartum. Conclusion: Health disturbances in women are associated with shifts in their gut microbiome composition in the postpartum. Further research of the gut microbiome's significance during this period could lead to new understanding of how to improve maternal health. Proteomic We and others have shown that, in mice, membrane proteins Xanthine dehydrogenase (Xdh), Butyrophilin (Btn1a1), Perilipin 2 (Plin2) and Cell death effector A (Cidea) facilitate the docking and envelopment of cytoplasmic lipid droplets at the apical plasma membrane and remain embedded in the MFG membrane (MFGM) following MFG secretion. Importantly, experimental manipulation of this complex disrupts milk production, highlighting its central role in mammary function. Substantial dissimilarity in milk fat content between mice (~30%) and humans (~4%) underscores the importance of determining cross-species conservation of the MFG secretion mechanism. Here, we describe the human MFG and MFGM proteomes and identify points of divergence with murine MFG docking and membrane trafficking components. Methods: We collected paired human MFG and MFGM samples from 13 women across mature lactation (2wks, 2mo and 4mo postpartum) in an ancillary study of women in a randomized controlled trial of diet during gestational diabetes. We conducted proteomics via liquid chromatography with tandem mass spectrometry and identified 1,976 MFG proteins and 1,997 MFGM proteins with Normalized Spectral Abundance Factor (NSAF) >0.01. We conducted overrepresentation pathway analysis of MFGM proteins with NSAF >0.1 using Reactome and compare our results with previous proteomic analyses of mouse MFG (n=3 mice) and MFGM (n=2 pooled from 10 mice) samples. Introduction: Accurate gestational age (GA) determination is crucial to optimal pregnancy management, including scheduling of GA-specific screening tests, assessment of fetal growth, interventions during pregnancy, and timing of delivery. Recent studies have investigated whether biomolecules originating from the placenta in the maternal circulation can be used for accurate and robust estimation of GA, functioning as a "placental clock." In this cross-sectional study, we conducted transcriptomic profiling of the miRNA landscape in early, mid, and late GA placentas in order to determine changes in placental miRNA expression throughout normal pregnancy. We hypothesized that GA-dependent changes in miRNA levels in placental miRNA expression can be used to accurately estimate GA, with the ultimate goal of detecting changes in placental miRNA circulating in maternal serum. Methods: Placental tissue was collected at early (5-12 weeks, n=26), mid (12-30 weeks, n=18), and late (30-42 weeks, n=54) gestational time points. RNA (RIN≥7) was isolated from placental tissue using the miRVana kit. Small RNA libraries were prepared using the NEB Next Small RNA Library Prep Kit and sequenced on an Illumina HiSeq 4000. Small RNAs were mapped and quantified using the Genboree exceRpt small RNA pipeline. Filtered miRNA raw counts were TMM-normalized prior to performing differential expression analysis with the limma-voom method in R. We assessed differences in miRNA expression between GA windows using pairwise comparisons, and longitudinally using GA as a continuous variable to identify miRNAs that changed over time. A P-value<0.05 adjusted for multiple testing was considered significant. Results: Pairwise comparisons indicated that 302 miRNAs were significantly differentially expressed (DE) between early to mid-gestation; 231 miRNAs were DE between mid to late gestation; and 416 miRNAs were DE between early to late gestation (p adj <0.05). Using GA as a continuous variable, 458 miRNAs were DE (p adj <0.05), including 70 miRNAs (15%) localized on the C19MC cluster on chromosome 19. The placenta contains miRNAs that display strong GAspecific patterns of expression and can be used for establishing a "clock" that can aid in dating of pregnancies where standard clinical criteria are unavailable. Future studies will focus on (1) correlating changes in placental miRNA with maternal serum and proteomic data, and (2) advanced computational approaches to develop multivariate assays for more accurate GA estimation. These studies are key to determining the potential roles of miRNAs in placental development and function. Introduction: Hemochorial placentation occurs in many mammalian species and is the pivotal event in the establishment of pregnancy. Trophoblast cells migrate into the uterus where they transform the uterine vasculature and facilitate the delivery of nutrients. The rat and human each possess a uterine-placental interface that is characterized by deep intrauterine infiltration of trophoblast cells. Invasive trophoblast cells, arise from the junctional zone (JZ) in the rat and a homologous structure in the human placentation site termed the extravillous trophoblast (EVT) column. Thus, development of these structures is vital to establishing the uterine-placental interface. Some insights into JZ development have arisen from mutagenesis of Phlda2 loci in the mouse. However, the mouse has limitations for investigating the uterine-placental interface. In this study we examined the biology of PHLDA2 in the development of the invasive trophoblast cell lineage in vitro using human trophoblast stem (TS) cells and in vivo using the rat. Methods: PHLDA2 expression was assessed in in first trimester human placentas and rat placentation sites during gestation using in-situ hybridization. A role for PHLDA2 in human TS cell differentiation was assessed using loss-of-function approaches via lentiviral delivered shRNA gene silencing. We also generated global loss-of-function rat models using CRISPR/Cas9 genome editing. WT and PHLDA2 mutant placentation sites were examined histologically and through biochemical and molecular analyses Results: PHLDA2 transcripts were prominently expressed in the base of the EVT column of the human placenta and in trophoblast progenitor cells within the ectoplacental cone of the midgestation rat placenta and subsequently within the labyrinth zone of the late gestation rat placenta. In human TS cells, the expression of PHLDA2 was upregulated during EVT cell differentiation. Effective PHLDA2 knockdown was achieved using shRNAs. A mutant Phlda2 rat founder was produced containing a 103 bp deletion in Exon 1 resulting in a frameshift and a premature stop codon. Conceptuses inheriting a maternal mutant Phlda2 allele exhibited placentomegaly characterized by an expanded JZ and abnormal infiltration of invasive trophoblast cells into the uterine compartment. PHLDA2 mutant placentas showed an upregulation of phospho-AKT as well as phospho-AKT-substrate levels within the junctional zone. PHLDA2 knockdown in human TS cells similarly altered AKT signaling. production via mitochondrial oxidative phosphorylation was lower in basal respiration and in presence of oligomycin, FCCP, and rotenone/antimycin in KD compared to CT cells. The basal respiration, maximal respiration, spare respiratory capacity, ATP production-coupled respiration, and non-mitochondrial oxygen consumption were reduced by 1.54-, 1.74-, 3-, 1.46-, 1.42-fold, respectively (P<0.05) , in KD compared to CT cells, while there was no difference in proton leak between these two cells. Conclusion: Taken together, reduced AMPK protein abundance impairs mitophagy mediated by PINK1/PARKIN and FUNDC1, and mitochondrial ATP production, and decreases mitochondrial mass, suggesting that AMPK plays a critical role in maintaining cellular homeostasis by regulating both mitophagy and mitochondrial functions. Introduction: Maternal diet is often a predictor of pregnancy outcome and fetal health as the placenta is sensitive to a variety of stimuli including diet, cigarette smoke, hypoxia, and oxidative stress. Indeed, women commonly employ prenatal vitamins, pregnancy supplements, and specialized diets to achieve a healthy pregnancy. Coumestrol is a phytoestrogen derived from spinach and soy that does not cross the fetal-placental barrier and induces the protective pregnancy hormone, adrenomedullin. Interestingly, vegetarians exhibit a decreased risk for developing preeclampsia, a hypertensive pregnancy disorder originating from abnormal placentation. Thus, coumestrol may be a valuable supplement to promote placental development, ensuring a healthy pregnancy. Due to the popularity of vegetarian and soy-based diets, we sought to examine how coumestrol affects the placenta. I hypothesized that plant-derived coumestrol promotes trophoblast cell function and migration, generating a favorable fetal and placental environment. Methods: An RNA microarray on coumestrol treated human trophoblast cells (HTR8/SVneo) was performed and Partek Genomics software was used for analysis. Differentially regulated genes were determined and Ingenuity Pathway Analysis was used to determine top pathways. Trophoblast cells were seeded to confluency, scratched, and allowed to migrate and average wound closure was determined. Total levels of reactive oxygen species (ROS) were measured in coumestrol exposed cells using Cell Rox Deep Red reagent. To assess murine in vivo effects, wildtype dams were treated with coumestrol from day 0.5 to 12.5 of pregnancy. Fetal and placental weights were recorded. Placentas were evaluated for gene expression and morphology. Fiji software was used to perform image analysis. The unpaired t-test with Welch's correction was used to measure differences between vehicle and coumestrol groups. Results: A total of 3,079 genes were found differentially regulated with top pathways including cell migration and the oxidative stress response. Coumestrol treatment decreased migration of trophoblast cells and caused accumulation of ROS. Within mice, fetal and placental weights were reduced significantly and the placentas exhibited normal morphology. Placental RNA levels for antioxidants and angiogenic genes were significantly decreased. microscopy was used to generate intensity ratios of active-to-inactive cell surface integrins. TIRF was also used to image FAs containing active integrins in migrating cells. The publicly available Focal Adhesion Analysis Server was used to analyze FA characteristics. Confocal microscopy was used to quantify the percent of endosomal vesicles containing an active integrin. T-tests and Mann Whitney U-tests assessed statistical significance (p<0.05). Results: When compared to controls, FGRadv ECs had significantly higher amounts of active integrin αvβ3 at the membrane (p=0.012). Yet there were no changes in the number of integrin αvβ3-positive FAs, suggesting that αvβ3 is not entering FAs at a rate similar to controls. Conversely, there was no difference in α5β1 activity, although FGRadv ECs displayed significantly more active integrin α5-positive FAs (p=0.028). When assessing differences in intracellular trafficking, there were significantly fewer early endosomal vesicles containing active α5 (p=0.002) and recycling vesicles containing active β1 (p=0.006). Conclusion: These data confirm that integrins αvβ3 and α5β1 are dysregulated in FGRadv placental ECs and that the mode of dysregulation is specific to the integrin heterodimer. These newly identified changes in FGRadv EC cellular processes represent a previously unidentified mechanism contributing to persistent angiogenic deficiencies in FGRadv. Introduction: Fetal growth and the incidence of pregnancy complications, such as preeclampsia and preterm birth, have been reported to exhibit racial/ethnic differences. The placenta is metabolically active and supports the growth of the fetus, and placental insufficiency is implicated in the pathogenesis of preeclampsia and preterm birth. We hypothesized that the placenta may exhibit racial differences in the capacity of bioenergetics and metabolism during mid gestation, which may underlie the racial disparity in fetal growth and pregnancy complications. Methods: Focused on self-identified Black and White populations, which show the most differences in fetal growth and pregnancy complication rates, we performed a metabolomics study (Metabolon Precision Metabolomics TM ) in both male and female placentas (n=9 per group for Black males and White males; n=10 per group for Black females and White females) from second trimester pregnancies that were electively terminated at 18-22 weeks to assess the similarities and differences of metabolomic signatures between Black and White patients as well as between males and female fetuses. Welch's two-sample t-tests were used to identify metabolites that differed significantly between experimental groups. Results: 866 metabolites were identified in the human placentas. Racial differences were profound in both genders. The levels of 317 and 332 metabolites were significantly different (p<0.05) between Black and White patients in male and female placentas, respectively, and nearly all of these metabolites were lower in Black compared to White placentas. Notable changes in metabolite levels that were significantly lower in Black vs. White placenta include metabolites in glycolytic and energy related pathways, fatty acid beta-oxidation, branched-chain amino acid metabolism, oxidative stress, steroid hormone metabolism, nucleotide metabolism, and cofactor and vitamin metabolism. More modest differences were observed between sexes, and the levels of 34 and 54 metabolites were significantly different (p<0.05) between males and females in White and Black patients, respectively. Levels of acylcarnitine species were significantly higher in Black females compared to Black males (p<0.05), however, no differences were found between White females and White males. Conclusion: Our results demonstrate significant and biologically relevant racial differences in the placental metabolome in Black and White populations. The observed diff erences in metabolites that impact placental and fetal development may contribute to the racial disparity in fetal growth and pregnancy complications. Modeling 1:25] ) to mimic maternal drug exposure during normal physiologic or oxidative stress (OS) associated pregnancy pathologies respectively. We determined statin pharmacokinetics and metabolism (LC-MS/MS), drug induced cytotoxicity (LDH assay), and effi cacy on reducing OS induced infl ammation (multiplex cytokine assay). Results: Both statins propagated through various maternal-fetal cell layers of the FMi-OOC and PLA-OOC within 4h (p<0.05) and produced cell and time-specifi c statin metabolites from various cell types in OOCs without causing any cytotoxicity ( Fig.1-top/middle) . OS did not aff ect statin kinetics but increased pro-infl ammatory cytokines compared to controls. Statins signifi cantly increased anti-infl ammatory cytokines across the FMi-OOC and PLA-OOC (p<0.05) ( Fig.1-bottom) in OS induced cells. Conclusion: Like placenta, fetal membranes-decidual interface also transport drugs and shows effi cacy. Our study showed, OOC devices can be used to study drug's pharmacokinetics, metabolism, safety, and effi cacy that is expected to limit the use of animal models in preclinical trials. Introduction: Obesity in women at reproductive age is increasing worldwide and is a risk factor for pregnancy complications. In the fi rst trimester (FT) of pregnancy the human placenta is rapidly growing making it sensitive to environmental infl uences. Therefore, we hypothesized that the altered intrauterine milieu associated with maternal obesity modifi es placental proteome and function. Methods: Maternal BMI was calculated and leptin, glucose, C-peptide and insulin sensitivity (IS HOMA ) quantifi ed in maternal serum. To identify obesity-associated changes in the placental proteome we run untargeted proteomics (LC-MS/MS) in placentas from lean (n=10) and obese (n=10) women (gestational age 5 +0 -6 +6 weeks). The results were validated using westernblotting and immunohistochemistry. The FT trophoblast cell line ACH-3P was used to perform functional assays in vitro. The cells were challenged with insulin (10nM) or palmitoleoyl ethanolamide (100nM), a metabolite found in higher concentrations in women with low insulin sensitivity (untargeted metabolomics) for 24h at physiological oxygen tension (6.5% O 2 ) in a hypoxia bench. Changes in the concentration of the top identifi ed proteins under exposure to the diff erent stimuli was quantifi ed by qPCR and westernblotting. Results: Placental proteome diff ered between lean and obese women with a stronger separation between those with low vs high insulin sensitivity (Principal Component Analysis). Proteins (n=89) In the guinea pig cytomegalovirus (GPCMV) model of congenital infection, maternal infection after mid-gestation causes a characteristic localization of infected cells in placentas and a transcriptional response indicative of immune activation. These observations led us to hypothesize that the guinea pig placenta becomes sensitized to infection-associated injury late in gestation that is caused by the maternal or fetal immune response to GPCMV and culminates in high rates of fetal growth restriction and stillbirth. In this study, we used spatial transcriptomics (ST) to quantify host and viral gene expression at the maternal-fetal interface and elucidate mechanisms underlying placental dysfunction post-infection. Methods: Time mated guinea pigs received a subcutaneous injection of GPCMV or saline at 35 days gestation and placentas were collected at 21 days post-infection. GPCMV viral loads and the localization of infected cells in placentas were assessed using virus-specific droplet digital PCR and RNA-Scope. Representative placentas were selected and sectioned onto Visium Spatial Gene Expression Slides (10X Genomics) so that gene expression in the placenta, subplacenta, and decidua could be assessed. Following on-slide cDNA synthesis, library preparation, and Illumina sequencing, data was analyzed using Partek® Flow® software (v10.0). Results: ST data was generated from twelve tissue sections (N=6 infected and normal placenta), resulting in data from 34,470 spots (each spot is 55 µm in diameter). Graph-based clustering of the batch-corrected host gene expression data identified 17 clusters of spots, which localized to similar regions in each section of placenta, and transcripts that can be used as biomarkers for each cluster. While an earlier bulk RNA-Seq experiment detected very few GPCMV reads in infected placentas, ST detected up to 2X10 5 viral reads in each section of infected tissue. These reads mapped to the 3' ends of most GPCMV transcripts, reinforcing that the placenta is a site of productive viral replication. We used ST to quantify gene expression at near singlecell resolution in the placenta of an unconventional model organism. Compared to bulk RNA-Seq, ST markedly improved our analysis of viral gene expression and understanding of the host response at sites of infection. The The placenta releases a range of proteins into the maternal and fetal circulations with potential effects on fetal development and maternal physiology. However, there is limited knowledge about these proteins and their functions. We aimed to identify proteins that the placenta releases into or takes up from the maternal and fetal circulations. Methods: We obtained blood samples from the maternal antecubital vein (AV M ), radial artery (RA M ) and uterine vein (UV M ) as well as the umbilical artery (UA f ) and vein (UV f ) during cesarean section in 75 healthy pregnancies in the 4-vessel cohort (figure part A). We measured proteins by the Somalogic 5000-plex platform, and defined them as released or taken up on the maternal and fetal sides of the placenta by paired Welch's t-tests between abundance in pairs of vessels (FDR<0.05). Proteins released into the maternal circulation were further defined as placenta specific and/or upregulated using the antecubital vein-radial artery difference as a negative control (Figure part B ). For these proteins, we determined changes across gestation using longitudinal AV M samples from 70 healthy pregnant women in a prospective cohort (Figure part C). Results: More proteins were taken up or released into the fetal circulation as compared to the maternal (Figure part D). Placental growth factor was released from the placenta to the maternal circulation in accordance with previous studies. The protein with most significant release to maternal side (lowest FDR) was Pleiotrophin. We found that Secreted frizzledrelated protein 3 was taken up by the placenta from fetal circulation and released to maternal side. Of 45 proteins released from placenta to both maternal and fetal side were Soluble fms-like tyrosine kinase-1 (sFlt-1), Tissue factor pathway inhibitor 2 and Fatty-acid amide hydrolase 2. The 101 placenta specific and/or upregulated proteins showed a diversity of trends across gestation, however most of these proteins increased steadily. The identification of proteins taken up and released by the human placenta, to both the mother and fetus, may facilitate improved understanding of normal physiology during pregnancy as well as pave the way for development of biomarkers of healthy and pathological placental function. Intrauterine Growth Restriction Increases Placental Retention of Lysophosphatidyl-DHA at the Expense of Fetal Acquisition in the Rat. Sophie Hochhauser †, Nicolette Jessen, J. Alan Maschek, James E. Cox, Haimei Wang, Lisa A. Joss-Moore. University of Utah, Salt Lake City, UT, United States. Introduction: Appropriate accretion of the ω-3 fatty acid docosahexaenoic acid (DHA) is critical for fetal brain development. Brain accretion of DHA is reduced in animal models of intrauterine growth restriction (IUGR), and human infants affected by IUGR have lower levels of circulating DHA. IUGR infants also have a higher risk of neurodevelopmental impairment, as do infants with prenatal DHA deficiency. DHA accretion in the developing brain is facilitated by circulating DHA moieties with selective transport across the blood-brain barrier, including lysophosphatidyl-DHA (LPC-DHA). Recent evidence suggests that placentally produced LPC-DHA is the preferred DHA moiety exported to the fetal circulation from the placenta. We hypothesize that IUGR will increase placental levels of LPC-DHA at the expense of fetal serum LPC-DHA acquisition in the rat. Methods: IUGR was generated in Sprague-Dawley rats by bilateral uterine artery ligation. Male and female feto-placental units were collected by c-section at term gestation from control and IUGR litters. Gas chromatography-mass spectrometry was used to measure levels of LPC-DHA in male and female matched placenta and fetal serum. Differences were assessed by one-way ANOVA and Fisher's post hoc test. Results: Results are IUGR as % of control±SD, *P<.05. In males, IUGR increased placental LPC-DHA levels (159±33%*) while decreasing serum LPC-DHA (58±16%*) compared to male control. In females, IUGR did not alter placental or serum LPC-DHA. In addition, across all groups, placental LPC-DHA was significantly correlated with serum LPC-DHA. Conclusion: We conclude that IUGR increases placental retention of LPC-DHA at the expense of fetal serum LPC-DHA. Ongoing studies are assessing the effect of IUGR on fetal brain LPC-DHA acquisition in matched samples. We speculate that placental retention of LPC-DHA in IUGR impairs fetal brain LPC-DHA acquisition and may contribute to impaired neurodevelopment. Methods: Female C57BL/6 dams (n=6-8) were mted with males and on gestational day (GD)12.5 were infected with a single iv injection of ZIKV (ZIKV PE243 strain) low dose (LD, 10 3 PFU) and high dose (HD, 5x10 7 PFU) or mock control (CT). On GD18, dams were euthanized, the fetuses and placenta were weighed and the placentas were subjected to protein expression analysis by western blot or to MALDI-FT-ICR for detection of UDP-GlcNAc. Data for each gender were analyzed using one-way ANOVA followed by Tukey's test. Experiments were approved by the Animal Care Committee (CEUA-036/16 and 104/16). The fetuses from dams infected with high dose of ZIKV presented lower weight than the fetuses from the control group (-16%, p=0.015). The placentas from dams infected with low or high virus doses also showed a significant reduced weight than those from the control group (ZIKV LD: -20%, ZIKV HD: -21%, p=0.039). In both female and male placentas, the expression of GFAT1 was not different among the experimental groups. Also, the O-GlcNAcylation was lower in female placentas from infected dams (ZIKV LD: -50.13%, p=0.016 and ZIKV HD:-60%, p=0.0053), although no differences in the expression of OGT and OGA in both genders. The spatial distribution of UDP-GlcNAc in specific placental tissue regions was different among infected and control groups. Conclusion: Placental distribution of UDP-GlcNAc is modulated by ZIKV infection. The O-GlcNAcylation is altered in female placentas infected with ZIKV, suggesting that the regulation of placental function in response to this infection is gender-specific. Introduction: Iron is an essential micronutrient for cell survival and growth, however excess concentrations of this metal -alongside lipid peroxidation -have also been identified as a driver of ferroptosis, a newly identified mode of programmed cell death. Previous work has established placental hypoxia and iron imbalance as independent contributors to the pathogenesis of preeclampsia (PE), yet the underlying relationship between iron, oxygen, and cell death remains elusive. Accordingly, we hypothesized that the coordination of iron and oxygen is essential in maintaining a healthy, physiological placenta, and disruption to this tightly regulated iron-oxygen axis increases susceptibility of ferroptosis in PE. Gene differential expression and gene ontology functional annotation that associated with specific cell cluster were analyzed. Results: Fifty cell clusters were classified into villi-, decidua-, and immune-categories from 20 placental tissues of 15 EPL (groups A, B, C) and five of elective termination of unplanned pregnancies (ET, group D). A total of 217,649 genes, including 40,705 from group A, 51,506 from B, 43,755 from C, and 75,683 from D, were sequenced. Both groups A and B comprised sporadic miscarriages, yet the period of intrauterine development of placental cells in group B could be longer than in group A. Group C was the recurrent miscarriages. Variant subtypes of cell clusters were characterized. E.g., CTB1, a subtype of very early VCT1, was identified from D-ET but not in EPL, and VCT2 was seen in A_SM (as well as C_RM), but not in B_SM (nor in D_ET). The top three most abundant cell clusters in EPL were VCT1, decidua macrophage (dM1), and HB1, which were different from EVT3, VCT1, and Epi1 (epithelial glandular 1 cell) in ET. Among the top-10-expressed marker genes in VCT cell clusters, seven genes (PAGE4, PEG10, SMAGP, VGLL1, ISYNA1, XAGE3, and DUSP9) were present in all EPL and in ET. An additional two genes, PHLDA2 and NGFRAP1, were only present in EPL, not in ET. Three genes, the HBB, MORC4, and ADIRF, were found to be specifically expressed in A_SM, B_SM, and C-RM, respectively, which suggested they may present as EPL-specific biomarkers. Several genes showed specific expression in EVT cells of variant EPL. Conclusion: Differential gene expression among cell clusters from miscarried and normal pregnancies and unique marker genes in trophoblasts that may lead to identification of biomarker for EPL were identified and characterized. Inflammasome Activation in Preeclampsia and Intrauterine Growth Restriction. Michal Silber * , 1,2 Nadav Dekel †, 1 Ishai Heuzler †, 1,2 Sivan Farladansky-Gershnabel * , 1,2 Tal Biron-Shental * , 1,2 Gil Shechter-Maor * , 1,2 Aliza Amiel * , 3 Avivit Weisz * , 1 Sydney Benchetrit * , 1,2 Tali Zitman-Gal * . The velamentous umbilical cord insertion (VCI) inserts into the fetal membranes instead of the placental disk. This abnormality is associated with SGA infants, stillbirths, congenital anomalies, and postpartum hemorrhage. Some VCIs may begin with suboptimal implantation sites and subsequently "migrate" to a more favorable location as the pregnancy progresses (trophotropism). This process may involve loss of placental parenchyma such that VCI placentas could end up smaller than their counterparts with a normally situated cord insertion. We tested the hypothesis that placentas with VCI would be lighter than placentas with normal cord insertions. Methods: This was a secondary analysis of a previously published casecontrol study to determine the accuracy of an ultrasound diagnosis of VCI. All placentas with a VCI were included in this study and compared to an equal number of randomly selected placentas that had a pathologic examination documenting either a central or eccentrically located umbilical cord insertion. The placentas were compared with respect to raw weight in grams as well as z scores for gestational age. A multiple linear regression was performed with placental weight in grams as the dependent variable and the independent variables of gestational age at delivery, maternal age, parity, smoking status, hypertension, Caucasian/ nonCaucasian race and VCI. A p value < 0.05 was considered signifi cant. The results are given in the The multiple linear regression was significant with an adjusted R square of 0.295 for the variables selected, p <0.001. Only the gestational age at delivery (p < 0.001) and the presence of a VCI (p=0.021) were significant predictors of placental weight at delivery. Conclusion: Placentas with VCI were significantly lighter than placentas with a normally located umbilical cord insertion. Although the absolute weight was only 10% lighter, the comparison placentas came from pregnancies considered to be high risk, and therefore would be expected to have lower placental weights than women with normal pregnancies. The difference between placental weights in VCI and low risk pregnancies with normal cord insertions may be further magnified. Nicolette Jessen †, Adrienne Cohen, Trey Benally, Ryan Gage, Sophie Hochhauser, Haimei Wang, Lisa A. Joss-Moore. University of Utah, Salt Lake City, UT, United States. Introduction: The placenta is an essential mediator of fetal lipid acquisition. In uteroplacental insufficiency (UPI), lipid droplets accumulate in the placenta, and fetal lipid acquisition is impaired. We recently identified a novel epigenetic pathway that mediates placental lipid accumulation regulated, in part, by the transcription factor peroxisomeproliferator-activated-receptor (PPARγ). We showed that UPI in the rat increases PPARγ activity in the male placenta, but not in the female placenta, with concomitant increased placental lipid accumulation. PPARγ variants, including the novel delta 5 splice variant (PPARγΔ5), can impact the downstream effects of PPARγ activation. As PPARγΔ5 is a dominant-negative variant, the result of reduced PPARγΔ5 is increased PPARγ signaling. Whether PPARγΔ5 is expressed in the rat placenta and the effect of UPI on expression is unknown. We hypothesize that PPARγΔ5 will be expressed in rat placenta and that UPI will decrease expression of PPARγΔ5 in the male placenta. Methods: UPI was generated in Sprague-Dawley rats by bilateral uterine artery ligation. Male and female placenta were collected by c-section at term gestation from control and UPI litters. Male and female placenta were treated as separate groups. PCR, gel electrophoresis, and sequencing were used to confirm the presence of PPARγΔ5 in the rat placenta. PPARγΔ5 We performed mRNA-sequencing on 124 first trimester and 43 third trimester placenta, with first trimester obtained from leftover tissue after chorionic villus sampling, and third trimester obtained after delivery. All pregnancies resulted in healthy live births. We performed differential expression analysis (adjusted for fetal sex) on the full cohort, as well as a subset of 23 pregnancies with matched first and third trimester placenta. Results: We identify 12,986 differentially expressed protein coding genes in the full analysis, and 12,533 in the subanalysis, at FDR<0.05 and study-specific threshold TPM>0.66. The fold-changes of genes significant in either analysis had a Pearson's correlation coefficient of 0.98 between analyses. To identify the most robust differences, we increased the stringency and obtained 6,449 genes that met FDR<0.001, fold-change>1.5, and TPM>0.66 in both analyses. Pearson correlation increased to 0.99. Of these, 2,919 were upregulated in first trimester and 3,530 were upregulated in third trimester. Next, we identified stable expression and low variability across gestation (TPM>0.66, P>0.05, fold-change<1.5, coefficient of variability<1.25). P-values above 0.05 vary widely between analyses for some genes, not others, and higher P-values alone do not indicate more "similar" genes for mRNA-seq. Low fold-changes and low coefficient of variability were better indicators of stable placenta genes. Conclusion: Despite differences in patient cohorts, results were similar to the subanalysis controlled for genetic background, suggesting that larger sample size can overcome patient variability. This may aid future study design and increase confidence in first vs third trimester placenta studies with variable patient cohorts. The The human placenta has a very large functional surface area, represented by the syncytiotrophoblast microvillous plasma membrane in contact with maternal blood. Plasma membranes consist of a lipid bilayer, requiring an active phospholipid biosynthesis. However, de novo synthesis (Kennedy Pathway) of phospholipids and remodeling (Lands Cycle) of phospholipids in human trophoblast has not been extensively studied. Our objective was to determine the acyl transferase and phospholipase enzyme isoforms present in human placenta that contribute to the formation of phospholipids. Methods: Immunoblotting was used to determine the expression of enzymes in the de novo biosynthesis and remodeling pathways in placental homogenates of 14 healthy term human placentas. The de novo enzymes studied were Glycerol Phosphate Acyl Transferase (GPAT3) and AcylGlycerol Phosphate Acyl Transferase (AGPAT2 and 4). The remodeling enzymes studied were Phospholipase A2 and group 4c (PLA2G4c) and Lyso-Phosphatidyl Choline Acyl Transferase (LPCAT4). Positive controls included human cerebellum for GPAT3, AGPAT2, PLA2G4c and LPCAT4, and human whole heart lysate for AGPAT4. Results: Detection of the enzyme in both the positive control and the human placental homogenate at the correct molecular weight was considered a positive result. GPAT3 (48 kDa), AGPAT2 (33 kDa), AGPAT4 (45 kDa), LPCAT4 (55 kDa) and PLA2G4c (59 kDa) proteins were all present in human placental homogenate across multiple pathologies. Conclusion: GPAT3, an enzyme catalyzing the first step of phospholipid synthesis by adding a fatty acid to glycerol-3-PO4, is likely to be responsible for the initiation of phospholipid de novo synthesis in the human placenta. Moreover, AGPAT2 and 4, enzyme isoforms for the second acyl transferase step, were identified in the human placenta. PLA2G4c cleaves a fatty acid from a formed phospholipid to release the fatty acid, which is critical for phospholipid remodelling. This cytosolic enzyme is a candidate for initiating phospholipid remodeling in the human placenta. Re-constituting a new phospholipid molecule is likely due to the action of the LPCAT4, which prefers to add long chain polyunsaturated fatty acids. We have identified enzymes responsible for phospholipid synthesis and remodeling in human term placenta, which will allow us to conduct studies of placental enzyme expression in pregnancy complications such as diabetes, obesity, and intrauterine growth restriction. Impacts Gestational hypertension and preeclampsia are prevalent vascular disorders that significantly increase the risk for both premature birth as well as perinatal maternal cardiac sequelae. Air pollution is a known inducer of systemic vascular damage and cardiac disease, and recent research has shown that gestational exposure to air pollution is correlated with increased incidence of preeclampsia. The current study seeks to examine how ozone, as a major constituent of air pollution, may cause systemic effects that are detrimental to normal fetoplacental growth and maternal cardiac function using a mouse model. Methods: FVB/N-Tg(Kdr-mCherry) mice were mated and exposed on gestational day (GD) 10.5 to either filtered air (N=3) or 1.0 ppm ozone (N=3). Placental tissues were taken at GD 18, dissociated, and processed for single-cell RNA sequencing using the 10X Genomics platform. Separately, C57BL/6 mice (1.0 ppm ozone N=3, filtered air N=5) were bred and exposed under the same paradigm and taken at GD18 for cardiac evaluation using a Vevo LAZR rodent ultrasound to evaluate fetal and maternal heart function. Results: Differential gene analysis of placentas revealed 304 differentially expressed genes between exposure conditions. Metascape pathway analysis of these genes highlighted cellular senescence, cellular response to lipid, and gas transport pathways being downregulated. Vascular endothelial growth signaling and toxic response pathways were shown to be upregulated. Fetal hearts from ozone exposed animals had significantly (P=0.05) decreased ejection fraction, fractional shortening, cardiac output, and left ventricular mass. Maternal cardiac output and stroke volume were increased due to pregnancy in filtered air controls, but this change appeared reduced in ozone-exposed mice. Conclusion: Gestational exposure to inhaled ozone produced a wide range of cellular responses within the placenta, reduced cardiac function in fetuses. Understanding the cellular mechanisms of air pollution induced gestational morbidities may help aid in identification and prevention of these pathologies. Disentangling Introduction: Early-onset fetal growth restriction (FGR) carries a significant risk of neonatal morbidity and mortality. Placental insufficiency forms its main pathophysiological mechanism, resulting from impaired remodeling of the spiral arteries. Several immune cells are involved in the development of the placental vasculature, and immune cell imbalances have been found in FGR. The Dutch STRIDER trial (Sildenafil Therapy In Dismal prognosis Early-onset intrauterine growth Restriction) investigated the effects of sildenafil, used to promote placental vasodilatation, on early-onset FGR. Clinical outcomes were not improved, however, the vasodilatory effects of sildenafil could influence local immune cells. Therefore, placental leukocyte and macrophage numbers were studied in early-onset FGR treated with sildenafil or placebo. Methods: Placental samples were included from the Dutch STRIDER trial, in which singleton pregnancies with a GA between 20+0 and 29+6 weeks with severe FGR of likely placental origin were treated with sildenafil or placebo until delivery or 32 weeks GA. Using immunohistochemistry (IHC), placental tissue was stained for leukocytes (CD45) and macrophages (CD68). Cell numbers were analyzed with computerized morphometry in the decidua basalis and villous areas and expressed as number of cells/mm 2 . Statistics were performed using the Mann-Whitney U test. Results: 149 cases were included, 79 treated with sildenafil and 70 with placebo. No statistical differences in baseline characteristics like GA and birthweight were found. Higher numbers of decidual CD45 + leukocytes and CD68 + macrophages were found in the sildenafil compared to the placebo group (p=0.017 and p=0.022). These differences were not found in the villous area. When comparing FGR cases with preeclampsia (PE) to FGR cases where women did not develop PE, higher numbers of decidual and villous CD45 + leukocytes, and villous CD68 + macrophages were found in cases of solitary FGR (p=0.026, p=0.020 and p=0.000). The higher numbers of decidual CD45+ leukocytes and CD68+ macrophages in early-onset FGR treated with sildenafil suggest an interaction between sildenafil and the maternal immune system. Sildenafil has earlier been found to alter endometrial and placental immune cells in infertility and miscarriage. However, it is unknown whether sildenafil leads to an increase or decrease in inflammatory parameters. To more specifically analyze the immunological effects of sildenafil in early-onset FGR, more IHC stainings will be performed to characterize the altered numbers of leukocytes and macrophages into specific subsets. (2) to explore the relationship between maternal hemodynamic and morpho-functional cardiac changes in pregnancies complicated by HDP. Methods: We recruited women with HDP and grouped them according to fetal growth pattern (AGA or FGR). Echocardiography data and sera sample were collected at the diagnosis. Women with an uneventful pregnancy were recruited as control group. Cultures of neonatal rat cardiomyocytes were used to investigate the role of the sera in their proliferation regulation. 5-ethynyl-2'-deoxyuridine was administered to label DNA synthesis in cells. The effect of these sera on apoptosis was also evaluated. Finally, we evaluated the relationship of cardiomyocytes and fibroblasts proliferation and apoptosis with maternal hemodynamic. The serum of women with HDP triggered a significant increase in the percentage of proliferating cardiomyocytes versus controls (p<0.001). Any toxic effect was not detected on cardiomyocyte cultures as no differences in terms of apoptosis were detected. In HDP-FGR group, cardiomyocyte proliferation was significantly associated with an increase in systemic vascular resistance index (SVRI) (p<0.02). In the HDP-AGA group, fibroblasts proliferation was significantly associated with a reduction in stroke volume (SV). In this group apoptosis was significantly and directly correlated with SV (p<0.01). Obesity had a significant impact on hemodynamic variables and as such introduced an additional effect on proliferation and apoptosis. We hypothesize that the transcriptional activity of ion channels associated with increased vasodilation is greater in normotensive control (C) vs. PE placenta among Andean residents of high altitude and directly associated to the degree of Andean ancestry. Methods: Placental biopsies were obtained from C (n=39) and PE (n=31) women residing in La Paz, Bolivia (3600-4100 m) undergoing Cesarean section (non-laboring, singleton pregnancies). We isolated total RNA from villous tissue and performed RT qPCR using custom plates to detect 26 genes encoding for ion channels (K + , Ca 2+ , Na + , Cland thermo/ mechanosensitive) that are relevant for placental function and/or PE. In those channels for which transcription differed between C and PE women, we performed immunofluorescence to determine their localization within the placenta. Genomic DNA was extracted from peripheral blood samples to determine maternal ancestry using the Multi Ethnic Genotyping Array and comparing them to known populations from the 1000 Genomes Project. qPCR results are expressed as deltaCt values and significant differences assessed by Student's t test. Results: From the 26 genes analyzed, the mRNA expression of nine ion channels (KCNQ1, KCNQ4, KCNE1, KCNJ8, ABCC9, ATP2A2, CACNA1C, TRPV6 and PKD2) was reduced in placentas from PE vs. C women, no change was observed in the other 17 genes analyzed. Initial immunofluorescence showed expression of KCNQ1, KCNQ4, KCNJ8 and PKD2 in syncytiotrophoblasts and chorionic plate vessels. The ancestry analyses indicated that our cohort was largely of high-altitude Amerindian origin (88%), with low-altitude Amerindian (5%), European (6%) and African admixture (2%). Conclusion: As expected, the expression of genes encoding for ion channels that promote vasodilation (e.g., K + channels) were reduced in PE, however, ion channels which activation opposes vasodilation (e.g., CACNA1C, TRPV6 and PKD2) was also reduced. These results suggest that cation channels may be involved in processes other than vasodilation (e.g., proliferation, metabolism, nutrient transport) in the placenta or that compensatory mechanisms are in place to oppose the reduced expression of K + channels. Further studies will determine whether ion channel expression is related to the degree of high-altitude Amerindian ancestry via functional studies in placental cell cultures and vessels. Postpartum Vascular Dysfunction After a High Cholesterol Diet-Induced Pregnancy Complication in Mice. Tamara Sáez †, Raven Kirschenman, Anita Quon, Floor Spaans, Sandra T Davidge * . University of Alberta, Edmonton, AB, Canada. Introduction: Women who have had preeclampsia are at risk of cardiovascular complications, potentially via vascular dysfunction that persists postpartum. We have previously shown that a high cholesterol diet (HCD) in late pregnancy in mice (a mouse model of dyslipidemia/ preeclampsia) induces vascular dysfunction via lectin-like oxidized lowdensity-lipoprotein (oxLDL) receptor-1 (LOX-1), and potential activation of the angiotensin II type 1 receptor (AT 1 ). However, whether LOX-1, and its potential interaction with AT 1 , may be involved in the vascular dysfunction that occurs postpartum after a complicated pregnancy, is unknown. We hypothesize that postpartum vascular dysfunction induced by a HCD in late pregnancy in mice is mediated by LOX-1 and involves AT 1 activation. Methods: Pregnant LOX-1 knock-out (LOX-1KO) and wild-type (WT) mice were fed either a HCD or standard control diet (CD) between gestational day 13.5 and term. After delivery, all females (n=3-9) were fed a CD for 3 months. Mice were euthanized and aortas were isolated to assess ex vivo vascular responses to methacholine (MCh) by wire myography. MCh-induced vasodilation was evaluated in the presence or absence of L-NAME (nitric oxide synthase inhibitor; 100 µM). Also, prior to the MCh response curve, some vessels were exposed to oxLDL (50 µg/mL) to induce LOX-1 activation, with or without the AT 1 antagonist candesartan (CS; 1 nM) to evaluate AT 1 involvement. Data were analyzed by two-way ANOVA followed by Sidak's post hoc testing and p<0.05 was considered statistically significant. Our preliminary data showed that the postpartum vascular dysfunction in WT mice, that were fed a HCD during late pregnancy only, is mediated by LOX-1 activation and appears to involve activation of the AT 1 receptor. Our data suggest that LOX-1 could be a potential therapeutic target to prevent the development of cardiovascular complications in women who experienced preeclampsia. Oxidative (mtDNA) is an indicator of cellular stress and systemic infl ammation. These properties are accentuated when mtDNA undergoes oxidative damage. In addition, toll-like receptor 9 (TLR9), a receptor of the innate immune system, is activated by mtDNA. Infl ammation, oxidative stress, and cell death are characteristics of placental ischemia, a common feature of preeclampsia. Recent work from our lab has shown dysregulation of circulating cell-free mtDNA in pregnancies with preeclampsia and association of this dysregulation with preeclampsia diagnosis. However, mechanisms underlying the release of mtDNA remain unclear. We hypothesized that human trophoblast cells exposed to oxidative stress via antimycin A, an inhibitor of complex III of the electron transport chain, would induce release of mtDNA via cell death-dependent mechanisms, leading to increased TLR9 activation. Methods: BeWo cells (ATCC, CCL-98) were treated with increasing concentrations of antimycin A (10, 50, 100, 320 µM) and vehicle (ethanol, 0.16% v/v) for 4 hours. Supernatants were collected and snap frozen in liquid nitrogen. Absolute real-time qPCR quantifi cation with TaqMan™ probes and chemistry was used to quantify cell-free mtDNA (amplifi cation target: MT-ND5 gene) and nuclear DNA (nDNA). Flow cytometry was used to assess the activation of cell death mechanisms in response to oxidative stress. To determine TLR-9-associated immunostimulatory potency of cell culture supernatants, we used an engineered cell line of human embryonic kidney 293 cells transfected with a human TLR-9 gene (HEK-Blue TM hTLR9 (Table 2) . However, these diff erences did not persist after adjusting for pre-existing hypertension. Diff erences were found in LV septal wall thickness during pregnancy but did not persist at follow-up. There were no diff erences in E/A ratio, E-wave deceleration time, or ejection fraction between groups at either time point. RHI at follow-up was not diff erent between the groups (controls 2.0 vs PEC 2.2, p=0.28). In this small longitudinal cohort, women with PEC had lower e' and higher E/e' at four years postpartum compared to controls, however the diff erence did not persist after adjusting for pre-existing hypertension. Endothelial function was not diff erent between the groups. Since PEC is known to have lifelong sequelae on cardiovascular outcomes, markers and timing of testing in larger cohorts are needed to stratify CV risk in these populations. Endothelial Dysfunction in Preeclampsia: The Story of the Interleukins. Rachel L Dahn †, Amanda C Ampey, Jason L Austin, Ian M Bird * . University of Wisconsin, Madison, WI, United States. Introduction: Preeclampsia (PE) is marked by the failure of pregnancy enhanced vasodilation and the loss of endothelial monolayer integrity giving rise to hypertension and proteinuria. PE is also associated with elevated TNF, and local uteroplacental production of VEGF. We have previously shown that TNF acts long term to disrupt cell-cell connectivity, this can in part be reversed by Src and ERK kinase inhibitors. While elevated TNF is associated with PE, it is not a reliable diagnostic predictor of PE. Innovation: Interleukins are also altered in the circulation of PE subjects. We propose that IL1B, IL6, IL8, associated with the Th1 phenotype seen in PE may act alone or with TNF to further modulate monolayer integrity. Objective: Establish both time and dose eff ects of TNF with IL1B, IL6 or IL8 to pinpoint the most damaging combination and identify possible synergy. Methods: Uterine Artery Endothelial cells from late pregnant sheep (P-UAEC) were grown to confl uence in 96-well ECIS impedance sensing plates which measures monolayer resistance. The higher the resistance, the better the monolayer integrity. Cells were serum starved and treated alone with diff erent doses (0.1, 1, 10ng/mL) of IL1B, IL6 or IL8, then +/-TNF (5, 10, 20, 50ng/mL) for 20 hours. Results: Please see Table 1 : The data is intriguing that IL1B and TNF can act alone, and together may have an additive negative impact on the monolayer. While increases in local IL6 may also enhance destruction by TNF, IL8 provides protection at the lower TNF doses (this eff ect is lost as TNF increases). i.q. 5-67.) the most values of sFlt/PlGF ratio were higher than the cut-off of the normal ratio. Figure 1 shows an association, in the whole cohort, between TVR and sFlt/PlGF ratio, this function being signifi cant ( p <0,005). Correlation index R 2 was 0.34 considering the two-variables adjusted for covariates. Our data confi rmed the association between the TVR and sFlt1/PlGF ratio in a large cohort of pregnancy at risk. They both could be useful in identifying the severity of placental oxidative stress and improve the management of these cases. In conclusion, we think that both these indices could give some important information about pregnancy complicated by HDP and/or FGR and thus improve their management. Introduction: Preeclampsia is a common complication of pregnancy with serious maternal and fetal risks. Although its pathogenesis remains elusive, there is evidence suggesting an association with environmental exposures such as temperature, sunlight, and humidity. We assessed in a region of the US that experiences all four seasons. Methods: We conducted a cross-sectional study of administrative data from 2017-2019 at a Midwestern tertiary care center. The proportion of patients with preeclampsia among all deliveries was compared across Summer (June-August), Fall (September-November), Winter (December-February), and Spring (March-May) seasons. The data were averaged across all years and secondarily stratified by year. We also performed a sensitivity analysis including only preterm preeclampsia defined as deliveries <37 weeks with preeclampsia per ACOG criteria. One-way ANOVA was used to compare the incidence of preeclampsia across the four seasons. Results: There were 10,338 deliveries from 2017-2019. The incidence of preeclampsia among all deliveries was significantly different across the four seasons, with the highest rate in the Winter and the lowest rate in the Summer (p=0.01; Figure 1 ). The trend was consistent across each individual year (Figure 2 ). The incidence of preterm preeclampsia among all deliveries did not significantly vary by season (4.3% v 4.6% v 5.5% v 5.5%, P=0.81). Conclusion: There is with the highest rates in Winter. Future work should investigate the role of physiologic responses to cold weather, such as vasoconstriction, the role of vitamin D through sunlight exposure, and viral illness in the pathogenesis of preeclampsia. differentially expressed microRNAs (miRNAs) in women with two extreme PCOS phenotypes to determine their role in the pathogenesis of disease. Methods: miRNA were extracted and isolated from 30 plasma samples of well-characterized women with PCOS. We compared differential expression of miRNAs from 15 women with PCOS phenotype A (ovulatory dysfunction, hyperandrogenism and polycystic ovary morphology on ultrasound) and 15 women with PCOS phenotype D (ovulatory dysfunction and polycystic ovary morphology only). Phenotype A carries the highest risk for metabolic disease compared with phenotype D, who are at lower risk. Demographic data including physical exam findings, clinical laboratory results and ultrasound data were compared between the phenotypes using standard descriptive statistics. miRNA were isolated, sequenced, and mapped to the human genome with miRBase v21. DESeq2 Bioconductor package and the Benjamini-Hochberg False Discovery Rate procedure were employed to find differences between phenotype A vs. D using a FDR<0.05. Results: 1993 miRNAs were identified to be expressed with a baseMean expression over 1 and 820 miRNAs were significantly different between phenotypes A and D with a fold-change over 2. Principal component analysis demonstrated separation of phenotypes A and D. There were 53 miRNAs upregulated in phenotype A and 767 upregulated in phenotype D. miR-411-5p had the greatest fold change (3.4-fold higher in phenotype A). miR-126-3p, miR-142-3p and let-7f-5p had the highest baseMeans (57945.9, 55295.1, 52991.8 respectively) and were upregulated in phenotype A compared with D. These miRNAs upregulated in phenotype A have potential roles in endothelial cell function, atherosclerosis and myocardial infarction. miR-126-3p has also been identified in PCOS and is associated with elevated antral follicle count and Antimullerian Hormone levels. Conclusion: Differences in miRNA expression may contribute to the variable presentation of PCOS phenotypes, including potential cardiovascular outcomes. Clinically significant miRNAs could serve as biomarkers for adverse metabolic risk. (tPNf), as well as the timing of reaching the 2 to 8-cell stage (t2 to t8), the start of blastulation (tSB) and reaching the full (tB) and expanded blastocyst stage (tEB). Oolemma area was measured at T0, tPNa and tPNf following ICSI (n=793), and only at tPNf following IVF (n=472). Adjusted linear mixed models and logistic regression were used to analyze oolemma area and pre-and post-implantation embryo outcomes in PCOS and non-PCOS women. Results: PCOS women had a higher number of metaphase-II oocytes (mean 8.9 vs 7.1 oocytes respectively, p=0.001) but fertilization rates did not differ compared to non-PCOS women. There was no difference in oolemma area shrinking rate from T0 to tPNf between PCOS (n=295) and non-PCOS (n=970) women (mean 29.17 µm 2 /hour and 28.15 µm 2 / hour, respectively), however oolemma area was smaller at T0 (10300 µm 2 vs 10564 µm 2 , P=0.02) and tPNf (9775 µm 2 vs 9888µm 2 , p=0.03) compared to non-PCOS oocytes. After adjusting for maternal age, ovarian stimulation and sperm retrieval method, oolemma area of PCOS women at tPNf demonstrated no associations with embryo morphokinetics except t8, whereas it was strongly associated with embryo morphokinetics from t2 to t8 in non-PCOS women (e.g. t8 β= -14.h, 95% CI -24.1, -4.4, P=0.005). Oolemma area at tPNf was not associated with the success of implantation or live birth rate independently from PCOS. Conclusion: PCOS women have smaller oolemma areas but equivalent shrinking rates as non-PCOS women. Associations with oolemma area and embryo morphokinetics disappeared in PCOS women compared to non-PCOS, indicating that the pathophysiology of PCOS may disrupt the additional contribution that oolemma area has for embryo development. Bromodomain extra terminal (BET) proteins have a significant role to play in cell proliferation and differentiation during normal development and growth. Several BET inhibitors have been developed to treat bromodomain linked diseases. JQ1, a potent bromodomain inhibitor, as shown promising outcomes in treating cancer. BET genes are highly expressed during oogenesis and spermatogenesis. Inhibition or mutation of BET genes is associated with abnormal spermatogenesis, decreased sperm motility, increased sperm morphological defects and also sterility. The present study was designed to understand the effect of JQ1 on various functional parameters in human spermatozoa under in vitro conditions. Methods: Normozoospermic semen samples were collected after the completion of routine analysis. Liquefied ejaculates were centrifuged to pellet the spermatozoa and the pellet was resuspended in Earle's Balanced Salt Solution (EBSS) medium. The suspension was split into 3 groups viz. control (EBSS), vehicle control (0.01% DMSO) and JQ1 (100 μM in EBSS). The motile spermatozoa were extracted by swim up technique. At 0, 4 and 24h after swim up, motility, sperm kinematics, mitochondrial membrane potential, DNA integrity and protein acetylation level was assessed. Results: A significant (p<0.05) decrease in progressive motility and survival was decreased in JQ1 group compared to control. Non-significant increase in the percentage of spermatozoa with mitochondrial damage was observed in the JQ1 exposed groups compared to control. Sperm kinematics assessed by CASA showed significant (p<0.05) decrease in the curvilinear velocity, amplitude of lateral head displacement and average path velocity in JQ1 exposed spermatozoa compared to control. Percentage of TUNEL positive spermatozoa was significantly higher in JQ1 group (p<0.05) compared to control. Further, a significant decrease in the lysine acetylation level was observed in the JQ1 exposed spermatozoa. Conclusion: JQ1 exposure to human spermatozoa significantly decreased sperm functional competence. However, further studies are required to understand the underlying mechanism of action and its consequence on early embryo developmen Fig. 1F , we found UP waves (green dots) from women with regular cycles tightly cluster within a regular DUR-MAG region (green ellipsoid), suggesting high UP magnitude and shorter duration. In comparison, UP waves (red dots) from women with irregular menstrual cycles scattered within a large DUR-MAG region (red ellipsoid), suggesting lower UP magnitude and a wider range of duration. The UP magnitude of women with regular cycles is significantly higher than patients with irregular cycles (p-value =0.009). Patients with irregular menstrual cycles have a significantly higher variance of DUR (p-value = 2.2e-7). Our preliminary longitudinal study supports the feasibility and reliability of imaging and quantifying UP patterns using EMMI in a noninvasive fashion. This developed and validated imaging system can be used to quantitatively define physiologically normal uterine peristalsis patterns throughout the regular menstrual cycle and provide detailed information about the uterine peristalsis patterns. In the presence of O 2 , the enzyme converts L-arginine into NO and citrulline. Previously, we have shown that NO is important in maintenance of oocyte quality, as it protects the oocyte from aging. In contrast, HOCl, the fi nal product of MPO, deteriorates oocyte quality independent of the presence of cumulus cells. We hypothesize that, in aged animals, 1) NO bioavailability will be decreased through HOCl mediated iNOS heme destruction and subunit dissociation accompanied by the release of H 4 B, and 2) aging-associated defi ciency of H 4 B can be related to NO defi ciency due to decreased iNOS dimerization. In addition, the incidence of NOS monomerization, subsequent protein nitration, and oxidative stress is presumably high thereby aff ecting oocyte quality. Methods: Using absorbance spectroscopy and gel filtration chromatography, we investigated the role of increasing concentrations of HOCl in mediating iNOS heme destruction and subsequent subunit dissociation and unfolding. Then, using oocytes (n=50) from B6D2F1 mice-young breeders (YB, (8) (9) (10) (11) (12) (13) (14) ), retired breeders (RB, 48-52w) and old animals (OA, 80-84w), measured concentrations of H 4 B and its metabolites through HPLC analysis. Results: Dimer iNOS dissociation occurred between 15 and 100 µM HOCl, accompanied by loss of heme content and NO synthesis activity, although the dissociated subunits maintained reductase activities. Unfolding of NOS occurred at 300 µM HOCl and above with a loss of reductase activities. These events can be prevented when the enzyme is preincubated with melatonin, an HOCl scavenger, prior to HOCl addition. Furthermore, HPLC analysis showed a significant decrease in H 4 B and its metabolites in the oocytes of OA (48%) and RB (73%) compared to YB (100%). Conclusion: Chronological aging which enhances oocyte deterioration could be due NO insufficiency in oocytes caused by HOCl mediated NOS heme destruction and H 4 B deficiency. Furthermore, based on our previous work, the presence of melatonin plays a crucial role as an MPO inhibitor and HOCl scavenger to protect the oocyte. Introduction: SAMP8 females show symptoms of premature ovarian senescence, similar to those occurring in women. Interestingly, some of these symptoms are shared with telomerase-deficient mice. Previously, we had described a possible altered telomere maintenance in the SAMP8 ovary and in the offspring of older SAMP8. The objective of this work is to study telomere maintenance in blood, to explore its potential role as biomarker of ovarian failure. Methods: Blood from 3-and 7-months-old SAMP8 and SAMR1 (control) females was collected, and peripheral blood mononuclear cells were isolated by Ficoll gradient. Telomere length (TL) was measured by high-throughput quantitative in situ hybridization using a telomeric probe. Telomere protection was measured by immunofluorescence against the TRF1 shelterin. Images were captured on a Perkin Elmer system and analysed with Acapella. Statistics were performed with Graph Pad Prism. Results: Although TL in blood cells remains stable during early reproductive lifespan (from 3 to 7-months old), telomeres tend to be shorter in blood from SAMP8 females compared to age-matched SAMR1 mice (287.3 vs 333.4 a.u. at 3 months-old,ns). In fact, 7-months old SAMP8 females show statistically significantly shorter telomeres than 7-months old SAMR1 females (281.1 vs 359 a.u.,p=0.0251). The percentage of critically short telomeres tends to increase in SAMP8 with age (22.91 vs 32.03%,ns), and is higher in 7-months old SAMP8 females compared to controls (32.03 vs 13.15%,p=0.0212). When we evaluated telomere protection, TRF1 levels were statistically significantly higher in 7-months old SAMP8 females compared to younger SAMP8 (584.2 vs 332.2 a.u.;p=0.00262) and tend to be higher than in age-matched controls (584.2 vs 429.7 a.u., ns). Conclusion: A lower TL in blood was found in SAMP8 females, indicating a poor telomere maintenance due to telomerase deficiency or a higher rate of telomere loss influenced by different factors such poor telomere protection at younger ages or excess of reactive oxygen species. Regarding telomere protection, TRF1 levels were higher in blood cells from older SAMP8 females (7-months old), suggesting an essential role for telomere protection in this model, particularly, because of the presence of a higher percentage of short telomeres observed at older ages. Introduction: Ovarian stimulation (OS) uses gonadotropins to develop multiple follicles simultaneously for oocyte retrieval. In women who respond robustly to gonadotropins, OS induces supraphysiological levels of estrogen (E2) and a premature rise in progesterone (P4) levels. It is hypothesized that this results in histologic and molecular endometrial changes that contribute to diminished endometrial receptivity and reduced embryo implantation. To understand how OS induces this negative impact, we employed RNA sequencing (RNA-Seq) to investigate the endometrial transcriptome during the window of implantation (WOI) in OS cycles versus natural cycles (NC). Methods: Eight subjects (four NC and four OS with a GnRH antagonist/ hCG trigger protocol, without embryo transfer) were enrolled. During the WOI, on LH+8 (NC) and hCG+9 (OS), serum E2 and P4 levels were determined at the time of endometrial biopsy. Pipelle biopsies were obtained and processed for analysis of mRNA expression by RNA-Seq and protein analysis by immunostaining. Next Generation Sequencing and bioinformatics were conducted at the Genomics Core, Albert Einstein College of Medicine, NY. Results: Mean E2 levels were 145.9 ± 28.5 pg/ml (NC) vs. 613.3 ± 168.1 pg/ml (OS), p=0.03, while the mean P4 levels were 10 ± 2.6 ng/ml (NC) vs. 17.9 ± 5.9 ng/ml (OS), p=0.27. When comparing hCG+9 to LH+8, 94 differentially regulated genes were identified (padj<0.05) where 25 genes were upregulated, and 69 genes were downregulated. PCSK1N was the most highly downregulated gene (-5-fold-change, padj=10 -5 ). PCSK1N encodes a potent inhibitor (called proSAAS) of prohormone convertase 1 (PC1), an enzyme that proteolytically processes several proteins into bioactive peptides, including the opioid peptide, met-enkephalin. PC1 is encoded by PCSK1 and interestingly, on hCG+9 vs. LH+8, PCSK1 expression was upregulated (5.3-fold-change, padj=10 -5 ). Immunostaining at LH+8 revealed that both proSAAS and PC1 protein are expressed in glandular epithelium of the endometrium. Conclusion: RNA-Seq analysis revealed that OS reduced PCSK1N and increased PCSK1 mRNA expression and we hypothesize that these changes result in increased endometrial opioid signaling. To date, the roles of endometrial opioid signaling in regulating embryo implantation are not well understood, but studies in the mouse have shown that enhanced opioid signaling impairs endometrial receptivity and decreases embryo implantation rates. Together, these data lay the foundation for investigating the role of glandular opioid signaling in endometrial receptivity in the mouse and human endometrium. whose expression peaked in estrous, while expression of markers such as Cyp19a1 decreased significantly between proestrus and estrus. Finally, we focused on secreted factors that varied significantly based on stage of the estrous cycle to identify candidate biomarkers. We evaluated Prss35, Nppc, Tinagl1, Inhba and their corresponding proteins in the plasma of staged mice by ELISA. Interestingly, we demonstrated that Tinagl1 levels peak at estrus/metestrus, Nppc at metestrus/diestrus, Prss35 at diestrus/ proestrus, and Activin A at proestrus/estrus. Conclusion: Single cell RNA sequencing of cycling murine ovaries revealed dynamic pathways across cell types which varied according to stage, and new secreted biomarkers whose concentration could be used to predict staging or ovulation. Pregnancy Related Hormones Regulate the Release of the Anti-Inflammatory Molecule sCD83. Pauline Krupa, Damián O. Muzzio, Jens Ehrhardt, Marek Zygmunt * . University Medicine Greifswald, Greifswald, Germany. Introduction: Anti-inflammatory factors are crucial to counterbalance pro-inflammatory conditions that might threaten pregnancy wellbeing. We have shown that mCD83 and its anti-inflammatory soluble form sCD83 are associated to healthy pregnancy in mice. Furthermore, the application of sCD83 can rescue mice from inflammation-mediated pregnancy loss. The aim of this work is to evaluate the expression and regulation of both forms CD83 in human pregnancies. The expression of mCD83 was characterized on dendritic cells, T and B lymphocytes from decidua parietalis and decidua basalis after caesarian sections at term (n6) by flow cytometry. The expression of mCD83 was assessed as well in vitro after 48 h stimulation with LPS, PMA and ionomycin. The role of pregnancy-related hormones on CD83 regulation was assessed in peripheral blood lymphocytes from healthy female donors (n7) after 48 h in the presence of LPS, PMA and ionomycin. The release of sCD83 on MACS-isolated T and B lymphocytes was assessed by ELISA. The data were analyzed with Student t-test and one-way ANOVA. A p < 0.05 was considered statistically significant. Results: The expression of mCD83 was significantly higher in B lymphocytes than in T lymphocytes and Dendritic cells in peripheral blood and both decidua basalis and parietalis. Decidua basalis B cells experienced the highest mCD83 upregulation after stimulation with LPS. TGF-β1, progesterone and hCG induced mCD83 expression in the presence of LPS, while dexamethasone induced a down-regulation of mCD83 in B lymphocytes. Furthermore, a decreased release of sCD83 and enhanced release of sCD83 was observed after treatment with dexamethasone and TGFβ-1, respectively. Conclusion: The membrane bound form of CD83 is expressed at the fetomaternal interface especially on B cells and can be induced upon pro-inflammatory stimuli as LPS. Pregnancy related hormones modulate both mCD83 expression and sCD83 release. We postulate that TGF-β1, progesterone and hCG promote pregnancy tolerance through induction sCD83, while cortisol, increasing during the pro-inflammatory phase of labor, reduces its release. Together, this data suggests that sCD83 might be a relevant factor collaborating to immune balance during human pregnancy. Ovarian Ovarian stimulation (OS), the process by which the ovaries are stimulated to develop multiple follicles synchronously, exposes the endometrium to supraphysiologic levels of estradiol (E2) and an early rise in progesterone (P4). In the mouse, it has been demonstrated that ovarian E2 and P4, acting via VEGF-VEGFR2, regulate angiogenesis in the endometrium, which is essential for proper endometrial decidualization and embryo implantation. We hypothesized that exposure of the endometrium to altered E2 and P4 levels associated with OS alters decidual angiogenesis. To test our hypothesis we measured expression of CD31 in the endometrium using flow cytometry and whole mount 3D imaging. Methods: 17 subjects [9 women in natural cycles (NC) and 8 women undergoing OS cycles without embryo transfer] were enrolled. Early secretory phase, periovulatory biopsies were performed after the luteinizing hormone (LH) surge at LH+1 (n=4) in NC or after hCG trigger at hCG+2 (n=3). Mid secretory phase, window of implantation (WOI) biopsies were performed at LH+8 (n=5) or at hCG+9 (n=5). Serum E2 and P4 levels were determined at the time of biopsy and reported as mean ± SEM. Tissue was processed for flow cytometry, and the percentage of CD31+CD45-cells was determined. Tissue fixed in DMSO:Methanol was stained for CD31 and for nuclei with Hoechst dye. Confocal images were analyzed using Bitplane Imaris software to create 3D reconstructions of the endometrial vasculature which enabled determination of vessel volume per volume of tissue. Statistical significance was defined as p<0.05. Results: At the time of biopsy, E2 levels (pg/ml) at LH+1 vs hCG+2 were 149.1±27.9 vs 1097.0±552.0 (p<0.01) and at LH+8 vs hCG+9 were 202.3±51.68 vs 623.9±130.7 (p=0.01). P4 levels (ng/ml) at LH+1 vs hCG+2 were 1.2±0.3 vs 12.6±1.8 (p<0.01) and at LH+8 vs hCG+9 were 8.6±1.9 vs 23.5±7. 3 (p=0.25) . In OS cycles compared to NC, 3D imaging and flow analysis suggest a trend towards increased vascular density in the early secretory phase but decreased vascular density in the mid-secretory phase endometrium. In NC, % CD31+ cells/live cells in the early and mid-secretory phase were similar (1.0 vs 0.3%, p=0.41). In OS cycles, % CD31+ cells/live cells were significantly higher in the early vs mid-secretory phase endometrium (2.4 vs 0.4%, p=0.04). Conclusion: CD31, as measured by flow cytometry, reflects the amount of vasculature, while 3D imaging of CD31 shows the amount and architecture of vasculature. These preliminary data on CD31 expression support the hypothesis that OS changes the endometrial vasculature. Further investigation using these modalities is needed to delineate the effects of OS on decidual angiogenesis. Introduction: Currently the rate of reproductive health problems such as infertility, premature ovarian failure, abnormal levels of sex steroid hormones, among others, has been increasing. One of the causes associated with these problems is the exposure to chemical substances such as endocrine disruptors (EDs), molecules capable of altering the hormonal balance and the regulation of embryonic development. Studies show that EDs are present in the leaching of additives from polypropylene containers used for food use. Therefore, the aim of this work is to analyze the effects of polypropylene leachates on the cellular characteristics of the ovarian follicle and the estrous cycle in a murine model. Methods: Twenty-two CD-1 strain female mice (14-week-old) were randomly selected to be separated into groups of 11 mice. The groups were divided into leachate ingestion group (GL) and control group (CG). Mouse body weight, feed and water consumption were evaluated during treatment. The treatment was carried out for 57 days, for the GC purified water was administered which was heated in a microwave oven at 1000 W power for 4 min in a glass container and, for the GL water with leachates was obtained by heating purified water in a polypropylene container, it was introduced into a microwave at 1000 W power for 4 min. During the treatment, vaginal smears were taken to determine the stages of the estrous cycle. At the end of the treatment, mating was carried out with males of proven fertility and the number of pregnant females, and the number of offspring was obtained. At the end of the experiment, the females were sacrificed, and the ovaries were obtained for histological analysis for the presence of apoptosis in the follicular cells. The results were analyzed using One sample t test and 2-way ANOVA, the differences were considered statistically significant with a p<0.05. The analysis of the effect of leachates on the number and latency of total estrous cycles, between GC and GL, showed a significant difference with a mean of 10.73 and 10.09 respectively (p=0.0001). An increase in the number of offspring was also observed in the GL compared to the group, 8.8 vs. 5.4 mice, respectively (p<0.0021). Finally, no effects on body weight, feed and water consumption were observed. It is relevant to mention that morphological alterations were presented in offspring of the GL. Conclusion: The endocrine disruptors found in polypropylene additives seem to cause alterations in the latency and number of estrous cycles and in the number of offspring per litter. However, histological analysis is needed to observe these changes. Therefore, there is not sufficient evidence to demonstrate a deleterious action of polypropylene leachates on reproductive health. Introduction: Gonadotropin receptors are expressed in the somatic cells of ovarian follicles. While theca cells mediate the initial steps in steroidogenesis, granulosa cells are involved in both steroidogenesis and oogenesis. As oocytes do not express the gonadotropin receptors, gonadotropin induced oocyte maturation is dependent on the somatic cells. Granulosa cells express the receptors for both follicle stimulating hormone (FSH) and luteinizing hormone (LH), and these cells are the major targets of gonadotropin responses. Although gonadotropin receptors are G-protein coupled receptors that predominantly signal through cAMP-dependent or independent activation of protein kinases, a crucial step in gonadotropin responses is the activation or inhibition of transcriptional regulators that induces or represses gene expression. Methods: We performed a time course analyses of granulosa cell transcriptomes using RNA-sequencing before and after administration of exogenous gonadotropins, PMSG and hCG that activate FSH receptor, and LH receptor respectively. All RNA-sequencing data were verified by RT-qPCR analyses. Results: PMSG administration stimulated follicle development to antral stage, whereas administration of hCG induced follicle maturation to preovulatory stage, oocyte maturation, and ovulation. RNA-seq analyses identified differential expression of a large number of genes following gonadotropin administration. Remarkably, 87% of the differentially expressed genes (2079 out of 2,386) after PMSG treatment, were found to be downregulated. Even though only 13% genes were upregulated by PMSG, it included genes like Lhcgr, Cyp19a1, and Star that are essential for hCG response. While PMSG treatment predominantly repressed the granulosa cell genes, approximately 60% of the differentially expressed genes were upregulated 4h after hCG treatment. Our findings indicate that temporal downregulation of basally expressed genes in granulosa cells is required for FSH-signaling mediated follicle development, however, subsequent upregulation of the downregulated genes are necessary for LH-signaling induced oocyte maturation and ovulation. Introduction: Organoids are considered 3D in vitro structures to study functional and morphological features of specific tissues. However, epithelial organoids derived from human endometrium do not recapitulate completely the specific microenvironment due to lack of stromal cells. In this context, endometrial assembloids fusing primary stromal cells and organoids just in one passage were reported recently. Here, we go one step further establishing human endometrial assembloids derived from epithelial organoids and stromal stem cell line (ICE7), providing an optimal model for long-term culture. Methods: To build assembloids, organoids from passages 3 to 7 obtained from human endometrial biopsies were mixed with ICE7 (Cervelló et al., 2011; human endometrial side population stromal stem cell line passages 7 to 15) in 1:1 condition (combining 1 volume part of organoids passaged at a ratio 1:3 with 1 volume part of 5·10 5 ICE7/ml) or in 1:4 condition (combining 1 volume part of organoids with 4 volume part of ICE7 at same concentrations), resuspended in 15% advanced DMEM/F12 -85% Matrigel and cultured in standard expansion medium (ExM). To check viability, LIVE/DEAD assay was employed. Proliferation was assessed by spheroid counting (n=16) at day 2, 4, and 7 validated by Quantitative MTS colorimetric assay . Presence of typical human glandular epithelial (cytokeratin 18, CK-18) and stromal (vimentin) markers were identified by immunocytochemistry (ICC). Kruskal-Wallis with Dunn's multiple comparison test was applied. Results: Cell survival was verified up to day 7 with 100% of greendyed assembloids in both co-culture conditions and controls. Day 2-7 proliferation rates showed significant larger proliferation in 1:4 coculture condition (1.76±0.32) with respect to endometrial organoids alone (1.29±0.21) and was even larger in 1:1 condition (2.23±0.40) (P <0.05). MTS assay results compared to organoids validated this difference showing higher cell proliferation of 2.35±0.69 in 1:1 and 1.50±1.28 in 1:4. Bright field images showed human gland spheroid-shaped coated in endometrial stromal cells; and ICC revealed positive staining for CK-18 and vimentin in both co-culture conditions. Conclusion: Our results showed an optimized model for the future establishment of human endometrial assembloid lines, not described previously. This study revealed that 1:1 co-culture condition with stromal stem cells is the most suitable scenario based on survival, proliferation rates, imaging, and specific endometrial markers. This new technology could provide pre-clinical testing platforms for the investigation of endometrial biology and human reproduction. SUPPORT: FPU19/04850; FPU18/06327; PI17/01039; PROMETEO/2018/137; CP19/00149. Ciliary Dysregulation, the Missing Link Between Uterine Disorders Affecting Endometrial Function. Almudena Devesa-Peiro †, 1 Carmen Gonzalez-Moncada †, 1 Patricia Sebastian-Leon, 1 Antonio Pellicer * , 2 Patricia Diaz-Gimeno * . 1 1 IVI Foundation -Instituto de Investigación Sanitaria La Fe (IISLAFE), Valencia, Spain; 2 IVIRMA, Rome, Italy. Introduction: Uterine disorders are complex, multifactorial and often comorbid conditions affecting female fertility. Despite their controversial effect on endometrium reported by individual studies, we previously described with powerful meta-analysis techniques uterine disorders affect molecular functions in endometrium related to embryo implantation (Devesa-Peiro et al., 2020) . However, applying these techniques for functional integration among uterine disorders to highlight shared molecular mechanisms involved in infertility remains unexplored. This study aims to identify these links at endometrial level, determining shared potential contributors to endometrial subfertility Methods: Functional integration of endometrial effects in adenocarcinoma (ADC), recurrent implantation failure (RIF), recurrent pregnancy loss (RPL) and endometriosis was done by integrating transcriptomic raw data of 9 case vs control studies (2 of RIF, 2 of RPL, 3 of ADC and 2 of eutopic endometriosis; 123 cases, 127 controls). The same preliminary analyses were applied in all studies and results were functionally integrated under de DerSimonian & Laird random effects model to account for study heterogeneity. Significant shared endometrial dysregulations amongst disorders (FDR< 0.05) were selected to identify major gene contributors. These genes were characterized using GeneCards, PubMed and specific databases and compared amongst uterine disorders Results: Six ciliary functions (cell projection assembly, cilium assembly, cilium organization, ciliary basal body, cilium and ciliary part) were downregulated in all uterine disorders (0.00224 hours, respiratory distress syndrome, macrosomia, small for gestational age, congenital anomalies, intrauterine fetal demise (IUFD), and infant death) using chisquare tests. Multivariable logistic regression models were then utilized to assess the association of mode of conception with the outcomes. Results: Among 64,500 neonates, 56,803 (88.07%) were spontaneous gestations, 5,808 (9.0%) were ART gestations, and 1,889 (2.93%) were fertility enhancer gestations. Pregnant patients who used fertility enhancing drugs or ART were more likely to be white, older, highly educated, on private insurance and have normal BMI (p<0.001for all). Multivariable logistic regression showed that pregnancies conceived through fertility enhancing drugs had signifi cantly higher odds of NICU Conclusion: Although patients who underwent ART or fertility enhancing treatments were more likely to have demographics that are associated with favorable neonatal outcomes, both methods of non-spontaneous twin gestations were more likely to have adverse neonatal outcomes in NICU admission and respiratory distress syndrome. The higher risk nature of pregnancies conceived through ART and fertility-enhancing drugs may necessitate closer follow ups throughout gestation to avoid adverse neonatal outcomes. Smokers. Lindsay K Doherty, Sean D. Cleary, Angelo Elmi, Debra Bernat, Lorien Abroms. George Washington University, Washington, DC, United States. Introduction: Many pregnant people smoke also report using e-cigarettes. Little information is known about the eff ects of e-cigarette use during pregnancy. Understanding who is most likely to use e-cigarettes during pregnancy could be helpful for risk monitoring. This analysis evaluates maternal predictors of e-cigarette use among pregnant cigarette smokers. Methods: This was a secondary analysis of a randomized trial of a text messaging program for smoking cessation during pregnancy (Quit4Baby). Pregnant smokers with information regarding e-cigarette use were included in this analysis (n=411). Participants were surveyed four times during the study period. Demographic characteristics, health concerns, stress, and feelings of depression were evaluated at baseline. The primary outcome was past 30-day e-cigarette use, defi ned as any self-reported use of e-cigarettes within the past 30 days during the pregnancy. Secondary outcome was past 7-day e-cigarette use. Odds ratios and 95% confi dence intervals were estimated from multivariable logistic regression models. Results: Overall, 228 (55%) reported past 30-day e-cigarette use. Higher proportions of e-cigarette users were non-Hispanic white and overweight, and lower proportions of e-cigarette users were obese and in their third trimester, compared with non-users (Table) . In multivariable analysis, past 30-day e-cigarette users had higher odds of being non-Hispanic white, overweight, and having some college education; and lower odds of earning over $30,000 annually and study enrollment in the third trimester. Past 7-day e-cigarette users had lower odds of obesity and higher odds of being non-Hispanic white and having greater health concerns (Table) . Maternal stress and feelings of depression were not associated with e-cigarette use. Conclusion: E-cigarette use was signifi cantly associated with maternal race, body mass index, education, income, greater health concerns, and trimester at enrollment in this sample of pregnant smokers. Introduction: Collecting growth measures of children up to at least two years of age showed growth in the first year on a logarithmic path, but after age 1 year, growth becomes linear. We analyzed growth as raw data adjusted for birth GA and infant sex, the differences between the actual Year 1 and Year 2 physician visit, the calculated logarithm in Year 1, and the linear slope of growth after Year 1 to at least Year 2. We also explored whether placental gross features can distinguish the at-risk child in a low-risk sample. A community hospital-based sample with universal placental examination was searched for those births followed to at least age 2 years at our institution. Autism Spectrum Disorder (ASD) was identified using billing codes in a patient population of the developmental pediatric group from the Department of Pediatrics. At least 2 diagnoses related to ASD as per Newschaffer et al were required to be considered an ASD case. Controls were selected from the infant of same gender, gestational age +/-2 weeks, and season of birth +/-2 weeks. Birth weight (BW), birth length (BL) and head circumference (HC) were recorded at birth. We identified weight measured at the visit closest to the child's first and second birthday. Gross placental examination was performed and trimmed placental weight (PW), major and minor disk axes, minimum and maximum disk thickness and cord eccentricity was recorded. Ellipsivity was defined as the ratio of major to minor axes. 165 ASD and 617 controls were included in the current analysis. Due to non-normal distribution of growth variables, non-parametric tests were used. Results: Weight at Year 1 and Year 2 were reduced in ASD cases compared to controls. Plots of head circumference show an intersection, a crossing of slope, and 95% CI when head circumference is plotted against gestational age in both male and female children with ASD. After stratification for sex, significant associations persisted in males, with increased ellipsivity. Effects in females were not statistically significant, likely due to the smaller sample size of females. Introduction: Endometrioses occur most commonly in women of working and productive age. Those are well known to cause dysmenorrhea and complicate the quality of their life. However, dysmenorrhea which affects presenteeism in the working time is yet to be elucidated. Here, we investigated interactions of presenteeism and severity of dysmenorrhea with endometriosis. Methods: Participants were extracted from working outpatients present to our university hospital due to dysmenorrhea associated with endometriosis. We examined the dysmenorrheal numerical rating scale (Dysmenorrheal-NRS; 1=no pain, 5=worst pain) during menstruation from the Japanese Version of Menstrual Distress Questionnaire and presenteeism by using Work Functioning impairment scale (Wfun) before initial treatment. We evaluated the prevalence rate of presenteeism and interactions of presenteeism due to dysmenorrhea. Results: A total of 25 women participated in this study. Wfun average score before initial treatment was 18.08, and nine women (9/25;30%) had no work impairment, seven (7/25;28%) had mild, six (6/25;24%) had moderate and three (3/25;12%) had severe work impairment. Dysmenorrheal-NRS was 3.92. More than half of working women who suffer from dysmenorrhea associated with endometriosis had work presenteeism and the rate was higher than general population. Conclusion: Working women who have dysmenorrhea associated with endometriosis have the risk of work functioning impairment during menstruation in addition to quality of life. Treatment for working women who have dysmenorrhea may be needed in occupational site. -1.18) to meet guidelines compared to normal weight patients (using prepregnancy weight calculations). Maternal age, race/ethnicity, education, and diabetes were not associated with meeting IOM guidelines. There was no difference in the association between residence in a food desert and the ability to meet IOM guidelines when comparing race/ethnic categories. The majority of patients did not meet IOM recommendations, regardless of food access designation. In this study, the most significant risk factor for inappropriate weight gain was pre-pregnancy BMI. Future studies are needed to help the most at-risk patients meet IOM gestational weight gain guidelines. Impact of Food Desert Residence on Peripartum Blood Transfusion Rates. Mary Ann Smith †, 1 Tanner Paul Jean, 2 Adrianna Campos, 2 Ronee E Wilson, 2 Salemi L Jason, 2 Peeraya Sawangkum, 2 Kimberly Fryer, 2 Adetola Louis Jacques * . 1 1 University of Florida, Gainesville, FL, United States; 2 University of South Florida, Tampa, FL, United States. Introduction: Food insecurity in pregnancy is associated with increased risk of iron deficiency anemia, which may increase the risk of requiring blood transfusions peripartum. Little research exists examining the relationship between residing in a food desert and peripartum interventions. This study assesses the influence of food insecurity on peripartum blood transfusion rates. Methods: Data from the Florida Department of Health for births occurring in 2019 were linked to data from the 2019 US Department of Agriculture Food Access Research Atlas. A food desert was defined as a census tract with 20% of the population having income less than 80% of the statewide median family income and with at least 500 people, or 33% of the population, living more than 1 mile (urban areas) or more than 10 miles (rural areas) from the nearest supermarket. Adjusted risk ratios (aRR) and 95% confidence intervals were calculated using modified Poisson regression models. Models were adjusted for covariates and data were stratified by race/ethnicity. Results: Of 209,087 live singleton births in 2019, 16% resided in a food desert. Patients over 35 years old and those who attended grade 9-12 (with no diploma) had an increased risk of blood transfusion (aRR: 1.52, CI: 1.09 -2.12; aRR: 2.12, CI: 1.33 -3.37, respectively). Patients living in a food desert were 1.5 times more likely to require a blood transfusion (CI: 1.11 -1.96). When stratified by race/ethnicity, this increased risk was highest and significant only among Black birthers (aRR: 1.83, CI: 1.13 -2.98). Conclusion: Birthers living in a food desert are more likely to receive blood transfusions during their delivery hospitalization. This association was significant only among Black birthers. Food insecurity is associated with anemia in pregnancy and may account for the higher rate of transfusions in those living in food deserts. Previous race-based guidelines for management of anemia made Black birthers more likely to be anemic at the time of delivery. However, further research is needed to determine if this disparity is due to a difference in treatment of Black birthers, underlying medical conditions in these mothers, or poor nutritional status from living in a food desert. Transcriptomic Dysregulation in the Endometrium of COVID-19 Patients: A Next-Generation Sequencing Approach. Lucía De Miguel-Gómez †, 1 Patricia Sebastián-León * , 1 Mónica Romeu * , 2,3 Amparo Faus * , 1 Antonio Pellicer * , 4,5 Patricia Diaz-Gimeno * , 1 Irene Cervelló * . 1 Introduction: COVID-19 pandemic has affected millions of people worldwide with common respiratory, musculoskeletal and digestive symptoms. However, COVID-19 is a systemic disease with manifestations in other tissues that would come from a global pro-inflammatory status rather than a direct viral infection (described virus absence in these other tissues). We previously reported that 14 endometrial biopsies from COVID-19 women tested negative for SARS-CoV-2. However, could this systemic inflammation affect the endometrium, despite the absence of the virus? This study aimed to elucidate, using RNA next-generation sequencing, if these virus-free endometria were indirectly affected by COVID-19. Methods: Endometrial biopsies were collected (project 2020-268-1) from COVID-19 patients (n=9; RNA quality-based selection of 9/14 samples) and healthy donors (n=9). After RNA extraction, libraries were constructed using TruSeq Stranded mRNA protocol and samples were sequenced with NextSeq 550 (Illumina). Principal component analysis (PCA) of sequencing quality reads was performed to explore potential bias/outliers; a cycle phase correction was also addressed for removing menstrual cycle progression variability. Then, differential expression analysis (DEA) and functional enrichment (GSEA) were performed consulting Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). All analyses were run in R statistical environment. Results: Clustering the 18 endometrial samples by whole transcriptome showed a clear effect of COVID-19 on global gene expression (1 outlier/ group was discarded). GSEA reported statistically significant (FDR<.05) up-regulated biological processes in the endometria of COVID-19 women related to response to other organisms (response to symbiont and virus) and the immune system and inflammation (innate immune response, T cell activation, type I interferon production regulation). These results linked to KEGG pathways, where COVID-19, cytokine-cytokine receptor interaction, neutrophil extracellular trap formation, and T cell receptor signaling paths were significantly up-regulated. In addition, 2/8 COVID-19 endometria were control-like in transcriptomic terms showing the population variability described in COVID-19 symptoms. When removing these control-like samples, we identified 235 affected genes (FDR<.05). Conclusion: 75% of COVID-19 women included in this study present an altered endometrial transcriptomic profile affecting COVID-19 pathway and immune system probably due to the extent of this systemic disease, despite no direct viral infection. The effect of pre-existing and COVID-19-related factors impact on neonatal auocomes remains elusive. We conducted a prospective observational cohort study including all consecutive pregnant or puerperal women with confirmed PCR for SARS-CoV-2 infection presenting at Hospital San José, in Santiago, Chile, from April 8th through August 31st, 2021, to compare negative deterinants of decreased neonatal weight and gestational age. Methods: All patients in both waves were managed with the same hospitalization criteria and the same follow-up protocol. Demographic, obstetric, and neonatal clinical characteristics were analyzed and compared by Chi-square/Fisher test, Lasso linear regression, and multivariable logistic regressions. Results: A total of 1036 women were recruited from which 724 neonates were included in this study, with 498 from the first and 225 from the second COVID-19 wave. Twenty-nine percent of women had complicated and 72.1% late (> 24 weeks of gestation) onset disease, and 9.9 % severe disease (e.g. mechanical ventilation or intubation). Within the first wave there was increased proportion of women with complicated disease onset (p < 0.05) and comorbidities (p < 0.01), but lower late-onset (p < 0.01), disease severity (p < 0.01) and obstetrics pathology (p < 0.01). No difference in birth weight and gestational age at delivery were observed between waves. Modeling by Lasso regression showed that disease severity, obstetrics pathology, and complicated disease onset were the main factors that negatively affect birth weight and gestational age at delivery. Conclusion: Altogether, this data shows that despite clinical differences between two consecutive Covid-19 waves in Chile, disease severity and complicated disease onset are the main drivers in the negative neonatal outcomes after SARS-CoV2 infection, resulting in decreased birth weight and prematurity. Ayomipo S Madein †, Lisbet Lundsberg, Moeun Son, Jennifer Culhane, Audrey Merriam. Yale University, New Haven, CT, United States. Introduction: Given the restrictions and stress caused by the COVID-19 pandemic, we investigated if women pregnant during the pandemic had higher gestational weight gain than women pregnant during pre-pandemic periods. Methods: A retrospective cohort study of women who delivered at an urban academic medical center before and during the COVID-19 pandemic using electronic medical record data. The pre-pandemic epoch included matched months (March to December) in 2017-2018, and the pandemic epoch included the same matched months in 2020. Women who delivered between 3/12/17-12/31/17 and 3/12/2018-12/31/2018 were included in the pre-pandemic group; women who delivered between 3/12/2020-12/31/2020 were included in the pandemic group. Women were included if they were ≤20 weeks' gestation at the start of the period time windows with singleton live gestations. The COVID-19 pandemic group was further stratified into those <14 weeks gestation and 14-≤20 weeks gestation at the start of the period window. The primary outcome was maternal weight gain at delivery, defined as below, meeting, and above Institute of Medicine recommendations based on pre-pregnancy BMI and adjusted for gestational age at delivery. Demographic characteristics included race/ethnicity (non-Hispanic white, non-Hispanic black, Hispanic, and Asian/other non-Hispanic), insurance type, smoking status, and presence of gestational diabetes or hypertensive disease of pregnancy. Univariate analyses were performed, and multivariate logistic regression modeling was used to examine pandemic exposure, both dichotomized and categorical (exposure <14weeks and 14≤20 weeks gestation), on maternal weight gain using those who met recommendations as the reference group. Results: Among 5,421 women, 67.37% were in the pre-pandemic group and 32.63 % were in the pandemic group. 58.6% were classified as having above recommended gestational weight gain, while 23.8% met recommendations, and 17.6% were below recommendations. No significant differences were observed in baseline characteristics between the pre-pandemic and pandemic groups. The pandemic period was not associated with weight gain below (OR 1.08, 95% CI 0.90-1.29) or above (OR 1.03, 95% CI 0.90-1.18) recommendations. This was also true when the pandemic group was further stratified into <14 weeks gestation (Below: OR 1.20, 95% CI 0.97-1.47; Above: OR 1.10 (5% CI 0.93-1.30) and 14-≤20 weeks gestation (Below: OR 0.89, 95% CI 0.68-1.16; Above: OR 0.92, 95% CI 0.75-1.13) per Institute of Medicine recommendations based on pre-pregnancy BMI. Conclusion: This study demonstrates no significant difference in maternal weight gain before and during the COVID-19 pandemic. Further studies should assess how activity levels changed during the pandemic period, differences in weight gain between women who did and did not have changes in employment status, and evaluation of psychiatric history or medications. Severity of Placental SARS-CoV-2 Infection is Associated with Adverse Pregnancy. Bingbing Wang, Liviu Cojocaro, Wei-Bin Shen, Hannah Shen, Peixin Yang, Shifa Turan. University of Maryland, Baltimore, MD, United States. Introduction: Pregnant women are at increased risk of COVID-19. This could be explained through the prism of multiple physiologic and immunologic changes in pregnancy. In addition, certain immunological reactions originate in placenta in response to viral infections. Historically, adverse pregnancy outcomes (APO) have been described even with asymptomatic maternal viral infections. We hypothesized that the degree of SARS-CoV-2 presence in the placenta is correlated with APO. We conducted a prospective cohort study in which we analyzed 22 placental specimens collected from pregnant women who had a laboratory confirmed SARS-CoV-2 infection. Institutional Review Board at the University of Maryland, Baltimore, approved this study. Placental specimens were immediately formalin-fixed and paraffin-embedded following delivery. We performed in situ RNA hybridization with use of RNAScope HD Reagent Kit-RED (Advanced Cell Diagnostics, CA). We categorized the cases in mild placental infiltration (PI) if the viral detection was limited to syncytiotrophoblast (STB) and severe PI if the virus was detected in cytotrophoblast (CTB) or fetal blood vessels (FBV). APO were defined as premature rupture of membranes, spontaneous preterm labor, hypertensive disorders of pregnancy, intra-uterine fetal demise (IUFD), or vertical SARS-CoV-2 infection. Severe maternal infection was defined by requirement of oxygen supplementation. Results: Out of 22 analyzed placenta, virus was detected in 17 cases, eleven being mild and six severe. Viral infection extended to FBV in four cases. There was a higher rate of APO with severe PI (100% vs. 27%, p=0.005). Moreover, two cases of IUFD and one of neonatal vertical infection occurred with severe PI. There was no correlation between the severity of PI and maternal oxygen requirement (16% vs. 31%, p=0.49). We found a significant correlation between PI and APO but no correlation with the severity of maternal infection. Additionally, the cases of IUFD and the neonatal vertical infection occurred with severe PI, underlining the importance of fetal-placental in cases of SARS-CoV-2 infection. Impact of Depression on Fertility Treatment Outcome During the COVID-19 Pandemic. Lauryn Welling †, Liubin Yang †, Janet Bruno-Gaston †, Elizabeth Kravitz †, Alvin To †, Amy Schutt * . Baylor College of Medicine, Houston, TX, United States. Introduction: Over one percent of the current United States population utilizes assistive reproductive technology (ART). ART is associated with increased rates of depression and anxiety, likely due to both the diagnosis itself as well as the stressful nature of the treatment. Prior studies have examined the impact of depression on fertility outcome following ART with conflicting results. More research is needed during COVID pandemic given the significance of infertility, limited number of studies, and contradictory findings. This study analyzed the correlation between positive depression screening and fertility outcome following assisted reproductive technology. Methods: Patients presenting for an initial infertility evaluation at the Family Fertility Center between March 2019 and September 2020 completed the Edinburgh Postnatal Depression Scale (EPDS). All female patients over the age of 18 who underwent infertility treatment, completed the EPDS, demographics data, and had outcome data collected were included in the study. Outcome data was measured in the number of months between the date of the patient's initial infertility visit and the date of their viability ultrasound as defined by positive fetal heartbeat. Study participants were divided into two groups based on their EPDS score, with a score of 10 used as a threshold to signify an increased risk of clinical depression. PSPP was used to analyze the data with an independent samples t-test and chi-square test to determine statistical significance (P less than 0.05). Results: A total of 150 women out of the 1,394 who presented for an initial infertility evaluation were included in the study. Only 27 participants screened positive for depression while 123 screened negative. The average number of months to achieve clinical pregnancy was 9.04 months for the group that screened positive for depression and 9.02 for the group that screened negative. The group who screened positive for depression was no less likely to have a positive pregnancy outcome (p-value of 0.321, 95% CI of -1.49 to 1.46). In secondary analysis, there was no difference in rate of positive depression screen between before the COVID-19 pandemic (before 03/2020) and during the pandemic (after 03/2020) (p=0.516). The results suggest that patients in this cohort who screened positive for depression were no less likely to have a positive fertility outcome following assisted reproductive technology during the COVID pandemic. T-191, T-194, F-195, F-196 F-111, T-190, F-191 Subject Index Poster T-128, T-130, T-131, T-132, T-136, T-140, T-141, T-144, T-147, T-149, T-152, T-153, T-155 , T-157, T-158, F-128, F-130, F-132, F-134, F-135, F-137, F-141, F-142, F-144, F-147, F-150, F-152, F-153, F-154, F-157, F-158 T-191, T-195, T-197, T-198, T-200, T-202, T-204, F-185, F-189, F-192, F-196, F-197, F-200 Herein, our aim was to further evaluate the safety of ES in pregnancy by examining whether such an eff ect was reproducible (in obese and/or healthy rat pregnancies) and, if so, whether it could negated by postponing ES until after implantation. Methods: 36 female Wistar rats (252±3g, aged 81d) were evenly allocated to HFD (TestDiet 58V8: 20% sucrose, 45% fat) or control diet (Con) for 59±4d pre-mating/throughout pregnancy (n=18 each) /15Hz frequency, 30min/day) of rectus abdominis, tibialis anterior and triceps surae under 2-3% isofl urane anesthesia from E1-4 (early ES, n=6) At necropsy on E19±1 (blind to group allocation), litter size and resorption rate (primary endpoints) were compared between groups by Kruskal-Wallis test. Secondary endpoints (normally distributed) were analyzed by two-way ANOVA with diet (Con/ HFD) and treatment (ES vs no ES) as fi xed factors. Results: At mating, body weight was higher in HFD vs Con groups (mean±SEM 314±9g vs 252±10g, p=0.007), reflecting higher pregestational weight gain (100±9g vs 59±3g, p<0.001) Resorptions (range 0-6) were equivalent (p=0.612) in ES-treated/untreated parameters diff ered between early and late ES subgroups Exposure to HFD enhanced maternal adiposity, refl ected by up to 2-fold increases in the weights of the following internal fat depots: mesenteric There were no signifi cant eff ects of ES or ES*diet interactions. Conclusion: We did not replicate the potentially adverse eff ect of ES on litter size observed in our prior study, which may have simply represented a chance observation in one of many secondary endpoints. Herein, we found no eff ect of ES on litter size Recent Trends in Preeclampsia/Eclampsia Based on Maternal Age, Race/Ethnicity, and Pre-Pregnancy Weight Categories Methods: We used electronic health records (EHR) of pregnancies delivered at the Kaiser Permanente Southern California healthcare system between 2011-2020 (n=387,553) for this study. Data on diagnosis of PE/E were extracted from EHR. We first examined the annual rate of PE/E, followed by the adjusted temporal trends by comparing rates in the earlier 2011 vs most recent 2020 periods. Relative risks were used to examined age, race/ethnicity and BMI disparities in PE/E diagnosis after adjusting for potential confounders. Results: Preeclampsia/eclampsia prevalence increased from 4 Elevated IFNγ levels are found at the maternal-fetal interface during early pregnancy and in preeclampsia as well as in serum of COVID-19 patients. Thus, HTR8 and PMVECs were treated with IFNγ (10 ng/ml) alone or in combination with 10 ng/ml S-protein. IFNγ alone significantly increased more than 3-fold of IL-6 and IL-8 mRNA levels in HTR8 as well as over 10-fold in PMVECs (P<0.05), while IFNγ + S-protein treatment further enhancing induced IL-6 and IL-8 mRNA levels (P<0.05) in both cell types. Similarly, ELISA detected significantly higher IL-6 (~1.5-fold in HTR8 and 2.5-fold in PMVECs; P<0.05) and IL-8 (~6-fold in HTR8 and 2-fold in PMVECs; P<0.05) secretion levels in culture supernatants of cells treated with 1000 ng/ml S-protein vs. control. Moreover, IFNγ also significantly increased IL-6 (~ 2-fold in HTR8 and 10-fold in PMVECs) and IL-8 (~3-fold in HTR8 and 1.5-fold in PMVECs) levels and the combination of IFNγ+S-protein treatment further elevating IL-6 and IL-8 levels in both cell types Differences in Circulating miRNA Signatures in Women with Extreme PCOS Phenotypes. Jessica L Chan, 1 Tania L Gonzalez, 1 Amy E Flowers, 1 Rae A Buttle †, 1 Yizhou Wang, 1 Chintda Santiskulvong, 1 Xue-Ying Song, 1 Rosemarie DiPentino †, 1 Ekaterina L Clark †, 1 Anna Dorfman †, 1 Allynson Novoa †, 1 Ricardo Azziz, 2 Margareta D Pisarska * . 1 Introduction: Ovulatory dysfunctions are the main cause of female infertility. Single cell RNA sequencing (scRNAseq) has given us new insights into the cell types of the ovary and their transcriptional program across various species. However, less is known regarding the evolution of these cell states during the estrous cycle. We hypothesized that a comprehensive dynamic atlas of murine ovarian cell transcriptomes during the estrous cycle may reveal new biomarkers reflective of staging. Methods: We monitored estrous cycle stages through vaginal smears, before harvesting and dissociating ovaries to perform scRNAseq and bioinformatic analyses. We validated cell type-specific transcripts and variable gene expression using qPCR and in situ RNA hybridization (RNAish). Finally, we evaluated secreted cycling biomarkers by ELISA of plasma samples. Results: Following scRNAseq analysis, we identified the cell types constituting the ovary, including subpopulations of granulosa cells, mesenchyme, oocytes, epithelium, endothelium, and immune cells which were validated based on gene expression by RNAish. The mesenchyme was subclustered in stroma, smooth muscle, immature theca, and steroidogenic theca, which were validated based on expression of Enpp2/Kcnk2, Mfap5, Hhip/Cd24a and Cyp17a1 respectively by RNAish. Granulosa cells were divided into mitotic, periovulatory, preantral/cumulus, atretic, antral/mural subtypes which were validated based on expression of Top2a, Runx1, Kctd14, Ghr, Mro and Onecut 2 respectively by RNAish. We then compared variable gene expression of cell types by stage of the estrous cycle to identify dynamic biomarkers. In granulosa cells, we identified genes including Id2, Rgcc, Star or Psap Using Live Imaging and FUCCI ESC to Distinguish G1 Cell Cycle Delays, General Stressors Like PFAS and Phthalates or G2 Cell Cycle Delay for Mutagenic Stressors Like Benzoapyrene. Ruden Ximena * , 1 Abdulhasan Mohammed * , 1 Sean Harris * , 2 Douglas Ruden * , 1 Awoniyi Awonuga * , 1 Elizabeth Puscheck * , 1 Daniel Rappolee * . 1 1 Wayne State University, Detroit, MI, United States; 2 University of Michigan, Ann Arbor, MI, United States. Introduction: Developmentally toxic stressors tend to delay cell cycle in G1phase as reported by Fluorescence ubiquitinated cell cycle indicator (FUCCI) embryonic stem cells (ESC) during assay in a live imager. Here we test with mutagenic stressor can be identified by FUCCI ESC by delay in G2phase of the cell cycle. Methods: FUCCI muESC were cultured in FluroBright DMEM in a % CO2 at 37℃ using the Cytation CO2 incubator Biospa 8 Automated with six log base10 doses from 1-100nM and 1-100uM of PFOA, DEP, Cortisol, BPA, BaP or control 300mM sorbitol in the presence of LIF or in the absence of LIF +/-1uM Retinoic acid. LOAEL or IC50 doses were determined for growth suppression, and these were used to do a 72hr exposure, isolate mRNA and do bulk RNAseq using barcoded libraries GnRH Effects on Trophoblast Migration and Proliferation at the Maternal-Fetal Interface. Catherine O'Byrne †, 1 Alyssa Williams †, 1, 2 Helen Jones * , 1,2 Rebecca Wilson * . 1,2 1 University of Florida, Gainesville, FL, United States; 2 University of Florida College of Medicine, Gainesville, FL, United States. Introduction: Placenta accreta spectrum (PAS) refers to morbidly adherent placentation which overly invades into the endometrium, myometrium, or beyond. PAS affects approximately 0.4% of deliveries in the United States; however, the mechanisms underlying PAS are poorly understood. In our animal model, Grb2-associated binding 3 protein (Gab3) facilitated proper maternal uterine natural killer, uNK, cell signaling at the maternal-fetal interface. Mutations in Gab3 result in abnormal trophoblast invasion and vascular remodeling, which may be involved in the development of PAS. Gab3 mutations also result in an upregulation of uNK gonadotropin releasing hormone (GnRH1), which has been implicated in facilitating trophoblast cell invasion. We aimed to evaluate the effects of GnRH1 on trophoblast cell migration and proliferation. Methods: HTR8 cells, an extravillous invasive trophoblast model, were cultured in 12 well plates to 90% confluency (n=3 passages). Cells were then treated in triplicate with increasing GnRH concentrations; 0.0 nM, 1.0 nM, 10.0 nM, 100 nM; over 48 h and scratch migration assays were performed simultaneously. The scratch width was measured at time of scratch (T 0 = 0 h) and then six time points post scratch: 2, 4, 8, 18, 24, and 48 h. Percent closure of the scratch was calculated from T 0 . At 48 h, a crystal violet assay was performed to determine cell number and thus proliferative capacity. Difference in scratch closure between GnRH concentration and time was determined using a two-way mix effect ANOVA analysis. Differences in proliferation between GnRH concentrations were compared using a non-parametric ANOVA analysis. Results: The GnRH treated cell groups did not have significantly different scratch closure percentages (p>0.05) compared to the control group when measured at the six time points. There was also no significant difference in the crystal violet optical density between the concentration groups (p>0.05), indicating no difference in cell number. Conclusion: Scratch migration assays of GnRH treated HTR8 cells did not demonstrate altered trophoblast migration rates or increased proliferation when compared to untreated HTR8 cells. The statistically similar percent closures at each time point and the number of cells at 48 h may be attributed to the GnRH1 concentrations studied. A more pronounced difference may be observed with larger concentrations of GnRH1. Additionally, differences may be seen in invasion assays, as the HTR8 cells are cultured in an environment that better models the uterine decidua and that allows both invasion and migration. Placental Phospholipid Synthesis is Increased in Obese Mothers with Gestational Diabetes. Theresa L Powell * , 1 Lana Madi, 1 Charis Uhlson, 1 Karin Zemski Berry, 1 Claire Palmer, 1 Veronique Ferchaud-Roucher, 2 Marisol Castillo-Castrejon * . 3 1 University of Colorado, Aurora, CO, United States; 2 University of Nantes, Nantes, France; 3 Oklahoma University Health Science Center, Oklahoma City, CO, United States. Introduction: Placental phospholipid synthesis is critical for the expansion of the exchange surface area across gestation and for production of critical signaling molecules. Pregnancies complicated by gestational diabetes (GDM) and obesity commonly have larger placentas but have lower levels of docosahexaenoic acid (DHA) in cord blood at birth. A specific phospholipid form, lysophosphatidylcholine-DHA is released by the placenta to the fetus. We hypothesized that placental phospholipid synthesis is enhanced in obese women with GDM, but that lipolysis, needed to produce lysophosphatidylcholine-DHA, is reduced. Methods: We isolated primary human trophoblast (PHT) cells from term placentas of healthy (n=10) and GDM/obese mothers (n=10) delivered at term. We incubated PHT with a mixture of 13 C labeled fatty acids (palmitic, oleic, linoleic, DHA) for 2 and 24 hrs, extracted the phospholipids and determined percent incorporation of 13 C labeled fatty acids into phosphatidic acid, phosphatidylcholine, and lysophosphatidylcholine using liquid chromatography mass spectroscopy. We determined expression of acyl transferase (GPAT3, AGPAT2 and 4) and phospholipase (PLA2G4c) enzymes for de novo synthesis and lipolysis using immunoblotting in 24 healthy and 24 Obese/GDM term placentas. Data were analyzed by Linear Mixed Models. Results: GDM/Obese mothers had greater body mass index by design. Gestational age, birth weight, birth length, and placental weight did not differ between groups. Trophoblast cells isolated from GDM/Obese pregnancy demonstrated a significantly higher capacity to synthesize phospholipids with increased 13 C labeled palmitic and linoleic acids incorporation into 3 of 14 phosphatidic acid species and 13 of 43 phosphatidylcholine species at both 2 and 24 hrs. Lysophosphatidylcholine 13 C linoleic acid and 13 C oleic acid were significantly higher in tophoblast cells from GDM/Obese mothers. Expression of acyl transferase and phospholipase enzymes responsible for phospholipid synthesis and lipolysis did not differ in GDM/Obese placentas. Conclusion: Increased biosynthesis of phospholipids may reflect an enlarged surface area in placentas of GDM/Obese mothers. Greater lysophosphatidylcholine 13 C linoleic and 13 C oleic acid demonstrates increased phospholipid lipolysis without an increase in lysophophatidylcholine 13 C DHA. These data suggest that critical long chain polyunsaturated fatty acids are preferentially incorporated into placental plasma membrane phospholipids rather than producing the form needed for fetal delivery in placentas of GDM/Obese mothers. These findings may contribute to understanding the lower cord blood DHA levels at birth in obese women with GDM. Related to Abnormal Cell Turnover. Zhiyong Zou, 1 Lynda K Harris * , 1, 2, 3 Karen Forbes * , 1,2,4 Alexander E.P. Heazell * . Introduction: Estrogen related receptor gamma (ESRRG), a nuclear receptor, is significantly decreased in placentas complicated with fetal growth restriction (FGR) and might be causally related to the observed placental dysfunction. miR-377 is predicted to be an upstream regulator of ESSRG and is associated with impaired cytotrophoblast proliferation in cultured first trimester villous explants. We hypothesised that increasing miR-377 expression would reduce expression of ESRRG and lead to abnormal cell turnover. Methods: Term placental explants (n=7) from appropriate for gestational age (AGA) infants were cultured for 48 hours with miR-377 mimics, or non-targeting controls. Levels of miR-377, ESRRG and its downstream genes were assessed by real-time QPCR. Expression and localization of ESRRG, and the numbers of apoptotic cells and cells in cycle were assessed by immunohistochemistry. Results: miR-377 was increased in the cultured explants transfected with 100nM miR-377 mimics without statistical significance (Median ± IQR, miR-377 mimic, 1.5±2.6, P=0.06, Figure1A). ESRRG mRNA (Median ± IQR, miR-377 mimic, 0.7±0.4, Figure 1B ) and protein expression (Median ± IQR, miR-377 mimic, 0.2±0.5, Figure1C) were significantly decreased in explants transfected with the miR-377 mimic (P<0.05), but downstream targets of ESRRG, HSD17B1, CYP191.1, HSD11B2, and PLAC1 were not reduced ( Figure 1E and F). The miR-377 mimic (100nM) reduced the number of cells in cycle (Median ± IQR, 0.9±0.5 (control) vs 0.1±0.1 (miR-377 mimic)) and increased apoptosis (Median ± IQR, 0.5±0.4 (control) vs 1.2±1.3 (miR-377 mimic)) in the cultured explants. Conclusion: These data suggest that miR-377 could alter cell turnover in term placental tissue by regulating the expression of ESRRG. The involvement of the ESRRG pathway in FGR merits further exploration. Membrane Lipids and Vitamin C Improves the Outcome of Whole Ovarian Tissue Vitrification. Guruprasad Kalthur * , Keerthana Karunakar Poojary †, Shweta Hegde †, Daphne Crasta †, Sandhya Kumari †, Reyon Dcunha †, Satish Kumar Adiga †. Kasturba Medical College, Manipal, Manipal, India. Introduction: In the recent years due to advancement in the cancer treatment approaches, the cancer free survival rate has improved dramatically. However, the current treatment regimens employed are known to be gonadotoxic resulting in compromised reproductive function among these cancer affected children. Due to uncertainty about the future fertility potential of these cancer affected children, fertility preservation is recommended to these individuals. For prepubertal girls, the only option left for the fertility preservation is ovarian tissue cryopreservation (OTC). In the present study, we explored the beneficial effects of supplementation of membrane lipid components (lecithin and cholesterol), Vitamin C and HPMC in the freezing medium during vitrification of ovarian tissue. Methods: Ovaries from prepubertal Swiss albino mice (2 week old) were collected in M2 media and frozen with control vitrification medium (CVM, 15% DMSO and 15% EG along with 0.6M sucrose in M2 media) and test vitrification medium (TVM, 2% lecithin, 0.02% cholesterol, 0.25%, HPMC and 0.5% vitamin C). Whole ovary vitrification was carried out by 2 step protocol by incubating in equilibration solution [7.5% Dimethyl sulfoxide (DMSO) and 7.5% Ethylene glycol (EG) in M2 media] for 15 min, followed by vitrification solutions for 30 min. After thawing, ovarian tissues were transferred to M2 media and the ovaries were teased to collect the GV stage oocytes. Oocyte survival was assessed. For comparison, oocytes were collected from fresh ovarian tissue (unfrozen, FC). Viable Germinal vesicle stage oocytes (GV) collected were subjected to in vitro maturation (IVM). Post thaw survival, maturation rate, mitochondrial membrane potential, intracellular ROS level, spindle integrity, actin distribution pattern and activation potential was assessed in the oocytes. Results: TVM had significant higher oocyte survival rate compared to CVM (p<0.001). Maturation rate in FC was significantly lower in CVM oocytes (p<0.001). Further, significant decrease in the ROS level was observed in oocytes from TVM group compared to CVM (p<0.001).The percentage of oocytes exhibiting uniform mitochondrial distribution (p<0.001) and filamentous actin (p<0.01) was significantly higher in the TVM group compared to CVM. Cryoinjury to meiotic spindle was lower in the TVM oocytes compared to CVM. When MII oocytes obtained after IVM were subjected to artificial activation with Strontium chloride (SrCl 2 ), both FC and TVM oocytes had similar activation rate, which was lower in CVM (63.23%). The presence of lecithin, cholesterol, HPMC and vitamin C in vitrification solution improved the cryosurvival of whole ovarian tissue and enhanced the functional competence of oocytes by helping membrane reorganization and reducing the ROS levels during freeze -thaw process. Roberto Gonzalez-Martin †, 1 Andrea Palomar, 1,2 Perez-Deben Silvia, 1 Salsano Stefania, 1 Alicia Quiñonero, 1 Juan Giles, 3 Carmen Vidal, 3 Bellver Jose, 3 Nicolas Garrido, 1 Dominguez Francisco * . 1,2 1 IVI Foundation, Valencia, Spain; 2 ISSLaFe Biomedical Research Institute, Valencia, Spain; 3 IVI-RMA Valencia, Valencia, Spain. Introduction: Phthalate exposure has been reported to be a risk factor that could threaten reproductive success, altering ovarian function and influencing embryo development and pregnancy outcomes. However, data about the association of phthalates exposure with clinical IVF outcomes are scarce. Among the phthalate metabolites detected in biological samples, the most prominent is mono(2-ethylhexyl) phthalate (MEHP). This research aimed to quantify MEHP in urine and follicular fluid (FF) samples of women undergoing infertility treatment to establish its impact on ovarian response and clinical outcomes. Methods: Phthalate exposure has been reported to be a risk factor that could threaten reproductive success, altering ovarian function and influencing embryo development and pregnancy outcomes. However, data about the association of phthalates exposure with clinical IVF outcomes are scarce. Among the phthalate metabolites detected in biological samples, the most prominent is mono(2-ethylhexyl) phthalate (MEHP). This research aimed to quantify MEHP in urine and follicular fluid (FF) samples of women undergoing infertility treatment to establish its impact on ovarian response and clinical outcomes. Results: MEHP was detected in urine at oocyte retrieval and embryo transfer in 88.2% and 96.3% of women, respectively, with a creatinine corrected geometric mean ± SD concentration of 0.018 ± 0.1086 and 0.030 ± 0.057µg/g CR. Regarding follicular fluid, MEHP was only detected in 13.33% of women, with a geometric mean ± SD 0.059 ± 0.271µg/L. Log transformed MEHP concentrations in urine collected at the oocyte retrieval slightly correlates with increased number of oocytes (r= 0.333, p = 0.019) and number of Metaphase II (r=0.360, p = 0.011). No further associations were identified between MEHP found and the other clinical outcomes in this matrice. No other significant associations between MEHP concentrations in follicular fluid or urine collected at the time of embryo transfer and clinical outcomes were found. Conclusion: Urinary higher MEHP at oocyte retrieval was associated with a slightly increase in total and mature number of oocytes, but not with other IVF clinical outcomes. Further work is needed to elucidate the potential hazard of phthalate exposure on female reproduction. Support: PFIS (PI/00009), APOTIP/2020/013, Miguel Servet Contract (CPII18/00002) and ISCIII FIS project (PI17/00931). Tatheer Adnan, Komal Peer, Shruthi Mahalingaiah. Harvard School of Public Health, Boston, MA, United States. Introduction: By allowing users to track their menstrual periods and the associated signs and symptoms, Menstrual Cycle Tracking Apps (MCTAs) may have potential in epidemiological studies of women's health. However, there is limited information about characteristics of MCTA users compared to both those who track their cycles in other ways, and those who do not track their cycles. Methods: We sought to examine diff erences between MCTA users and non-users in the Ovulation and Menstruation Health Pilot Study survey. Our study collected survey responses from 263 participants recruited from a gynecology clinic (n=36), community fair (n=61), and from the internet (n=166). We then compared diff erences in demographic/socioeconomic/ lifestyle factors, health conditions, and menstrual cycle characteristics between MCTA users and non-users and between women who tracked their cycles (with any forms of tracking) and those who did not Results: Overall, participants were mostly white, had attained 4-years of college education or higher, and had a median annual household income between $50,000-$74,999. Among all participants, 103 were MCTA users. Among the 160 non-users, 63 used other forms of cycle tracking (i.e., paper or digital calendar, software, birth control, and memory). No statistically signifi cant diff erences in race/ethnicity, household income, and education level between MCTA users and non-users and between cycle trackers and non-trackers were found. Menstrual cycle characteristics, such as cycle length, frequencies of normal and abnormal cycles, time to cycle regularity, and age at menarche were also comparable between MCTA users and non-users and between cycle trackers and non-trackers. Compared to users, MCTA non-users were more likely to smoke (18% vs 6%), use hormonal birth control (89% vs 72%) and had a higher prevalence of endometriosis (5% vs 1%) but lower prevalence of heartburn (17% vs 26 %). Meanwhile, cycle trackers were more likely to have high cholesterol (14%v 7 %), diabetes (5% v 0 %), and anxiety disorder (41% v 27%) compared to non-trackers. Conclusion: Our results suggest that MCTA users and non-users and menstrual cycle trackers and non-trackers were largely comparable in demographic factors, socioeconomic factors and menstrual cycle characteristics. Non-users and non-trackers, however, may have a more significant health burden. Nonetheless, our findings strengthen the generalizability of epidemiological results drawn from studies utilizing MCTAs. Within studies using MCTA sourced data, the addition of survey questions or health data may further facilitate whether diff erences exist between MCTA sourced data and comparison populations Introduction: Latin America has the highest regional cesarean birth rates, globally. Rates are driven in part by repeat cesarean birth once a woman has a history of prior cesarean. Trial of labor after cesarean is a safe alternative to repeat cesarean; obstetricians have used vaginal birth after cesarean (VBAC) calculators to counsel patients on the risks and benefi ts of mode of birth after a prior cesarean. The objective of this analysis was to develop a VBAC predictive model specifi c to Latin America to assist in counseling and assessment of risk in this population. Methods: We used data from the WHO Global Survey on Maternal and Perinatal Health (WHOGS), which includes data from 19 countries, but our sample was limited to the 8 Latin American Countries: Cuba, Argentina, Brazil, Mexico, Paraguay, Ecuador, Nicaragua, and Peru. We constructed a predictive model for two types of VBAC outcomes, "successful VBAC" and "successful VBAC without major adverse maternal or perinatal outcomes". Receiver operating characteristic curves and area under the curve were utilized in assessing the predictive properties of our proposed models. Results: From the original 290,610 women included in WHOGS, 98,072 women were Latin American, 13,314 had a cesarean as their most recent mode of birth, and 4,535 underwent trial of labor after cesarean with a singleton gestation at a gestational age of at least 24 weeks, which was our fi nal analytic sample. Variables that were signifi cant in mixed modeling of "successful VBAC" included maternal age, maternal BMI, previous infant birthweight, and previous uterine surgery. The AUC for this model was .61 (Table 1A ). Variables that were signifi cant in the mixed modeling of "VBAC without adverse outcomes" included maternal age, maternal BMI, number of prior pregnancies, previous infant birthweight, previous surgery, hemorrhage, obstetric complications, and total number of prenatal visits. The AUC for this was .599 (Table 1B) . Conclusion: The variables used in these predictive models are moderate in their predictive ability with the best model predicting the right outcome only 61% of the time. We hypothesize this dataset does not include relevant clinical variables that might produce a more predictive Latin American model to guide physicians and women in the decision to attempt a VBAC. Food Desert Residence Does Not Influence Meeting Maternal Weight Gain Recommendations. Adetola Louis Jacques * , 1 John Paul Tanner, 2 Mary Ann Smith, 1 Adrianna Campos, 2 Jason L Salemi, 2 Peeraya Sawangkum, 2 Kimberly Fryer, 2 Ronee E Wilson * . 2 1 University of Florida, Gainesville, FL, United States; 2 University of South Florida, Tampa, FL, United States. Introduction: Weight gain during pregnancy is an important factor aff ecting short and long-term maternal and fetal health. Due to these impacts, the Institute of Medicine (IOM) publishes guidelines regarding gestational weight gain. However, many social and medical factors may impact whether a patient meets the IOM recommendations. This study explored the infl uence of food desert residence on the ability to meet the IOM gestational weight gain guidelines. Methods: Individual level data from the Florida Department of Health for births during 2019 were linked food access data from the 2019 United States Department of Agriculture (USDA) Food Access Research Atlas. Food desert census tracts were identifi ed per the USDA defi nition. Adjusted risk ratios (aRR) and 95% confi dence intervals were calculated using modifi ed Poisson regression models. Models were adjusted for known confounders and data were stratifi ed by race/ethnicity. Results: During 2019 there were 209,087 total live singleton births in Florida, of which sixteen percent resided in a food desert. IOM weight gain guidelines were not met by 69% of patients. Residence in a food desert was not associated with ability to meet IOM guidelines (aRR: 1.01, CI: 1.00 -1.01). Patients considered underweight were more likely to meet guidelines (aRR: 0.94, CI: 0.92 -0.96), while overweight (aRR: 1.14, CI: 1.13 -1.15) Bunkyo-ku Japan Chiba University, Graduate SOM Chiba Japan Tokai University SOM Isehara Japan Hamamatsu University SOM Hamamatsu Oral