key: cord-034648-vfqth54o
authors: nan
title: WITHDRAWN
date: 2020-10-12
journal: Fertil Steril
DOI: 10.1016/j.fertnstert.2020.08.1019
sha: 
doc_id: 34648
cord_uid: vfqth54o

nan

dysregulated in 2 nd trimester chorionic villous samples (CVS) from PE pregnancies relative to CVS from healthy pregnancies. DESIGN: Laboratory in vitro studies using the first-trimester TB cell line, HTR-8/SVneo . RNA-sequencing (RNA-seq) of HTR-8 cells and CVS.

MATERIALS AND METHODS: HTR-8 cells were exposed to hypoxia (2.5% O 2 ) or normoxia (21% O 2 ) for 6hrs and RNA-seq was performed. Significant differentially expressed genes (SDEGs) and upregulated Gene Ontology (GO) cellular pathways were identified. To determine if gene expression changes were associated with changes in cellular response, transwell assays were performed to assess HTR-8 cell migration and invasion after exposure to 2.5% O 2 or 21% O 2 for 6hrs and 24hrs. For transwell migration assays (n¼6), number of migrated cells through culture inserts were counted. For invasion assays (n¼6), number of cells that invaded into Matrigel and collagen I matrices were counted. Means were compared using unpaired t tests and statistical significance was defined as p <0.05. To determine if changes in gene expression induced by exposure of HTR-8 to 2.5% O 2 were present in 2 nd trimester CVS from PE pregnancies relative to healthy pregnancies, CVS were collected and analyzed under an IRB approved protocol. RNA-seq of CVS from PE (n¼2) and healthy (n¼4) pregnancies was performed and DEGs were identified.

RESULTS: GO analysis showed that exposure of HTR-8 to 2.5% O 2 for 6hrs upregulated cellular migration pathways. SDEGs included genes involved in TB migration and invasion (MMP9, TIMP1, and PAPPA), as well as genes involved in tumor migration and invasion (ACTA2, MFAP4, SNAI2, SLCO4A1, GDF15, KLF5, ZBTB20, and ZNF703). HTR-8 cell migration and invasion, through both collagen I and Matrigel matrices, was significantly increased after exposure to 2.5% O 2 for 24hrs but not 6hrs. Analysis of RNA-seq data from CVS of PE and healthy pregnancies identified 17 significant DEGs, 4 of which (FAT2, SPON2, RASGRF2, and SCLO4A1) were decreased in CVS from PE pregnancies and are involved in cellular migration and invasion.

CONCLUSIONS: Exposure of 1 st trimester TBs to physiologic hypoxic conditions induces expression of genes associated with cellular migration and invasion and increases 1 st trimester TB migration and invasion in vitro. Decreased expression of migration and invasion genes in CVS from PE pregnancies may impair TB migration and invasion in the 2 nd trimester of pregnancy, resulting in inadequate spiral artery transformation and prolonged hypoxia, preceding the development of PE. OBJECTIVE: In vitro culture systems that preserve 3-D follicle architecture and oocyte-granulosa cell interaction may be the key to in vitro oocyte maturation (IVM). Hyaluronan is one component of natural extracellular matrices that provide support to cells in vivo. This report describes the application of hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes.

DESIGN: Pre-antral follicle culture in vitro. MATERIALS AND METHODS: Ovaries of 14-16 day old B6D2F1 mouse pups were treated with collagenase. Released pre-antral follicles with 2-3 layers of granulosa cell were collected. The hyaluronan hydrogel (HA) was prepared by mixing 25 ml of the HA gel (3 mg/ml) with 1 ml of 0.03% hydrogen peroxide to initiate cross-linking. Microdrops of HA gel (5-7 ml) were placed in individual wells and rapidly seeded with 4 follicles before gelling was completed. Culture medium (100 ml) was added to each well and the dish was overlaid with oil. Follicle culture was performed in f-MEM supplemented with 5% FBS, 100 mIU/ml FSH, 10 mIU/ml LH and ITS. Dishes were incubated at 37 C with 6% CO2. Final maturation was triggered with hCG (1.5 IU/ml) and EGF (5 ng/ml) after 12 days of growth. Follicles were monitored daily. Mature oocytes were inseminated with epididymal sperm. Fertilization was assessed the following morning based on presence of 2 pronuclei or cleavage to 2-cell. Outcome parameters monitored were estradiol levels, follicle morphology and survival throughout the in vitro culture interval (IVC), germinal vesicle breakdown (GVBD), oocyte maturation to metaphase II stage, fertilization and development to blastocyst. Cell number in blastocysts was determined by Hoechst staining RESULTS: Pre-antral follicles measured 121.9 AE 40.9 mM with oocyte diameters of 61.1AE 4.1 mM at time of seeding. Mean oocyte diameter, measured after maturation was 90.2 AE 8.0 mM. IVM data from three experiments were pooled. A total of 402 pre-antral follicles were embedded in HA gel. Antrums were observed in 55% of follicles by day 8. Post-hCG trigger 314 oocyte-cumulus complexes ovulated from the cultured follicles and 84% (264/314) underwent GVBD. The maturation rate to MII was 72.6%. (228/314). The fertilization rate was 82.5% (188/228). The subsequent blastulation rate with IVM oocytes was 46.3% (87/188). Blastocysts were cryopreserved for future transfer experiments to determine their ability to produce live offspring. Seven blastocysts were subsequently thawed and stained with Hoechst to determine cell number. Mean cell number was 55 AE 7.5.

CONCLUSIONS: HA was an excellent biomaterial for 3-D culture, allowing follicle growth, antrum formation and ovulation of oocyte-cumulus complexes upon hCG trigger. This matrix was easy to use, its rigidity could be adjusted and its transparent nature allowed optimal visualization. Functional competence of HA-matured oocytes was evidenced by their ability to fertilize and continue development to the blastocyst stage. Live birth data are needed to further validate this 3-D culture system. Testing of HA with follicles from larger animal models is warranted.

REFERENCES: Desai N, Alex A, AbdelHafez F, Calabro A, Goldfarb J, Fleischman A and Falcone,T. Three-dimensional in vitro follicle growth: overview of culture models, biomaterials, design parameters and future directions. Reprod Biol Endocrinol. 2010 Oct 14; 8:119 Desai N, Abdelhafez F, Calabro A, Falcone T. Three dimensional culture of fresh and vitrified mouse pre-antral follicles in a hyaluronan-based hydrogel: a preliminary investigation of a novel biomaterial. Reprod Biol Endocrinol. 2012 Jun 13;10 (1) OBJECTIVE: To identify cell types in male and female reproductive systems at risk of SARS-CoV-2 infection based on expression of viral host entry proteins.

DESIGN: Transcriptomic and proteomic analysis. MATERIALS AND METHODS: Publicly available single cell RNA sequencing (scRNAseq) data in human testis and non-human primate ovary was analyzed, as well as bulk RNA and proteomic data in human testis and ovary. Additionally, novel RNA sequencing data from 18 samples of human cumulus cells was generated. Tissue-and cell-type specific risk of viral infection was predicted based on the co-expression of host receptor angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) which are required for viral binding and cleavage, respectively. Expression of receptor basigin (BSG) and cysteine protease cathepsins L (CTSL) were also assessed as they are hypothesized to facilitate viral entry.

RESULTS: Based on scRNAseq data in non-human primate ovarian tissue, ACE2 and TMPRSS2 co-localize in a sub-population of oocytes in antral follicles (62% of cells, Pearson correlation¼0.37), but to a lesser extent in less mature oocytes and not at all in ovarian somatic cells. While ACE2 transcripts were detected in human cumulus cells (mean 24.03 transcripts per million), TMPRSS2 expression was absent in 15/18 samples and low in the others (0.13 TPM). Consistent with these findings, bulk RNA and protein data showed ACE2 expression in ovarian tissue, but no TMPRSS2 expression. In testicular cells, including sperm, co-expression of ACE2 and TMPRSS2 was not detected (Pearson correlation¼-0.01). Bulk RNA and protein data revealed ACE2 but no TMPRSS2 expression. BSG was more broadly expressed in testis than ACE2 and was co-expressed with CSTL in early (78.7%) and late (90.8%) primary spermatocytes.

CONCLUSIONS: Given that known COVID-19 symptoms are associated with tissues co-expressing ACE2 and TMPRSS2 (e.g. lung, heart, kidney, gut), it is conceivable that reproductive function could be effected if constituent cells co-express these genes. Based on these results, testicular cells including sperm are not at risk of ACE2-TMPRSS2-mediated viral entry, however low levels of SARS-Cov-2 in human semen have been reported and may suggest alternative routes of entry. The cells predicted to have the greatest susceptibility to infection are antral oocytes which are either ovulated or atrophy within several days of appearance each cycle and are therefore unlikely to have sustained impact on female fertility if infected. Moreover, the lack of ACE2-TMPRSS2 expression in cumulus cells may act as a barrier to infection. Therefore, procedures in which cumulus cell-enclosed oocytes are collected and fertilized outside the female reproductive tract (e.g. IVF) may not pose a risk. IVF success rates decline with increasing female age and it is therefore imperative that patients are allowed to access fertility treatments if safe to do so. The results presented here are indicative, but ovarian pathology data from women of reproductive age with COVID-19 at time of death is needed for definitive confirmation. OBJECTIVE: SEPT12, a member of the Septin gene family, has expression restricted to the testis and is critical for normal spermatogenesis. Septin genes code for polymerizing guanosine triphosphate (GTP) binding proteins whose homologs have been conserved throughout evolution from yeast to humans (1) . Roles assigned to Septin genes include cell division, cytoskeletal organization, and membrane-remodeling events including the epithelialmesenchymal transition in metastatic cancers (2) . A study that investigated azoospermia and teratozoospermia in a mouse knockout model demonstrated oocytes fertilized via ICSI with sperm targeted SEPT12 antisense alleles resulted in embryo arrest by the morula stage (3) . The application of RNA sequencing to elucidate the expression profile of SEPT12 in human preimplantation embryos may unlock insights into the transcriptional events of early embryogenesis.

DESIGN: Prospective cohort study on human, donated embryos. MATERIALS AND METHODS: The study included patients who donated fresh embryos at the blastocyst stage during an IVF cycle between January, 2016 and June, 2016. Embryos were biopsied, and approximately 2-4 cells were removed for preimplantation genetic testing for aneuploidy (PGT-A) by next generation sequencing (NGS) using the ReproSeq assay to assess copy number variants (CNVs). The remaining cells of the embryo were designated for RNA Sequencing. Read counts per gene were summed across embryo cohorts and normalized using the median of ratios. Differential gene expression between embryo cohorts was calculated using DESeq2, in order to estimate variance-mean dependence and evaluate differential gene expression using a negative binomial distribution. A likelihood ratio test was used to account for heterogeneity due to patient, batch, and ploidy and growth status (arrested/ongoing). The adjusted threshold for significance was p<0.05.

RESULTS: 43 blastocysts underwent PGT-A assessment and RNA sequencing. 36 showed expression of SEPT12, 6 of the 7 blastocysts that failed to show SEPT12 expression had poor trophectoderm morphology grade. The expression of SEPT12 was further examined in 15 embryos, 9 were enriched (>90%) for trophectoderm cells (TE) and 6 enriched (>80%) for inner cell mass cells (ICM). SEPT12 expression was significantly higher in TE cells than ICM cells, where P<0.0001.

CONCLUSIONS: Septins were first discovered nearly fifty years ago however their function remains poorly understood (4) . The importance of this gene family has been indicated by the conservation of the functional domains throughout evolution. SEPT12 has been shown to be critically important in spermatogenesis. This study is the first to characterize SEPT12 expression in the human embryo. Our data supports the findings that wild type SEPT12 expression was preferentially associated with blastocyst formation compared to arrest at the morula stage for embryos that contained the antisense SEPT12 allele (3). Our current studies are focused on characterizing SEPT12 expression in cleavage and morula stage human embryos to further elucidate this gene's role in early embryogenesis.

The septin family of GTPases: architecture and dynamics

The pathophysiology of the septin gene family

SEPT12 deficiency causes sperm nucleus damage and developmental arrest of preimplantation embryos

Genetic control of the cell division cycle in yeast

IV Genes controlling bud emergence and cytokinesis