Function and interaction of coronavirus ion channel proteins

Objective: COVID-19 has emerged as a significant global health crisis causing devastating effects on world population accounting for over 6 million deaths worldwide. Although acute RTI is the prevalent cause of morbidity, kidney outcomes centered on a spectrum of AKI have evolved over the course of the pandemic. Especially the emerging variants have posed a daunting challenge to the scientific communities, prompting an urging requirement for global contributions in understanding the viral dynamics. In addition to canonical genes, several subgroup- specific accessory genes are located between the S and E genes of coronaviruses regarding which little is known. Previous studies have shown that accessory proteins (aps) in viruses function as viroporins that regulate viral infection, propagation and egress [1]. In this study we attempted to characterize the function of aps of coronavirus variants as ion channels. Furthermore, we also probed the interaction of ap4 with the host system. Method(s): Serial passaging (selection pressure), growth kinetics, confocal imaging, genome sequence analysis and proteomics were performed in Huh-7, MRC5 cells and/or human monocyte derived macrophages. Potassium uptake assay was performed in a Saccharo myces cerevisiae strain, which lacks the potassium transporters trk1 and trk2. Ion conductivity experiments were performed in Xenopus laevis oocytes using Two Electrode Voltage Clamp (TEVC) method. Result(s): Serial passaging demonstrated the acquisition of several frameshift mutations in ORF4 resulting in C-terminally truncated protein versions (ap4 and ap4a) and indicate a strong selection pressure against retaining a complete ORF4 in vitro. Growth kinetics in primary cells illustrated a reduction of viral titers when the full-length ap4 was expressed compared to the C-terminally truncated protein ap4a. Confocal imaging showed that ap4 and ap4a are not exclusively located in a single cellular compartment. Potassium uptake assay in yeast and TEVC analyses in Xenopus oocytes showed that ap4 and ap4a act as a weak K+ selective ion channel. In addition, accessory proteins of other virus variants also elicited microampere range of currents. Conclusion(s): Our study provides the first evidence that ap4 and other accessory proteins of coronavirus variants act as viroporins. Future studies are aimed at demonstrating the role of ap4 during the viral life cycle by modulating ion homeostasis of host cell in vivo (interacting proteins obtained from proteomic studies) and thereby serve as a tool for potential drug target.

Applications of advances in mRNA-based platforms as therapeutics and diagnostics in reproductive technologies.

The recent COVID-19 pandemic led to many drastic changes in not only society, law, economics, but also in science and medicine, marking for the first time when drug regulatory authorities cleared for use mRNA-based vaccines in the fight against this outbreak. However, while indeed representing a novel application of such technology in the context of vaccination medicine, introducing RNA into cells to produce resultant molecules (proteins, antibodies, etc.) is not a novel principle. It has been common practice to introduce/inject mRNA into oocytes and embryos to inhibit, induce, and identify several factors in a research context, while such aspects have also been proposed as potential therapeutic and diagnostic applications to combat infertility in humans. Herein, we describe key areas where mRNA-based platforms have thus far represented potential areas of clinical applications, describing the advantages and limitations of such applications. Finally, we also discuss how recent advances in mRNA-based platforms, driven by the recent pandemic, may stand to benefit the treatment of infertility in humans. We also present brief future directions as to how we could utilise recent and current advancements to enhance RNA therapeutics within reproductive biology, specifically with relation to oocyte and embryo delivery.

SARS-CoV-2 E-PROTEIN VIROPORIN INHIBITOR BIT225 ACTIVE IN hACE2 TRANSGENIC MICE

Background: The CoV-2 envelope (E) protein plays an important role in virus assembly, budding, immunopathogenesis and disease severity. E protein has ion channel activity, is located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we report that BIT225, an investigational HIV clinical compound, inhibits E ion channel activity and prevents body weight loss and mortality and reduces inflammation in lethally infected K18-hACE2 transgenic mice. BIT225 efficacy was observed when dosing was initiated before or 24 h or 48 h after infection. Method(s): SARS-CoV-2 E protein ion channel activity and Xenopus TMEM16A were measured in Xenopus oocytes. K18-hACE2 transgenic mice were infected intranasally with 104 pfu SARS CoV 2 (US-WA1/2020) and dosed orally twice daily with BIT225 for up to 12 Days. Dosing was initiated 12 h pre-infection or 24 h or 48 h post-infection. Disease parameters measured were survival, body weight, viral RNA by qPCR and infectious virus titre (plaque assay) in lung tissue homogenates and serum. In addition, levels of pro-inflammatory cytokines (IL-6, IL-1alpha, IL-1beta, TNFalpha & TGFbeta, MCP-1) were measured in lung and serum samples. Result(s): BIT225 inhibited ion channel activity of E-protein, but not that of TMEM16A in Xenopus oocytes. BIT225 dosed at 300mg/kg BID for 12 days starting 12 h pre-infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (n=12), while all vehicle-dosed animals reached a mortality endpoint by day 9 across two studies (n=12). Figure 1 shows results from a time of addition study: When treatment with BIT225 started at 24 h post-infection, body weight loss and mortality was also prevented (100% survival, n=5). In the group of mice where treatment started at 48 h after infection, body weight loss and mortality were prevented in 4 of 5 mice. Treatment efficacy was associated with significant reduction in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. Conclusion(s): BIT225 is an inhibitor of SARS-CoV-2 E-protein viroporin activity. In the K18 model BIT225 protected mice from weight loss and death, inhibited virus replication and reduced inflammation. These effects were noted when treatment with BIT225 was initiated before or 24-48 hours after infection and validate viroporin E as a viable antiviral target and support the clinical study of BIT225 in treatment of SARS-CoV-2.

Detection of SARS-CoV-2 in Follicular and Endocervical Fluid of in Vitro Fertilization Candidates with Positive Polymerase Chain Reaction Tests

Background: The coronavirus disease 2019 (COVID-19) pandemic was a unique global challenge with a wide range of severity extending from an asymptomatic form to a serious intense respiratory syndrome. This study aimed to detect SARS-CoV-2 in the follicular and endocervical fluid of in vitro fertilization (IVF) candidate patients with a positive polymerase chain reaction (PCR) test for SARS-CoV-2. Method(s): All participants and their partners, who were a candidate to start assisted reproductive technology (ART) from April 2020 to October 2020, completed a triage questionnaire two weeks before starting the ART cycle. According to Avicenna center protocol, a diagnostic test for COVID-19 using real-time PCR of nasopharyngeal swabs was performed on all ART candidates, 48 hours before the day of the oocyte trigger. In the operating room, sterile swabs were used to provide cervicovaginal specimens to determine SARS-CoV-2 in cervicovaginal fluid. Moreover, the first aspirated follicular fluid was referred to the lab to assess the presence of SARS-CoV-2. Result(s): A positive PCR test for SARS-CoV-2 was verified in 32 participants. In this study, virus particles were not detected in the follicular and endocervical fluid of the women with positive PCR tests. Conclusion(s): We are still at the beginning of the road and need reliable data on the safety of ART at the time of the pandemic. The risk of infection during all processes of ART including oocyte retrieval needs attention. The fluid from mature follicles is a potential site to be infected and the human cumulus cells could not be a deterrent factor to the entrance of the virus in the oocyte. This process may lead to gametes infection. We did not detect virus RNA in the follicular and endocervical fluid of the patients with a positive PCR test. Although, more studies with a larger sample size are mandatory in this field.

22nd International Congress of Iranian Society for Reproductive Medicine

The proceedings contain 158 papers. The topics discussed include: the success of various endometrioma treatments in infertility: a systematic review and meta-analysis;cell therapy accompanied by natural biomaterials, a novel therapeutic strategy for primary ovarian insufficiency treatment;ovarian hyperstimulation syndrome: a new look at an old problem;role of doppler ultrasonography and 3D ultrasound in female infertility;clinical outcome of artificial oocyte activation following intracytoplasmic sperm injection;the research priorities in infertility;how old is too old for infertility treatment?;the role of sexual dysfunction in men's health;recombinant follicle-stimulating hormone in treatment of sperm DNA fragmentation;the effect of zinc on tetrahydrocannabinol-induced Sertoli cells apoptosis;and detection of SARS-CoV-2 in follicular and endocervical fluid of in vitro fertilization candidates with positive polymerase chain reaction tests.

Detection of SARS-CoV-2 in Follicular Fluid of COVID-19 Infected Women

Background: The coronavirus disease-2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now spread all over the world. This study was designed to assess the possibility of SARS-CoV-2 presence in follicular fluid, which may have harmful effects on normal ovulation and fertility. Method(s): Five women who were candidates for Assisted Reproductive Technique (ART) and had a COVID-19 PCRpositive test on the day of oocyte retrieval participated in the study. SARS-CoV-2 tests were performed on the follicular fluid obtained from these women. Result(s): SARS-CoV-2 RNA was detected only in one follicular fluid sample, and other follicular fluid samples were negative. Conclusion(s): Because the COVID-19 effect on human reproduction is unknown, exact precautions should be taken during this pandemic, especially for women applying for ART.

The BISTIM study: a randomized controlled trial comparing dual ovarian stimulation (duostim) with two conventional ovarian stimulations in poor ovarian responders undergoing IVF.

STUDY QUESTION: Is the total number of oocytes retrieved with dual ovarian stimulation in the same cycle (duostim) higher than with two consecutive antagonist cycles in poor responders? SUMMARY ANSWER: Based on the number of total and mature oocytes retrieved in women with poor ovarian response (POR), there is no benefit of duostim versus two consecutive antagonist cycles. WHAT IS KNOWN ALREADY: Recent studies have shown the ability to obtain oocytes with equivalent quality from the follicular and the luteal phase, and a higher number of oocytes within one cycle when using duostim. If during follicular stimulation smaller follicles are sensitized and recruited, this may increase the number of follicles selected in the consecutive luteal phase stimulation, as shown in non-randomized controlled trials (RCT). This could be particularly relevant for women with POR. STUDY DESIGN, SIZE, DURATION: This is a multicentre, open-labelled RCT, performed in four IVF centres from September 2018 to March 2021. The primary outcome was the number of oocytes retrieved over the two cycles. The primary objective was to demonstrate in women with POR that two ovarian stimulations within the same cycle (first in the follicular phase, followed by a second in the luteal phase) led to the retrieval of 1.5 (2) more oocytes than the cumulative number of oocytes from two consecutive conventional stimulations with an antagonist protocol. In a superiority hypothesis, with power 0.8 alpha-risk 0.05 and a 35% cancellation rate, 44 patients were needed in each group. Patients were randomized by computer allocation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eighty-eight women with POR, defined using adjusted Bologna criteria (antral follicle count ≤5 and/or anti-Müllerian hormone ≤1.2 ng/ml) were randomized, 44 in the duostim group and 44 in the conventional (control) group. HMG 300 IU/day with flexible antagonist protocol was used for ovarian stimulation, except in luteal phase stimulation of the duostim group. In the duostim group, oocytes were pooled and inseminated after the second retrieval, with a freeze-all protocol. Fresh transfers were performed in the control group, frozen embryo transfers were performed in both control and duostim groups in natural cycles. Data underwent intention-to-treat and per-protocol analyses. MAIN RESULTS AND THE ROLE OF CHANCE: There was no difference between the groups regarding demographics, ovarian reserve markers, and stimulation parameters. The mean (SD) cumulative number of oocytes retrieved from two ovarian stimulations was not statistically different between the control and duostim groups, respectively, 4.6 (3.4) and 5.0 (3.4) [mean difference (MD) [95% CI] +0.4 [-1.1; 1.9], P = 0.56]. The mean cumulative numbersof mature oocytes and total embryos obtained were not significantly different between groups. The total number of embryos transferred by patient was significantly higher in the control group 1.5 (1.1) versus the duostim group 0.9 (1.1) (P = 0.03). After two cumulative cycles, 78% of women in the control group and 53.8% in the duostim group had at least one embryo transfer (P = 0.02). There was no statistical difference in the mean number of total and mature oocytes retrieved per cycle comparing Cycle 1 versus Cycle 2, both in control and duostim groups. The time to the second oocyte retrieval was significantly longer in controls, at 2.8 (1.3) months compared to 0.3 (0.5) months in the duostim group (P < 0.001). The implantation rate was similar between groups. The cumulative live birth rate was not statistically different, comparing controls versus the duostim group, 34.1% versus 17.9%, respectively (P = 0.08). The time to transfer resulting in an ongoing pregnancy did not differ in controls 1.7 (1.5) months versus the duostim group, 3.0 (1.6) (P = 0.08). No serious adverse events were reported. LIMITATIONS, REASONS FOR CAUTION: The RCT was impacted by the coronavirus disease 2019 pandemic and the halt in IVF activities for 10 weeks. Delays were recalculated to exclude this period; however, one woman in the duostim group could not have the luteal stimulation. We also faced unexpected good ovarian responses and pregnancies after the first oocyte retrieval in both groups, with a higher incidence in the control group. However, our hypothesis was based on 1.5 more oocytes in the luteal than the follicular phase in the duostim group, and the number of patients to treat was reached in this group (N = 28). This study was only powered for cumulative number of oocytes retrieved. WIDER IMPLICATIONS OF THE FINDINGS: This is the first RCT comparing the outcome of two consecutive cycles, either in the same menstrual cycle or in two consecutive menstrual cycles. In routine practice, the benefit of duostim in patients with POR regarding fresh embryo transfer is not confirmed in this RCT: first, because this study demonstrates no improvement in the number of oocytes retrieved in the luteal phase after follicular phase stimulation, in contrast to previous non-randomized studies, and second, because the freeze-all strategy avoids a pregnancy with fresh embryo transfer after the first cycle. However, duostim appears to be safe for women. In duostim, the two consecutive processes of freezing/thawing are mandatory and increase the risk of wastage of oocytes/embryos. The only benefit of duostim is to shorten the time to a second retrieval by 2 weeks if accumulation of oocytes/embryos is needed. STUDY FUNDING/COMPETING INTERESTS: This is an investigator-initiated study supported by a research Grant from IBSA Pharma. N.M. declares grants paid to their institution from MSD (Organon France); consulting fees from MSD (Organon France), Ferring, and Merck KGaA; honoraria from Merck KGaA, General Electrics, Genevrier (IBSA Pharma), and Theramex; support for travel and meetings from Theramex, Merck KGaG, and Gedeon Richter; and equipment paid to their institution from Goodlife Pharma. I.A. declares honoraria from GISKIT and support for travel and meetings from GISKIT. G.P.-B. declares Consulting fees from Ferring and Merck KGaA; honoraria from Theramex, Gedeon Richter, and Ferring; payment for expert testimony from Ferring, Merck KGaA, and Gedeon Richter; and support for travel and meetings from Ferring, Theramex, and Gedeon Richter. N.C. declares grants from IBSA pharma, Merck KGaA, Ferring, and Gedeon Richter; support for travel and meetings from IBSA pharma, Merck KGaG, MSD (Organon France), Gedeon Richter, and Theramex; and participation on advisory board from Merck KGaA. E.D. declares support for travel and meetings from IBSA pharma, Merck KGaG, MSD (Organon France), Ferring, Gedeon Richter, Theramex, and General Electrics. C.P.-V. declares support for travel and meetings from IBSA Pharma, Merck KGaA, Ferring, Gedeon Richter, and Theramex. M.Pi. declares support for travel and meetings from Ferring, Gedeon Richetr, and Merck KGaA. M.Pa. declares honoraria from Merck KGaA, Theramex, and Gedeon Richter; support for travel and meetings from Merck KGaA, IBSA Pharma, Theramex, Ferring, Gedeon Richter, and MSD (Organon France). H.B.-G. declares honoraria from Merck KGaA, and Gedeon Richter and support for travel and meetings from Ferring, Merck KGaA, IBSA Pharma, MSD (Organon France), Theramex, and Gedeon Richter. S.G. and M.B. have nothing to declare. TRIAL REGISTRATION NUMBER: Registration number EudraCT: 2017-003223-30. ClinicalTrials.gov identifier: NCT03803228. TRIAL REGISTRATION DATE: EudraCT: 28 July 2017. ClinicalTrials.gov: 14 January 2019. DATE OF FIRST PATIENT'S ENROLMENT: 3 September 2018.

Does mRNA COVID-19 vaccination in oocyte donors impact ovarian stimulation parameters or IVF outcomes for recipients?

RESEARCH QUESTION: What is the effect of mRNA severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in young oocyte donors in terms of ovarian response to stimulation, fertilization rate, embryo development and clinical outcomes in recipients? DESIGN: This retrospective, multicentre cohort study evaluated 115 oocyte donors who had undergone at least two ovarian stimulation protocols (before and after complete SARS-CoV-2 vaccination) between November 2021 and February 2022. Comparisons were made of the primary outcomes of days of stimulation, total dose of gonadotrophins and laboratory performance in ovarian stimulation in oocyte donors before and after vaccination. A total of 136 cycles in matched recipients were analysed as secondary outcomes and, from those, 110 women received a fresh single-embryo transfer, with analysis of biochemical ß-human chorionic gonadotrophin concentrations and rates of clinical pregnancy with heartbeat. RESULTS: Longer stimulation was required in the post-vaccination than pre-vaccination group (10.31 ± 1.5 versus 9.51 ± 1.5 days; P < 0.001) along with higher gonadotrophin consumption (2453.5 ± 740 versus 2235.5 ± 615 IU; P < 0.001) with a similar starting dose of gonadotrophins in both groups. More oocytes were retrieved in the post-vaccination group (16.62 ± 7.1 versus 15.38 ± 7.0; P = 0.02). However, the number of metaphase II (MII) oocytes was similar between groups (pre-vaccination 12.61 ± 5.9 versus post-vaccination 13.01 ± 6.6; P = 0.39) and the ratio of MII/retrieved oocytes favoured the pre-vaccination group (0.83 ± 0.1 versus 0.77 ± 0.2 post-vaccination; P = 0.019). In recipients with a similar number of provided oocytes, the fertilization rate, total number of obtained blastocysts, number of top-quality blastocysts, and rates of biochemical pregnancy and clinical pregnancy with heartbeat were not significantly different between groups. CONCLUSIONS: This study shows no adverse influence of mRNA SARS-CoV-2 vaccination on ovarian response in a young population.

Is There any Effect of COVID-19 on Follicular Environment in Women Underwent ART?

There is insufficient data on the impact of severe acute respiratory coronavirus-2 (SARS-CoV-2) on the reproductive tissues, its possible risk of cross-contamination, transmission and adverse effect on in vitro fertilization (IVF) outcome. Until today, there is no report associated with viral RNA in both follicular fluid and embryo culture medium from SARS-COV-2 positive women. In this case report, a 24-year-old woman with SARS-CoV-2 was presented. We investigated the SARS-COV-2 positivity in the follicular fluid and embryo culture medium of mildly symptomatic woman on oocyte pick up (OPU) day. We could not detect viral RNA in neither the follicular fluid nor the embryo culture medium. In addition, although the response of ovarian stimulation was normal, the number and maturity of the retrieved oocytes were low.

Successful Pregnancies, Births, and Children Development Following Oocyte Cryostorage in Female Cancer Patients During 25 Years of Fertility Preservation.

The preservation of fertility in cancer patients is a crucial aspect of modern reproductive medicine. Amenorrhea and infertility often occur after cancer therapy, worsening the quality of life. Cryopreservation of oocytes in young cancer patients is a therapeutic option for preserving fertility. A prospective study was conducted on 508 cancer patients who underwent oocyte cryopreservation to preserve fertility between 1996 and 2021 including the COVID-19 pandemic period. Patients underwent ovarian stimulation, followed by egg retrieval, and oocytes were cryopreserved by slow freezing or vitrification. Sixty-four thawing/warming cycles were performed. Survival, fertilization, pregnancy, and birth rate over the thawing/warming cycles were obtained. The data were compared with those from a group of 1042 nononcological patients who cryopreserved supernumerary oocytes. An average of 8.8 ± 6.9 oocytes were retrieved per cycle, and 6.1 ± 4.2 oocytes were cryopreserved. With their own stored oocytes, 44 patients returned to attempt pregnancy. From a total of 194 thawed/warmed oocytes, 157 survived (80%). In total, 100 embryos were transferred in 57 transfer/cycles, and 18 pregnancies were achieved. The pregnancy rate per transfer and pregnancy rate per patient were 31% and 41%, respectively. No statistically significant differences were observed between oncological patients and nononcological patients. A total of 15 babies were born from oncological patients. Children born showed normal growth and development. One minor malformation was detected.

SARS-CoV-2 RNA in Follicular Fluid, Granulosa Cells, and Oocytes of COVID-19 Infected Women Applying for Assisted Reproductive Technology

Background: The Coronavirus disease 2019 (COVID-19) pandemic has raised concerns regarding the application of assisted reproductive technology (ART) in the world. Many ART programs have been delayed or continued with new precautions due to the ambiguity about vertical transmission and pregnancy safety. Regarding the possible risks of SARS-CoV-2 infection on ART and the resultant embryos, this study aimed to investigate the presence of SARS-CoV-2 in follicular fluid, granulosa cells, and oocytes of COVID-19-infected women undergoing ART. Materials and Methods: COVID-19-positive polymerase chain reaction tests were reported for five women undergoing ART cycles on the day of oocyte retrieval. SARS-CoV-2 tests were performed on oocytes, granulosa cells, and follicular fluid obtained from these COVID-19-infected women. Results: SARS-CoV-2 RNA was detected only in one follicular fluid sample;however, other follicular fluid samples, granulosa cells, and oocytes were negative regarding viral RNA. Conclusion: Given the unknown effects of COVID-19 on human reproduction and ART, strict precautions should be taken during the COVID-19 pandemic. [GMJ.2022;11:e2638] DOI:10.31661/gmj.v11i.2638

An Update Review of the Pathogenesis Hypothesis in Preeclampsia

Objectives: Hypertensive disorders occur in approximately 12% to 22% of pregnancies and cause substantial perinatal morbidity and mortality of both mother and fetus. Hypertensive disease is directly responsible for approximately 20% of maternal deaths and can be classified as chronic hypertension, gestational hypertension, preeclampsia-eclampsia, and chronic hypertension with superimposed preeclampsia. At present, the pathogenesis of preeclampsia is still unclear, we wrote this article to make a uptodate review of this disease. Mechanism: A comprehensive search of several databases was conducted from inception up to March 2022. The searched databases were Web of Science, MEDLINE, Ovid, and Cochrane Database of Systematic Reviews. The search strategy included the combinations of the following medical terms: Hypertensive disorders;preeclampsia;mechanism;pathogenesis hypothesis. Findings in Brief: At present, the pathogenesis of preeclampsia is still unclear, the theory of Genetic, Inflammatory Response, Immune Imbalance in Maternal-Fetal Interface, Oxidative Stress, Vascular Endothelial Cell Damage are supposed involved in the progress of preeclampsia. Conclusions: Although there are various theories mentioned above, none of the hypothesis can fully explain preeclampsia. More research is needed on the mechanism of preeclampsia.

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.

From insult to injury: war and cross border reproductive care (CBRC). Difficulties for patients and collaborators

From Insult to Injury: War and Cross Border Reproductive Care (CBRC) after Covid Ukraine has had a thriving fertility sector for many years, both for its citizens and foreigners, for whom it has been a very popular destination for cross-border commercial surrogacy, with an estimate of between 2000 and 2500 babies born each year. Such cross border reproductive activities had already been complicated by the Covid pandemics of the last 2 years, but when Russia invaded the country in February 2022, intended parents, surrogates, egg donors, agencies and local clinics faced an emergency which led to acute distress in addition to that already felt by all with a sudden war in Europe. We discuss the available facts with regards to the disruption to fertility services for all concerned, as well as the complex legal and ethical issues surrounding this emergency. Neighboring countries have been accepting (mostly women) refugees, who may need health care in a system stressed by extra numbers of patients. Intended parents have been trying to ensure their plans are confirmed whilst their surrogate has probably moved to a different jurisdiction. Above all, the voice of collaborators may have been overwhelmed by that of the (future) families they are contracted to help. All this, will leave its mark on CBRC in an area which has been a traditional European and recently Chinese hub for years to come.

The BISTIM study: first RCT comparing dual ovarian stimulation on the same cycle (duostim) vs 2 conventional ovarianstimulations in poor ovarian responders undergoing IVF

Study question: Is the number of cumulated oocytes with dual ovarian stimulation on the same cycle (duostim) higher compared to 2 consecutive antagonist cycles in poor responders? Summary answer: Considering the number of total and mature oocytes collected in poor ovarian responders, there is no benefit of duostim vs two consecutive antagonist cycles. What is known already: Several waves of follicular development exist on the same cycle. Recent studies have shown the ability to obtain oocytes with equivalent quality in the luteal phase, even after a previous ovarian stimulation in the follicular phase. During stimulation, smaller follicles are recruited and sensitized, which may increase the selection of follicles available on the second stimulation. In poor ovarian responders (POR) this potentialization may have a great interest, as 2 stimulations on the same cycle could give a higher number of oocytes compared to two conventional stimulations. However, these preliminary data need to be confirmed with a randomized controlled trial. Study design, size, duration: This is a multicenter, open-labeled randomized control trial (2018, september-2021, march). The primary objective was to demonstrate that two ovarian stimulations within the same cycle (first in the follicular phase, followed by a second in the luteal phase) lead to the retrevial of 1.5 more oocytes than the cumulative number of oocytes from two consecutive conventional stimulation, in POR women. According to this hypothesis, 44 patients were needed in each group. Participants/materials, setting, methods: 88 POR women, defined with Bologna criteria (AFC≤5 and/or AMH≤1.2ng/ml and ≤3 oocytes if previous IVF) were randomized, 44 in duostim group (D) and 44 in conventional group (C). Fertistart Kit®300IU/day with antagonist protocol was used except in luteal phase stimulation of group D. In group D, oocytes were pooled and inseminated after the second retrieval, with freeze all embryos. Fresh transfer was performed in group C. The analysis is presented in intention to treat. Main results and the role of chance: There was no difference between the groups regarding demographics, ovarian reserve markers (AFC, AMH) and stimulation parameters. The mean number of cumulated oocytes retrieved with 2 ovarian stimulation was not statistically different in group D and C, respectively 5.0+/-3.4 and 4.6+/-3.4 (p=0.56). The mean number of cumulated mature oocytes was not statistically different, 3.7+/-3.3 in group D vs 3.1+/-3.0 in group C (p=0.38). The mean number of embryos was significantly lower in the group D, 0.8+/-1.3 vs group C 1.6+/-1.3 (p<0.01). There was no statistical difference of the mean number of oocytes retrieved per cycle in cycle 1 vs cycle 2 in both group D and C. The delay, between the first and the second day 1 of stimulation was statistically different in group D 14.4 days (10-19) vs group C 90.6 (28-232). The ongoing pregnancy rate in group D 17.9% (7/39) was not statistically different with group C 29.3% (12/41), (p=0.23). And the mean time to ongoing pregnancy tends to be longer in group D (144 days) vs group C (115 days) but was not statistically different (p=0.21). Limitations, reasons for caution: The RCT was impacted by Covid pandemia and stop of IVF activities for 10 weeks. Delays were recalculated to exclude this period, however one women in group D cannot have the luteal stimulation. We also faced unexpected good ovarian responses and pregnancies after the first oocyte pick-up in group C. Wider implications of the findings: In routine practice, the benefit of duostim in patients with POR is not confirmed. Firstly, because there is no potentialization on the number of oocyte retrieved in luteal phase after follicular phase stimulation. Secondly, because the freeze all strategy avoids a pregnancy with fresh embryo transfer after the first cycle.

Pfizer SARS-CoV-2 BNT162b2 mRNA vaccination (BNT162b2) has no adverse effect on elective oocyte cryopreservation outcomes.

RESEARCH QUESTION: Do elective oocyte cryopreservation outcomes in women 1-13 months after SARS-CoV-2 vaccination alter compared with unvaccinated women and do different time intervals between vaccination and ovarian stimulation impact these outcomes? DESIGN: This retrospective cohort study, conducted in a university-affiliated IVF centre, included 232 elective oocyte cryopreservation cycles of vaccinated and unvaccinated patients, without previous infection with the SARS-CoV-2 virus, between December 2020 and January 2022. Two control groups - pre-pandemic (January 2019 to February 2020) and intra-pandemic (December 2020 to January 2022) unvaccinated groups - were compared with the vaccinated group, further divided into four subgroups (under 3, 3-6, 6-9 and 9-13 months). The primary outcome was the elective oocyte cryopreservation cycle outcomes - number of retrieved and number of mature oocytes. RESULTS: The vaccinated group demonstrated comparable outcomes with regards to number of retrieved and mature oocytes compared with the pre-pandemic and intra-pandemic unvaccinated groups (12.6 ± 8.0 versus 13.0 ± 8.2 and 12.5 ± 7.4 retrieved and 10.1 ± 6.9 versus 9.5 ± 6.4 and 10.1 ± 6.3 mature oocytes, respectively; not significant for both). Similar results were noted in a comparison between the intra-pandemic unvaccinated group and the four vaccinated subgroups. No correlation was found between the parameter of days from vaccination and cycle outcomes. Similarly, analysis of covariance showed no association between vaccination status and timing and number of mature oocytes. CONCLUSIONS: The SARS-CoV-2 vaccination does not alter the outcomes of elective oocyte cryopreservation procedures. This is true even in a relatively long time interval of 9 to 13 months from vaccination.

COVID-19 infection control protocol and acceptance in an ART in vitro fertilization hospital.

Purpose: In Tokyo, where the highest coronavirus disease 2019 (COVID-19) infection rates have been reported nationally, we introduced and performed polymerase chain reaction (PCR) testing on the patients prior to them coming for oocyte retrieval (OR) or embryo transfer (ET) procedures. In addition, we recommended that patients self-inject ovarian stimulation drugs to reduce the number of hospital visits required. We aimed to assess the patient acceptance of these measures and the change of treatment number. Methods: We conducted a retrospective study examining the patients coming for OR or ET, from the first time a state of emergency was declared in Japan, May 2020, until September 2021. Results: A total of 79 out of 94 (94%) patients complied with the measures. This may reflect that PCR universal screening was accepted by most patients as necessary for reducing infection spread. In addition, the number of patients receiving OR and ET increased. The widespread adoption of work-from-home practices during the pandemic has made outpatient visits more acceptable to the general public. Conclusions: Universal screening and self-injection are accepted and effective infection measures in patients presenting for OR and ET.

American College of Obstetricians and Gynecologists 2022 Annual Clinical and Scientific Meeting

The proceedings contain 344 papers. The topics discussed include: estimating in vivo levonorgestrel release rate and exposure over eight years with levonorgestrel releasing intrauterine system 52 mg use with population pharmacokinetic approach;immediate vs routine postpartum intrauterine device placement after teen pregnancy: a cost-effectiveness analysis;examining the association of immediate postpartum long-acting reversible contraception payment carve-outs and postpartum LARC use in Medicaid;contraceptive congruence: a novel measure of contraceptive use that acknowledges pregnancy ambivalence;charm 2: a gender synchronized family planning intervention for couples in rural India, a cluster randomized trial;telehealth follow-up after medical management for early pregnancy loss;providing mifepristone and misoprostol in emergency departments during the COVID-19 pandemic;and medical students' knowledge of and attitudes towards oocyte cryopreservation.