In clinical practice, treatment cycles are routinely started without the intention of undertaking fresh embryo transfer, as the freeze-all strategy is suitable for a multitude of alternative stimulation protocols as well as preimplantation genetic testing of embryos. Based on strengths, weaknesses, opportunities and threats (SWOT) analysis, it was evaluated whether a freeze-all strategy may be advised for all patients regardless of their (predicted) ovarian response. Therefore, the latest literature including the outcome of all randomized controlled trials (RCT) and their combined analysis were reviewed. Only two large trials, both from China, were considered conclusive and showed (following first embryo transfer) a higher chance of live birth following elective vitrification in comparison with fresh embryo transfer in women with polycystic ovary syndrome and in ovulatory women with a good prognosis. The other six trials showed either no difference in clinical outcome or were inconclusive by design. It is concluded that elective vitrification is likely favourable for high responders as it results in an improved chance of live birth following single embryo transfer, and is likely equally successful for normal responders. One trial in low responders indicated that the live birth rate may be compromised, which needs further confirmation in the future. This SWOT analysis of embryo transfer deferral also concluded that this approach allows the number of oocytes per stimulation cycle to be maximized safely in order to improve the chance of pregnancy. To minimize the risk for obstetric and perinatal complications in frozen-thawed embryo transfer cycles, (modified) natural cycles should be considered as the preferred approach for regular cycling women.

Fertilization involves dynamic sperm-egg interactions, yet has been primarily studied in static samples. Here, we use high-resolution live imaging to capture fertilization from the moment of sperm binding in zona-intact mouse oocytes. We identify two phases of sperm remodeling: a static phase, during which sperm remain beneath the oocyte cortex as DNA decondensation and histone loading occur, and a mobile phase characterized by stereotyped sperm movement. Initial displacement away from the spindle is driven by cytoplasmic streaming, with manipulations in mouse indicating that sperm movement requires chromatin decondensation and oocyte polarization. Subsequently, polar body cytokinesis generates convergent cortical flows that draw sperm toward the emerging female pronucleus. Finally, we capture sperm-egg fusion in human oocytes and characterize post-fusion events including meiotic resumption and sperm movement, offering a live-imaging description of human fertilization dynamics. Together, these findings provide a continuous spatiotemporal framework for mammalian fertilization, extended by initial observations in human oocytes.

RESEARCH QUESTION:What are the effects of integrating preimplantation genetic testing for aneuploidy into preimplantation genetic testing for monogenic/single gene disorders (PGT-M), termed 'comprehensive PGT' (coPGT-M)? DESIGN:A retrospective cohort of frozen embryo transfer cycles, derived from intracytoplasmic sperm injection, with single embryo transfers (SET) performed between 2019 and 2022 in patients undergoing their first round of assisted reproductive technology. CoPGT-M was implemented from July 2021. Only cycles with at least one biopsied blastocyst were included. Outcomes were compared between 701 embryos from 126 PGT-M cycles and 801 embryos from 131 coPGT-M cycles. RESULTS:The median female age at oocyte retrieval was similar between the PGT-M and coPGT-M cohorts [29.5 (IQR 27.0-33.0) versus 29.5 (IQR 28.0-33.0) years], as were male age, body mass index, and anti-Müllerian hormone concentration. The PGT-M cohort had a significantly lower median number of cumulus-oocyte complexes [15.0 (IQR 10.0-20.0) versus 15.5 (IQR 11.0-26.0); P = 0.039], while the number of zygotes, blastocysts biopsied, and blastocysts eligible for transfer after genetic analysis were comparable. The percentage of cycles resulting in at least one live birth (PGT-M 42.1% versus coPGT-M 51.9%) and the median number of embryo transfers required to reach the first live birth among patients who achieved a live birth [PGT-M 1 (IQR 1-2) versus coPGT-M 1 (1-2)] were comparable. Generalized estimating equation analysis, adjusted for embryo quality, showed higher odds of live birth per SET [adjusted OR (aOR) 1.86, 95% CI 1.21-2.87; P = 0.005] and lower odds of pregnancy loss (aOR 0.50, 95% CI 0.27-0.93; P = 0.028) in the coPGT-M cohort. CONCLUSIONS:While cumulative outcomes per cycle were similar, coPGT-M was associated with significantly higher odds of live birth per embryo transfer and lower risk of pregnancy loss, indicating clinical benefit, even in younger patients.

RESEARCH QUESTION:Can a random start natural micronized progestin-primed ovarian stimulation (NPPOS) protocol provide an equivalent and flexible alternative strategy for elective freeze-all stimulation cycles compared with a conventional start gonadotrophin-releasing hormone (GnRH) antagonist protocol? DESIGN:This retrospective cohort study included 1202 elective freeze-all cycles between 2022 and 2024. Four groups were compared: GnRH-antagonist early follicular (n = 503), NPPOS early follicular (n = 319), NPPOS late follicular (n = 163) and NPPOS luteal (n = 217) starts. The primary endpoint was the number of metaphase II (MII) oocytes retrieved. The secondary endpoints included the follicular output rate (FORT), follicle-oocyte index (FOI) and the duration of stimulation. A predefined subgroup analysis was conducted in poor ovarian responders. RESULTS:The concentration of anti-Müllerian hormone was comparable across groups, and the median number of MII oocytes retrieved was similar: NPPOS late follicular group, 8 (IQR 5-15); GnRH-antagonist group, 10 (IQR 6-16); NPPOS early follicular group, 10 (IQR 6-16); and NPPOS luteal group, 10 (IQR 5-16) (P = 0.20). A generalized estimating equation Poisson regression model analysis confirmed equivalence. FORT and FOI-MII remained stable across the NPPOS early and late follicular, and luteal groups: estimated marginal mean ± SD FORT, 37.1 ± 9.9%, 40.4 ± 24.8% and 39.8 ± 24.9 (P = 0.30); and FOI-MOI, 70.8 ± 38.7%, 73.5 ± 44.2% and 75.0 ± 43.6% (P = 0.60), respectively. The NPPOS luteal group required a longer mean ± SD duration of stimulation (11 ± 2 days) compared with the NPPOS early follicular group (9 ± 2 days; P < 0.001), NPPOS late follicular group (10 ± 3 days; P = 0.004) and GnRH-antagonist group (10 ± 2 days; P < 0.001). In poor ovarian responders, the MII yield was comparable across all groups. CONCLUSIONS:Random start NPPOS using micronized progesterone is an effective and flexible alternative to a conventional GnRH-antagonist protocol in freeze-all cycles. The ability to initiate stimulation irrespective of menstrual phase combined with the use of oral progesterone may reduce treatment delays and improve convenience without compromising ovarian response.

RESEARCH QUESTION:Do FSH types (recombinant FSH [rFSH] versus highly purified human menopausal gonadotrophin (HP-HMG) versus corifollitropin alfa (CFA) differ in the efficiency of ovarian response, as assessed by follicular output rate (FORT) and follicle-to oocyte index (FOI)? DESIGN:Retrospective cohort study analysing 4525 antagonist IVF and intracytoplasmic sperm injection cycles using CFA (n = 728), rFSH (n = 1848) or HP-HMG (n = 1949). The FORT and FOI indices were calculated as ratios of follicle or oocyte yield to antral follicle count (AFC). Generalized estimating equations were used to account for clustering by patient with covariates for age, anti-Müllerian hormone (AMH), weight, stimulation duration, gonadotrophin dose and elective freeze. RESULTS:The CFA yielded the highest ovarian response indices (FORT 16-22 [61.6%], FOI-COC [106.4%], FOI-MII [81.1%]), compared with rFSH (49.3%, 98.0%, 74.6%) and HP-HMG (39.6%, 71.5%, 46.5%). Stratified analyses confirmed this ranking across AMH strata. In low AMH (<1.1 ng/ml), FOI-MII was 81.4% with CFA, 64.5% with rFSH and 49.2% with HP-HMG. In normal/high AMH (1.1-3 ng/ml) FOI-MII was 86.3%, 64.5% and 53.1%. Generalized additive model analyses demonstrated a modest non-linear AMH effect only for CFA, peaking at about 1.5-2.0 ng/ml before declining, whereas rFSH rose gradually and HP-HMG remained quite flat. Age-stratified analyses showed a uniform decline across FSH types. The risk of OHSS (>18 follicles ≥11 mm) was lowest with CFA (6.5%), compared with 19.8% for HP-HMG and 39.2% for rFSH. CONCLUSION:After adjusting for AFC, CFA achieved the highest FORT and FOI across most AMH strata, consistently outperforming HP-HMG and matching or exceeding rFSH, while maintaining the lowest follicle-count-based OHSS risk.

STUDY QUESTION:Can a single-step warming protocol for vitrified blastocysts provide comparable or superior outcomes to a conventional multi-step warming approach in a clinical IVF setting? SUMMARY ANSWER:Single-step warming resulted in higher blastocyst survival, intactness, and transfer rates compared to the conventional multi-step protocol, with comparable ongoing pregnancy rates and a consistent trend toward lower miscarriage rates across all subgroups. WHAT IS KNOWN ALREADY:While vitrification has become the gold standard for embryo cryopreservation, the warming process remains critical to embryo survival and implantation potential. Traditional warming relies on multi-step dilution protocols to minimize osmotic stress. Preliminary studies have suggested that single-step rehydration protocols may be equally effective, but data on clinical validation remain scarce. STUDY DESIGN, SIZE, DURATION:A three-phase validation study was conducted at a single university-based IVF centre including (i) risk analysis, (ii) preclinical validation (n = 246 blastocysts), and (iii) a clinical comparison of outcomes over one year (March 2024-March 2025) between single-step warming (n = 1925 cycles) and the conventional multi-step protocol (n = 1744 cycles, March 2023-March 2024). PARTICIPANTS/MATERIALS, SETTING, METHODS:In the preclinical phase, vitrified surplus and PGT blastocysts were rewarmed using the single-step protocol and compared to historical controls. Survival (≥50% intact cells), intactness (100% intact), and transfer suitability were assessed. Single-step warmed blastocysts were monitored for 24 h post-warming in a time-lapse incubator, and viability was further evaluated using live/dead fluorescent staining to quantify cell damage. In the clinical phase, outcomes (survival, transfer rate, pregnancy, ongoing pregnancy, miscarriage) were retrospectively compared across protocols, stratified by PGT status and day of vitrification. MAIN RESULTS AND THE ROLE OF CHANCE:In the preclinical phase, blastocyst survival was ≥50% in 99.1% of cases after single-step warming versus 96.8% after multi-step warming (P = 0.0924). Fully intact blastocysts were significantly more frequent in the single-step group (85.4% vs. 76.3%, P = 0.0058), and transfer suitability at 2 h post-warming was also higher (96.7% vs. 91.2%, P = 0.0076). Time-lapse monitoring confirmed a high re-expansion rate (93.9%) with a mean re-expansion time of 3.3 ± 2.7 h. Fluorescent viability staining showed that 90.5% of blastocysts exhibited no or minimal cell damage. In the clinical cohort, significantly higher survival rates were seen in the single-step group across multiple subgroups, including non-PGT Day 5 (98.5% vs. 96.1%, P = 0.0003) and PGT Day 5 (100% vs. 98.2%, P = 0.0407) blastocysts. Fully intact rates were significantly higher in all subgroups and transfer rates were significantly higher in the single-step group for non-PGT Day 6 (97.2% vs. 92.8%, P = 0.0060) and PGT Day 5 cycles (100% vs. 97.8%, P = 0.0209). While some early pregnancy outcomes (e.g. clinical pregnancy in non-PGT Day 5) favoured the multi-step protocol (40.5% vs. 35.7%, P = 0.0286), ongoing pregnancy rates were not significantly different in any subgroup. Miscarriage rates showed a consistent trend in favour of single-step warming but did not reach statistical significance. LIMITATIONS, REASONS FOR CAUTION:This was a single-centre study, and the control group was retrospective. WIDER IMPLICATIONS OF THE FINDINGS:The single-step warming protocol is a clinically validated, safe, and efficient alternative to conventional multi-step warming. Its implementation may improve workflow and streamline blastocyst handling without compromising clinical outcomes. STUDY FUNDING/COMPETING INTEREST(S):No external funding was received. The authors declare no conflicts of interest apart from KT, who has received travel support related to the manuscript from Fuijifilm Irvine Scientific, in the form of payment to her institution. TRIAL REGISTRATION NUMBER:N/A.

Diminished ovarian reserve (DOR) is common in women with infertility and is associated with poorer in vitro fertilization (IVF) outcomes. Testosterone is widely used off-label in this patient group, although evidence for its efficacy and safety is limited. To address this, we conducted a triple-blind, placebo-controlled, randomized clinical trial evaluating whether transdermal testosterone gel prior to IVF improves clinical pregnancy rates in women with DOR. Females aged 18-43 with infertility and DOR according to the Bologna criteria were recruited at 10 fertility clinics in Europe between April 2015 and November 2022. Of 316 assessed for eligibility, 290 were enrolled and randomized. Two were excluded from the primary analysis as their treatment coincided with the onset of the COVID-19 pandemic, and they did not start ovarian stimulation, leaving 288 participants. Participants were randomized to 5.5 mg of transdermal testosterone or matching placebo once daily for ~9 weeks prior to ovarian stimulation. All participants received ovarian stimulation in a long GnRH-agonist cycle with 300IU/day of highly purified human menopausal gonadotropin; fresh embryo transfer was performed if an embryo was available. The primary outcome was clinical pregnancy rate following fresh embryo transfer, defined as an intrauterine gestational sac with an embryo demonstrating cardiac activity at ≥7 weeks' gestation. Of the 288 participants, 134 were randomized to testosterone and 154 to placebo. Clinical pregnancy rates did not differ significantly, occurring in 21 women (15.7%) in the testosterone group and 23 (14.9%) in the placebo group (risk ratio (RR), 1.05; 95% confidence interval (CI) 0.61 to 1.81, p = 0.86). The study was terminated for futility at the prespecified interim analysis based on a conditional power calculation once 70% of the target sample size were randomized. In this study, transdermal testosterone did not improve clinical pregnancy rates compared with placebo in patients with infertility and DOR. Trial Registration: ClinicalTrials.gov: NCT02418572, EudraCT: 2014-001835-35.

STUDY QUESTION:Does transdermal testosterone treatment improve fertility-related quality of life (QOL) in women with diminished ovarian reserve (DOR)? SUMMARY ANSWER:Transdermal testosterone for 9 weeks at a dose of 5.5 mg per day did not result in improved fertility-related QOL compared with placebo in women with DOR. WHAT IS KNOWN ALREADY:Reduced QOL is prevalent in women with infertility, many of whom have DOR. Several studies have shown a correlation between DOR and lower testosterone levels, and testosterone is frequently prescribed to women with DOR undergoing fertility treatment. Some studies have reported that testosterone therapy may improve wellbeing in pre- and post-menopausal women, though others have found no benefit. There are no studies evaluating the effect of testosterone on QOL in women undergoing fertility treatment. STUDY DESIGN, SIZE, DURATION:Pre-planned secondary analysis of a double-blind placebo-controlled randomized controlled trial that included 288 participants recruited between April 2015 and August 2022. Of these, 213 completed QOL surveys both before and after treatment and were eligible for inclusion in this analysis. PARTICIPANTS/MATERIALS, SETTINGS, METHODS:Participants were women aged 18-43 years with DOR according to the Bologna criteria and planning to undergo IVF treatment at one of eight fertility clinics in Spain, Belgium, and Denmark. Participants were randomized to 5.5 mg of transdermal testosterone per day as 1% gel (n = 106) or an identical placebo (n = 107), applied for a median of 60 days prior to commencing ovarian stimulation. QOL was assessed using the FertiQoL instrument prior to commencing the intervention, and at the completion of the intervention but prior to commencing ovarian stimulation. QOL scores were compared using a one-way ANCOVA adjusted for age, BMI, parity, history of IVF treatment, and baseline FertiQoL scores. MAIN RESULTS AND THE ROLE OF CHANCE:There were no significant differences in baseline characteristics between the testosterone (n = 106) and placebo (n = 107) groups. After adjustment, testosterone showed no benefit over placebo for the Total FertiQoL score (F(1,204)=0.07, P = 0.79), the Core and Treatment scores, nor for any of the included FertiQoL subscales. Total testosterone levels were higher in the testosterone group than the placebo group at the end of the treatment (3.2 ± 2.7 nmol/l vs 0.6 ± 0.4 nmol/l, P < 0.001). LIMITATION, REASONS FOR CAUTION:QOL was a secondary outcome in this trial, and participants were not recruited based on a low QOL. WIDER IMPLICATIONS OF THE FINDINGS:Considering the available evidence, including the current study, premenopausal women are unlikely to benefit from testosterone treatment with regard to wellbeing and QOL. This study provides further evidence that testosterone should not be seen as a treatment for low wellbeing or QOL. STUDY FUNDING/COMPETING INTEREST(S):The study was supported by unrestricted grants and support from Besins Healthcare, Roche Diagnostics, and Ferring Pharmaceuticals. The study medication and placebo were provided by Besins Healthcare. Funders had no access to patient data and had no role in the interpretation of the data, nor in the writing or approval of the final manuscript. The researchers were independent of the funders and had full access to all the data in the study. S.J.L. has received honoraria from Merck, Organon, and Hologic, consulting fees from Merck, and travel support from Merck, Organon, Besins Healthcare, and Ferring Pharmaceuticals. S.R.D. has received grants from NHMRC Australia, MS Australia, MRFF Australia, the Australian Heart Foundation, and Lawley Pharmaceuticals, consulting fees from Besins Healthcare, Astellas, and Abbott, honoraria from Theramex, Astellas, and Bayer, travel support from Astellas, and drugs/placebo from Lawley Pharmaceuticals for clinical trials; she is an Executive Board Member of the Australian Academy of Health and Medical Sciences. C.B. has received honoraria from Ferring Pharmaceuticals, IBSA, Organon, Merck A/S, and Abbott. A.G. has received honoraria from Lab Seid and travel support from Merck Serono. P.H. has received honoraria from Merck, IBSA, Gedeon Richter, and Besins Healthcare. L.D.L.F. has received consulting fees from Gedeon Richter, Ferring Pharmaceuticals, and Organon, travel support (personal and to institution) from Gedeon Richter, Ferring Pharmaceuticals, IBSA, Merck, Organon, and Theramex, and educational support (to institution) from Gedeon Richter and Merck. A.P. has received grants from Gedeon Richter, Ferring Pharmaceuticals, and Merck A/S, consulting fees from Gedeon Richter and Ferring Pharmaceuticals, honoraria from Ferring Pharmaceuticals, Gedeon Richter, Merck A/S, Abbott, and Organon, and travel support from Gedeon Richter. D.S. has received grants from Organon, Ferring Pharmaceuticals, Besins Healthcare, Gedeon Richter, and Vitrolife, honoraria from Organon, Ferring Pharmaceuticals, Besins Healthcare, Gedeon Richter, and Merck, travel support from Organon, Ferring Pharmaceuticals, Besins Healthcare, Gedeon Richter, and Merck, and is President of the Belgian Society for Reproductive Medicine. N.P.P. has received grants from Merck Serono, Ferring Pharmaceuticals, Theramex, Organon, Besins Healthcare, and Gedeon Richter, consulting fees from Merck Serono, Besins Healthcare, Organon, IBSA, FertilAI, and Alife, and honoraria from Merck Serono, Theramex, IBSA, Ferring Pharmaceuticals, Organon, Roche Diagnostics, and Besins Healthcare. S.G.M., F.M., and F.F. have no interests to declare. TRIAL REGISTRATION NUMBER:NCT02418572 (ClinicalTrials.gov).

Research question Do FSH types (recombinant FSH (recFSH) vs. human menopausal gonadotropin (HP-hMG) vs. corifollitropin alfa (CFA)) differ in the efficiency of ovarian response, as assessed by Follicular Output Rate (FORT) and Follicle-to-Oocyte Index (FOI)? Design This retrospective cohort study analyzed 4525 antagonist IVF/ICSI cycles using CFA (n=936), recFSH (n=2156) or HP-hMG (n=1433). FORT and FOI indices were calculated as ratios of follicle or oocyte yield to antral follicle count (AFC). Generalized Estimating Equations (GEE) were used to account for clustering by patient with covariates for age, AMH, weight, stimulation duration, gonadotropin dose, and elective freeze. Results CFA yielded the highest ovarian response indices (FORT16–22: 61.6%, FOI-COC: 106.4%, FOI-MII: 81.1%), compared with recFSH (49.3%, 98.0%, 74.6%) and HP-hMG (39.6%, 71.5%, 46.5%). Stratified analyses confirmed this ranking across AMH strata. In low AMH (<1.1 ng/mL), FOI-MII was 81.4% with CFA, 64.5% with recFSH, and 49.2% with HP-hMG. In normal/high AMH (1.1–3 ng/mL) FOI-MII was 86.3%, 64.5%, and 53.1%. GAM analyses demonstrated a modest non-linear AMH effect only for CFA, peaking at ∼1.5–2.0 ng/mL before plateauing, while recFSH rose gradually and HP-hMG remained flat. Age-stratified analyses showed a uniform decline across FSH types. OHSS risk (>18 follicles ≥11 mm) was lowest with CFA (6.5%), compared with 19.8% for HP-hMG and 39.2% for recFSH. Conclusion After adjustment for antral follicle count, CFA achieved the highest FORT and FOI across most AMH strata, consistently outperforming HP-hMG and matching or exceeding recFSH, while maintaining the lowest follicle-count–based OHSS risk.

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

What is the function of GATA3 in mouse and human trophectoderm (TE) formation, and does its function differ between species? GATA3 is dispensable for mouse blastocyst formation, whereas GATA3 is suggested to be important for human trophoblast stem cell (TSC) differentiation, resembling TE in vitro. GATA3 and the highly similar transcription factor (TF) GATA2 are key TE markers in mouse and human embryos. Gata3 knockout (KO)/knockdown studies suggested its crucial role in mouse blastocyst formation, with embryos arresting at the morula stage. However, other studies reported lethality only at 11–12 dpf. In human, GATA3 is detected in a subset of morula cells, suggesting an early role in TE formation. Overexpression of GATA2 and GATA3 in human embryonic stem cells (hESCs) drives differentiation into TSCs, indicating their necessity for human TE induction. However, GATA3’s exact function in mouse and human TE formation is still unclear. Gata3 KO mouse embryos were assessed morphologically until E4.5 and immunofluorescent (IF) stained to confirm GATA3’s absence (n = 69). Besides, the impact of the KO on total cell numbers and TE cells was assessed by IF staining Gata3 KO blastocysts for nuclei (n = 16,DAPI) and CDX2 (n = 15, TE marker). Naïve hESCs were targeted for GATA3 with CRISPR/Cas9 and were differentiated towards TSCs (in vitro counterpart of human TE), to check GATA3’s necessity for TSC differentiation/human TE formation. Following delivery of Gata3 targeting CRISPR/Cas9 ribonucleoprotein complexes in mouse zygotes, embryos were cultured and morphologically observed until E4.5, alongside media and Scrambled (inactive gRNA) controls. Targeted embryos were IF stained and genotyped by next generation sequencing. Single-cell RNA sequencing data from Petropoulos et al.(2016) was analyzed to assess GATA3’s expression throughout human preimplantation development, followed by IF of human blastocysts. Naïve hESCs were targeted for GATA3 with CRISPR/Cas9 through nucleofection and differentiated towards TSCs. IF staining of WT mouse embryos (n = 16) revealed that GATA3 is expressed in TE progenitor cells in the morula, and its expression persists until the late blastocyst stage. Majority (94%, n = 65) of Gata3-/- embryos (n = 69) successfully formed blastocysts, with no significant morphological difference compared to Scrambled embryos (n = 148). However, Gata3 -/- mouse embryos showed a reduced number of total cells (n = 16) and CDX2 + (TE marker) cells (n = 15). Single-cell RNA sequencing data of human embryos of Petropoulos et al. (2016) revealed that GATA3 is expressed in a subset of morula cells, increases in early TE progenitors, and persists in the mature TE in human blastocysts. GATA2 (TF highly similar to GATA3) expression onsets later in the early TE and predominates in the mature TE. This suggests an earlier role of GATA3 in the human TE than GATA2, which can be considered as a mature TE marker. IF confirmed GATA3’s expression in the human TE at the blastocyst stage and co-expression with TEAD4, GATA2 and CDX2. Differentiation of mosaic GATA3 KO naïve hESCs towards TSCs (in vitro counterpart of human TE) revealed a hampered differentiation, with some colonies unable to form flattened TSC colonies, suggesting GATA3’s crucial role in human TE differentiation. CRISPR/Cas9 editing faces challenges like mosaicism, off-target effects, and chromosomal rearrangements. In addition, limited availability of human gametes hinders studying the role of GATA3 in the human blastocyst formation. Therefore, in vitro stem cell models are used as a first step to investigate GATA3’s role in human TE/TSC formation. Our goal is to identify the molecular pathways crucial for TE formation in mouse and human embryos. By using CRISPR/Cas9 to generate KO embryos/naïve hESCs, we gain insight into key molecular mechanisms, potentially shedding light on pregnancy failure due to defects in the TE specification, formation or maintenance. No

Can C-type natriuretic peptide (CNP) based CAPA-IVM (Capacitation in-vitro maturation) improve ovarian tissue oocyte (OTO) maturation competence in transgender patients? CAPA-IVM improves oocyte maturation but shows comparable developmental potential in transgender OTO when compared with standard and in-house IVM. OTO-IVM could be an additional method of fertility preservation in patients where prior ovarian stimulation is undesired. OTO-IVM has resulted in live births in cancer and polycystic ovarian syndrome (PCOS) patients. CAPA-IVM has further improved oocyte competency in these patients by better synchronizing nuclear and cytoplasmic maturity. Similarly, OTO can be collected from transgender men without ovarian stimulation during gender reassignment surgery. While oocytes do survive and mature, OTO-IVM in transgender men is characterized by a decreased fertilization rate and severely compromised developmental competency. Hence, this study investigated whether CAPA-IVM can improve cytoplasmic maturity for transgender OTO. Patients were recruited from July 2022 to December 2024. Ovaries were collected from 21 transgender patients (age= 20-24 years, mean age=21 years) who underwent gender reassignment surgery after testosterone treatment (mean treatment duration=33 months). All ovaries were collected in a cold medium (4oC) and manipulation was performed within 30 minutes of the collection for the retrieval of cumulus oocyte complexes (COCs). Collected COCs were cultured either in in-house IVM and Standard IVM medium for 48 hours or in biphasic CAPA-IVM for 54 hours (24 hours pre-maturation culture and 30 hours standard IVM). Following maturation assessment, oocytes were analysed for calcium-releasing potential, and developmental competency after ICSI. In vitro matured MI (metaphase I) oocytes from stimulated IVF patients served as controls. Shallow whole genome sequencing was performed on subsequent embryos to detect chromosomal abnormalities in all groups. After culturing 716 COCs, the survival rate following maturation was comparable between In-house and CAPA-IVM (76% vs 80% p = 0.2661), but significantly higher in CAPA-IVM and in-house IVM when compared to Standard IVM (64% p < 0.0001 and p = 0.007 respectively). CAPA-IVM and in-house IVM showed comparable maturation rates (46% vs 45% p = 0.782). However, maturation was significantly improved in CAPA-IVM when compared to standard IVM (46% vs 36%, p = 0.027). The average calcium release (in arbitrary units) for CAPA-IVM (1.40AU); standard IVM (1.25AU), and in-house IVM (2.34AU) were significantly lower compared to controls (5.40AU). Following ICSI, 26/45(58%) CAPA-IVM, 21/34(62%) Standard IVM, and 25/42(60%) in-house IVM oocytes were normally fertilized, which was comparable with ICSI-control 29/38(76%). Blastocyst rates were significantly lower in Standard IVM (0/21,p= 0.007) and CAPA-IVM (1/26, p = 0.015), but comparable in in-house IVM (3/25, p = 0.190) when compared to controls (10/29). Shallow whole genome sequencing of developed embryos (Day 2- Day 5) showed that 8/13 (61.5%) in CAPA-IVM, 4/8 (50%) in In-house IVM, 1/6 (17%) in Standard IVM, and 4/12 (33%) in the control group were chromosomally normal. The limited inclusion of patients warrants caution in the interpretation of the results. Moreover, the lack of a better control group might mask the properties of OTO-IVM. Our results demonstrate an improvement in oocyte survival and maturation rates with CAPA-IVM. Nonetheless, no increase was observed in embryo development after CAPA-IVM. Ongoing proteomic and transcriptomic analysis will shed more light on the advantages of the current IVM systems and decipher the true potential of OTO-IVM for transgender men. No

BACKGROUND:Transgender and gender diverse (TGD) people seek gender-affirming care at any age to manage gender identities or expressions that differ from their birth gender. Gender-affirming hormone treatment (GAHT) and gender-affirming surgery may alter reproductive function and/or anatomy, limiting future reproductive options to varying degrees, if individuals desire to either give birth or become a biological parent. OBJECTIVE AND RATIONALE:TGD people increasingly pursue help for their reproductive questions, including fertility, fertility preservation, active desire for children, and future options. Their specific needs certainly require more insight into the effects of GAHT on gonads, gametes, and fertility. This systematic review aims to provide an overview of the current knowledge on the impact of GAHT on gonads, gametes, fertility, fertility preservation techniques, and outcomes. SEARCH METHODS:This review was registered in the PROSPERO registry under number CRD42024516133. A literature search (in PubMed, Embase, and Web of Science) was performed with a medical information specialist until 15 November 2024. OUTCOMES:In all TGD people using GAHT, histological changes have been reported.Using testosterone GAHT, ovarian cortical and stromal changes were reported by various studies. In most studies, persistent activity in folliculogenesis can be concluded based on the descriptions of the follicle count, distribution, and oocyte retrieval yield. However, there may be a negative effect on the fertilization rate in the presence of testosterone. Reports of successful ovarian stimulation, fertilization, pregnancies, and live births have been published, describing cases with and without testosterone discontinuation.After using oestrogen GAHT, testes are reported to be more atrophic, including smaller seminiferous tubules with heavy hyalinization and fibrosis. Spermatogenic levels varied widely from complete spermatogenesis to meiotic arrest with spermatids, to spermatogonial arrest, Sertoli cells only, or even tubular shadows. Oestrogen and anti-androgen treatment causes higher proportions of sperm abnormalities (i.e. low total sperm count, low sperm concentration, poor sperm motility) or azoospermia. However, after cessation, this may be restored. WIDER IMPLICATIONS:Although knowledge of the effect of GAHT is growing, blind spots remain to be uncovered. Therefore, additional research in this specific population is needed, preferably comparing outcomes before and after the start of GAHT. This may help to reveal the pure impact of GAHT on reproductive functioning. Research suggestions also include investigations into the reversibility of the GAHT effect, especially for those who start transition at a young age. Looking carefully at the presented data on GAHT effects on gonads and gametes, the correct advice is to assess and reassess reproductive wishes and preferences repeatedly, and also to explore individual fertility preservation needs during gender-affirming treatment, given the expanding knowledge and therapy opportunities. Finally, concerns regarding long-term health outcomes and quality of life of children born by the use of gametes preserved after exposure to GAHT require prospective follow-up studies.

Does transdermal testosterone treatment improve markers of ovarian reserve compared to placebo in infertile women with diminished ovarian reserve (DOR) according to the Bologna criteria? Transdermal testosterone treatment did not result in significantly increased AMH levels, antral follicle count (AFC), or number of oocytes or mature oocytes retrieved. DOR is a common cause of infertility and represents one of the most challenging patient groups to treat. Testosterone treatment has been proposed to improve outcomes in this population, with animal studies showing that testosterone promotes oocyte activation and FSH responsiveness whilst reducing apoptosis and follicular atresia. The most recent meta-analysis suggested testosterone pretreatment could result in ∼1 additional oocyte being retrieved. However, this was based on studies of low-to-moderate quality, resulting in substantial uncertainty regarding the effect of testosterone on ovarian response. This study aims to determine whether testosterone pretreatment improves markers of ovarian reserve in women with DOR. Between April 2015 and August 2022, 288 participants were randomised in a placebo-controlled RCT of transdermal testosterone to improve clinical pregnancy rates. This is a secondary analysis of that trial and includes 273 participants who had results available for ovarian reserve testing with AMH and/or AFC. Inclusion criteria were age 18-43 with infertility and DOR according to the Bologna criteria. Participants included in this analysis were recruited from eight fertility units in Spain, Belgium and Denmark. Participants were randomised to ∼9 weeks of transdermal testosterone (5.5mg/day) or placebo prior to commencing ovarian stimulation. AMH, AFC and serum total testosterone were measured at study entry and at the completion of the study drug but prior to commencing ovarian stimulation. Baseline characteristics were similar between groups, including ovarian reserve markers. After ∼9 weeks of treatment, there was no difference between the testosterone and placebo groups in either AMH levels (0.66 ± 1.30 ng/ml vs 0.55 ± 0.34 ng/ml, mean difference (MD) 0.11 (95% CI -0.15 to 0.37)) or AFC (5.0 ± 2.8 vs 5.0 ± 2.3, MD 0.0 (95% CI -0.6 to 0.7)). In the entire study population (n = 288) there was no difference between the testosterone and placebo groups in either the number of oocytes retrieved (MD 0.34, 95% CI -0.36 to 1.0) or the number of mature oocytes retrieved (MD 0.23, 95% CI -0.40 to 0.86). In subgroup analyses by age and baseline testosterone level, no population was identified that benefited from testosterone treatment. ANCOVA analysis was performed to adjust for age, BMI, baseline serum AMH level or AFC respectively, and baseline testosterone level. After adjustment, testosterone treatment compared to placebo was not associated with either AMH (marginal means 0.64 (95% CI 0.47 to 0.81) versus 0.60 (95% CI 0.44 to 0.76)) or AFC (marginal means 5.0 (95% CI 4.6 to 5.5) versus 5.0 (95% CI 4.6 to 5.4). As intended, the testosterone dose used achieved levels at the upper end of the reference range, however some previous studies have advocated supraphysiological targets. The duration of treatment aligns with knowledge of ovarian physiology, however we cannot exclude longer treatment, higher doses or different androgens having different effects. Compared to placebo, transdermal testosterone treatment did not improve biochemical or sonographic markers of ovarian reserve in women with DOR according to the Bologna criteria. These results do not support testosterone treatment in this population to improve ovarian response. Yes

Can single-cell, mass spectrometry-based proteomics reveal proteins underlying the reduced fertilizing capacity of Patl2-/- MII mouse oocytes? Is it possible to treat Patl2-related infertility? Reduced protein synthesis from maternal mRNAs is detected in Patl2-/- MIIs, which can be rescued by spindle transfer (ST), but not assisted oocyte activation (AOA). PATL2 is an RNA-binding protein that represses maternal mRNA translation during oocyte maturation. PATL2 mutations in humans often cause GV arrest, although some patients produce MII oocytes with reduced fertilization and developmental potential. Consequently, oocyte donation is required. The Patl2-/- knockout mouse model offers a unique opportunity to study Patl2-related infertility and potential treatments. To gain a deeper understanding of the role of Palt2 at the MII stage, we evaluated various oocyte quality markers and conducted single-cell proteomic analysis. Proposed treatments to overcome low fertilization, including AOA, targeting calcium release, and ST, replacing suboptimal cytoplasm, were investigated. Palt2 -/- mice (C57BL/6NTac-Patl2tm1a) with a deletion of Patl2 exon 7 were used. Breeding of heterozygous mice from April 2021 to October 2023 produced 271 pups, of which 36 were homozygous Palt2-/- females. MII oocytes were collected from Patl2-/- and Patl2+/+ females for the evaluation of oocyte quality markers, and for the assessment of AOA and ST efficacy. Moreover, some MII oocytes were vitrified for subsequent single-cell proteomics. At least three replicates were conducted per experiment. Four- to 12-week-old mice underwent superovulation and oocyte collection to assess maturation rate. Spindle configurations, calcium releasing capacity after SrCl2 exposure, as well as activation (AR) and blastocyst rates (BR) after PIEZO-ICSI were evaluated in MII oocytes. AOA was performed by SrCl2 exposure, and ST involved transferring the Patl2-/- spindle to Patl2+/+ enucleated cytoplasm followed by SrCl2 exposure. Vitrified-warmed oocytes were used for single-cell proteomics using a timsTOF Ultra mass spectrometer operated in diaPASEF mode. The MII rates were higher in Patl2+/+ (89.39%) than Patl2-/- mice (79.63%, p = 0.015). Nuclear maturation was not compromised, as normal spindle rates were similar in Patl2+/+ (86.00%) and Patl2-/- oocytes (78.57%, p = 0.414) The total calcium released during oocyte activation did not significantly differ between Patl2+/+ (AxF=4.59) and Patl2-/- oocytes (AxF=6.15, p = 0.145) . After PIEZO-ICSI with wild-type sperm, reduced activation (Patl2-/-=31.71%, Patl2+/+=76.74%, p < 0.0001) and blastocyst rates (Patl2-/-=7.69%, Patl2+/+=42.42%, p = 0.035) were observed in knockout oocytes. Single-cell proteomics identified 4939 proteins and confirmed absence of Patl2 in knockout oocytes, analyzing 25 Patl2+/+ and 27 Palt2-/- MIIs. After filtering, 3777 proteins were used for statistical analysis, revealing 1511 differentially expressed proteins (q-value < 0.05; 999 downregulated, 512 upregulated in Patl2-/- oocytes). Multiple proposed Patl2 interactors (Cpeb1, Eif4e1b) and maternal effect genes (MEGs) implicated in RNA regulation (Zar1), meiosis progression (Wee2), cell division (Cdk1), and microtubule organization (Tuba4a) were significantly reduced. Additionally, proteins involved in calcium dynamics were upregulated (Pde1b, Tmco1, Micu). Consistent with these observations, AOA did not lead to comparable activation (Patl2-/-=75.95%, Patl2+/+=95.97%, p < 0.0001) or blastocyst rates (Patl2-/-=45.00%, Patl2+/+=79.83%, p < 0.0001). However, ST treatment in Patl2-/- oocytes restored activation (100%) and blastocyst rates (75.00%) to levels observed in Patl2+/+ oocytes (AR = 96.15%, p = 1 and BR = 96.00%, p = 0.049). The Patl2-/- mice exhibits a less severe phenotype compared to patients carrying PATL2 variants. Patl2-/- oocytes display high MII rate without significant spindle abnormalities, which contrasts with a previous published report. Additionally, the comparison between AOA and ST treatments was conducted using parthenogenetically activated oocytes, rather than biparental embryos. ST could treat PATL2-related female-infertility in patients with MII oocytes, whereas AOA is unlikely effective, as calcium release in Patl2-/- oocytes is slightly increased. Single-cell proteomics reveals cytoplasmic deficiencies in Patl2-/- MII oocytes linked with abnormal regulation of MEGs, some already associated to fertilization failure and embryo arrest in humans. No

STUDY QUESTION:What information does an international group of professionals and egg donors consider relevant and morally necessary for prospective egg donors to provide valid informed consent? SUMMARY ANSWER:Participants considered 80% of all concrete information items (CIIs) to be relevant (e.g. all legal aspects) and 67% to be morally necessary. WHAT IS KNOWN ALREADY:Studies indicate that egg donors are not always adequately informed and have expressed a desire for more comprehensive information. This highlights the need for a comprehensive guideline of essential information for prospective egg donors. STUDY DESIGN, SIZE, DURATION:This modified Delphi study used a survey in an iterative process of three rounds to reach a consensus on what information items are relevant and morally necessary for a valid informed consent of candidate egg donors. Invitations to participate were sent out in November 2023 and the final round closed in November 2024. PARTICIPANTS/MATERIALS, SETTING, METHODS:The 35 participants were experienced egg donors and professionals from a range of disciplines (social and medical sciences, bioethics, psychology, fertility medicine and law) from 14 countries. The survey consisted of 13 categories and 133 CIIs, which participants scored for relevance via a 4-point Likert scale and moral necessity on a dichotomous scale (yes/no). Content Validity Index (CVI) was calculated for measuring relevance and percentage of agreement for moral necessity. A comment section was available. MAIN RESULTS AND THE ROLE OF CHANCE:Consensus was indicated as an I-CVI (CVI per item) of 0.78 or higher. The same cut-off was used to indicate consensus for moral necessity. For 27 out of 133 CIIs, the I-CVI was lower than 0.78. The percentage of moral necessity was below 0.78 for 44 CIIs. Four CIIs reached a I-CVI of 1: all experts thought it was relevant for a candidate donor to know (i) the need to undergo ovarian stimulation and (ii) a retrieval procedure, as well as (iii) her legal rights over the donated eggs after the retrieval procedure and (iv) her legal right to withdraw consent. The latter is the only CII that scored 100% on moral necessity. The CII with the lowest I-CVI is 'The family type and characteristics of the potential recipients of the donor eggs' (0.32). The CII with the lowest percentage of agreement for moral necessity was 'An egg donor's social circle might give negative feedback/opinions on the donation' (36.36%). In several categories (e.g. 'Physical side-effects and risks'), almost all CIIs reached a consensus among experienced egg donors, bioethicists, and humanities and social sciences experts, while hardly any CII reached a consensus among fertility experts, lawyers, and academic medical doctors. LIMITATIONS, REASONS FOR CAUTION:Despite our efforts, we were unable to obtain input from registered nurses and midwives. Not all participants remained engaged through all the iterative rounds, which may weaken the results of the study. However, the dropout rates between rounds in this study were within the acceptable 20-30% range. WIDER IMPLICATIONS OF THE FINDINGS:Our results give fertility professionals a standard of essential information to make sure that prospective egg donors are adequately informed and know what to expect when they decide to donate. It also gives researchers a potential standard to evaluate the quality of the information provision in fertility clinics. STUDY FUNDING/COMPETING INTEREST(S):This study is funded by the Special Research Fund, Bijzonder Onderzoeksfonds of Ghent University (BOFSTG2020000901). The authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER:N/A.

Is additional ploidy screening (PGT-A) on blastocysts screened for monogenic conditions (PGT-M) of any added value? Additional ploidy screening improved live birth rates per single embryo transfer and significantly reduced pregnancy loss odds, with no impact on cumulative cycle outcomes. Numerical chromosomal abnormalities in embryos are key contributors to implantation failure, miscarriage, and delayed pregnancy. Preimplantation genetic testing for aneuploidy (PGT-A) aims to improve outcomes by excluding aneuploid embryos, though its utility remains debated. Since July 2021, our center has implemented comprehensive PGT-M (coPGT-M) to simultaneously screen for monogenic conditions and aneuploidies, allowing only unaffected, euploid embryos for transfer. This approach may shorten time to pregnancy and reduce miscarriage risk. This single-center retrospective cohort study analyzed first-rank cycles conducted from December 2019 to October 2022. Outcomes - including biochemical pregnancy, clinical pregnancy, live birth rates, and pregnancy loss - were compared between 126 PGT-M and 131 coPGT-M cycles (226 PGT-M and 177 coPGT-M single embryo transfers [SETs]). Clinical follow-up extended through December 31, 2024. Exclusively first-rank cycles with ≥1 biopsied blastocyst were included. Exclusion criteria encompassed cycles with no oocytes retrieved, no embryos suitable for biopsy, use of non-autologous or cryopreserved oocytes, and translocation carriers. Statistical analyses included Student’s t-tests, Chi-square tests, Fisher’s exact tests, and generalized estimating equations (GEE) models for transfer-specific comparisons. A total of 126 PGT-M and 131 coPGT-M cycles were included, with both groups demonstrating comparable baseline characteristics, including female age (30.4±4.2 vs. 30.3±3.9 years, p = 0.810), AMH (3.00±2.58 vs. 2.93±2.22 mcg/L, p = 0.805), BMI (23.6±4.2 vs. 24.6±4.4 kg/m², p = 0.095), and mode of inheritance of the monogenic condition. Laboratory outcomes were comparable, with the mean number of biopsied blastocysts being 5.6±3.8 in the PGT-M group and 6.1±4.7 in the coPGT-M group (p = 0.308), while the mean number of embryos eligible for transfer were also similar (resp. 2.6±2.2 vs. 2.4±2.0, p = 0.424). At least one ongoing pregnancy or live birth was achieved by 42.1% of patients after PGT-M and 51.9% after coPGT-M (p = 0.114), with a comparable mean number of transfers required (1.55±0.75 vs. 1.47±0.80, p = 0.592). The odds of having an ongoing pregnancy or live birth per SET was 86% higher in the coPGT-M group compared to the PGT-M group (odds ratio 1.86 [CI 1.21;2.87], p = 0.005). Additionally, the odds of experiencing a pregnancy loss were 50% lower in the coPGT-M group compared to the PGT-M group (odds ratio 0.5, [CI 0.27;0.93], p = 0.028). These findings highlight the enhanced outcomes associated with coPGT-M in terms of higher live birth rates and reduced miscarriage odds per single embryo transfer. Not all eligible embryos had been transferred at the time of analysis, and ongoing follow-up will provide more comprehensive cumulative data, likely further strengthening the conclusions. Cumulative outcomes per cycle were comparable; however, coPGT-M showed higher per-SET pregnancy success and lower miscarriage risk. These findings support the potential clinical benefits of incorporating PGT-A into PGT-M protocols, even in younger patients. No

Can variant screening identify the genetic causes of female-related oocyte/zygote/embryo maturation arrest (OZEMA) and male-related failed fertilization (FF)? Genetic analysis revealed CHEK1/WEE2/TUBB8 mutations in three OZEMA/FF female patients, while PLCZ1/ACTL9/IQCN variants were found in 6 out of 17 male patients with FF. OZEMA, a condition characterized by oocyte, zygote, or embryo maturation arrest, can contribute to female infertility, while FF can be attributed to both male and female factors. Recent advancements in next-generation sequencing (NGS) have facilitated the identification of genetic variants associated with these conditions, enhancing diagnostic precision, prognostic prediction, and the potential for personalized treatment strategies. Consequently, a targeted gene panel was employed to enhance the efficiency of genetic screening for OZEMA and FF. From April 2023 to December 2024, whole-exome sequencing (WES) analysis using a 51-gene panel was conducted in 29 females with OZEMA/FF to identify potential genetic variants associated with their phenotypes. In a second cohort, 17 male individuals exhibiting FF or suboptimal fertilization after intracytoplasmic sperm injection (ICSI) were specifically screened for PLCZ1, ACTL9, and IQCN variants. Twenty-nine female patients with OZEMA or FF underwent genetic screening. Genomic DNA from peripheral blood was extracted and subjected to WES to identify the potential variants. Seventeen male patients with suboptimal or FF after ICSI were screened for variants in PLCZ1/ACTL9/IQCN. DNA was extracted from saliva samples, followed by NGS analysis. Variants identified through NGS were validated via Sanger sequencing. Subsequently, assisted oocyte activation (AOA) during ICSI was performed in patients with identified male mutations. Among the 29 female patients, three were found to harbor pathogenic variants. The first patient, a 37-year-old with zygote cleavage failure (0/4 ICSI, 0/5 IVF), carried a heterozygous mutation in CHEK1 (p.Arg442Gln). The second patient, a 30-year-old with total FF after two ICSI cycles (0/9 MII oocytes) and FF after AOA during ICSI (0/6 MII oocytes), exhibited a homozygous WEE2 mutation (p.Arg200*). The third patient, a 32-year-old with oocyte maturation arrest after two ICSI cycles (25 MI oocytes, 2 GV oocytes that did not mature further after extended in vitro culture), carried a homozygous variant in TUBB8. In the second cohort, six male patients were identified to carry variants in PLCZ1, ACTL9, or IQCN. One patient with total FF carried a compound heterozygous variant in PLCZ1 (p.His233Leu, p.Gln94*), while another male with total FF carried a heterozygous variant in ACTL9 (p.Arg271Pro), both males also displayed an IQCN variant. IQCN variants were detected in four other male patients. Although these IQCN variants were predicted to be benign, their high detection rate (6/17, 35.3%) suggests a potential role in impaired fertilization. Application of assisted oocyte activation in five of these male patients resulted in two ongoing pregnancies. The pathogenicity of the identified variants was assessed using predictive bioinformatics tools, but the findings lack comprehensive experimental validation. These results enhance our understanding of the genetic underpinnings of human infertility. The findings suggest that genetic screening could have a broader clinical impact, aiding in the diagnosis and management of patients with previous unexplained infertility. No

What are the experiences, barriers and needs of pregnant trans masculine individuals and their healthcare providers within preconception and prenatal care? Trans masculine people and their healthcare providers experience various barriers regarding access, gender-inclusive language, trans-inclusive care and information, transphobia, and anticipatory anxiety towards each other. Research on the experiences of trans masculine people and their health care providers within pregnancy-care is limited. Pregnancy-related care has an exclusionary focus on femininity and (cisgender) women, resulting in trans erasure and cisnormativity in ideas, spaces, systems, and laws. This is a qualitative study. A thematic analysis on data from in-depth interviews with four trans masculine individuals and ten healthcare providers was performed. The inclusion of both groups aimed to gather sufficient insight into the needs and barriers to provide adequate recommendations. The respondents consist of two groups: (1) Trans masculine individuals who are or have been pregnant, (2) healthcare providers with experience in taking care of trans masculine patients during their pregnancy or fulfilling their pregnancy wish. Trans masculine respondents were suitable if they identify as trans masculine; have a masculine or androgynous gender expression, first name and/or pronouns according to society; are or have been pregnant; the pregnancy happened after a (partial) social transition. The analysis resulted in 5 key themes: (1) Femininity and pregnancy; (2) Gendered language; (3) Needs, barriers, and information resources; (4) Stress and anxiety, (5) Trans-inclusive care. All interviewed participants described a primary focus on women or femininity within pregnancy-related care and societal views, resulting in difficulties and challenges for both trans masculine patients and their healthcare providers within pregnancy-related spaces, language and ideas resulting in transphobia, discrimination and distress. Both trans masculine individuals and healthcare providers feel the necessity for more trans-specific information and scientific knowledge regarding pregnancy-related care. Almost all respondents experienced anticipatory anxiety towards each other. For all interviewed trans masculine patients, stress was caused by the anticipation of which healthcare providers and the corresponding care experience they would receive. A significant number of healthcare providers experienced anticipatory anxiety, mostly linked to misgendering and being pointed out by the patient or their partner. There are several barriers to trans-inclusive preconception and prenatal care such as openness and attitude of both healthcare providers and trans masculine patients towards each other. Nevertheless, individual and organizational good practices of trans-inclusive care were identified by most participants such as gender-inclusive language, trauma-informed care practices and trans-inclusive measures at the department level. This study used the opting-in principle. This may be revolved in a recall bias, where only healthcare providers who already felt competent/confident in their care for trans masculine individuals may have enrolled. Nevertheless, the current pool of healthcare providers also experienced barriers and challenges, making this study representable and valuable. This is the first study to develop insights into the care experiences of trans masculine individuals and their healthcare providers. This dual focus is particularly relevant, as most previous research only focuses on one group. This combination makes it possible to unravel similarities and differences in their perceptions and experiences. No