key: cord-0827596-wg3tif39
authors: Porcu, Eleonora; Tranquillo, Maria Lucrezia; Notarangelo, Leonardo; Ciotti, Patrizia Maria; Calza, Nilla; Zuffa, Silvia; Mori, Lisa; Nardi, Elena; Dirodi, Maria; Cipriani, Linda; Damiano, Giuseppe; Labriola, Francesca Sonia
title: HIGH SECURITY CLOSED DEVICES ARE EFFICIENT AND SAFE FOR VITRIFICATION TO PROTECT HUMAN OOCYTES FROM THE RISK OF VIRAL CONTAMINATION ESPECIALLY DURING THE COVID-19 PANDEMIC
date: 2020-09-30
journal: Fertility and Sterility
DOI: 10.1016/j.fertnstert.2020.08.522
sha: f29c4aabdac76bdf5436dc634cfaa4107000ea6e
doc_id: 827596
cord_uid: wg3tif39

nan

commercial standard vitrification media; Tvitri-4, produced in small scale for research by INVITRAÒ, based on the standard composition with four modifications including carbohydrate trehalose instead sucrose, reduced non-permeant cryoprotectant concentration, and addition of two aminoacids; and Tvitri-4 supplemented with L-carnitine (LC) and oleic and linoleic fatty acids (FA).

DESIGN: Experimental study. MATERIALS AND METHODS: 23 C57BL/6J females were superovulated with 5UI eCG followed by 5UI hCG and oocytes (n¼562; 4 replicates) were randomly divided in 4 groups: fresh control group (FC), vitrified using Irvine (IRV), Tvitri-4 (T4), or supplemented Tvitri-4 (T4-LC/FA) media. Fresh or vitrified-thawed oocytes were inseminated with 1x10 6 sptz/ml and cultivated for 96 or 120 hours in KSOM (Cosmo bio co., LTD) incubated at 37 o C with 5% CO 2 . Blastocysts of each group were individually fixed in methanol/water. Lipids were extracted using One Step Methanol protocol (9 blastocysts/group) and flow injected into the triple quadrupole spectrometer equipped with an electrospray ion source. Lipids were analyzed using the multiple reaction monitoring profiling (MRM-profiling) method and values of relative intensities of ions detected in each group were compared using univariate (one-way ANOVA, volcano plot) and multivariate analysis (PLS-DA).

RESULTS: One-way ANOVA (p-value %0.05) showed that 90 out of the 125 lipids were differently expressed among the four groups, while a comparison between the vitrified groups showed no difference. Two by two comparisons between the control and vitrified groups using volcano plot (p-value %0.05, fold-change R1.5) detected 55, 17, and 11 significant features between FC vs. IRV, FC vs. T4, and FC vs. T4-LC/FA, respectively; all of them more abundant in the FC group. Partial least square discriminant analysis (PLS-DA) variables of importance (VIP scores) higher than 1.3 followed the same pattern and identified the phosphatidylinositol containing 36 carbon and one unsaturation in the fatty acyl residues -PI(36:1), and phosphatidylcholines PC(38:4), PCo(36:5), PC(34:1), and PC(30:0) among the top features; the exception being the free stearic acid (C18:0), which was the top feature in the FC x T4-LC/FA comparison, being more abundant in the latter.

CONCLUSIONS: Vitrification changed the lipid profile of mice blastocysts causing an overall reduction on lipid abundances that affected PC lipids the most. This effect was more apparent in IRV, followed by T4, and T4-LC/ FA suggesting that supplementation of media with L-carnitine and unsaturated fatty acids may have protective effects on lipid content of blastocysts from vitrified oocytes, whose impacts needs further investigation.

CNPq ( OBJECTIVE: Human ovarian tissue cryopreservation, an essential method of fertility preservation, has led to the birth of more than 180 healthy babies around the world. Although there are many reports on cryopreservation and transplantation of ovarian tissues, the optimum storage conditions after thawing are still unclear. In this study, we performed xenotransplantation of ovarian tissue into nude mice to determine what kind of storage conditions are appropriate.

DESIGN: Prospective controlled animal study. MATERIALS AND METHODS: We used ovaries derived from four SD rats at 10 weeks of age. Each ovary was cut in half, then 16 slices were cry-opreserved by the slow freezing method (using 1.5M DMSO as cryoprotectant). Then, the tissues were thawed and stored at 4 C, RT (24 C), or 37 C for 2.5 hours in DPBS buffer. They were grafted under the kidney capsule of an ovariectomized nude mice (8-20 weeks old). Five IU PMSG and 5 IU hCG was injected once into mice at about 4 weeks after transplantation. Engraftment and follicle development in each group was compared with that in the control group (immediately grafted after being thawed).

RESULTS: The engraftment rates of frozen-thawed ovarian tissues were assessed in the control group (100%), 4 C group (87.5%), RT group (84.6%), and 37 C group (50.0%). The engraftment rate was significantly decreased when the tissues were stored at 37 C compared with those of the other groups (P < 0.05). The rates of engrafted ovarian tissues with macroscopically confirmed follicles were assessed in the control group (61.5%), 4 C group (71.4%), RT group (45.5%), and 37 C group (33.3%). Those rates in the RT and 37 C groups decreased compared with the control and 4 C groups although there was no significant difference. The follicles were aspirated and oocytes were collected in the control group (9), 4 C group (12), RT group (3), and 37 C group (1).

CONCLUSIONS: Storing the frozen-thawed tissues at 4 C led to a successful engraftment and follicle development. Although the tissues were well engrafted, there was little follicular growth in the RT group compared with the 4 C group. The storage at 37 C resulted in poor engraftment and follicle development. This study showed that the storage temperature of frozen-thawed ovarian tissues affects the engraftment and/ or follicle development. Frozen-thawed ovarian tissues should be transplanted immediately after being thawed. In case they need to be kept a while before transplantation, storage at lower temperatures is recommended. Table 1 . CONCLUSIONS: The efficacy of vitrification was assessed in vitro using survival, fertilization and cleavage rates and in vivo after embryo transfer by pregnancy, implantation and miscarriage rates.

Results shows no statistically significant differences using HSV or CryotopÒ for oocytes vitrification. Therefore, in order to ensure safety, especially during the current COVID-19 pandemic, the use of the closed device eliminates the potential sample contamination during vitrification and storage without compromising its in vitro and in vivo survival and development. OBJECTIVE: The current global pandemic has triggered concerns regarding the potential infectivity of the SARS-CoV-2 virus to blastomeres known to possess ACE-2 receptors. In 2010, Pomeroy and coauthors reviewed the negligible risks associated with the potential cross contamination of human reproductive tissues, gametes and embryos in cryostorage. The purpose of this investigation is to explore changes in ART lab practices over the last decade that could warrant a reassessment of the latter AAB/CRB embryo cryopreservation guidelines relative to disease transmission potential.

DESIGN: Retrospective analysis of clinical practices that may alter the way we look at acceptable risks in embryo vitrification (VTF) and cryostorage methods. Specifically, we will investigate the effectiveness of a validated closed VTF system relative to zona pellucida (ZP)-intact and non-intact blastocyst cryopreservation. Additionally, we will discuss the merits and need for safer cryostorage systems.

MATERIALS AND METHODS: Human blastocysts were vitrified in a closed, aseptic device system and rapidly-warmed and sucrose diluted using standard procedures. From 2009 to 2012, 90% of all vitrified blastocysts had an intact ZP without the need for pre-VTF collapsing due to the use of I.C.E. non-DMSO solutions (>7.9M glycerol/EG). Between 2012-2014 we transitioned into 100% of all embryos experiencing laser ZP ablation and or blastocyst biopsying procedures by 2015. The latter trophectoderm exposed blastocysts were effectively contained in flexipettes which were weld-sealed into CBS straws without risk to possible pathogen exposure in liquid nitrogen cryostorage. Chi-squared analysis was used to assess differences (p<0.05) in survival and pregnancy outcome data.

RESULTS: The routine application of ZP-exposed trophectoderm and blastocyst biopsying improved (p<0.05) our survival rates from 95% (1066 of 1126 BL) to 99.4% (3352 of 3373 BL) with increased (p<0.05) embryo implantation efficiency (46% vs 69% implantation using 1.91 vs 1.07 blastocysts/FET, respectively).

CONCLUSIONS: The protective barrier of an intact ZP to potential pathogen exposures is no longer a clinical reality for cryopreserved blastocysts. Although we agree that the relative risks of embryo disease transmission in cryostorage remain negligible, why take any risks when highly effective closed VTF systems (ICE straw, HSV, mS-VF, VitriSafe) have been established over the last decade? Alternatively, we question whether the use of LN 2 -vapor storage tanks for open-VTF systems alleviates potential airborne viral cross-contamination, while they most certainly create a greater risk for potential embryo wastage as discussed by Pomeroy et al. (2010) and overtly realized by recent tank failure experiments and known catastrophic events. Finally, it is worth noting that embryos vitrified in an insulated straw environment are more resistant to detrimental additive temperature fluxes that can occur under sub-optimal cryostorage handling procedures. So, we ask, is it time to reconsider the status quo of embryo good tissue practices when viral pandemics are a reality?

References: Pomeroy OBJECTIVE: Keeping liquid nitrogen (LN 2 ) tank properly is extremely important for an ART clinic. As the accident in the U.S. in 2018 showed, a tank failure causes serious damage for embryos and patients. However, there's no detailed information as to what to do when tanks are damaged. In our previous study, we indicated that a damaged 10L tank can keep freezing for 7-8 hours if it retains a certain level of LN 2 . In this study, we analyzed the influence of tank capacity on the estimated embryo salvage period in a simulated tank failure.

DESIGN: Prospective experimental trial. MATERIALS AND METHODS: We prepared 3 tanks of different capacity (XT10, HC20, HC35, Taylor-Wharton, USA). All tanks were filled up to full with LN 2 . To simulate tank failure, we drilled a 2mm diameter hole in the vacuum valve of each tank. A temperature prove was set in a plastic sleeve of cane in the tanks. We measured the temperature and LN 2 levels every 15 min for the first hour. Then, they were measured every hour to until the rise in temperature began. After the temperature initiated to rise, they were measured every 15 min to until the temperature reached -80 C, which is the temperature at which embryos start to get damaged. Before tank failure simulation, temperatures and LN 2 level of each tank were measured every 24 hours for 7 days to see the temperature and LN 2 volume shift without tank damage.

RESULTS: Speed of LN 2 level decrease of the 10, 20 and 35L tank was 4.6, 4.5, and 2.8 cm/h, respectively. The temperature at the start of measurement for all tanks was -196 C. In the 10L tank, the rise in temperature began when the remaining LN 2 level was 1cm. In the 20 and 35L tanks, it began 

Oocytes frozen (mAEds)