Abstract STUDY QUESTION What are the mechanisms underlying the complex interactions between polyendocrine metabolic ovarian syndrome (PMOS) and obesity as studied by a multi-omics integration approach? SUMMARY ANSWER Multi-Omics Factor Analysis v2 (MOFA+) suggested that PMOS was characterized by an androgen-driven metabolic reprogramming in which sexual dimorphism was reshaped in an obesity- and omics-specific manner, consisting of strong associations of proinflammatory proteins and adipokines with gut microbiota genera and metabolomics variables that were amplified by obesity. WHAT IS KNOWN ALREADY PMOS is a common metabolic-endocrine disorder in which androgen excess, weight excess and obesity complex interactions define a particularly unfavorable cardiometabolic phenotype. STUDY DESIGN, SIZE, DURATION Cross-sectional study with 2 control groups involving 46 Caucasian young adults, conducted at an Academic Hospital in Madrid, Spain. PARTICIPANTS/MATERIALS, SETTING, METHODS We recruited 15 women with PMOS, 16 healthy control women, and 15 control men of similar mean age and BMI. Participants were further grouped into non-obese (BMI < 30 kg/m²) or obese (BMI ≥ 30 kg/m²) subjects. We integrated serum metabolomics and circulating proteins with gut microbiota composition, using MOFA+. MAIN RESULTS AND THE ROLE OF CHANCE MOFA+ identified five latent factors explaining the main sources of variation across omics layers. Results showed sexual dimorphism in an omics- and obesity-specific manner: the metabolomics profile presented sexual dimorphism in non-obese individuals, whereas circulating proteins and gut microbiota differed between women with or without PMOS and men only in obese subjects. Correlation analyses revealed a global weakening of associations in men, particularly among metabolites, a reinforcement of protein-related associations with obesity, and a distinct PMOS-specific pattern characterized by strengthened correlations within and between gut microbiota and proteomic features. Women with PMOS exhibited the strongest metabolomics-proteomics correlations, further exacerbated by obesity. LIMITATIONS, REASONS FOR CAUTION The relatively small sample size of our study might have missed relevant associations that could have reached statistical significance in larger samples. Also, we only included women with hyperandrogenic phenotypes of PMOS. Hence, our results may not apply to non-hyperandrogenic phenotypes of the syndrome. WIDER IMPLICATIONS OF THE FINDINGS PMOS showed a metabolic profile similar to that of obesity even in the absence of excess weight, supporting the concept that adipose tissue dysfunction, proinflammatory proteins and adipokines, rather than fat mass excess per se, was a central driver of the metabolic disturbances of the syndrome. Altogether, these findings highlight the complex interplay between androgen excess, adiposity and host–microbiota interactions, and provide a systems-level framework that may facilitate the identification of novel biomarkers and targeted therapeutic strategies in PMOS. Also, these findings highlight the importance of considering sex, sex hormones and obesity-specific contexts in biomarker discovery and mechanistic studies. FUNDING This research was funded by Instituto de Salud Carlos III grants PI11/0357, PI18/01122 and PI21/00116, and co-funded by the European Union. Francisco Garcia-Garcia was supported by CIAICO/2023/149 funded by the Consellería de Educación, Cultura y Universidades de la Generalitat Valenciana, and PID2021-124430OA-I00 funded by MICIU/AEI/10.13039/501100011033 and by ERDF/EU. CIBERDEM and IRYCIS are also initiatives of Instituto de Salud Carlos III. DISCLOSURES The funding organizations played no role in the study design; collection, analysis, and interpretation of data; the writing of the report; or the decision to submit the report for publication. The Authors have no competing interests to disclose TRIAL REGISTRATION NUMBER N/A
Abstract STUDY QUESTION In best-prognosis preimplantation genetic testing (PGT) cycles—defined by the availability of multiple transferable embryos—can polygenic embryo screening (PES) provide meaningful, context-dependent stratification that may influence embryo prioritization? SUMMARY ANSWER In a sister-pair breast cancer validation cohort, the polygenic risk score (PRS) showed modest predictive performance, and when applied retrospectively to embryos, differences in modelled susceptibility were context-dependent, primarily influenced by the number of available embryos, parental polygenic risk profile, the limited discriminative power of the PRS, and the presence of disease-related monogenic variants. WHAT IS KNOWN ALREADY The use of PES as an embryo-ranking tool within PGT remains controversial. Its potential utility depends on embryo availability and clinical context, yet empirical evaluations in real-world PGT settings remain limited. STUDY DESIGN, SIZE, DURATION This retrospective study combined (i) family-based validation using 184 affected–unaffected sister pairs, and (ii) embryo-level analysis of 310 preimplantation genetic testing for monogenic disorders (PGT-M) cycles comprising 1,722 embryos. Simulation modelling estimated differences in modelled embryo-level PRS percentile ranking under varying clinical and genetic scenarios. PARTICIPANTS/MATERIALS, SETTING, METHODS Family-based performance of a breast cancer PRS was evaluated using affected and unaffected sister pairs. Embryo genotype data from PGT-M cycles were used to model PES under varying conditions, including the number of transferable embryos, the presence of pathogenic monogenic variants, and parental PRS percentile strata. MAIN RESULTS AND THE ROLE OF CHANCE Within-family PRS discrimination was modest: sisters in the top 10% of the PRS distribution had 4.07-fold higher breast cancer odds than their siblings (95% CI: 1.55–6.33). In the PGT-M cohort, 55% of cycles produced ≥3 transferable embryos, with a median within-cycle spread of approximately 25 PRS percentiles. In BRCA1/2-related cycles, PRS did not introduce distinct risk strata but revealed dispersion in modelled susceptibility among embryos sharing the same monogenic background. Embryo PRS distributions were concordant with parental profiles: 71% of embryos from couples with parental PRS ≥80th percentile were classified as high-PRS, compared with 7% when at least one parent was ≤20th percentile. Simulation analyses indicated that PRS-based embryo prioritization was associated with a 12-point lower mean embryo PRS percentile compared with morphology-based embryo prioritization in PGT-M cycles, attenuating to 10 points when PGT-A was incorporated. These differences reflect a statistical surrogate outcome only and do not demonstrate reduction in lifetime disease incidence or other clinical benefits. LARGE SCALE DATA Clinical data from 310 PGT-M cycles involving 1,722 embryos and a family-based validation cohort of 184 affected–unaffected sister pairs were analyzed. No population-scale dataset was generated. LIMITATIONS, REASONS FOR CAUTION These findings derive from high-prognosis PGT-M cycles and may not generalize to other PGT settings. Current PRS models have modest performance and variable transferability across ancestries. The retrospective design, limited subgroup sizes, and modelling assumptions (e.g., aneuploidy and sex distribution) further constrain interpretation. The primary simulation outcome—PRS percentile shift—is a hypothesis-generating surrogate endpoint, not a clinical outcome; clinical benefit was not assessed or demonstrated. These findings should not be interpreted as supporting routine clinical implementation of PES; premature use may increase parental anxiety, inequitable access, and pressure to rank embryos using predictions that remain unvalidated at the embryo level. WIDER IMPLICATIONS OF THE FINDINGS This study provides an empirical framework for evaluating the current limits of PES within PGT. The observed PRS percentile shifts appear concentrated in best-prognosis cycles with multiple transferable embryos and elevated parental PRS, whereas little change is observed when embryo numbers are limited or risk is dominated by high-penetrance monogenic variants. These findings are hypothesis-generating and may inform future prospective research and ethical discussion, while underscoring that clinical utility and implementation require further validation within appropriately governed settings. FUNDING This study was funded by Major Scientific Program of CITIC Group (No. 2023ZXKYB34100), the Science Foundation of Hunan Province (Grant 2023JJ30422), and Health Research Project of Hunan Provincial Health Commission (grant number: W20243089). DISCLOSURES The authors declare no conflicts of interest.
STUDY QUESTION:Does late-night, modified-release hydrocortisone (MR-HC) improve biochemical control and restore menstrual regularity in women with non-classic congenital adrenal hyperplasia (NCCAH)? SUMMARY ANSWER:Once-daily MR-HC substantially improved biochemical androgen control and rapidly restored menstrual cyclicity in women with NCCAH, including those with longstanding irregular cycles. WHAT IS KNOWN ALREADY:While pregnancy rates in women with NCCAH are generally comparable to those of the general population, time to conception is often prolonged and assisted reproductive techniques are frequently required. Optimizing adrenal androgen suppression through the superior pharmacokinetic profile of MR-HC may shorten this interval and reduce the need for fertility interventions. So far, MR-HC has not been tested in NCCAH. STUDY DESIGN SIZE DURATION:Prospective observational cohort study conducted between September 2021 and November 2025 at a tertiary referral center and European Reference Network (Endo-ERN) hub for congenital adrenal hyperplasia. Thirty women with NCCAH were enrolled and followed for a median of 22.5 months (interquartile range [IQR] 8.25-36.75), with up to five study visits. Only women with at least 3 months of follow-up were included in the analysis. PARTICIPANTS/MATERIALS SETTING METHODS:The cohort comprised 30 women with NCCAH. At initiation of MR-HC, 20 women transitioned from conventional glucocorticoid therapy, and 10 were treatment-naïve. Sixteen women had an active desire to conceive, while 15 presented with irregular menstrual cycles or secondary amenorrhea at baseline. MAIN RESULTS AND THE ROLE OF CHANCE:Among 14 evaluable women, ovulatory function, defined as return of regular menstrual cycles or conception, was restored in 13 (92.6%) of women within 6 months of MR-HC initiation. One woman with concomitant hypothalamic hypogonadism remained amenorrhoeic and commenced hormone replacement therapy. Cycle normalization occurred at a median MR-HC dose of 10 mg (IQR 10-15 mg), with all but one woman receiving a single bedtime dose. Among 16 women seeking pregnancy, ten conceived within a median of 2.65 months (IQR 2.0-12.4) compared to 8 months (IRQ 1.0-24.0) in a historic cohort of NCCAH women before the approval of MR-HC (not significant). Among those seeking pregnancy, 10 women had been attempting to conceive unsuccessfully for a median of 28.5 months (IQR 12-46) before. Six women did not become pregnant during follow-up (13.5 months; IQR 6.25-23). The hydrocortisone equivalent dose (HCeq) at the time of conception was significantly lower under MR-HC (10.0 mg (IQR 10-12.5) vs. 16.9 mg (IQR 15-25), P = 0.02) in the historic cohort. Transition to MR-HC led to marked reductions in early-morning salivary 17-hydroxyprogesterone and serum testosterone, independent of prior glucocorticoid (GC) exposure. Metabolic effects were minimal: body weight decreased slightly, glycated hemoglobin (HbA1c) rose transiently, predominantly in treatment-naïve women, before returning to baseline. Blood pressure, fasting glucose, and lipid profiles remained stable. LIMITATIONS REASONS FOR CAUTION:While the rapid normalization of, in part, long-standing menstrual irregularities was striking, the absence of a control group does not allow causal inferences, and the single-center design within a specialized tertiary setting may limit generalizability. WIDER IMPLICATIONS OF THE FINDINGS:A single nightly dose of MR-HC appears sufficient to achieve hormonal control and subsequent menstrual-cycle restoration in most women with NCCAH and may improve time to conception. These findings support MR-HC as a promising therapeutic option in the management of NCCAH. FUNDING:This work was supported by the Deutsche Forschungsgemeinschaft (Heisenberg Professorship 325768017, project 314061271 TRR205 to NR and FBCRC-1665 -515637292 to NR and UK). LT was supported by the LMU Munich funding scheme (FöFoLe) and by the IFCAH (International Fund Congenital Adrenal Hyperplasia) grant 2023. HN was supported by the Clinician Scientist Program RISE, supported by the Eva Luise und Horst Köhler Stiftung & Else Kröner-Fresenius-Stiftung (2019_KollegSE.03 to HN). DISCLOSURES:N.R. was Principal Investigator and consulted for Neurocrine Biosciences and Diurnal Ltd. L.T., M.K. A. und H.F.N. were Sub-Investigators for Neurocrine Biosciences and Diurnal Ltd. TRIAL REGISTRATION NUMBER:N/A.
STUDY QUESTION:Does postmenopausal hormone replacement therapy (HRT), initiated within 1 year after menopause diagnosis, confer differential long-term health effects according to age at menopause? SUMMARY ANSWER:Long-term postmenopausal HRT was associated with age-dependent clinical outcomes, with broadly consistent cardiovascular and skeletal associations across age groups, but more variable metabolic and oncological associations according to age at menopause. WHAT IS KNOWN ALREADY:The 'timing hypothesis' suggests that HRT initiated closer to menopause may have more favorable cardiometabolic associations than later initiation, but evidence across distinct age-at-menopause strata remains limited. STUDY DESIGN SIZE DURATION:This retrospective new-user cohort study utilized de-identified data from the TriNetX Research Network, which provided access to electronic medical records for ∼111 million patients globally. To establish a relatively healthy baseline cohort, we included women aged 30-90 years without prior major systemic diseases who had a documented diagnosis of menopause. To ensure adequate time for the development of hard clinical endpoints, we required that the index event (initial menopause diagnosis) occurred at least 10 years before data extraction (31 December 2025), thereby guaranteeing a minimum follow-up period of one decade. To reduce immortal time bias and survivor bias inherent in prevalent-user designs, we employed a new-user, time-to-event cohort design. Women with a pre-existing history of type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD), osteoporosis, compression fracture, venous thromboembolism (VTE), breast cancer, or colorectal cancer on or before the index date were excluded. PARTICIPANTS/MATERIALS SETTING METHODS:Women who initiated postmenopausal HRT containing estrogen and/or progestogen within 1 year after menopause diagnosis were propensity score-matched 1:1 with non-users using baseline demographic, clinical, and laboratory covariates. Primary matching yielded 83 670 matched pairs in women who experienced menopause at age ≤60 years and 12 175 matched pairs in women who experienced menopause at age >60 years. Time-to-event analyses using Cox proportional hazards regression were conducted across four clinically defined menopausal age strata: premature ovarian insufficiency (POI, <40 years), early menopause (40-45 years), normal menopause (46-60 years), and late menopause (>60 years). Hazard ratios (HRs) were calculated for incident T2DM, CVD, compression fracture, breast cancer, colorectal cancer, and VTE. Interaction P-values were calculated using the normal menopause group (46-60 years) as the reference for interaction testing. Sensitivity analyses were conducted in both the overall ≤60-year and >60-year cohorts using an alternative 1:1 matching strategy based only on age, race, and baseline BMI, excluding laboratory parameters. MAIN RESULTS AND THE ROLE OF CHANCE:In the overall matched cohort of women experiencing menopause at age ≤60 years, postmenopausal HRT was associated with lower hazards across all evaluated systemic endpoints. Compared with non-users, postmenopausal HRT was associated with lower hazards of T2DM (events: 3517 vs 4580; crude incidence: 4.20% vs 5.47%; HR: 0.595, 95% CI: 0.575-0.615) and CVD (events: 3960 vs 4413; crude incidence: 4.74% vs 5.29%; HR: 0.699, 95% CI: 0.688-0.709). Skeletal health was preserved, evidenced by a lower hazard of compression fractures (events: 426 vs 441; crude incidence: 0.51% vs 0.53%; HR: 0.780, 95% CI: 0.719-0.846). Furthermore, postmenopausal HRT was associated with lower hazards of breast cancer (events: 1281 vs 1504; crude incidence: 1.53% vs 1.80%; HR: 0.786, 95% CI: 0.743-0.831), colorectal cancer (events: 222 vs 276; crude incidence: 0.27% vs 0.33%; HR: 0.847, 95% CI: 0.738-0.971), and VTE (events: 1247 vs 1346; crude incidence: 1.49% vs 1.61%; HR: 0.859, 95% CI: 0.811-0.910). In the primary age-stratified model, the >60-year cohort showed a higher breast cancer hazard (HR: 1.151, 95% CI: 1.057-1.255); in the corresponding >60-year group sensitivity analysis, using alternative matching, the estimate remained directionally similar (HR: 1.218, 95% CI: 1.027-1.444). Across the remaining >60-year sensitivity outcomes, the direction of association was preserved, including continued lower hazards of T2DM, CVD, VTE, and compression fracture, while the colorectal cancer association remained neutral. LIMITATIONS REASONS FOR CAUTION:Important limitations include residual confounding, incomplete socioeconomic and screening data, limited details on HRT formulation, route, dose and duration, unavailable breast cancer subtype information, unavailable exact maximum follow-up duration, and the inability to directly incorporate standardized patient-level geography into the matching or regression models. Region-restricted inference was additionally constrained because the Europe, Middle East, and Africa (EMEA) > 60-year HRT cohort contained only 48 exposed users, precluding stable matched analysis. WIDER IMPLICATIONS OF THE FINDINGS:While cardiovascular and skeletal associations appeared broadly consistent across menopausal ages, the associations with breast cancer in the older age group highlight the critical need for individualized prescribing. These real-world findings are most consistent with earlier initiation of postmenopausal HRT for symptomatic women and those with POI, while urging extreme caution with initiation in late-onset menopause. FUNDING:No specific external funding was received. The study was supported by departmental funds from the Department of Obstetrics and Gynecology, Taichung Veterans General Hospital. DISCLOSURES:The authors declare no competing interests. TRIAL REGISTRATION NUMBER:N/A.
STUDY QUESTION:What factors are associated with couples' returning to have ART after having a first ART-conceived child? SUMMARY ANSWER:Among returning couples, return was independently associated with younger maternal age, a singleton rather than twin first birth, and the availability of cryopreserved embryos and/or oocytes. WHAT IS KNOWN ALREADY:Long-term family-building trajectories after an initial ART birth remain poorly characterized, despite being essential for counseling couples aiming to achieve their intended family size in the context of declining fertility rates and delayed parenthood in high-income countries. Age-related biological constraints, limited fertility awareness, and overestimation of ART effectiveness contribute to unrealized reproductive intentions; nevertheless, the determinants of couples' return to ART after a first live birth and their association with subsequent live births remain largely unexplored. STUDY DESIGN SIZE DURATION:Monocentric retrospective cohort study of 5852 couples achieving a first ART-conceived live birth (index birth) from cycles initiated between 2010 and 2020, with follow-up through December 2024 to evaluate return to ART for further family building and subsequent live birth outcomes. As data were retrieved from a complete institutional database, no loss to follow-up occurred. PARTICIPANTS/MATERIALS SETTING METHODS:Couples achieving a first ART-conceived live birth between 2010 and 2020 at a single fertility center in Italy were included. Donor cycles and PGT were excluded. Participants were followed until December 2024. Couples were classified as returners or non-returners and stratified according to cryopreserved oocyte and/or embryo storage status. Outcomes of interest were return to ART for further family building and live birth achievement among returning couples. MAIN RESULTS AND THE ROLE OF CHANCE:Overall, 1978 couples (33.8%) returned for further ART cycles; among returners, a second live birth was achieved by 49.3%. Return was significantly higher among couples with cryopreserved reproductive material compared with those without stored embryos or oocytes (41.0% vs 23.3%, P < 0.001), particularly among couples with cryopreserved embryos only or with both embryos and oocytes stored (41.6% and 44.3%, respectively). At multivariable analysis, independent predictors of return included lower maternal age (aOR 0.93 per year increase), male-factor infertility (aOR 1.26), singleton rather than twin first live birth (aOR 0.12 for twins), presence of cryopreserved material (aOR 2.21), and higher gestational age at delivery (aOR 1.03). Among couples who returned, the proportion achieving a second live birth varied markedly by cryopreservation status, ranging from 39.5% in couples without stored material to 64.6% in those with both embryos and oocytes available (P < 0.001). Few couples returned after a second live birth; however, the live birth proportion for a third child exceeded 50% when both embryos and oocytes were present. LIMITATIONS REASONS FOR CAUTION:Psychosocial and economic determinants potentially influencing non-returners were not assessed, data on spontaneous pregnancies were unavailable, and cross-center migration could not be excluded. In addition, this monocentric study was conducted in Italy, where regulatory and cultural aspects of ART may influence treatment access and reproductive decision-making, potentially limiting generalizability. WIDER IMPLICATIONS OF THE FINDINGS:ART counseling may benefit from being tailored to couples' long-term reproductive goals. Treatment strategies aimed at increasing the number of embryos and/or oocytes cryopreserved at younger ages can facilitate their use at more advanced reproductive ages. This may help couples achieve their desired family size. FUNDING:This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors, and was supported by institutional resources from the Division of Gynecology and Reproductive Medicine, Department of Gynecology, Fertility Center, IRCCS Humanitas Research Hospital, Rozzano. Milan, Italy. DISCLOSURES:The authors declare no competing interests. TRIAL REGISTRATION NUMBER:NCT07458165.
Abstract STUDY QUESTION Is prolonged cryopreservation associated with adverse clinical pregnancy and live birth after frozen-thawed embryo transfer? SUMMARY ANSWER Prolonged embryo cryopreservation is associated with lower clinical pregnancy and live birth rates, particularly within the first 3–12 months of storage, but rates plateau after two years. WHAT IS KNOWN ALREADY Evidence on the impact of cryopreservation duration on reproductive outcomes remains scarce and conflicting, with some studies reporting no adverse effects and others suggesting increased risks of pregnancy loss, preterm birth, and epigenetic alterations after prolonged storage. STUDY DESIGN, SIZE, DURATION Systematic review and meta-analysis of studies comparing pregnancy outcomes by cryopreservation duration. PubMed, Embase, and the Cochrane Library were searched from inception to December 31, 2025, for English-language articles. The search strategy combined terms for embryos (e.g., "Embryo," "Blastocyst"), cryopreservation techniques (e.g., "vitrification," "cryopreservation," "freezing"), and pregnancy outcomes (e.g., "pregnancy," "live birth") using Boolean operators. A total of 5,208 records were identified from PubMed, 9,272 from Embase, and 331 from the Cochrane Library. PARTICIPANTS/MATERIALS, SETTING, METHODS Two independent reviewers screened studies, extracted data, and assessed quality using the Newcastle–Ottawa Scale. Inclusion required reporting of clinical pregnancy rate (CPR) or live birth rate (LBR) stratified by cryopreservation duration. Studies on donated embryos, preimplantation genetic testing, or gamete cryopreservation were excluded. Pooled odds ratios (ORs) with 95% CIs were calculated using fixed- or random-effects models based on heterogeneity (I²). Subgroup analyses were performed for neonatal outcomes (singleton vs. multiple births). Publication bias was assessed via funnel plots. MAIN RESULTS AND THE ROLE OF CHANCE A total of 14,811 records were screened (5,208 from PubMed, 9,272 from Embase, 331 from Cochrane). After removal of duplicates, 11077 records were screened by title and abstract, and 785 full-text articles were assessed for eligibility. Twenty-three studies (254,017 transfer cycles/patients) were included. Embryos cryopreserved >5 years had lower survival rates (OR = 0.84, 95% CI: 0.72–0.98, P = 0.03; I2=0%, P = 0.99). Compared with ≤3 months storage, longer storage was associated with reduced CPR (>3 months: OR = 0.87, 95% CI: 0.78–0.96, P = 0.005; >6 months: OR = 0.70, 95% CI: 0.66–0.74, P < 0.00001; >12 months: OR = 0.71, 95% CI: 0.61–0.82, P < 0.00001; >2 years: OR = 0.77, 95% CI: 0.65–0.91, P = 0.003), reduced LBR (>3 months: OR = 0.90 95% CI: 0.84–0.95, P = 0.0008; >6 months: OR = 0.74, 95%CI: 0.69–0.80, P < 0.00001; >12 months: OR = 0.72, 95% CI: 0.63–0.84, P < 0.0001; >2 years: OR = 0.80, 95% CI: 0.72–0.89, P < 0.0001) and reduced biochemical pregnancy rate (>3 months: OR = 0.82, 95% CI: 0.73–0.92, P = 0.0007; >6 months: OR = 0.70, 95% CI: 0.63–0.77, P < 0.00001; >12 months: OR = 0.72, 95% CI: 0.65–0.80, P < 0.00001; >2 years: OR = 0.74, 95% CI: 0.61–0.90, P = 0.002). Miscarriage rate (OR = 1.16, 95% CI: 1.09–1.25, P < 0.0001) and preterm birth rate (OR = 1.10, 95% CI: 1.00–1.20, P = 0.04) was higher after >12 months storage. High birth weight and malformation rates were not significantly increased, but low birth weight was less frequent after >6 (OR = 0.83, 95% CI: 0.70–0.99, P = 0.04) or > 12 months (OR = 0.75, 95% CI: 0.57–0.98, P = 0.03), and the proportion of male infants was lower after >3 months (OR = 0.92, 95% CI: 0.86–0.98, P = 0.02) and >2 years (OR = 0.79, 95% CI: 0.64–0.97, P = 0.02), compared with those cryopreserved for ≤3 months. No significant publication bias was detected. LIMITATIONS, REASONS FOR CAUTION Limitations include substantial heterogeneity across studies (design, cryopreservation method, embryonic stage, confounder adjustment), inability to evaluate frozen-thawed embryo transfer (FET) cycle type, maternal age, and embryo quality due to inconsistent reporting, retrospective observational design precluding causal inference, residual confounding, potential survivor bias, and combined singleton/multiple-birth data for neonatal outcomes. Despite a large sample size (254,017 cycles), these factors warrant cautious interpretation. WIDER IMPLICATIONS OF THE FINDINGS This comprehensive meta‑analysis reveals a non‑linear relationship between cryopreservation duration and outcomes, reconciling previous conflicting reports by demonstrating early declines (first 3–12 months) followed by a plateau beyond two years. The stability of outcomes in ultra‑long storage is reassuring for patients requiring delayed transfer. These findings support clinical counselling that encourages early transfer when feasible, but provides reassurance that ultra‑long storage does not lead to progressive deterioration in pregnancy or neonatal outcomes. FUNDING This work was funded by First-class discipline innovation-driven talent program of Guangxi Medical University (to Jingzhen Lai) and College Students’ Innovation Training Project of Guangxi Medical University (X202410598348). DISCLOSURES The authors declare no conflicts of interest. REGISTRATION NUMBER CRD42024555362.
Abstract STUDY QUESTION Does geographic proximity to a centralised cryobank reflect age-dependent differences in ovarian tissue cryopreservation (OTC) utilisation? SUMMARY ANSWER Paediatric patients resided significantly closer to the cryobank than adolescents and adult women, suggesting age-dependent geographic differences in the utilisation of fertility preservation services. WHAT IS KNOWN ALREADY OTC is an established fertility preservation option, and previous studies have primarily focused on return rates of cryopreserved ovarian tissue and subsequent pregnancy and live birth outcomes. However, less is known about utilisation patterns of fertility preservation, particularly for OTC in paediatric oncology, where utilisation may be influenced by factors such as clinician-initiated counselling and local referral pathways. STUDY DESIGN, SIZE, DURATION This retrospective nationwide multicentre cohort study included 2,426 patients referred from 124 centres who underwent OTC at a central cryobank between 2000 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients were stratified into children (<15 years, n = 149), adolescents (15–19 years, n = 296), and adults (≥20 years, n = 1,981). Residential addresses were geocoded, and distances to the cryobank were calculated. Regional distribution was categorised by population size. Statistical analyses included Chi-square and non-parametric tests with Bonferroni correction. MAIN RESULTS AND THE ROLE OF CHANCE Median distance to the cryobank increased with age (children: 136 km; adolescents: 183 km; adults: 222 km), with children living significantly closer than adolescents (p = 0.03) and adults (p = 0.001). Overall, 44.8% of patients resided in large cities, with significant age-group differences (p < 0.001). Paediatric cases were concentrated in urban areas, whereas adults were more geographically dispersed. Among adults, distance varied by indication (p = 0.021), but not in younger groups. This pattern persisted despite the nationwide referral network. LIMITATIONS, REASONS FOR CAUTION The retrospective design precludes causal inference. Distances were calculated as straight-line measures instead of real travel time, and socioeconomic factors were not available. Furthermore, only patients who successfully underwent OTC were available for analysis. Consequently, the study cannot assess true access to fertility preservation, referral rates, counselling rates, or the number of eligible patients who did not proceed to treatment. Although patients were referred from centres across Germany, cryopreservation was centralized in Bonn. Regional referral relationships and the structure of the referral network may therefore have influenced the observed geographic distribution, particularly in paediatric patients, and may limit generalisability to the entire German population. WIDER IMPLICATIONS OF THE FINDINGS The findings demonstrate age-dependent geographic utilisation patterns among patients who underwent OTC. These patterns may reflect differences in referral pathways, counselling practices, and utilisation of fertility preservation services. However, because only patients who successfully underwent OTC were included, the study cannot directly assess referral rates, unmet need, or access barriers. Expanding structured referral pathways and reimbursement policies may improve equitable utilisation of fertility preservation services. FUNDING Intramural funding from the University Hospital Bonn. This publication was supported by the Open Access Publication Fund of the University of Bonn. No external grant funding was received from any funding agency in the public, commercial, or not-for-profit sectors. DISCLOSURES The authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER N/A
Abstract The pathogenesis of endometriosis and adenomyosis remains incompletely understood, and a notable gap persists between the reported heritability and the disease variation explained by identified susceptibility loci. In this manuscript, we showcase key epidemiological and clinical observations—including associations with early menarche, shorter anogenital distance, specific childhood growth trajectories, hypospadias in males, and developmental exposure to endocrine-disrupting chemicals-—that collectively point to the influence of early-life developmental programming. Recent evidence revealing a hyperestrogenic/hypoandrogenic hormonal imbalance in the umbilical cord blood of pregnant women with endometriosis provides a critical link between these seemingly disparate findings. We propose a novel hypothesis of hyperestrogenic imprinting: Female offspring of women with endometriosis and/or adenomyosis are exposed in utero to a distinct hormonal environment, which induces lasting epigenetic changes in the developing reproductive tract, thereby imprinting a lifelong susceptibility to these two diseases. This mechanism offers a cohesive explanation for the familial aggregation of endometriosis and possibly adenomyosis, addresses the issue of “missing heritability”, and integrates various risk factors rooted in early development. A limitation of this opinion piece is that its hypothesis is primarily informed by animal studies and conceptual synthesis, and would benefit from further validation through original human data. As a falsifiable hypothesis, it presents a clear pathway for future validation and, if proven, opens avenues for novel preventive strategies.
STUDY QUESTION:Do particular pairs of individuals modeling sexual pairs exhibit differences in the induction of semen-mediated long-lasting tolerance in support of pregnancy? SUMMARY ANSWER:Yes, factors from both sexes contribute to the variability in the induction of long-lived regulatory T cells (Tregs) in response to semen, with factors related to the recipient seeming to slightly outweigh the factors related to the semen donor. WHAT IS KNOWN ALREADY:Semen contains multiple immunomodulatory factors that can influence reproductive tract immunity in the recipient, promote tolerance to antigens from the semen donor (designated henceforth as 'paternal'), and ultimately support successful pregnancy. These factors include the soluble fraction containing prostaglandins and cytokines, as well as a very high concentration of extracellular vesicles (EVs). Both soluble and vesicular fractions of semen have been shown to promote tolerogenic phenotypes and migration in antigen-presenting cells (APCs), and to induce Tregs. This study was undertaken to understand the variation in tolerance induction between pairs of individuals modeling sexually active couples. STUDY DESIGN SIZE DURATION:We designed an in vitro pairing study using random semen donors (N = 11) and peripheral mononuclear cells (PBMCs) from random self-identified female recipients (N = 3). Semen was fractionated into vesicle-enriched (semen extracellular vesicles; SEVs) and soluble (vesicle-depleted semen plasma; VDSP) fractions. Monocyte-derived dendritic cells (MoDCs) from each of the recipients were exposed to semen fractions from each donor, then cocultured with autologous naïve T cells to assess tolerogenic responses, specifically, generation of Tregs (FOXP3+ CD25Hi CD127Lo CD4+ T cells) and their phenotypic differences (TIGIT+, PD-1+ CTLA-4+, or AREG+ Tregs). These cells were then screened for T cell receptor-dependent activation by activation-induced marker (AIM) expression assay. These responses were compared across all pairs of semen donors and recipient cells. In addition, semen fractions were screened for candidate mediators of tolerance, such as human leukocyte antigen G (HLA-G) and adenosine-generating enzymes. PARTICIPANTS/MATERIALS SETTING METHODS:This study used an in vitro human cell culture approach in which primary blood-derived immune cells were exposed to semen-derived vesicular and non-vesicular fractions. Blood samples were obtained from healthy self-identified female donors, and semen samples were obtained from healthy participants who provided initial screening specimens for a contraceptive study. Semen-derived vesicular and non-vesicular fractions were characterized by western-blot analysis for markers associated with immune tolerance. Primary immune cells were exposed to these semen fractions, and the major endpoints included changes in Treg phenotypes and subset frequencies, as well as AIM expression. These outcomes were assessed by flow cytometry. No animal models were used. MAIN RESULTS AND THE ROLE OF CHANCE:HLA-G content in semen (SEV and VDSP) varied markedly across donors, ranging from undetectable to high concentrations. Similarly, semen's capacity to generate adenosine from ATP differed by ∼4-fold among donors. Induction of Tregs by SEV and VDSP from 11 semen donors was highly variable, from 2.8% to 63.4% of CD4+ T cell population. Semen fractions from a few donors consistently elicited lower tolerogenic responses across all recipients. However, when we considered the combined effects of both semen fractions, none of the semen donors failed to elicit a measurable tolerogenic response. On the other hand, one recipient was a consistently low responder to tolerance induction. Thus, both recipient immune cells' features and semen-intrinsic properties are significant determinants of tolerogenic responsiveness. Semen re-exposure enhanced Treg AIM expression. The strongest effect was observed when Tregs were exposed to VDSP-loaded MoDCs. In contrast, SEV-loaded MoDCs caused a much weaker response. LARGE SCALE DATA:N/A. LIMITATIONS REASONS FOR CAUTION:One limitation of our study is that we used semen and circulating recipient cells from random donors not selected for any clinical characteristics, such as history of infertility. In addition, we did not correlate HLA-G content and adenosine-generating enzyme expression levels with the Treg and AIM readouts. We show, via Treg activation, that semen induces tolerance to its antigens, but we do not demonstrate which specific antigens are recognized. We posit that the variation in semen-induced tolerance observed between couples plays a role in reproductive outcomes; however, larger studies in clinically characterized cohorts and using cells from the tissue microenvironment will be required to establish this link. WIDER IMPLICATIONS OF THE FINDINGS:Our findings demonstrate that semen-induced tolerance is highly variable and may be more strongly shaped by recipient factors than by semen donor factors. In addition, the generation of Tregs with enhanced activation to previously encountered semen supports the concepts of trained immunity and antigen-specific tolerance that arise cumulatively following exposure to semen from the partner. These Tregs are poised to expand and support pregnancies with fetuses expressing paternal alloantigens. FUNDING:This work was supported by the following grants: National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01AI153342) to L.V., National Institutes of Health/National Institute on Drug Abuse (R01DA040386) to F.H., National Institutes of Health/National Center for Advancing Translational Sciences (KL2TR002317) to G.G.G. DISCLOSURES:The authors have no conflicts of interest to declare.
STUDY QUESTION:How does benzodiazepine use during an IVF cycle affect pregnancy outcomes? SUMMARY ANSWER:Benzodiazepine use was neither positively nor adversely associated with IVF pregnancy outcomes overall. WHAT IS KNOWN ALREADY:Benzodiazepine use during oocyte retrieval, primarily with midazolam, and diazepam use at the time of embryo transfer (ET) have been reported to yield comparable procedural success rates to those of non-users, respectively. However, these studies focus on exposure at a single time point, necessitating a more comprehensive investigation of benzodiazepine use across the entire IVF treatment period. STUDY DESIGN SIZE DURATION:A retrospective cohort study was conducted using Korea's Health Insurance Review and Assessment (HIRA) database between October 2017 and June 2024. A total of 99 484 women with a first completed IVF cycle were included. PARTICIPANTS/MATERIALS SETTING METHODS:Benzodiazepine users were defined as individuals who had at least one prescription during an IVF cycle (from the last menstrual period to ET) (user group, n = 28 649). Non-users were defined as those who had no benzodiazepine prescriptions within 365 days prior to ET to minimize exposure misclassification (non-user group, n = 61 433). The outcomes of interest were four IVF outcomes: clinical pregnancy rate, miscarriage rate, live birth rate, and preterm birth rate. To compare the two groups, propensity score overlap weighting was applied to adjust for potential imbalances in confounders. Relative risks (RRs) with 95% CIs were estimated using Poisson regression in the overlap-weighted at-risk population cohort for each outcome. MAIN RESULTS AND THE ROLE OF CHANCE:Compared with non-users, benzodiazepine users had a lower prevalence of prior live birth and a higher prevalence of anxiety disorders, along with greater use of antidepressants before adjustment. The absolute event rates of each outcome were as follows: (i) clinical pregnancy rate: 36.0% of users versus 35.7% of non-users, (ii) miscarriage rate: 14.9% versus 15.2%, (iii) live birth rate: 99.3% versus 99.3%, (iv) preterm birth rate: 13.3% versus 11.6%. Most IVF outcomes were comparable between groups, though a marginally increased risk of preterm birth was observed among users (clinical pregnancy: adjusted RR 1.02 [95% CI 1.00 to 1.05]; miscarriage: 0.99 [0.91 to 1.08]; live birth: 1.00 [1.00 to 1.00]; preterm birth: 1.10 [0.99 to 1.21]). However, stratified analyses by cumulative dose showed no dose-response relationship (≤1.2 mg and >2 mg, respectively-preterm birth: 1.09 [0.99 to 1.21] and 1.01 [0.87 to 1.17]). Additionally, the point estimate was closer to the null among users prescribed both injectable and oral agents during the cycle (preterm birth: 1.04 [0.86 to 1.26]). The robustness of the main findings was supported by analyses accounting for benzodiazepine exposure during pregnancy and by multiple sensitivity analyses. LIMITATIONS REASONS FOR CAUTION:Despite rigorous statistical adjustment, residual confounding by unmeasured factors and potential exposure misclassification due to discrepancies between prescription timing and actual medication use cannot be completely ruled out. WIDER IMPLICATIONS OF THE FINDINGS:This large population-based study found no association between benzodiazepine use throughout the IVF cycle and pregnancy outcomes. Although a small increase in preterm birth risk was observed, there was no evidence of a dose-response relationship or additional risk among patients with exposure to both oral and injectable formulations during the exposure assessment period. However, further studies are needed to evaluate the potential association. FUNDING:This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2026-25474077); This research was supported by a grant (RS-2026-25511535) from Ministry of Food and Drug Safety in 2026. DISCLOSURES:J-YS received grants from the Ministry of Food and Drug Safety, the Ministry of Health and Welfare, the National Research Foundation of Korea, and pharmaceutical companies, including LG Chem, GSK, Pfizer, Handok, JnJ, and SK Bioscience, outside the submitted work. TRIAL REGISTRATION NUMBER:N/A.
STUDY QUESTION:How can rare spermatozoa be identified efficiently during microsurgical testicular sperm extraction (micro-TESE)? SUMMARY ANSWER:An artificial intelligence (AI)-assisted system was developed to flag candidate rare spermatozoa in real time during micro-TESE, and it may support embryologists as a decision-support tool. WHAT IS KNOWN ALREADY:Patients with non-obstructive azoospermia (NOA) can obtain sperm for procreation through micro-TESE. During this procedure, sperm retrieval primarily relies on embryologists or laboratory technicians visually searching for sperm under a microscope, which is not only laborious and inherently subjective but also susceptible to errors. Although AI technology has been applied to identify trace amounts of sperm, existing models lack sufficient efficiency and true real-time performance. STUDY DESIGN SIZE DURATION:This study included model development followed by a single-centre clinical evaluation. An improved YOLO (You Only Look Once)-based rare sperm detection model, termed YOLOv11-RSD, was developed using microscopy data from 1165 surgical patients, comprising 1932 image samples containing a total of 5032 annotated sperm objects with confirmed identification. Clinical evaluation was performed between May 2024 and July 2025. Performance was assessed across confidence thresholds in obstructive azoospermia (OA) patients with normal spermatogenesis, and the system was then applied during micro-TESE in NOA patients and compared with routine embryologist assessment. PARTICIPANTS/MATERIALS SETTING METHODS:The model was developed using testicular sperm microscopy images collected at a single hospital. Real-time clinical feasibility was evaluated in 10 OA cases and 30 NOA cases. Embryologist assessment was used as the reference standard, and performance was assessed using PPV, sensitivity, F1-score, and 95% confidence intervals. Discordant AI-assisted detections were reviewed by embryologists in real time. MAIN RESULTS AND THE ROLE OF CHANCE:YOLOv11-RSD achieved real-time detection of candidate spermatozoa in microscopy images with high sensitivity and acceptable PPV under the selected operating threshold. Compared with baseline YOLOv11, YOLOv11-RSD showed improved overall detection performance across representative evaluation settings. In OA cases, the system achieved high sensitivity for sperm detection, reaching up to 96.7% across evaluated thresholds. During micro-TESE in NOA patients, at a confidence threshold of 0.50, positive predictive value (PPV), sensitivity, and F1-score were 80.58%, 96.11%, and 87.66%, respectively. The system highlighted candidate spermatozoa that were not identified during the initial manual assessment in six NOA cases, including two cases initially classified as sperm-negative; these findings were confirmed upon immediate re-review. Follow-up reproductive outcomes were available for six cases in which AI-assisted detection contributed to the search-and-confirmation workflow: embryo cleavage was achieved in all six cases, and three cases ultimately resulted in live births. Notably, among the two cases initially classified as sperm-negative, one case resulted in a singleton live birth. LARGE SCALE DATA:N/A. LIMITATIONS REASONS FOR CAUTION:This was a single-centre clinical evaluation with a limited clinical cohort. Although model inference was rapid, procedure-level efficiency was constrained by image acquisition and scanning logistics, and no definitive reduction in total procedure time was demonstrated. External multi-centre validation is required. WIDER IMPLICATIONS OF THE FINDINGS:AI-assisted sperm detection may support embryologists during micro-TESE by flagging candidate rare spermatozoa for rapid review. Further prospective multi-centre validation is required to determine whether this approach improves procedure-level efficiency or clinical outcomes. FUNDING:This work was supported by grants from National Natural Science Foundation of China (82301794), Shanghai Science and Technology Innovation Action Plan (24Y12800702), Natural Science Foundation of Shanghai (25ZR1401300), National Key Research and Development Program of China (2022YFC270300), China Jiliang University Research Grant (No. H251120), and Shanghai General Hospital Basic and Clinical Collaborative Research Program (JC202612). DISCLOSURES:The authors declare no competing interests.
STUDY QUESTION:Is there an association between the body roundness index (BRI) and pregnancy outcomes of IVF, and can it provide complementary information beyond body mass index (BMI)? SUMMARY ANSWER:Higher BRI was associated with a lower likelihood of live birth following IVF, particularly among women with normal BMI. WHAT IS KNOWN ALREADY:BMI is widely used to assess obesity-related reproductive risk but does not adequately characterize body fat distribution. BRI, a novel anthropometric index that may better reflect central adiposity, has been associated with cardiometabolic risk, but its relationship with IVF outcomes remains unclear. STUDY DESIGN SIZE DURATION:This study was a secondary analysis of data from two multicentre randomized controlled trials (RCTs) in China that evaluated live birth rates between freeze-only and fresh embryo transfers. The first trial enrolled 1508 women with polycystic ovary syndrome from 14 centres between June 2013 and May 2014, with follow-up completed in July 2015. The second trial recruited 2157 women with regular menstrual cycles from 20 centres between March 2015 and November 2015, with follow-up completed in March 2017. PARTICIPANTS/MATERIALS SETTING METHODS:After exclusions, 3523 women who underwent embryo transfer and had complete BRI data were included in the analysis. BRI was calculated from height and waist circumference measured prior to IVF. In the absence of universally accepted clinical thresholds for BRI, the participants were categorized into three groups according to the 33rd and 66th percentiles: <2.54 (n = 1171), 2.54 to <3.54 (n = 1174), ≥3.54 (n = 1178). The primary outcome was live birth, which was defined as the delivery of neonate(s) with signs of life at ≥28 weeks' gestation. Modified Poisson regression with robust variance was used to estimate adjusted risk ratios (aRRs) and 95% confidence intervals (CIs). The models were adjusted for age (continuous), duration of infertility (continuous), cause of infertility, type of embryo transfer, diagnosis of polycystic ovary syndrome (PCOS), number of oocytes retrieved (continuous), and number of embryo(s) transferred. These covariates were selected based on clinical relevance and prior literature to reduce potential confounding. MAIN RESULTS AND THE ROLE OF CHANCE:The rate of live birth was lower among women in the highest BRI group (group 3) compared with those in the lowest group (group 1) (45.2% vs 53.6%; aRR, 0.89; 95% confidence interval [CI], 0.81-0.97; P = 0.008). Women in group 3 also had an increased risk of total pregnancy loss compared with those in group 1 (aRR, 1.31; 95% CI, 1.07-1.61; P = 0.009). In BMI-stratified analyses, higher BRI was associated with a reduced likelihood of live birth (group 2, aRR, 0.89; 95% CI, 0.81-0.98; P = 0.019; group 3, aRR, 0.84; 95% CI, 0.73-0.96; P = 0.009) among women with normal BMI (18.5 to <24 kg/m2). Furthermore, each one-unit increase in BRI was associated with a 7% lower likelihood of live birth (aRR, 0.93; 95% CI, 0.87-0.98; P = 0.009) among women with normal BMI. No significant association between BRI and live birth was observed among women who were overweight or obese (BMI ≥ 24 kg/m2). LIMITATIONS REASONS FOR CAUTION:In this post-hoc analysis, causality cannot be established, and residual confounding cannot be ruled out. The small-sized subgroups, particularly among women with overweight or obesity, may have limited statistical power. WIDER IMPLICATIONS OF THE FINDINGS:These findings suggest that BRI may capture adiposity-related risk not reflected by BMI alone, particularly among women with normal BMI. BRI may serve as a complementary anthropometric measure alongside BMI for identifying women at higher risk of adverse IVF outcomes. Further studies are needed to validate these findings across BMI categories and clarify the clinical utility of BRI in IVF populations. FUNDING:This study was supported by the National Key Research and Development Program of China (2023YFC2705502) and the National Natural Science Foundation of China (82495194 and 82421004). DISCLOSURES:There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER:N/A.
STUDY QUESTION:Are pathogenic variants in homeodomain-interacting protein kinase (HIPK4) associated with sperm head abnormalities that cause male infertility? SUMMARY ANSWER:HIPK4 is a novel candidate gene associated with sperm head defects and human male infertility. WHAT IS KNOWN ALREADY:Numerous genes have been described in which pathogenic variants cause male infertility due to multiple morphological abnormalities of the sperm flagella (MMAF), but the genetic basis of sperm head defects is less well understood. STUDY DESIGN SIZE DURATION:This study included four infertile brothers displaying varying degrees of quantitatively and/or qualitatively impaired spermatogenesis, their parents, and their fertile brother. We also queried the Male Reproductive Genomics (MERGE) cohort comprising exome/genome sequencing data of >3300 men. PARTICIPANTS/MATERIALS SETTING METHODS:We performed exome sequencing in all five brothers and their parents. To characterize sperm phenotypes, we carried out standard semen analysis, immunofluorescence staining, and transmission electron microscopy (TEM). Further, we evaluated the impact of the HIPK4 variant in cell culture experiments using HEK293T cells. MAIN RESULTS AND THE ROLE OF CHANCE:By analysing the exome data, we could not identify a common genetic cause in all four affected brothers. However, one of the affected brothers was compound heterozygous for two loss-of-function variants in DNAH17 (c.1076_1077dup p.(Lys360*) and c.7752+2T>A p.?), associated with markedly reduced sperm motility and MMAF. The variants' pathogenicity was further validated by TEM of flagellar cross sections revealing an outer dynein arm defect and axonemal disruption. In contrast, his three infertile brothers were homozygous for the start-loss variant c.1A>G in HIPK4. This gene is expressed during spermiogenesis and is reportedly involved in sperm head shaping in mice. Heterologous expression of (partial) HIPK4 variant cDNA showed that translation was being initiated at an alternative in-frame start codon located 35 amino acids downstream, resulting in an N-terminally truncated protein p.(Met1_Glu35del). The truncated HIPK4 protein lacks parts of its kinase domain and shows reduced protein stability. Corresponding with published mouse models, all three brothers displayed 100% abnormal sperm head morphology with variable defects. Importantly, one brother affected by HIPK4 variants fathered a child after successful ICSI, demonstrating a successful treatment option for HIPK4-related teratozoospermia. No further men from the MERGE cohort were affected by biallelic HIPK4 variants. Taken together, HIPK4 is an autosomal recessive candidate gene in which pathogenic variants are associated with sperm head defects and male infertility. LARGE-SCALE DATA:The reported variants in DNAH17 and HIPK4 have been published in ClinVar. LIMITATIONS REASONS FOR CAUTION:Independent replication is required to assess the phenotypic spectrum and the reproductive outcome associated with biallelic HIPK4 variants and to formally establish the gene-disease relationship for male infertility. WIDER IMPLICATIONS OF THE FINDINGS:This study raises awareness of the significant genetic heterogeneity of male infertility. The described family highlights that distinct genetic causes may underlie a seemingly similar phenotype. Exome sequencing of families is helpful to efficiently disentangle individual causes among affected family members. FUNDING:N.N., J.R., H.O., S.L., C.F., and F.T. were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the Clinical Research Unit 'Male Germ Cells' (CRU326, project number 329621271). R.T.-W., N.N., J.R., H.O., and F.T. were supported by the Federal Ministry of Research, Technology and Space (BMFTR) as part of the project ReproTrack.MS (grant 01GR2303). S.A.K. was supported by the DFG Clinician Scientist programme CareerS Münster (project number 493624047). A.S.G. was supported by the Medical Faculty Münster via an Innovative Medical Research (IMF) grant (GA-122104). DISCLOSURES:The authors declare no conflicts of interest.
STUDY QUESTION:What are the risk factors associated with meiotic errors in blastocysts with mosaic biopsy results? SUMMARY ANSWER:Meiotic errors were identified in 3.9% of blastocysts with mosaic biopsy results and were correlated with high-level mosaicism, maternal origin, and advanced maternal age. WHAT IS KNOWN ALREADY:Chromosomal mosaicism mainly arises from postzygotic mitotic errors, except for rare events from rescued meiotic errors. Preimplantation genetic testing for aneuploidy origin (PGT-AO) can distinguish meiotic errors from mitotic errors; however, the clinical value of PGT-AO remains to be explored. STUDY DESIGN SIZE DURATION:A retrospective cohort of 391 blastocysts with mosaic biopsy results from a university-based fertility centre in China between January 2020 and December 2024 was analysed by PGT-AO. Risk factors associated with meiotic errors in blastocysts with mosaic biopsy results were explored. Pregnancy outcomes following 96 mosaic embryo transfers (METs) of embryos with mitotic errors were compared with those following transfer of 288 matched euploid embryos. Additionally, 7 donated blastocysts were separated for single-cell DNA sequencing. PARTICIPANTS/MATERIALS SETTING METHODS:The parental origin and cell-division origin were analysed in 391 blastocysts classified as euploid-aneuploid mosaic from a cohort of 8932 blastocysts detected using the single-nucleotide polymorphism (SNP) array. In the prospective study, pregnancy outcomes following METs of embryos with mitotic errors were compared with those following transfer of matched euploid embryos at a 1:3 ratio using propensity score matching. The primary outcome was the live birth/ongoing pregnancy rate (LB/OPR) of METs involving mitotic errors. Multivariate logistic regression analysis was used to evaluate risk factors for pregnancy outcomes. Prenatal and placental samples were analysed by SNP array and/or FISH for genetic verification. Single-cell DNA sequencing of 314 cells separated from 7 donated blastocysts (4 with high-level mosaicism, 2 with meiotic aneuploidy, and 1 with low-level mosaicism) was conducted to assess actual mosaicism. The chromosomal constitution of the blastocysts was comprehensively evaluated at the single-cell level, and concordance with the initial PGT-A results was assessed. MAIN RESULTS AND THE ROLE OF CHANCE:A SNP-based mosaicism quantification platform was established and validated using mixtures of single cells of varying ploidy to mimic clinical mosaic samples. In the retrospective cohort, the error origin was successfully determined for 384 of the 391 blastocysts with euploid-aneuploid mosaicism. The overall meiotic error rate was only 3.9% (15/384). Meiotic error was identified in 9.6% of blastocysts with mosaicism from women of advanced maternal age, which was significantly greater than the 3.0% observed in blastocysts with mosaicism from young women (OR = 3.43, 95% CI 1.12-10.46; P = 0.039). In addition, meiotic error was identified in 7.2% (10/139) of blastocysts with high-level mosaicism but 2.0% (5/245) of blastocysts with low-level mosaicism (OR = 3.72, 95% CI 1.25-11.12; P = 0.012). Meiotic error was also significantly greater in blastocysts with maternal-origin mosaicism than in those with paternal-origin mosaicism (8.9% vs 0.4%, OR = 21.66; P < 0.001). Clinical outcomes were comparable between METs and euploid embryo transfers, with no significant difference in LB/OPR (47.9% vs 52.8%, P = 0.409). Single-cell sequencing of donated blastocysts with high-level mosaicisms of mitotic errors demonstrated highly variable mosaic levels, ranging from a reproducibility rate of 8.7-100% for initial mosaic abnormalities at the single-cell level. LARGE SCALE DATA:Due to the individual privacy of the patients, the data are not publicly available. LIMITATIONS REASONS FOR CAUTION:Intrinsic technical noise cannot be completely distinguished from genuine mosaicism in the PGT-AO platform. The sample size of the MET cohort was limited, with few cases undergoing prenatal/postnatal genetic validation. In the single-cell study, the number of verified embryos was relatively small, and the threshold of copy number variation (CNV) detection was 10 Mb, which may have led to the underestimation of CNVs under 10 Mb. WIDER IMPLICATIONS OF THE FINDINGS:PGT-AO analysis demonstrated a low prevalence of meiotic errors in human blastocysts with mosaic biopsy results. PGT-AO is recommended for embryos from patients of advanced maternal age, embryos with high-level mosaicism, and embryos with maternal-origin mosaicism in PGT cycles. FUNDING:This study was supported by grants from the National Key Research and Development Program of China (No. 2023YFC2705503), National Natural Science Foundation of China (No. 82071716), Natural Science Foundation of Guangdong Province (No. 2025A1515010982), Key Clinical Technique of Guangzhou (No. 2023P-ZD19), and Medical Scientific Research Foundation of Guangdong Province (No. A2025203). DISCLOSURES:All authors declare no conflicts of interest.
STUDY QUESTION:Is exome sequencing (ES) an efficient approach for simultaneous analysis of causative single gene defects and copy number variants (CNV) in unexplained premature ovarian insufficiency (POI)? SUMMARY ANSWER:Among 51 idiopathic POI cases, a conservative molecular diagnostic yield of 12% was achieved with equal contribution of pathogenic or likely pathogenic (P/LP) monogenic causes and pathogenic microdeletions. WHAT IS KNOWN ALREADY:POI is a clinically and genetically heterogeneous condition, yet most cases remain idiopathic. Although numerous monogenic causes and CNVs have been implicated in POI, standardized clinical guidelines for comprehensive genetic analysis are still lacking. STUDY DESIGN SIZE DURATION:This observational study of 51 unexplained POI cases implemented an all-in-one ES-based approach to analyze P and LP variants in 288 POI candidate genes and large (>0.5 Mb) pathogenic CNVs leading to POI. PARTICIPANTS/MATERIALS SETTING METHODS:Patients were recruited and phenotyped at two tertiary care women's health and endocrinology centers in Estonia. Bioinformatic processing of ES data and assessment of rare causal variants in POI were performed using an in-house analysis pipeline including automated filtering and manual assessment for pathogenicity, followed by experimental validation via Sanger sequencing (monogenic findings) and chromosomal microarray analysis (CNVs). MAIN RESULTS AND THE ROLE OF CHANCE:Six of 51 unexplained POI cases had confident molecular findings, including three cases with P/LP monogenic variants and three carrying large pathogenic CNVs. Overall molecular diagnostic yield was estimated to be 12% (6 of 51). There was a statistically significant overrepresentation of likely causal genetic findings in primary amenorrhea (5 of 9 cases, 56%) compared to secondary amenorrhea/oligomenorrhea (1 of 42 cases, 2%) (P = 3.0 × 10-4). Primary amenorrhea cases presented diverse findings-TP63 p.(Arg97Gly) in three sisters (including one identified by cascade screening), 15q25.2 microdeletion in two unrelated subjects (POI relevant haploinsufficient genes: CPEB1, BNC1), and NR5A1 p.(Gly328Arg) in one case. A secondary amenorrhea case with ∼8.5 Mb microdeletion at Xq27.3-Xq28 enabled us to narrow down a critical region for non-recurrent Xq terminal losses causing POI (relevant haploinsufficient genes FMR1, AFF2, CETN2, HAUS7, and EMD). Additionally, heterozygous variants TBX6 c.622-2A>T, EXO1 c.136del, and NR2F2 p.(Val307Ala), as well as 1q21.1 microdeletion (two carriers), 1p36.22, and 12q21.1 microduplications were classified as potentially interesting Variants of Uncertain Significance (VUS) that require validation before their causal link to isolated POI can be assigned. TBX6 c.622-2A>T and 1q21.1 del have been implicated in other primary phenotypes, Mayer-Rokitansky-Küster-Hauser syndrome (MRKH, Müllerian anomalies), and 1q21.1 microdeletion syndrome (MIM #612474), respectively. A novel variant NR2F2 p.(Val307Ala) was identified in an isolated oligomenorrhea patient; the same substitution was independently detected in an unrelated male subject presenting oligozoospermia and unilateral cryptorchidism. No autosomal recessive POI cases were identified in our cohort, supporting a high level of heterogeneity and population-specificity in the genetic etiologies of POI, likely shaped by diverse demographic histories. LARGE SCALE DATA:All variants linked identified in this study have been submitted to the NCBI ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) and FerGI (https://www.eshre.eu/Specialty-groups/Special-Interest-Groups/Reproductive-Genetics/FeRGI) databases. LIMITATIONS REASONS FOR CAUTION:All recruited participants were of white European ancestry and living in Estonia. Thus, the results might not apply to other ethnic groups. This study was conducted in a relatively small and well-selected cohort. Validation in larger and more diverse cohorts is needed to further assess the utility of ES in solving idiopathic POI cases. WIDER IMPLICATIONS OF THE FINDINGS:The study's findings support the efficient use of ES as a comprehensive, all-in-one genetic test to achieve molecular diagnosis in unexplained POI, capturing both monogenic variants and pathogenic CNVs. Expanded genetic testing in POI is conceptually and clinically justified to reduce idiopathic cases and enable timely personalized management of reproductive and general health, including cascade testing of at-risk relatives. Given that ES enables the detection of both monogenic variants and CNVs, stepwise strategies may no longer be the most optimal, underscoring an urgent need to develop a standardized pipeline and guidelines for the generation, analysis, and interpretation of NGS data as well as for the counseling and management of patients based on their molecular findings. FUNDING:This study was funded by the Estonian Research Council grants PRG1021 and PRG3041 (to M.L.). DISCLOSURES:The authors declare no conflicts of interest.
