Is there evidence of a plateau in the cumulative live birth rate after a certain number of consecutive transfers of untested embryos? In 11 463 women, cumulative live birth rates increased with each additional untested embryo transferred, reaching 68.3% after six and 78.0% after ten blastocyst transfers. European IVF-monitoring data report pregnancy rates per transfer around 33% in 2020. Despite advances, not every embryo leads to live birth, and rising success rates may create unrealistic patient expectations. Recurrent implantation failure (RIF), often diagnosed after multiple failed cycles, highlights the need to assess cumulative live birth rates (CLBR). A reported 98% CLBR after five euploid blastocyst transfers suggests most failures are embryonic, not endometrial. Theoretical models using blastocyst euploidy rates by female age estimate cumulative implantation probabilities, but real-world data on cumulative success in patients without preimplantation genetic testing for aneuploidy (PGT-A) remain lacking. Non-interventional retrospective cohort study including records of all completed IVF/ICSI cycles (including fresh and frozen-thawed transfers of one oocyte retrieval cycle) at the Ghent University Hospital between January 2010 and December 2022. After excluding treatments involving PGT, egg donation and surrogacy, or a mix of cleavage stage and blastocyst stage transfers, our dataset consisted of 11 463 women who underwent in total 19 378 IVF/ICSI cycles, resulting in 31 478 embryo transfers. The number of embryos transferred ('time') until achieving live birth ('event') was analysed using a Kaplan–Meier approach with Inverse Probability Weighting (IPW). Additionally, logistic regression analysis was conducted to assess the predictive value of the number of previously transferred embryos on the live birth rates of the second and subsequent transfers, adjusting for female age, quality of previously transferred embryos, and stage of embryos transferred (cleavage stage versus blastocyst stage). Kaplan-Meier estimates using an IPW approach showed cumulative live birth rates (cLBR) increasing from 51.1% (95% CI: 49.2–53.0%) after three blastocyst transfers to 68.3% (95% CI: 64.6–72.0%) after six and 78.0% (95% CI: 69.5–86.5%) after ten transfers. Higher numbers of blastocysts are required to achieve the same cLBR as maternal age increases, with no age group reaching 80% cLBR until after eight transfers. After the fourth transfer, cLBRs were 68.9% (<35 years), 57.6% (35–37 years), 42.9% (38–40 years), 16.3% (41–42 years), and 13.5% (>42 years). Adjusted logistic regression showed no significant odds decrease per additional embryo transferred (OR = 0.91; 95% CI: 0.86–1.07). Maternal age was a significant predictor of live birth rate (OR = 0.92; 95% CI: 0.91–0.93), as were blastocyst transfer strategy over cleavage stage (OR = 1.34; 95% CI: 1.20–1.51) and the proportion of excellent/good-quality embryos transferred (OR = 1.21; 95% CI: 1.06–1.38). Live birth rate correlated with response to stimulation (p = 0.016) and blastocyst formation rate (p < 0.0001). Live birth rates after unsuccessful blastocyst transfers did not significantly differ based on the number of oocyte collection cycles needed to reach that number of blastocysts. The observational retrospective design limits the results, and residual confounding may remain despite adjustments for potential confounders. Patients with factors linked to less favourable outcomes were included, reflecting the heterogeneity of treatments and aiming to evaluate real-world clinical practice. Our data emphasize the potential for successful live birth even after multiple unsuccessful transfers. Factors such as age, embryo quality, response to ovarian stimulation, and rate of blastocyst formation influence outcomes. By addressing these multifaceted influences, our research provides valuable insights and hope for individuals experiencing repeated implantation failure. No

The composition of fatty acids (FA) in follicular fluid (FF) can be influenced by both dietary FA and endogenous lipid mobilization. Since the latter predominantly occurs during early lactation, we compared the impact of dietary lipid supplementation on the FA composition in FF lipid classes at two distinct time points in lactation. Furthermore, we investigated how the correlations between plasma and FF FA were affected by lipid supplementation and postcalving time. Twenty Holstein multiparous cows were randomly assigned to 200 g/d of prilled saturated fat in the rumen (n = 10) or sea buckthorn oil (SBT) in the abomasum (n = 10); specially, prilled saturated fat supplements enriched in palmitic acid (82 % 16:0; PA treatment) while SBT treatment included 27 % 16:1n-7, 28 % 16:0, 7.8 % 18:1n-7, 22 % 18:1n-9 and 11 % 18:2n-6. The treatment period ranged from 20 +/- 5 d precalving to 67 +/- 2 d (mean +/- standard deviation) postcalving. Plasma and FF samples were collected from 2 sequential sessions of ovum pick-up (OPU-1 and OPU-2) at 46 and 67 +/- 2 d postcalving. Except for 18:1n-9, proportions of the main unsaturated FA, supplied by SBT (i.e. 16:1n-7, 18:1n-7, and 18:2n-6) were increased (P-fat < 0.05) in most lipid fractions of plasma and FF as compared with PA group. Nevertheless, - particularly for 16:1n-7 - the response depended on the sampling time (P-fat x time < 0.10) was of lower magnitude at OPU-1. Despite the 18:1n-9 supply to the SBT group, proportions of this FA in the phospholipid (PL) and cholesterol esters (CHE) fractions of plasma and FF were lower as compared with the PA group (P-fat < 0.001). However, in the plasma PL fraction the difference between fat sources was greater at OPU-1 than at OPU-2 (P-fat x time < 0.05). Additionally, the ratio of 18:1n-9-18:0 decreased in the plasma non-esterified FA (NEFA) and PL fractions from OPU-1 to OPU-2 (P-time < 0. 10), while the ratio increased in FF NEFA and CHE (P-time < 0.05), irrespective of the fat source (P-fat x time > 0.05 except for plasma PL). Differences between lactation stages, either or not dependent on fat supplementation, were hypothesized to be related to the (need for) alleviation of lipotoxicity through endogenous desaturation. Furthermore, except for 16:1n-7, plasma and FF NEFA were uncorrelated, while esterified plasma and FF FA showed higher correlations at OPU-2 than OPU-1. As such, (dietary) interventions with MUFA-enriched supplements potentially are less effective earlier after calving.

STUDY QUESTION:Is there evidence of a plateau in the cumulative live birth rate (cLBR) after a certain number of consecutive transfers of untested embryos? SUMMARY ANSWER:In our cohort of 11 463 women, the cLBR continues to increase with each additional transfer of an untested embryo, reaching 68.3% after six blastocyst transfers and 78.0% after 10 blastocyst transfers. WHAT IS KNOWN ALREADY:While cumulative success rates in ART are rising, implantation failure remains a persistent challenge. The actual frequency of recurrent implantation failure (RIF) and whether RIF surpasses the inherent implantation potential of transferred embryos remains a matter of debate. A recent study reported a cLBR of 98% after five euploid blastocyst transfers, suggesting that most implantation failures are likely embryonic rather than endometrial. However, it remains unclear how these findings can be extrapolated to patients who did not undergo preimplantation genetic testing for aneuploidy (PGT-A). While theoretical models estimate cumulative implantation probabilities based on published blastocyst euploidy rates by female age, real-world data on cumulative success in routine clinical practice remain limited. STUDY DESIGN, SIZE, DURATION:This non-interventional retrospective cohort study included records of all completed IVF/ICSI cycles (including thus fresh and frozen-thawed transfers of one oocyte retrieval cycle) in women who underwent IVF/ICSI at the Ghent University Hospital between January 2010 and December 2022. After excluding treatments involving PGT, oocyte donation and surrogacy, or a mix of cleavage stage and blastocyst stage transfers, our dataset consisted of 11 463 women who underwent a total of 19 378 IVF/ICSI cycles, resulting in a total of 31 478 embryo transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS:The number of embryos transferred ('time') until achieving live birth ('event') was analysed using a Kaplan-Meier approach with inverse probability weighting (IPW). Additionally, logistic regression analysis was conducted to assess the predictive value of the number of previously transferred embryos on the live birth rates (LBRs) of the second and subsequent transfers, adjusting for female age, quality of previously transferred embryos, and stage of embryos transferred (cleavage stage versus blastocyst stage). MAIN RESULTS AND THE ROLE OF CHANCE:Kaplan-Meier estimates using an IPW approach showed cLBRs increasing from 51.1% (95% CI: 49.2-53.0%) after a third, up to 68.3% (95% CI: 64.6-72.0%) after a sixth and even as high as 78.0% (95% CI: 69.5-86.5%) after a tenth blastocyst transfer, respectively. As maternal age increases, higher numbers of blastocysts are required to achieve the same cLBR. Moreover, no age category achieves an 80% cLBR until after the transfer of eight blastocysts. Maternal age has a considerable effect, as illustrated by cLBR after the fourth blastocyst transfer of 68.9% (95% CI: 65.8-71.8%) for <35 years; 57.6% (95% CI: 50.4-64.8%) for 35-37 years; 42.9% (95% CI: 37.5-48.4%) for 38-40 years; 16.3% (95% CI: 10.7-21.8%) for 41-42 years; and 13.5% (95% CI: 3.2-23.7%) for >42 years, respectively. In the adjusted logistic regression analysis, the odds for achieving live birth are estimated to decrease for each additional embryo transferred; however, this effect is not statistically significant (OR = 0.91; 95% CI: 0.86-1.07). Female age, as expected, was a significant predictor of implantation rate with subsequent transfers (OR = 0.92; 95% CI: 0.91-0.93). Additionally, being assigned to a blastocyst transfer strategy rather than to a cleavage stage transfer strategy was also a significant predictor (OR = 1.34; 95% CI: 1.20-1.51), as was proportion of embryos classified as excellent or good quality based on predefined morphological criteria out of the total number of embryos previously transferred (OR = 1.21; 95% CI: 1.06-1.38). Implantation rate is also correlated with the response to stimulation (P = 0.016) and the blastocyst formation rate (P < 0.0001). There was no significant difference in LBR after an equal number of previously unsuccessful blastocyst transfers, depending on how many oocyte collection cycles it took to reach that number of blastocysts. LIMITATIONS, REASONS FOR CAUTION:The results are limited by the observational retrospective design, and while regression analyses were adjusted for potential confounding factors, residual confounding may persist, particularly given the considerable heterogeneity in treatments. We did not exclude patients with factors associated with less favourable reproductive outcomes because our goal was to evaluate actual clinical practice. WIDER IMPLICATIONS OF THE FINDINGS:Our data emphasize the potential for successful live birth even after multiple unsuccessful transfers. Factors such as age, embryo quality, response to ovarian stimulation, and rate of blastocyst formation influence outcomes. By addressing these multifaceted influences, our research provides valuable insights and a hopeful outlook for patients undergoing fertility treatment. STUDY FUNDING/COMPETING INTEREST(S):L.D. received a grant from the Agency for Innovation through Science (IWT SB-141441). The funder did not have any role in the study design; data collection, data analysis, and interpretation of data; the writing of the report; nor the decision to submit the paper for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. TRIAL REGISTRATION NUMBER:N/A.

Introduction Although sexual health has been holistically defined to include sexual satisfaction, it has been largely absent in health services and sexual and reproductive health and rights programmes in many parts of the world. We propose sexual satisfaction as a useful indicator, as one of the proxy measures for sexual health and well-being and as a component of well-being in general.Methods The Sialon II project is a multicentre biological and behavioural cross-sectional community-based survey implemented across 13 European cities during 2013–2014 among men who have sex with men. Sexual satisfaction was explored using one single item: ‘How satisfied are you with your sex life?’ A multivariable multilevel logistic random-intercept model was estimated to identify factors associated with reporting positive sexual satisfaction versus negative sexual satisfaction.Results Age, the number of partners and self-reported HIV status were not significantly associated with sexual satisfaction in the multivariate model. Participants reporting an insertive role or reported both an insertive and receptive role during the last anal intercourse were more likely to be sexually satisfied, compared with a receptive role. Participants reporting anal intercourse with a condom were more likely to be satisfied than those declaring no anal intercourse in the last 6 months, but no significant association was found compared with anal intercourse without condom. Knowledge of HIV-serostatus concordance with the last sexual partner was positively correlated with sexual satisfaction. Having had sexual intercourse with non-steady partners only in the last 6 months was negatively correlated. The more positive participants perceived their work/school, parents and friends/acquaintances’ attitudes towards gay or bisexual persons, the higher the odds they were satisfied with their sexual life.Conclusion Using a single item on sexual satisfaction in a bio-behavioural study, our analysis has shown that it is associated with individual, interpersonal and social/structural factors and has proven its usefulness as a sexual health indicator among men who have sex with men.

Fatty acids (FA) in follicular fluid (FF) are present in an esterified form [triglycerides, cholesterol esters and phospholipids] or as non-esterified FA, which partly originate from blood. However, a comprehensive comparison of blood vs. FF FA in various lipid classes is missing. The aim of this study was to determine the distribution of the FA composition in each lipid class of serum and FF, and to investigate their mutual correlations. A total of 74 patients undergoing assisted reproductive technology treatment were involved in the study. Both in serum as well as FF, saturated FA and mono-unsaturated FA were predominant in non-esterified FA and triglycerides fractions while poly-unsaturated FA were mainly present in phospholipids and cholesterol esters fractions, although phospholipids also contained high proportions of saturated FA. Irrespective of the lipid class, the FA proportions differed between serum and FF (P < 0.05). Despite these differences, most of the FA in triglycerides, phospholipids and cholesterol esters of FF were well correlated with their proportions in serum. Nevertheless, only weak to moderate associations (r < 0.60) were observed for the majority of the FA in the non-esterified FA fraction. Differences in FA product/precursor-ratios were found between serum and FF, such as higher C20:4n-6 to C18:2n-6 and C20:5n-3 to C18:3n-3 in FF. FA metabolism (e.g. desaturation and elongation) takes place in cells of the intrafollicular micro-environment. Moreover, good correlations between esterified FA in serum and FF suggest esterified FA in blood could be representative of esterified FA in FF.

STUDY QUESTION Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER NCT03354013.

Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targeted GENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.

STUDY QUESTION:Can spindle transfer (ST) overcome inferior embryonic development of in vitro matured ovarian tissue oocytes (OTO-IVM) originating from testosterone-treated transgender men?SUMMARY ANSWER:ST shows some potential to overcome the embryo developmental arrest observed in OTO-IVM oocytes from transgender men.WHAT IS KNOWN ALREADY:OTO-IVM is being applied as a complementary approach to increase the number of oocytes/embryos available for fertility preservation during ovarian tissue cryopreservation in cancer patients. OTO-IVM has also been proposed for transgender men, although the potential of their oocytes remains poorly investigated. Currently, only one study has examined the ability of OTO-IVM oocytes originating from transgender men to support embryo development, and that study has shown that they exhibit poor potential.STUDY DESIGN, SIZE, DURATION:Both ovaries from 18 transgender men undergoing oophorectomy were collected for the purposes of this study, from November 2020 to September 2022. The patients did not wish to cryopreserve their tissue for fertility preservation and donated their ovaries for research. All patients were having testosterone treatment at the time of oophorectomy and some of them were also having menses inhibition treatment.PARTICIPANTS/MATERIALS, SETTING, METHODS:Sibling ovaries were collected in either cold or warm medium, to identify the most optimal collection temperature. Cumulus oocyte complexes (COCs) from each condition were isolated from the ovarian tissue and matured in vitro for 48 h. The quality of OTO-IVM oocytes was assessed by calcium pattern releasing ability, embryo developmental competence following ICSI, and staining for mitochondrial membrane potential. In vitro matured metaphase I (MI) oocytes, germinal vesicle (GV) oocytes, and in vivo matured oocytes with aggregates of smooth endoplasmic reticulum (SERa) were donated from ovarian stimulated women undergoing infertility treatment and these served as Control oocytes for the study groups. ST was applied to overcome poor oocyte quality. Specifically, enucleated mature Control oocytes served as cytoplasmic recipients of the OTO-IVM spindles from the transgender men. Embryos derived from the different groups were scored and analysed by shallow whole genome sequencing for copy number variations (CNVs).MAIN RESULTS AND THE ROLE OF CHANCE:In total, 331 COCs were collected in the cold condition (OTO-Cold) and 282 were collected in the warm condition (OTO-Warm) from transgender men. The maturation rate was close to 54% for OTO-Cold and 57% for OTO-Warm oocytes. Control oocytes showed a calcium releasing ability of 2.30 AU (n = 39), significantly higher than OTO-Cold (1.47 AU, P = 0.046) oocytes (n = 33) and OTO-Warm (1.03 AU, P = 0.036) oocytes (n = 31); both values of calcium release were similar between the two collection temperatures. Mitochondrial membrane potential did not reveal major differences between Control, OTO-Warm, and OTO-Cold oocytes (P = 0.417). Following ICSI, 59/70 (84.2%) of Control oocytes were fertilized, which was significantly higher compared to 19/47 (40.4%) of OTO-Cold (P < 0.01) and 24/48 (50%) of OTO-Warm oocytes (P < 0.01). In total, 15/59 (25.4%) blastocysts were formed on Day 5 in the Control group, significantly higher than 0/19 (0%) from the OTO-Cold (P = 0.014) and 1/24 (4.1%) in OTO-Warm oocytes (P = 0.026). Application of ST rescued the poor embryo development, by increasing the Day 5 blastocyst rate from 0% (0/19) to 20.6% (6/29) (P = 0.034), similar to that in the ICSI-Control group (25.4%, 15/59). A normal genetic profile was observed in 72.7% (8/11) of OTO-Cold, 72.7% (8/11) of OTO-Warm and 64.7% (11/17) of Control Day 3-Day 5 embryos. After ST was applied for OTO-IVM oocytes, 41.1% (7/17) of the embryos displayed normal genetic patterns, compared to 57.1% (4/7) among ST-Control Day 3-Day 5 embryos.LARGE SCALE DATA:N/A.LIMITATIONS, REASONS FOR CAUTION:Due to the limited access to human oocytes and ovarian tissue, our results should be interpreted with some caution, as only a limited number of human oocytes and embryos could be investigated.WIDER IMPLICATIONS OF THE FINDINGS:The results of this study, clearly indicate that OTO-IVM oocytes originating from transgender patients are of inferior quality, which questions their use for fertility preservation. The poor quality is likely to be related to cytoplasmic factors, supported by the increased blastocyst numbers following application of ST. Future research on OTO-IVM from transgender men should focus on the cytoplasmic content of oocytes or supplementation of media with factors that promote cytoplasmic maturation. A more detailed study on the effect of the length of testosterone treatment is also currently missing for more concrete guidelines and guidance on the fertility options of transgender men. Furthermore, our study suggests a potentially beneficial role of experimental ST in overcoming poor embryo development related to cytoplasmic quality.STUDY FUNDING/COMPETING INTEREST(S):A.C. is a holder of FWO grants (1S80220N and 1S80222N). A.B. is a holder of an FWO grant (1298722N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) and 2018000504 (GOA030-18 BOF) funding. B.H. has additional grants from FWO-Vlaanderen (Flemish Fund for Scientific Research, G051516N and G1507816N) and Ghent University Special Research Fund (Bijzonder Onderzoeksfonds, BOF funding (BOF/STA/202109/005)), and has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest.TRIAL REGISTRATION NUMBER:N/A.

On Friday, June 24, 2022, the US Supreme Court officially reversed Roe v. Wade declaring that the constitutional right to abortion, upheld for nearly a half century, no longer exists. This constituted a chilling wakeup call for us all. Indeed, Roe's reversal abruptly unveiled that our long cherished—we had believed ever-lasting and ever-broadening—tolerance for reproductive choices and practices could suddenly crumble before our eyes. Yet, most of us had been oblivious of mounting ominous signs of rising intolerance that had been openly brewing before us, hiding in plain sight, but ignored by most. The time of horrendous and menacing dystopias was gone, we believed relegated to the darkest of all dust beans of history. The year 1984 had come and gone without leaving any ripple over the ocean of our certitude regarding reproductive freedom. Ignoring mounting threats, we remained comforted with the belief that tolerance and the governing case law/ruling was here to be and stay. And yet, today in the aftermath of Roe's reversal, we witness that our cherished reproductive tolerance is being infringed from all directions and in great peril everywhere. Generations upon generations, we have surfed over the wave of expanding all-embracing reproductive tolerance, which developed in post war times. President Joe Biden may best reflect the gradual transformation and evolution of reproductive views that has flourished over the past 50 years. Mr. Biden, now 80 years of age, was raised in a time when much of the country was less tolerant of people's sexual orientations. Perhaps his early policy choices in the Senate reflected those times, often siding with those who proposed restrictions, or limits, on gay men and lesbians. But a keen observer of the ways that society was changing, he modified his positions, as so many of us did. Today reflecting a sea change in public opinion—nearly 70% of Americans support same-sex marriage—the President, a practicing Catholic, openly supports gay rights including marriage and the rights of women to choose to have an abortion. (https://www.nytimes.com/2022/08/07/us/politics/biden-abortion-catholic-history.html). This evolution seemed so general that we took for granted that reproductive tolerance was here to stay. Indeed, the world bore witness of the progresses accomplished in reproductive tolerance. In December 1917, 2 months after the October Revolution, the new Soviet Republic legalized homosexuality, discarding the ban enacted by the Russian Empire. In the Western World, increasing reproductive tolerance has been general and overwhelming. In particular, same-sex marriage now has been recognized in 33 countries, with broadening reproductive tolerance implemented in all Western countries. The shock of Roe's reversal abruptly revealed that reproductive freedom as a whole—not just abortion rights—could suddenly be jeopardized. Although the Supreme Court ruling of June 24, 2022 only affects abortion rights, Justice Clarence Thomas raised the possibility of other tenable restrictions of reproductive freedom after Roe's reversal. https://www.nytimes.com/2022/06/24/us/clarence-thomas-roe-griswold-lawrence-obergefell.html. Indeed, the same judicial intricacies linked to interpretations of the 14th Amendment—one of the 3 post-Civil War reconstruction amendments—stand as the kingpin of other reproductive rights in the United States. These notably include the right to same-sex marriage, gay relationship, contraception, and other reproductive rights. In 1973, Justice Harry Blackmun said that a woman's access to an abortion was implicit in the right to privacy protected under the 14th Amendment. In 2022, however, Justice Samuel Alito ruled that this view was "egregiously wrong." Based on the bluntness of this latter judgment publicly expressed, one can rightfully question how far these reversals of reproductive tolerance can go? https://www.nytimes.com/2022/05/11/us/politics/roe-wade-supreme-court-abortion.html. The dominoes have started to fall, and they may not just stop at one. Reproductive intolerance is today reemerging from everywhere. First there were parts of the world, which had never been reached by the wave of reproductive tolerance, something we mostly ignored. Communities in Africa and in predominantly Muslim countries remain among the least accepting of homosexuality. In sub-Saharan Africa, at least 9 in 10 in Nigeria (98%), Senegal (96%), Ghana (96%), Uganda (96%) and Kenya (90%) believe homosexuality should not be accepted by society (1Abu-Ras W. Suárez Z.E. Breiwish R.R. Beyond the axes of inequality: religion, race, and everything in between.Am J Orthopsychiatry. 2021; 91: 217-235Crossref PubMed Scopus (5) Google Scholar). This likely constitutes remnants of Western colonization. Second, in the Soviet Union the first wave of tolerance for homosexuality enacted after the revolution was eradicated by Joseph Stalin. Under his tenure, male homosexuality was recriminalized and punished by up to 5 years of hard labor in prison. After a short return to tolerance with the dissolution of the Soviet Union, today however, under Vladimir Putin, ever-increasing laws have been enacted against lesbian, gay, bisexual, transgender, queer, intersex, and more (LGBTQI+) people and culture in Russia. The new laws effectively outlaw any public expression of LGBTQI+ lifestyle in Russia, to protect "traditional Russian values." Mr. Putin has long cast LGBTQI+ life mode as a Western intrusion into Russia's traditional society and values and has done so for political reasons. The current reversal in reproductive tolerance is not limited to Russia. In Poland, the Catholic Church—sapped by multiple sexual abuse scandals—inspired Poland's Constitutional Court to impose new restrictions on the right to abortion. This turned the country's abortion prohibition into a near-total ban. To this date, several women have died of septic shock because of these drastic measures. In Poland too, the ban on abortion sparked to initiate a broadening of reproductive intolerance. Hostile attitudes toward LGBTQI+ led several regions and municipalities to declare themselves "LGBTQI+ Ideology Free," calling for the exclusion of LGBTQI+ people from the Polish society. Hungary as well has enacted similar restrictions on reproductive freedom. These sparked a reaction of the European Union direction to override the restrictions based on fundamental European Union principles. This outcome of this legal action is however still uncertain, threatening, if it is lost, to leave the gains made in the last 50 years for LGBTQI+ people in Europe on shaky ground. In the rest of Europe, reproductive intolerance is gaining momentum as well. Gestational surrogacy for which the American Society for Reproductive Medicine recently provided recommendation for practices through a committee opinion (2Practice Committee of the American Society for Reproductive Medicine and Practice Committee of the Society for Assisted Reproductive Technology. Recommendations for practices using gestational carriers: a committee opinionFertil Steril. 2022; 118: 65-74Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) is banned in several European countries. In France, gestational surrogacy is not only forbidden but criminalized with prison terms for those who might help the process. This precludes any reproductive option for male homosexual couples. In France again, a Catholic-inspired group lobbies for forbidding preimplantation genetic testing for aneuploidy (https://www.genethique.org/dpi-a-le-comite-dethique-de-linserm-contourne-le-legislateur/), probably being oblivious of the call for tolerance expressed by the supreme Catholic leader, Pope Francis, in his famous "Who am I to judge?" Facing mounting reproductive intolerance abruptly revealed by Roe's reversal, the whole community of care givers in the field of reproductive medicine should unite and speak of one voice to support the plea for tolerance that stands at the very core of all medical acts. Transgender issues have existed since antiquity (3de Ziegler D. de Sutter P. Transgender men: clinical care and implications in reproductive medicine: introduction.Fertil Steril. 2021; 116: 919-921Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar) and are here to stay unharmed. Let's not allow reproductive medicine to become an exception in medicine by becoming the focus of intolerance and all sorts of haltering. We who in our work become confident of people's reproductive issues and aspiration should support with all our heart and soul the diversities of human reproductive quests and intrinsic differences. Together with Pope Francis, we should spearhead the essence of tolerance that he humbly, but forcefully, proffered: "Who am I to judge?" The authors are indebted to Prof Catherine Racowsky for her thoughtful editorial comments.

Abstract Study question How do patients perceive the pre-IVF treatment communication by the nursing and midwifery team and how do they evaluate implemented optimization? Summary answer Patient satisfaction with the pre-IVF treatment communication by the nursing and midwifery team improved from 86% to 98% after implementation of standardized written patient information. What is known already Nurses are the main point of contact for patients undergoing medically assisted reproduction techniques (Applegarth et al., 2008). Morris (2001) suggested that infertility nurses could more specifically be involved in informative counselling in anticipation of the fertility treatment. Specific training for nurses and midwives would be required in order to achieve that central role in effective fertility counselling (Applegarth et al., 2012). However, little has been published in peer reviewed literature on how to assess and improve these pre-IVF treatment information sessions. Study design, size, duration The pre-IVF communication by the nurse/midwife was assessed by female patients with a self-developed questionnaire (cohort 1). The nursing and midwifery team was subsequently informed about the results of the questionnaire and efforts were undertaken to optimise future patient communication. Four years later, a follow-up questionnaire assessed patient satisfaction about the intervention (cohort 2). Each cohort comprised fifty patients and nurses and midwives were blinded for patient participation to the study. Participants/materials, setting, methods The study was performed at an academic fertility centre and was approved by the Investigational Review Board. Data were collected with SurveyMonkey (pre intervention) and REDCap (post intervention). A descriptive analysis of the cohorts and patient feedback was performed. The intervention consisted of (1) the optimization of patient information documents if needed (2) providing a training to the nursing and midwifery staff, and (3) a follow-up questionnaire in a second cohort of female fertility patients. Main results and the role of chance The first questionnaire revealed that overall, patients were satisfied with the informative counselling session in anticipation of the IVF treatment. However, some patients indicated that they received a lot of information at once and that specific information that was of relevance for the further treatment (e.g. oocyte pick-up) was sometimes missed. Also, the data showed that not all patients received the same information. Therefore, all information that patients needed during an IVF treatment was gathered into a patient binder. It was supplemented with a timeline of the whole IVF trajectory, QR codes to movies explaining the administration of medication, and answers to common patients’ questions. It was possible to individualize the content of the binder per patient. Ideally, patients received this binder before the informative counselling session so they could read the content in advance. The binder was developed by a member of the nursing and midwifery team and was reviewed by the medical, laboratory, and administrative staff. Then, the nursing and midwifery team was trained on the use of the patient binder. The follow-up questionnaire revealed that patient satisfaction with the pre-IVF treatment counselling was 98% after the optimization of patient documentation (compared to 86% before the optimization). Limitations, reasons for caution Eligible patients were recruited by the treating physician. As no records were kept of the number of solicited patients for this study, no assessment of the response rate is possible. The cohorts were self-selected and limited in size and could therefore not reflect the general patient population. Wider implications of the findings A patient questionnaire and follow-up is a useful tool for a centre specific assessment and improvement of pre-IVF nurse/midwife communication with patients. Clinics could make efforts to invest in complete and written information and ask colleagues of the nursing and midwifery team to be involved in its creation. Trial registration number NCT04420169

Providing additional insights on the efficacy of human nuclear transfer (NT). Here, and earlier, NT has been applied to minimize transmission risk of mitochondrial DNA (mtDNA) diseases. NT has also been proposed for treating infertility, but it is still unclear which infertility indications would benefit. In this work, we therefore additionally assess the applicability of NT to overcome failed fertilization. Patient 1 carries a homoplasmic mtDNA mutation (m.11778G > A). Seventeen metaphase II (MII) oocytes underwent pre-implantation genetic testing (PGT), while five MII oocytes were used for spindle transfer (ST), and one in vitro matured (IVM) metaphase I oocyte underwent early pronuclear transfer (ePNT). Patients 2–3 experienced multiple failed intracytoplasmic sperm injection (ICSI) and ICSI-assisted oocyte activation (AOA) cycles. For these patients, the obtained MII oocytes underwent an additional ICSI-AOA cycle, while the IVM oocytes were subjected to ST. For patient 1, PGT-M confirmed mutation loads close to 100%. All ST-reconstructed oocytes fertilized and cleaved, of which one progressed to the blastocyst stage. The reconstructed ePNT-zygote reached the morula stage. These samples showed an average mtDNA carry-over rate of 2.9% ± 0.8%, confirming the feasibility of NT to reduce mtDNA transmission. For patient 2–3 displaying fertilization failure, ST resulted in, respectively, 4/5 and 6/6 fertilized oocytes, providing evidence, for the first time, that NT can enable successful fertilization in this patient population. Our study showcases the repertoire of disorders for which NT can be beneficial, to overcome either mitochondrial disease transmission or failed fertilization after ICSI-AOA.

Abstract Background Advanced maternal age and obesity are associated with impaired female fertility. Moreover, fatty acids (FA) in follicular fluid (FF) play important roles in oocyte maturation and embryo development. However, the effects of body mass index (BMI), age, and FF FA composition on embryo development between days 3 and 5 and blastocyst stage on day 5 are still unclear. Methods This study included 138 patients undergoing assisted reproductive technology (ART), which were divided into three BMI groups (18.5–24.9 kg/m2 vs. 25.0–29.9 kg/m2 vs. ≥ 30.0 kg/m2) and three age-related groups (20–30 years vs. 31–34 years vs. ≥ 35 years) which were compared for ART outcomes. Further, observations were divided into quartiles based on either of three parameters related to embryo outcome, i.e. (i) embryos developing between days 3 and 5 (ED3-5) and (ii) expanded blastocysts on day 5 (EB5), both expressed proportionally to the number of oocytes with two pronuclei (2PN), as well as (iii) the embryo utilization rate (EUR). Proportions of FF FA were then compared between Q1 and Q4, representing the quartile with the worst vs. the best embryo outcome, respectively. Finally, regression models were created to assess the relationships between BMI, age, FF total FA (TFA) concentration, relative proportions of specific FA and embryo outcome. Results Patients of Q1 had higher proportions of FF C20:5n-3, C22:6n-3 and total n-3 PUFA than Q4 patients. Furthermore, Q4 patients tended to be younger than Q1 patients. Within the whole cohort, the proportion of C20:5n-3 negatively correlated with ED3-5/2PN and EUR, while EB5/2PN tended to be negatively correlated with age. Regression models within the overweight and obese group confirmed the negative relation between C20:5n-3 and ED3-5/2PN, but also indicated additional associations: C18:1n-9 and C20:4n-6 were positively associated with ED3-5/2PN and EUR, respectively while the proportion of C18:0 was negatively associated with EUR. Conclusion The proportions of n-3 PUFA, particularly C20:5n-3 and C22:6n-3 were reduced in the patients’ quartile with the best embryo outcome. This group of patients was also younger. However, the embryo quality parameters of overweight/obese patients were not associated with age but were positively associated with FF C18:1n-9 and negatively with the proportions of C18:0 or C20:5n-3. Trial registration This study’ registration number was B670201627735.

STUDY QUESTION:What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse?SUMMARY ANSWER:TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner.WHAT IS KNOWN ALREADY:Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet.STUDY DESIGN, SIZE, DURATION:The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29).PARTICIPANTS/MATERIALS, SETTING, METHODS:A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis.MAIN RESULTS AND THE ROLE OF CHANCE:NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human.LARGE SCALE DATA:N/A.LIMITATIONS, REASONS FOR CAUTION:CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes.WIDER IMPLICATIONS OF THE FINDINGS:Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for ∼15% of human conceptions.STUDY FUNDING/COMPETING INTEREST(S):The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests.TRIAL REGISTRATION NUMBER:N/A.

STUDY QUESTION Is the cumulative live birth rate (CLBR) per oocyte collection cycle (OCC) comparable after cleavage-stage or blastocyst-stage transfer in combination with supernumerary blastocyst vitrification on Day 5 (D5) in patients with four or fewer zygotes on Day 1? SUMMARY ANSWER The CLBR in a fresh blastocyst-transfer or cleavage-stage transfer policy followed by vitrification on D5 is comparable in patients with four or fewer zygotes. WHAT IS KNOWN ALREADY Blastocyst transfer enhances the self-selection of the embryo and shortens the time to pregnancy in patients with normal or high ovarian response. Whether these advantages are also present in patients with a low ovarian response and/or a limited number of available zygotes is a continuous debate. STUDY DESIGN, SIZE, DURATION This was a retrospective, observational cohort study of 2359 consecutive OCCs between January 2014 and December 2018. According to a shift in transfer policy in our center, 571 OCCs had been scheduled for a fresh transfer on Day 3 (D3) and 1788 on D5. The D5 group was matched to the D3 group by propensity score (PS) matching according to multiple maternal baseline covariates. After PS matching, there were 571 OCCs in each group. PARTICIPANTS/MATERIALS, SETTING, METHODS OCCs scheduled for a D3 transfer (n = 571) or for a D5 transfer (n = 1788) were matched by PS matching in a 1:1 ratio accounting for potential confounding factors associated with CLBR. The model included patient characteristics, such as maternal age and cycle rank, as well as treatment characteristics such as GnRH analog regimen and ovarian response. Embryological variables included the number of zygotes and the number of 6- to 7- and 8-cell embryos on D3. The delivery outcomes of the fresh treatment cycle and the consecutive vitrified-warmed embryo transfers were analyzed up to the first live birth. The primary endpoint of this study was CLBR per OCC. Secondary outcomes were live birth rate per fresh transfer and embryo implantation rate per transferred embryo. MAIN RESULTS AND THE ROLE OF CHANCE The CLBR per OCC was comparable between the D5 and D3 groups (16.8% versus 17.7%, respectively, P = 0.600). Live birth rates per OCC did not differ between a cleavage-stage transfer and blastocyst-stage transfer policy (15.2% versus 12.4%, respectively, P = 0.160). In the D5 group, 201 cycles did not result in a blastocyst to perform an embryo transfer or cryopreservation; in the D3 group, only 59 cycles did not have an embryo transfer because of poor embryo quality (35.2% versus 10.3%, respectively; P < 0.001). A significantly higher number of fresh double embryo transfers were performed in the D3 group compared to D5 (23.8% versus 7.0%, respectively, P < 0.001). LIMITATIONS, REASONS FOR CAUTION Although adjusted for important confounders in the PS matching, BMI and embryo quality of the transferred embryo(s) were not taken into account. This study is limited by its retrospective design and is a single-center study, which may limit the generalizability of our findings. WIDER IMPLICATIONS OF THE FINDINGS The CLBR in a fresh blastocyst-transfer or cleavage-stage transfer policy followed by vitrification on D5 is comparable. A fresh embryo transfer on D3 can still be considered in patients with a poor ovarian response and/or limited number of zygotes when combined with blastocyst vitrification without impacting the overall CLBR of the cycle. STUDY FUNDING/COMPETING INTEREST(S) No external funding was obtained for this study. There are no conflicts of interest to declare.

Abstract Study question Could collection temperature and spindle transfer (ST) potentially improve development of embryos derived from in vitro matured (IVM) ovarian tissue oocytes (OTO) of transgender men? Summary answer Spindle transfer, but not collection temperature, significantly improved embryo development of OTO-IVM oocytes from transgender men. What is known already For transgender men, the fertility preservation strategy of ovarian stimulation may interfere with the desired masculine characteristics and enhance gender dysphoria. Alternatively, ovarian tissue oocytes collected ex vivo could serve as potential gametes, not requiring ovarian stimulation. Oocytes can be collected during gender affirming surgery, matured, and vitrified. Ovarian tissue oocyte in vitro maturation (OTO-IVM) has successfully been used for cancer patients, as live births have been reported. OTO-IVM in transgender men demonstrated sufficient maturation rates and survival following vitrification. Nevertheless, a decreased fertilization potential of these oocytes and severely compromised embryonic development have been observed. Study design, size, duration Patients between 18-24 years were recruited for this study from November 2020 to September 2021. Ovaries from 14 transgender men were collected in either cold (4oC, OTO-Cold) or warm (37oC, OTO-Warm) collection medium, to verify the best collection method. Following ovarian manipulation, cumulus oocyte complexes (COCs) were harvested from spent medium and underwent maturation for 48hrs. ST was performed to overcome inferior fertilization and embryonic development. Participants/materials, setting, methods Injected IVM oocytes underwent calcium imaging or were monitored for embryonic developmental potential. In vitro matured GV (germinal vesicle), MI (metaphase I) and in vivo matured oocytes with clusters of smooth endoplasmic reticulum (SERa) served as controls and cytoplasmic recipients for ST. OTO-IVM or control oocytes were used as spindle donors (ST-OTO or Control-ST respectively). Genetic analysis was performed to detect chromosomal abnormalities in embryos from all groups. Main results and the role of chance In total, we collected 252 OTO-Cold and 230 OTO-Warm oocytes, showing similar maturation rates (53%). For calcium imaging, 39 control, 33 OTO-cold and 31 OTO-warm oocytes were analysed, determining the product of amplitude per frequency, in arbitrary units (AU). The average value for control oocytes was 2.30AU, significantly higher than OTO-Cold (1.47AU, p=0.046) and OTO-Warm oocytes (1.03AU, p=0.036). Calcium release was similar between OTO-Cold and OTO-Warm oocytes. Following ICSI, 19/47 OTO-Cold and 24/48 OTO-Warm oocytes normally fertilized, significantly lower than the control group (42/52) (p < 0.001 and p = 0.001 respectively). Blastocyst formation was significantly higher in control oocytes (13/42,31%) when compared to OTO-Cold (1/19, p=0.027) and OTO-Warm (2/24, p=0.035). No statistically significant difference in fertilization rate and embryo development was detected between OTO-Cold and OTO-Warm oocytes. ST was performed to overcome poor embryo development in the OTO-Cold group. Following ST, 12/19 ST-OTO-Cold and 24/38 Control-ST oocytes were normally fertilized. Blastocyst development was similar between the two groups (4/12 and 7/24 respectively), but significantly higher than blastocyst development in OTO-Cold ICSI oocytes (p = 0.038 and p = 0.045). Genetic analysis revealed that 4/10 OTO-Cold, 4/11 OTO-Warm, 4/11 ICSI control, and 4/7 Control-ST embryos were chromosomally abnormal while 6/8 OTO-ST were abnormal, and 2/8 showed a suggestive low-grade mosaicism. Limitations, reasons for caution A major limitation of our study is the lack of ovaries from cis women. Control oocytes used in this study originate from infertility patients that underwent ovarian stimulation. High abnormality rate in ST-OTO embryos might be concerning for the safety of ST, but the number of embryos analysed is limited. Wider implications of the findings Our data indicate that OTO-IVM oocytes from transgender men display poor cytoplasmic quality, demonstrated by embryonic arrest and calcium imaging. ST was able to overcome poor embryo development, and it could be of interest to use freshly donated oocytes as cytoplasmic recipients for this. Trial registration number Not applicable

Abstract Study question Can clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing result in the correction of a single base pair substitution that causes male infertility? Summary answer CRISPR/Cas9 administration during intracytoplasmic sperm injection (ICSI) leads to correction attempts of mutant phospholipase C zeta (PLCζ), howeverc loss-of-heterozygosity (LOH). What is known already Failed fertilization after ICSI can be caused by mutations in the sperm-related oocyte factor PLCζ which can be overcome by assisted oocyte activation (AOA). In this way, children may inherit the infertility-causing mutation. Mutation transmission can be overcome through CRISPR/Cas9 delivery during ICSI. In previous studies using CRISPR/Cas9 in the human germline for mutation correction, loss-of-heterozygosity (LOH, loss of the allele of one of the parents) was observed. Two different explanations were given, namely partial or complete paternal chromosomal loss or the correction of the mutation by using the maternal wild-type allele instead of the exogeneous supplied repair template. Study design, size, duration We injected a gRNA-Cas9 protein complex to target the PLCζ mutant allele, a repair template harboring the desired nucleotide substitution and an additional synonymous variant to track template usage, together with patient’s sperm. To overcome fertilization failure, AOA was applied during ICSI. After a culture period of maximal 6 days the embryos were collected. At day 3, some embryos were dissociated in individual blastomeres. The extracted DNA was analyzed through different genetic sequencing techniques. Participants/materials, setting, methods Donated sperm of a patient experiencing complete fertilization failure after routine ICSI, harboring a heterozygous base pair substitution in PLCZ1 (c.136-1G>C), was utilized. Sperm was injected in donated in vitro matured oocytes or in vivo matured oocytes containing clusters of smooth endoplasmic reticulum. Next-generation sequencing was used to assess correction potential. Short tandem repeat (STR) and single nucleotide polymorphism (SNP) assays were used to determine whether the sperm contained the mutation and to evaluate LOH. Main results and the role of chance CRISPR/Cas9 injections had no significant impact (p > 0.05) on embryonic development. Due to the heterozygous nature of the mutation, 47% (27/58) of the embryos originated from mutated sperm injection. The CRISPR components showed a high specificity with absence of insertions/deletions in 97% of the embryos originating from wild-type sperm (n = 31). Embryos originating from mutant sperm (n = 27) fall into three categories:(1) 22% showed the untargeted mutant allele, (2) 52% showed additional mutagenesis and (3) 26% showed the wild-type allele, which could be explained by correction. Mosaicism, defined as various editing events, was present in 17% (1), 21% (2) and 71% (3) of the embryos. The low occurrence of the synonymous variant, incorporated in the repair template, suggests that the template is not used during correction attempts. In only 29% (2/7) and 14% (1/7) of the ‘corrected embryos’, respectively long (>18Mb) or medium width LOH (4Mb) was observed through STR analysis. SNP analysis in closer proximity showed in 71% (5/7) of the embryos LOH, even in the absence of LOH through STR, suggesting also the occurrence of short width LOH. These results will be studied in more detail before definitive conclusions can be made. Chromosomal LOH will be studied by ddRADseq. Limitations, reasons for caution The occurrence of mosaicism and LOH might complicate the use of traditional CRISPR/Cas9 in human embryos and should be studied in detail to draw definite conclusions on its potential future use. To this end, genomic data have been produced from both individual blastomeres and whole-embryos which will be further analyzed. Wider implications of the findings Our findings demonstrate caution to use CRISPR/Cas9 to correct mutations in the germ line. They seem to contradict other reports that show predominant lack of mosaicism and presence of long width LOH. A deeper evaluation will be undertaken to define the length and type of LOH in this study. Trial registration number Not Applicable