THE IMPACT OF COVID-19 ON THE OUTCOMES OF ASSISTED REPRODUCTIVE TECHNOLOGY PROGRAMS

Objective: To investigate the outcomes of assisted reproductive technology (ART) programs in patients with a history of COVID-19 of various severity. Material(s) and Method(s): This prospective study enrolled 240 infertile patients. They were divided into group 1 comprising patients without a history of COVID-19 (n=105) and group 2 (n=135) including patients who less than 12 months before the ART cycle had mild (subgroup 2a, n=85) or moderate (subgroup 2b, n=50) COVID-19. The level of specific antibodies to SARS-CoV-2, parameters of oogenesis, early embryogenesis, and clinical outcomes of HRT were evaluated. Result(s): The parameters of oogenesis and embryogenesis, pregnancy and delivery rates did not differ between groups 1 and 2. A weak negative correlation was detected between the level of IgG-antibodies to SARS-CoV-2 and the number of obtained oocytes and embryos. Patients with an interval between COVID-19 and ART cycle <=6 months had a significantly higher relative number of poor-quality blastocysts than women with >6 months interval. Patients who experienced moderate COVID-19 had a high early miscarriage rate of (12%). Conclusion(s): COVID-19 can adversely affect reproductive outcomes, lead to a decrease in the number of oocytes and embryos obtained in ART cycles and their quality, and increase the risk of early miscarriage. More research is needed to investigate the mechanisms underlying the adverse effects of COVID-19 and the post-COVID syndrome. Copyright © A group of authors, 2022.

Covid-19 Vaccination and Assisted Reproduction Outcomes: A Literature Review and Meta-Analysis

Objective: The COVID-19 pandemic remains a significant global health risk and poses an increased danger to pregnant mothers. There remains significant vaccination hesitancy amongst many in the population, in part due to unfounded claims regarding its effect on fertility (1). We conducted a literature review and meta-analysis comparing the available data on effects of COVID-19 vaccination on both ovarian stimulation and embryo transfer outcomes. Material(s) and Method(s): A literature search of PubMed was conducted using the search terms COVID-19, SarsCov2, vaccines, bnt162 vaccines, 2019-ncov vaccine mrna-1273, in vitro fertilization, egg retrieval, oocyte retrieval and embryo transfer. Reported ovarian stimulation outcomes included number of oocytes retrieved, number of mature oocytes, number of fertilized oocytes, and number of blastocysts formed, and were compared using Student's t-test. Embryo transfer outcomes included implantation rate, clinical pregnancy rate, and ongoing pregnancy rate, and were compared using chi square. Meta-analysis was performed by Standard Mean Difference method for oocyte outcomes and the Cochran-Mantel-Haenszel method for embryo transfer outcomes. Result(s): Our search retrieved 12 studies conducted between August 2021 and March 2022. Of them, only 6 compared outcomes between COVID-19 vaccinated and unvaccinated patients. Two included only ovarian stimulation outcomes, two included only embryo transfer outcomes, and two studies included outcomes for both. Of the ovarian stimulation outcomes reported, data adequate for meta-analyses were only included for the number of oocytes retrieved and number of mature oocytes. There were no statistically significant differences reported between vaccinated and unvaccinated patients amongst the pooled data from the four studies for the ovarian stimulation parameter - number of oocytes retrieved (mean 10.6 vs 10.6, 95% CI -0.144 - 0.157), or the number of MII oocytes (mean 7.43 vs 7.95, 95% CI -0.055 - 0.247). There were similarly no statistically significant differences amongst the pooled data from the four studies for any of the embryo transfer parameters - implantation rate (OR 0.97, 95% CI 0.76 - 1.24), clinical pregnancy rate (OR 0.88, 95% CI 0.70 - 1.11), or ongoing pregnancy rate (OR 1.22, 95% CI 0.78 - 1.91). Conclusion(s): The current literature demonstrates no differences in either ovarian stimulation or embryo transfer outcomes following COVID-19 vaccination. There remain several key parameters, however, that would benefit from additional investigation. Impact Statement: Patients can be reassured the current evidence reaffirms the safety profile of the mRNA COVID-19 vaccine and does not affect fertility. References: 1. Hsu AL, Johnson T, Phillips L, Nelson TB. Sources of Vaccine Hesitancy: Pregnancy, Infertility, Minority Concerns, and General Skepticism. Open forum infectious diseases 2022;9:ofab433-ofab. Copyright © 2022

Investigating impacts of CoronaVac vaccination in males on in vitro fertilization: A propensity score matched cohort study

Study question: Does a history of SARS-CoV-2 vaccination (CoronaVac) in males influence male fertility, gamete and embryo development, and in vitro fertilization (IVF) outcomes? Summary answer: CoronaVac vaccination in males may not have an adverse effect on patient's performance or the gamete and embryonic development potential during ART treatments. What is known already: Vaccines against COVID-19 have been approved for emergency use in several countries and regions, while concerns about the potential negative effect of vaccines on fertility contributed to vaccine hesitancy. It is urgent to explore the effect of CoronaVac on human fertility to help to overcome vaccine hesitancy about possible fertility impairment. Study design, size, duration: A retrospective cohort study enrolled couples undergoing IVF cycles between June and August 2021 at Reproductive Medicine Centre, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. According to the history of SARS-CoV-2 vaccination in males, the participants were divided into the vaccination group and the non-vaccination group. Participants/materials, setting, methods: A self-controlled study of semen analyses for males before and after CoronaVac vaccination was conducted. Baseline characteristics were matched using propensity score matching. Participants were categorized into the unexposed group (non-vaccination) and exposed group (vaccination), and the population was 271 for each. Semen parameters and IVF outcomes were the main outcomes. Main results and the role of chance: Generally, no statistically significant differences were exhibited between the matched cohorts regarding embryo developmental parameters, including fertilization rate, cleavage rate, high-quality embryo rate, blastocyst formation rate, and available blastocyst rate, as well as clinical outcomes, such as implantation rate, biochemical pregnancy rate, and clinical pregnancy rate. Moreover, males after vaccination seemed to have fluctuated semen parameters including increased semen volume, lower motility, and decreased normal forms of sperms, while the motile sperm counts were similar. In addition, all semen parameters were above the lower reference limits. Limitations, reasons for caution: It was a single-center retrospective cohort study with a small sample size, and the men enrolled were suffering from infertility, which limited the generalizability of the conclusions. In addition, the endpoint of the current is a confirmation of clinical pregnancy, a study with a longer period of follow-up was urgent. Wider implications of the findings: Our findings suggested that CoronaVac vaccinations in males may not have adverse effects on patient's performance or the gamete and embryonic development potential during ART treatments. Larger studies among a wider population with longer followup in the future are required to support and validate our observations.

Is children's health up to two years of age affected by embryo vitrification?

Study question: Does embryo vitrification affect children's health including growth, up to 2 years of age when compared to fresh embryo transfer? Summary answer: While embryo vitrification had an impact on birth parameters, no differences in growth or health outcomes were found up to 2 years of age. What is known already: Vitrification has become the preferred cryopreservation method for embryos. Frozen embryo transfer has been repeatedly associated with altered health outcomes when compared with fresh transfer including a decreased risk for small-for gestational age (SGA) and an increased risk for large-for-gestational-age (LGA) and macrosomia. Not only there is uncertainty which factors are responsible for the observed differences, also the heterogeneity among studies limits overall conclusions. Notwithstanding the observed differences at birth, little is known about growth and health of children born after embryo vitrification beyond birth while aberrant growth trajectories have been linked to cardiometabolic morbidity later in life. Study design, size, duration: This single-center cohort study compared anthropometry and health outcomes in singletons conceived after cleavage-stage or blastocyst-stage embryo vitrification with results after fresh embryo transfer between 2014 and 2018. Pregnancies after PGT, IVM, oocyte vitrification or oocyte/embryo donation were excluded. Eligible singletons living in Belgium and randomly selected for continued follow- up were invited for examination in our center at 2 months (infancy) and 2 years of age (early childhood). Participants/materials, setting, methods: Birth characteristics were available for 1237 and 2063 children born after embryo vitrification and fresh embryo transfer, respectively. Follow-up data were available for 582 and 757 children at 2 months and for 233 and 296 children at 2 years. Growth parameters were adjusted for neonatal, treatment and maternal characteristics. Subgroup analysis according to cycle regimen (HRT versus NC) and strategy (freeze-all versus previous fresh cycle) was performed. In addition, outcomes restricted to blastocysts are presented. Main results and the role of chance: Mothers giving birth to a child conceived after embryo vitrification presented more often with pregnancy-induced hypertensive disorders than controls (P<0.001). Birthweight, height and head circumference SDS of children born after embryo vitrification were higher than for children born after fresh embryo transfer (all P<0.001) even after adjustment for neonatal, treatment and maternal characteristics. Embryo vitrification was also associated with a decreased risk of SGA (AOR 0.48;0.00, 0.44) and an increased risk of macrosomia and LGA (AOR 3.59;1.12, 11.59)(all P<0.05). Restricting the sample to blastocysts (n=1795), we found a higher birthweight SDS and increased risks of LGA, macrosomia and pregnancy-induced hypertensive disorders after vitrification (all P<0.05). At infancy, weight and height SDS were larger for children born after embryo vitrification, but not after adjustment for co-variates. At childhood, no differences in anthropometrics were found between the groups. Weight and height gain from birth to infancy and from infancy to early childhood were comparable between the groups. Until 2 years, comparable rates of severe developmental problems, hospital admissions, surgical interventions and of chronic medication intake were found between the groups. Subgroup analysis showed that growth parameters at all ages were not affected by cycle regimen or cycle strategy. Limitations, reasons for caution: Participation rate at 2 years was lower than expected in both groups, probably due to cancellation/postponement of the visit related to the corona pandemic. Furthermore, although cycle strategy was not found to affect growth parameters, the sample size of the subgroup analysis remains rather small to draw firm conclusions. Wider implications of the findings: When adjusted for co-variates including birthweight, the observed differences in anthropometrics at birth in hildren born after embryo vitrification attenuated by 2 years of age. This suggests that outcomes in early childhood are determined by size at birth.

Vulnerability of human preimplantation embryos to SARSCoV-2 infection

Presence of SARS-CoV-2, the virus responsible for COVID-19, has been reported in numerous organs and tissues of infected patients, including the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood. While cases of intrauterine transmission between expectant mother and fetus have been documented, the impact of SARS-CoV-2 infection on early embryogenesis and establishment of a pregnancy are not known. This prompted us to ask if SARS-CoV-2 can infect embryos, since such an event could impact embryo viability and affect a subsequent pregnancy. We used a three-pronged approach to investigate this possibility: 1) Using RNA-seq and immunofluorescence, we learned that ACE2 and TMPRSS2, two factors required on target cells for SARS-CoV-2 entry, are coexpressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos;2) Using fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, we observed robust infection of trophectoderm cells, and this permissiveness could be attenuated with blocking antibodies targeting S or ACE2;and 3) Exposing human blastocysts to live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath.

38th Hybrid Annual Meeting of the European Society of Human Reproduction and Embryology

The proceedings contain 1092 papers. The topics discussed include: semen impairment and occurrence of SARS-COV-2 virus in semen after recovery from COVID-19;the SELECTIMO study - clinical outcomes of uninterrupted embryo culture with or without time-lapse based embryo selection versus interrupted standard culture: a randomized controlled trial;the bias is out of the bag: IVF culture dish well number influences embryo selection decision-making and implantation outcome;reducing inter-observer and intra-observer variability of embryo quality assessment using deep learning;annotation-free embryo score calculated by Idascorecorrelated with live birth and has no correlation with neonatal outcomes after single vitrified-warmed blastocyst transfer;simplifying the complexity of time-lapse decisions with AI: CHLOE (fairtility) can automatically annotate morphokinetics and predict blastulation (at 30HPI), pregnancy and ongoing clinical pregnancy;and controlled ovarian stimulation (COS) protocols for assisted reproduction: a Cochrane systematic review and network meta-analysis.

Effect of hydroxychloroquine on an overactive endometrial immune profile: A Pilot Case Study

Problem: A37 y.o, P1+1 presented with a 1.5 year Hx of secondary infertility. Initial ovulation induction with clomifene citrate was unsuccessful, followed by a single IUI attempt with gonadotropin stimulation. The couple progressed to IVF treatment with 3 failed embryo transfers. There were no endocrinological abnormalities, endometrial/ uterine anatomy was normal on transvaginal USS and saline infusion sonograpy, so an Endometrial Immune Profile(EIP) and Receptivity Array(ERA) were performed prior to further treatment. ERA was in the receptive range, and EIP demonstrated an overactive profile with high IL15:Fn14, suggestive of NK overactivation. In the absence of other pathology this was hypothesised as a potential cause for implantation failure. Immunotherapy options were discussed, including IVIG and adalumimab. Risks of these during the Covid pandemic resulted in the decision to try oral hydroxychloroquine, with cost benefits and potentially less adverse side effects. Unfortunately there is a paucity of published data and outcomes, but proposed benefits of this treatment were based on demonstration of improvemed serum TH1:TH2 cytokine ratios (reduction in TNFa and increase in IL:10), and demonstration of a reduction in miscarriage rate. Method of Treatment: Hydroxycholoroquine 200mgPOBD was commenced 6 weeks prior to commencing treatment, followed by a frozen transfer of a single blastocyst (5AA). Unfortunately this transfer after 8/52 treatment was unsuccessful. A repeat cycle was scheduled after a further 8 weeks, continuing the hydroxychloroquine for >3 months. Initial hCG 13 days post transfer was 2934, but the patient presented with sudden PV bleeding after 6 days, follow up hCG was only 3650. Transvaginal USS demonstrated a collapsed intrauterine gestation sac in keeping with a non-viable pregnancy. Onward referral to an Early Pregnancy unit for follow up confirmed a miscarriage. PGS was not incorporated into the cycles to assess for embryo aneuploidy. Results: Overall the implantation rate had increased from 0 (0/3) to 50% (1/2), but due to sample size this was not statistically significant (p = 0.81). Pregnancy rate per embryo transfer also increased form 0/3 to 1/2, but again was not significant because of low numbers. Due to the failure to achieve an ongoing pregnancy, a repeat biopsy was performed while using hydroxychloroquine to assess its effects on the endometrial immunological environment. This showed a normalisation of the IL15:Fn14 ratio (5.680 to 0.831), but with a slight elevation in the IL18:Tweak ratio (0.088 to 0.114).CD56 remained in the normal range (0.993 to 1.344). Conclusion: Although prescribed for inconsistent indications, there is little published data on hydroxychloroquine use for adverse reproductive outcome. This case report demonstrates the effect of oral hydroxychloroquine therapy on an overactive endometrial profile, leading to a major reduction in IL15:Fn4 ratio, suggesting a potential role in reducing uNK cytotoxicity. Anecdotally a 3 month course is recommended prior to transfer, which would be supported by these events. Unfortunately there is limited ability to make treatment recommendations based on a single sample, however, the findings suggest that a larger study to explore if this pattern is reproducible would have important clinical value.

Stem cell-derived trophoblast organoids model human placental development, X chromosome inactivation, and susceptibility to emerging pathogens

Problem: Trophoblast organoids derived from human placental villi provide a powerful 3D model system of placental development, but access to first-trimester tissues is limited due to ethical and legal restrictions. Here we sought to establish a methodology for establishing 3D trophoblast organoids from naïve human pluripotent stem cells (hPSCs), which have an expanded potential for extraembryonic differentiation. Method of Study: We previously demonstrated that naïve hPSCs readily give rise to self-renewing human trophoblast cells (hTSCs) that resemble post-implantation cytotrophoblast (CTB) progenitors and can further differentiate into specialized trophoblast lineages. Here we examined whether hTSCs derived from three distinct sources (naïve hPSCs, human blastocysts, and first-trimester placental tissues) have the potential to self-organize into 3D trophoblast organoids by transfer to Matrigel droplets in the presence of trophoblast organoid medium. The expression of protein markers in the resulting stem cellderived trophoblast organoids (SC-TOs) was examined by immunofluorescence and light-sheet microscopy, while their single cell transcriptome was analyzed using the 10X Genomics platform. We also investigated the X chromosome inactivation (XCI) status of organoids derived from female naïve hPSCs and their ability to differentiate into invasive extravillous trophoblast (EVT) organoids. Finally, we evaluated whether SC-TOs are susceptible to infection by various emerging pathogens (SARS-CoV-2 and Zika virus), as a basis for establishing a stem cell-based model system of placental infections during the first trimester. Results: Trophoblast organoids generated from naïve and primary hTSCs displayed comparable tissue architecture, placental hormone secretion, microRNA expression, and capacity for long-term selfrenewal. In-depth single cell transcriptome profiling revealed that SCTOs encompass a variety of trophoblast identities that closely correspond to CTB progenitor, syncytiotrophoblast (STB) and EVT cell types found in human post-implantation embryos. Interestingly, the cellular composition in trophoblast organoids derived from naïve and primary hTSCs was highly similar, which suggests that trophoblast organoid culture represents a powerful attractor state in which the influence of subtle epigenetic differences between naïve and primary hTSCs is mitigated. These organoid cultures displayed clonal XCI patterns previously described in the human placenta.Upon differentiation into specialized EVT organoids, extensive trophoblast invasion was observed in co-culture assays with human endometrial cells. We further demonstrated that SC-TOs display selective vulnerability to infection by SARS-CoV-2 and Zika virus, which correlated with the expression levels of their respective entry factors. Conclusions: The generation of trophoblast organoids from naïve hPSCs provides an accessible and patient-specific 3D model system of the developing placenta and its susceptibility to emerging pathogens. The ability to genetically manipulate naïve hPSCs prior to differentiation into SC-TOs enables functional interrogation of regulatory factors implicated in placental organogenesis.

DOES ZYMOT SPERM SEPARATION IMPROVE EM- BRYO DEVELOPMENT OUTCOMES WHEN APPLIED TO ALL INFERTILITY PATIENTS COMPARED TO DENSITY GRADIENT WASHING OR SURGICALLYATTAINED SPERM?

OBJECTIVE: The ZyMot sperm separation device has proven favorable for use in elevated DNA fragmentation index (DFI) male factor patients, as an alternative to density gradient (DG) washing or surgically attained testicular sperm. In 2020, without fully understanding the infectivity and transmission potential of SARS-CoV2 in semen, a more liberal application of a timed ZyMot microfluidic swim-up was applied to our IVF patients to dilute out and minimize potential pathogens. This study aimed to evaluate whether the use of ZyMot sperm improved normal embryo development. MATERIALS AND METHODS: Retrospective analysis of PGT-A/ICSI cycles (N= 3219) between 2016-2020 was conducted to assessed fertilization rates (FR), blastocyst development/utilization rates (BUR) and genetic outcomes. Sperm preparations were performed per standard operating or manufacturer advised (i.e., ZyMot) procedures. Cumulus oocyte complexes were harvested 36h post-hCG, stripped and ICSI performed 3-5hr later. Zygotes were assessed at 16-18hr post-ICSI, and embryos cultured under humidified tri-gas incubation for up to 7 days. Blastocyst (BL) development as evaluated, and expanded BL or greater were biopsied on Days 5, 6 or 7. All BL were vitrified and genetics determinations for euploidy, aneuploidy and mosaicism were contrast. Applying Chi-squared analysis, we compared potential differences (p<0.05) between oocytes inseminated by DG wash (n=23,549), ZyMot wash (n=7,331) or testicular sperm (n=815). RESULTS: No difference in FR (76%), D5 BL formation (52-56%) or BUR (52-53%) was detected between DG and ZyMot washed sperm, respectively. Meanwhile, testicular sperm had a lower FR (70%;p<0.05), fewer BL forming on D5 (48%;p<0.05) and a lower overall BUR (41%;p<0.05). In addition, fewer testicular-derived BL were euploid (39%;p<0.05) with more aneuploidy (54%;p<0.05) than DG wash (50%, 39%;respectively) or ZyMot swim-up (45%, 37%;respectively) derived-embryos. No difference in potentially viable BL (Euploidy+Mosiac outcome) was observed between DG or ZyMot wash groups (63-64%). CONCLUSIONS: Application of the ZyMot device in the general IVF population offered no benefit to embryo development outcomes compared to standard sperm wash procedures. Our data does support that microfluidic separation of sperm using ZyMot for male factor patients with elevated DFI is a more favorable and cost-effective approach to surgically attaining testicular sperm when ejaculated sperm is possible. However, when insufficient motile and or morphologically normal sperm are available in an ejaculate further analysis is needed to elucidate the benefit of testicular biopsy treatment, as our assessments in this study may be biased by including men with non-obstructive azoospermia. IMPACT STATEMENT: The timed selection of morphologically normal, highly progressive sperm by ICSI, PVP-swim-out likely mimics the potential benefits the ZyMot device may offer infertile men with elevated sperm DNA fragmentation generating similar blastocyst development and euploidy outcomes.

EXPOSURE OF OVARIES TO COVID-19 VACCINATION DOES NOT IMPAIR FERTILITY

OBJECTIVE: Misinformation regarding Covid vaccination has contributed to vaccine hesitancy. Initially, there were claims that immune cross reactivity between the SARS CoV-2 spike protein and syncytin-1 would prevent embryo implantation. We previously demonstrated no difference in implantation and sustained implantation rates between previously vaccinated or infected women compared to other women.1 More recently, misinterpretation of vaccine biodistribution data has led to a second claim that mRNA containing lipid nanoparticles are concentrated in the ovaries and spike protein produced there would also cause infertility. The purpose of this study is to determine whether prior in vivo ovarian exposure to lipid nanoparticle-mRNA vaccination against SARS-CoV2 spike protein reduces subsequent fertility in women. MATERIALS AND METHODS: This is an ongoing observational study of women undergoing frozen embryo transfer with a single expanded blastocyst. This is an interim report (n =128) encompassing transfers between Jan 1 and Jul 02. All patients had serum analyzed prior to starting stimulation for egg retrieval to quantitatively determine the level of AntiSARS-CoV-2 Spike IgG. Reactive (antibody positive) patients were questioned to determine a history of vaccination or infection. Patients were divided into three groups based on their status. Women who were vaccinated (n = 26);women who had previous infection with SARS-CoV-2 (n=11) and women without a history of either vaccination or infection (n=91). Only patients receiving the mRNA vaccines from BioNTech / Pfizer (BNT162b2) and Moderna (mRNA-1273) were analyzed. Outcome measure for the three groups were initial implantation rate (serum hCG level > 5 mIU/mL obtained 8 days after embryo transfer followed by a rising level two to three days later), sustained implantation rate (transvaginal ultrasound documented positive FHTs at two time points at least one week apart) and miscarriage rate (the difference between initial and sustained implantation rates). Baseline characteristics were analyzed using ANOVA. Chi square analysis was used to compare pregnancy rates. RESULTS: CONCLUSIONS: Embryos produced from oocytes exposed in vivo to lipid nanoparticles containing mRNA for the SARS CoV-2 spike protein are not less likely to produce pregnancy or more likely to miscarry. IMPACT STATEMENT: This data refutes the rumors that Covid-19 vaccinations are “toxic” to the ovaries & adds to the growing body of evidence that vaccinations do not cause infertility. (Table Presented).

IVF/OOF OUTCOMES DURING THE COVID-19 PANDEMIC

OBJECTIVE: COVID-19 has affected nearly every facet of modern life, and has left many wondering what implications, if any, the virus has on reproductive health. Increased levels of psychological stress, concern for viral contamination in embryology labs, and reports of decreased male fertility following COVID infection, have also been thought to contribute negatively to ART outcomes.We sought to determine whether the pandemic resulted in any differences in IVF/OOF outcomes. MATERIALS AND METHODS: Patients who tested negative for COVID-19 and underwent GnRH-antagonist IVF and OOF cycles from January 2020 through December 2020 at NYU Fertility Center, a period marked by the COVID-19 pandemic, were separated by month of treatment and compared with patients from the corresponding month in the prior year. In patients with multiple cycles over this time period, only the first cycle was used. Patient age, AMH, #oocytes retrieved, #oocytes matured, #fertilized, #blastocysts, and #euploid embryos were compared using Student's T-test. RESULTS: 2,467 patients were compared. While the number of cycles were remarkably decreased over March and April of 2020 (59 and 25 respectively), the total number of cycles were very similar for the entire year (1,239 in 2019;1,228 in 2020). There were no consistently significant differences in age, AMH, #oocytes retrieved, #oocytes matured, #blastocysts formed, or #euploid embryos formed, between the two years. CONCLUSIONS: Despite initial concerns, and prior research suggesting otherwise, we did not detect any consistent quantitative or qualitative differences in retrieval outcomes amongst COVID negative patients receiving care during the pandemic. IMPACT STATEMENT: These results can reassure patients and their providers that IVF/OOF cycles can be continued safely during the pandemic without compromising outcomes.

Remdesivir impairs mouse preimplantation embryo development at therapeutic concentrations.

Remdesivir (RDV) is the first antiviral drug to be approved by the US Food and Drug Administration for the treatment of COVID-19. While the general safety of RDV has been studied, its reproductive risk, including embryotoxicity, is largely unknown. Here, to gain insights into its embryotoxic potential, we investigated the effects of RDV on mouse preimplantation embryos cultured in vitro at the concentrations comparable to the therapeutic plasma levels. Exposure to RDV (2-8 µM) did not affect the initiation of blastocyst formation, although the maintenance of the cavity failed at 8 µM due to increased cell death. While exposure to 2-4 µM permitted the cavity maintenance, expressions of developmental regulator genes associated with the inner cell mass (ICM) lineage were significantly diminished. Adverse effects of RDV depended on the duration and timing of exposure, as treatment between the 8-cell to early blastocyst stage most sensitively affected cavity expansion, gene expressions, and cell proliferation, particularly of the ICM than the trophectoderm lineage. GS-441524, a major metabolite of RDV, did not impair blastocyst formation or cavity expansion, although it altered gene expressions in a manner differently from RDV. Additionally, RDV reduced the viability of human embryonic stem cells, which were used as a model for the human ICM lineage, more potently than GS-441524. These findings suggest that RDV is potentially embryotoxic to impair the pluripotent lineage, and will be useful for designing and interpreting further in vitro and in vivo studies on the reproductive toxicity of RDV.

SARS-CoV-2 host receptors ACE2 and CD147 (BSG) are present on human oocytes and blastocysts.

PURPOSE: To visualize SARS-CoV-2 host receptors ACE2 and CD147 on human oocytes and blastocysts. METHODS: Immunohistochemistry and confocal microscopy on human primary oocytes and pre (5 days post fertilization (dpf5) and (dpf6))- and peri (dpf7)-implantation blastocysts donated to research. RESULTS: SARS-CoV-2 host receptors ACE2 and CD147 are present on the membrane of trophectoderm, epiblast and hypoblast cells in human blastocysts. CD147 is also present on the oolemma. CONCLUSION: Theoretically, the earliest stages of embryonic development may be vulnerable for SARS-CoV-2 infection.

Absence of SARS-CoV-2 (COVID-19 virus) within the IVF laboratory using strict patient screening and safety criteria.

RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.

Influence of seasonal differences on semen quality and subsequent embryo development of Belgian Blue bulls.

Belgian Blue bulls are more susceptible to high temperature and humidity index (THI) than most other cattle breeds. Here, we investigated whether high ambient temperature during summer affected semen quality and subsequent embryo development in Belgian Blue cattle. For this purpose, semen samples were collected from six healthy mature Belgian Blue bulls in March (Low THI group; THI between 30.6 and 56.4) and August 2016 (High THI group; maximum THI of 83.7 during meiotic and spermiogenic stages of spermatogenesis; 14-28 days prior to semen collection) respectively. Motility, morphology, acrosome integrity, chromatin condensation, viability, and reactive oxygen species production were assessed for frozen-thawed semen. Moreover, the efficiency of blastocyst production from the frozen-thawed semen samples of the two groups was determined in vitro. Blastocyst quality was determined by assessing inner cell mass ratio and apoptotic cell ratio. Fresh ejaculates showed a higher sperm concentration in low THI when compared to the high THI group (P ≤ 0.05), whereas semen volume, subjective motility, and total sperm output were not affected (P > 0.05). In frozen-thawed semen, total and progressive motility, viability, and straight-line velocity were lower in high THI compared to the low THI group (P < 0.05), while H2O2 concentration, aberrant chromatin condensation, and abnormal spermatozoa were higher in the high THI group (P < 0.05). Blastocyst rates were significantly higher when low THI samples were used (P < 0.05). Moreover, the total cell number and trophectoderm cells were significantly higher (P < 0.05) in blastocysts derived from low THI samples, whereas the apoptotic cell ratio was significantly higher (P < 0.01) in blastocysts derived from high THI spermatozoa. In summary, our data show that elevated ambient temperature and humidity during summer can decrease the quality of frozen-thawed spermatozoa in Belgian Blue bulls and also affect subsequent embryo development.