Human Reproduction

BackgroundPolygenic embryo screening (PES) has recently become available to in-vitro fertilization (IVF) patients, allowing them to evaluate the genetic risk of each of their embryos for polygenic conditions such as heart attack or diabetes. Initial modeling predicted that transferring the embryo with the lowest genetic risk for one or more diseases would substantially reduce prevalence in the next generation, with relative risk reductions up to 50%. However, these models assumed the availability of a prespecified number of embryos and that the embryo with the most favorable polygenic risk is born once transferred to the uterus. In reality, a large percentage of embryo transfers do not lead to live births, and IVF frequently results in no or only a single live birth. MethodsTo quantify the expected risk reduction in the context of IVF, we used two datasets: 6944 ovarian stimulation cycles from 4452 Italian infertility patients and 2138 stimulation cycles of egg donors. In both datasets, we simulated the hypothetical application of PES in these cycles by assigning patients and their embryos randomly drawn polygenic risk scores for a given disease, assuming that embryos have been transferred in increasing order of their risk, and tracing their birth outcomes. We then compared the risk of the embryo born after hypothetical PES to the risk of an embryo born without PES. We either considered only completed cycles or integrated over possible birth outcomes of non-transferred embryos in incomplete cycles. ResultsIn stimulation cycles in infertility patients in which all embryos have been transferred and at least one child was born, we estimate that PES will result in relative risk reductions of just {approx}1-3%. In an intention-to-screen analysis of all completed cycles (regardless of birth outcomes), relative risk reductions are under 0.5%. The risk reductions increase, as expected, with more euploid blastocysts and with younger maternal age. Including incomplete cycles (in which not all embryos have been transferred) increases risk reductions to {approx}2-5%, due to the availability of more euploid blastocysts and a higher live birth rate per transfer in these cycles. Pooling all embryos from all cycles of the same patient increases risk reductions to {approx}5-10%. Relative risk reductions in egg donor cycles reach {approx}20% even with a single stimulation cycle per donor. ConclusionsWith the exception of particularly good-prognosis patients or cycles, typical infertility patients would benefit little from PES. In fertile patients, as represented by egg donors, PES is predicted to achieve greater relative risk reductions. However, even though these reductions are still substantially lower than prior estimates that did not account for realistic live birth rates. Ethical, social, and clinical issues associated with offering PES in the general population should be prioritized in future research.

Study questionis fetal exposure to paracetamol associated with markers of ovarian function in infancy? Summary answerMild to moderate doses of prenatal paracetamol exposure, assessed by detailed maternal reports and urinary measurements, is associated with ovarian morphology and activity as well as reduced size of estrogen-responsive tissues in infant girls. What is known alreadyMaternal use of paracetamol is widespread. Across multiple independent animal studies, fetal exposure consistently impairs the formation of primordial ovarian follicles, causing subfertility and premature estropause in female offspring. Study design, size, durationThe Copenhagen Analgesic study (COPANA) is a single center, prospective, observational cohort study conducted at the Copenhagen University Hospital - Rigshospitalet, Denmark (March 2020 to November 2022). Participants/materials, setting, methods;O_ST_ABSCOPANA cohortC_ST_ABS3425 eligible participants. In total, 685 healthy, singleton pregnant women of Caucasian origin were enrolled in the first trimester of pregnancy, 302 girls examined at follow up. Exclusion criteria: maternal diabetes or thyroid disease, pre- or post-term delivery, or severe infant illness. Exposure: pregnant women reported paracetamol use biweekly and provided first-trimester urinary samples which were analyzed for paracetamol levels (LC-MS/MS) and adjusted for urinary osmolarity (n = 299). Girls were classified by timing of exposure: early fetal life (<17 weeks, n = 92), mid-late fetal life ([&ge;]17 weeks, n = 67), or unexposed controls (n = 143). A subgroup of girls was exposed exclusively in early fetal life (n = 22). Independent confirmatory cohort1210 girls followed from infancy to adolescence. Exposure: maternal self-reported any use of paracetamol during pregnancy reported in early third trimester (yes/no). Main results and the role of chanceEarly fetal exposure was associated with reduced ovarian volume (-0.11 cm3, 95% CI -0.19 to -0.03) and uterine volume (-0.16 cm3, -0.32 to -0.01), whereas mid-late fetal exposure was associated with fewer ovarian follicles (-1.05, -1.71 to -0.39) compared to unexposed girls. AMH levels were lower in girls exposed exclusively in early fetal life (-0.45 SDS, -0.87 to -0.03) compared to unexposed girls. Maternal urinary paracetamol concentrations were inversely associated with ovarian and uterine volume as well as breast tissue diameter. In an independent cohort, fetal paracetamol exposure was associated with reduced uterine volume at puberty (-4.11 cm3, -7.29 to -0.92) and smaller ovarian volume in adolescence (-2.76 cm3, -4.82 to -0.70). Limitations, reasons for cautionThe design of the study allows evaluation of exposure- outcome associations, while causality is strengthened by experimental models demonstrating comparable effects. Residual confounding by indication cannot be completely excluded, although results were robust after accounting for fever and other maternal factors. The analytic design of the current study limited our ability to evaluate whether frequency or patterns of paracetamol use influenced the observed associations. Wider implications of the findingsThe consistency of findings across different assessment measures and cohorts, in combination with parallel evidence from animal studies, suggests potential long-term implications for female reproductive health. Study funding/competing interest(s)This research was supported by Rigshospitalets Research Council under grant (E-22717-21), Laege Sofus Carl Emil Friis og hustru Doris Friis Legat (F-23936-01), Aase og Ejnar Danielsens Foundation (20-10-0367), Helsefonden (20-B-0388), Axel Muusfeldt Foundation (2020-0385) and The Danish Centre for Endocrine Disrupting Substances (CeHoS) (2022-23219). The authors have nothing to declare. Trial registration numberClinicalTrials.gov ID: NCT0436922

Background: Male factor infertility is present in around 40% of couples utilising assisted reproductive technology (ART). However, it is unclear how specific causes of male infertility impact the chance of successful ART treatment, with most research either treating male infertility as single diagnostic group or being isolated smaller-scale studies focused on the treatment and outcomes of specific diagnoses. Objective: To study the impact of eleven specific aetiologies of male infertility on the chance of a clinical pregnancy in couples with known causes of male or female infertility following their first ART cycle. Material and methods: Population-based (initiated ART in Australia and New Zealand in 2020- 2022) cohort study assessing the impact of eleven male infertility diagnoses (idiopathic, Klinefelter syndrome, Y chromosome microdeletions, testis damage from cancer, testis damage from other causes, gonadotropin deficiency, congenital absence of the vas deferens/cystic fibrosis (CBAVD), other obstruction disorder, erectile dysfunction, and ejaculatory disorder) on the chance of a clinical pregnancy following a couple's first complete ART cycle (all fresh and frozen-thawed embryo transfers arising from one episode of ovarian stimulation). Adjusted risk ratios comparing a couples undergoing ART solely for treatment of male infertility with couples undergoing ART solely for treatment of tubal disease were calculated for the chance of a clinical pregnancy following a complete ART cycle and following an attempted fertilisation procedure. Results: A total of 39,053 couples were included, with male infertility present in 42.7% of cases, and the only cause of infertility in just under half of these cases. In more than three-quarters of male infertility cases the cause of infertility was unknown (idiopathic) or undiagnosed. Most couples undertaking ART for treatment of male infertility can expect similar success rates to couples seeking treatment for good prognosis female infertility diagnoses. However, those with Klinefelter syndrome and Y chromosome microdeletions had a 59.5% (aRR: 40.5% [95% CI: 16.9%-64.1%]) and 28.9% (aRR: 71.1% [95% CI: 45.5%-96.7%]) lower chance of a clinical pregnancy per initiated stimulation cycle compared to those with tubal disease as the only source of infertility. However, there was no difference once sperm was retrieved compared to other diagnoses tending to require surgical sperm retrieval, use frozen oocytes and necessitating ICSI. Discussion and Conclusion: In this population-based study most couples undergoing ART because of male infertility had similar success rates to those undergoing ART for treatment of female tubal disease, except for patients with Klinefelter syndrome and Y chromosome microdeletion who had approximately half and three-quarters the chance of a clinical pregnancy due to failed sperm retrieval/survival, but no difference (accounting for the use of surgical sperm, ICSI and potentially frozen oocytes) in outcomes once sperm were available. While these finding are reassuring for most men presenting to an ART clinic with male infertility, with more than three-quarters of male infertility cases reported as being idiopathic, there is an urgent need for greater research on the causes, diagnosis and implications of male infertility.

1.Study questionDo singletons conceived by medically assisted reproduction (MAR) experience an elevated incidence of childhood cancers and are they at a greater risk of such cancers compared to naturally-conceived singletons? Summary answerWe found no strong evidence the adjusted risk of childhood cancers is increased for MAR-conceived singletons. What is known alreadyThere is longstanding concern children conceived via MAR may be at increased risk of childhood cancer. Current epidemiological evidence does not support such a relationship. Study design, size, durationWe conducted a retrospective population-based cohort study of 5,104,121 singletons born in Australia between 1991 and 2019. Median follow-up time varied from 4 to 10 years depending on mode of conception. Participants/materials, setting, methodsWe linked birth records to public medical insurance data of the mother to ascertain MAR conception. We classified treatment as ovulation induction/intrauterine insemination (OI/IUI) or assisted reproductive technology (ART; IVF/ICSI), with ART coded as either fresh embryo transfer or frozen embryo transfer. The cohort included 4,924,354 naturally-conceived singletons and 179,767 singletons conceived via MAR. We calculated standardised incidence ratios (SIRs) to ascertain differences in population incidence of childhood cancer, and generated hazard ratios (HRs) using flexible parametric survival models controlling for key confounders. We report absolute incidence and risk differences for both statistical approaches. Main results and the role of chanceThere was no increase in the incidence or risk of all childhood cancers combined for singletons conceived via MAR, either any MAR or specific MAR types. There was some evidence the incidence of leukemias, myeloproliferative diseases, and myelodysplastic diseases was increased after ART compared to the general population (SIR: 1.32, 95% CI 1.02-1.68; equating to 2.09, 95% CI 0.13-4.44 extra cancers per 100,000 person-years), but no increased risk after adjusting for available confounders (HR: 1.04, 95% CI 0.73-1.46). These cancers showed increased incidence and risk for those conceived via IVF (SIR: 1.54, 95% CI 1.01-2.26; HR: 1.77, 95% CI 1.06-2.95), but not ICSI (SIR: 1.27, 95% CI 0.83-1.85; HR: 0.76, 95% CI 0.48-1.22). Incidence of renal tumours was elevated after IVF (SIR: 2.37, 95% CI 1.02-4.67; equating to 1.83, 95% CI 0.03-3.99 extra cancers per 100,000 person-years) and frozen transfer ART (SIR: 2.52, 95% CI 1.09-4.97; equating to 2.12, 95%CI 0.12-5.53 extra cancers per 100,000 person-years), however risk was not elevated after adjusting for available confounders (HR: 1.06, 95% CI 0.47-2.38; and HR: 1.63, 95% CI 0.73-3.61 respectively). Limitations, reasons for cautionWe did not have information on parental cause of infertility, which could be a confounder for childhood cancer, although we did adjust for parental history of cancer. For many specific cancer types, fewer than 50 cases were observed in total. Given the number of comparisons reported and closeness of the lower-bound confidence interval to 1, we cannot exclude that a significant association between conception via IVF and leukemias, myeloproliferative diseases, and myelodysplastic diseases reflects a type I error. Wider implications of the findingsOur findings align generally with published meta-analyses on the risk of childhood cancers following MAR conception and reinforce the need for very large studies to increase confidence. Parents who have conceived via MAR and their offspring can be reassured there is not strong evidence the treatments increase the overall incidence or risk of childhood cancer. Study funding/competing interest(s)This work was funded by the National Health and Medical Research Council (NHMRC: APP1164852). Dr ARW declares that their involvement in this work was supported by employment at UNSW Sydney. Prof CMV declares payment to their institution from the National Health and Medical Research Council (APP1164852). Prof NH declares payment to their institution from the National Health and Medical Research Council (APP1164852); royalties and licenses for Berek and Hackets Gynecologic Oncology (Walters Kluwer); royalties and licenses for Hacker and Moores Essentials of Obstetrics and Gynecology (Elsevier); consulting fees from Darwin Hospital and Gold Coast University Hospital; support for attending the British Gynaecological Cancer Society meeting in Aberdeen, UK, Jun 2023; support for attending the Symposium on Gynaecological Cancer in Budapest, Hungary, Nov 2023; support for attending the International conference of the Rajiv Gandhi Cancer Centre in Delhi, India, Mar 2025; and membership of the Medical Advisory Committee for TruScreen (Australia and New Zealand). A/Prof SO declares that they received payment to their institution from the National Health and Medical Research Council (APP1164852); they received a grant from the European Society for Human Reproduction and Embryology (Open call 2022) including payment to their institution; and that they are a member of the Advisory Board of the Cervical Screening Program in Norway through The Norwegian Institute of Public Health (NIPH), for which they were reimbursed travel expenses to their institution. Prof MC declares support for Theramex European Society for Human Reproduction and Embryology registration and Fertility Society of Australia and New Zealand registration and accommodation. A/Prof USD declares that her involvement in this work was supported via an Early Career Fellowship from the Cancer Institute NSW (ID: 2020/ECF1163) and employment at UNSW Sydney. A/Prof USD also declares payment to their institution from the National Health and Medical Research Council (APP2035240) and the Medical Research Future Fund (APP2032214; APP2038377), and the Australian Research Council (DP240100072) as well as current grants from NSW Health, Prince of Wales Hospital Foundation, and unpaid involvement as an Associate Editor for the "Journal of Psycho-Oncology Research and Practice". Prof LJ declares payment to their institution from the National Health and Medical Research Council (APP1164852). Prof RJN declares they are the Chair of the Clinical Advisory Committee, Westmead Fertility; External mentor at VinMec hospital; Editorial Editor at the journal "Fertility and Sterility"; and has received funding from the National Health and Medical Research Council (NHMRC) for the NHMRC Centre for Research Excellence in Womens Health in Reproductive Life (CRE WHiRL). A/Prof CS declares stock or stock options associated with CSL Ltd, Sigma Healthcare Ltd, Resmed Inc, Medical Developments International Ltd, Vitrafy Life Sciences Ltd, Intuitive Surgical, and Steris PLC. Prof GMC declares payment to their institution from the National Health and Medical Research Council (APP1164852). Prof CV declares payment to their institution from the National Health and Medical Research Council (APP1164852); research grants receive from Merck KGaA and Ferring; payments for honoraria from Merk Ltd, Merk Sharpe & Dohme, Ferring, Organon, Gedeon-Richter for being an invited lecturer in scientific meetings/conferences on multiple occasions as well as member of advisory boards for these companies who have a commercial portfolio in the field of assisted reproduction technology (ART); and speaking fees from IBSA, Vianex, Sonapharm; travel support for their participation in scientific meetings/conferences both nationally and internationally, usually as an invited speaker for the following companies - Merck Ltd, Merck Sharpe & Dohme, Ferring, Organon, Gedeon-Richter; unpaid involvement as a Board member of the Hellenic Society of Fertility and Sterility, Member of the Editorial Board of the journal "Human Reproduction", Senior Deputy of the Coordination Committee of the Special Interest Group "Reproductive Endocrinology" of the European Society for Human Reproduction and Embryology, Member of the Editorial Board of the journal "F&S Reviews", Member of the Editorial Board of the journal "RBM Online", Member of the Editorial Board of the journal "Reproductive Biology & Endocrinology", Member of the Editorial Board of the journal "Frontiers in Endocrinology", and Member of the Editorial Board of the journal "Reproductive Sciences". SubjectReproductive epidemiology

Background Endometriosis is associated with adverse pregnancy outcomes in standard observational studies, including placental complications, preterm birth, and caesarean delivery. However, causal inference from these studies is complicated by residual confounding, differential clinical management, and the presence of intermediate factors such as subfertility and the use of assisted reproductive technologies, which may lie on the causal pathway between endometriosis and adverse outcomes. We applied Mendelian randomization (MR) to estimate the causal effects of genetic liability to endometriosis on a broad range of maternal and perinatal outcomes. Methods We conducted a two-sample MR study using summary-level GWAS data. Forty-one independent genetic instruments for endometriosis were derived from the largest available GWAS meta-analysis (60,674 cases; 701,926 controls; mean F-statistic = 279). SNP-outcome associations were obtained for 30 outcomes from the MR-PREG collaboration, FinnGen Release 12, and a postpartum haemorrhage GWAS meta-analysis, spanning placental disorders, pregnancy timing, labour and delivery, hypertensive disorders, fetal growth, and neonatal outcomes. Primary analyses used the inverse-variance weighted method, complemented by MR-Egger, weighted median, weighted mode, and MR-PRESSO. Trio-based models disentangled maternal from fetal genetic contributions. Multiple testing was addressed using false discovery rate correction. Findings Across 30 outcomes, only placenta praevia reached FDR-corrected significance, with a robust and consistent causal signal across four of five sensitivity methods (IVW OR 1.62, 95% CI 1.33-1.97; q<0.001). Within the placental disorders domain, estimates for premature placental separation and the broader placental disorders phenotype were directionally concordant but imprecise. For premature rupture of membranes, estimates were concordant across three methods, though the association was sensitive to cohort exclusion and did not survive multiple testing correction and should be interpreted cautiously. By contrast, hypertensive disorders, gestational diabetes, postpartum haemorrhage, stillbirth, and most neonatal outcomes showed estimates consistently close to the null across all methods. Trio-based analyses suggested predominantly maternal genetic pathways for most outcomes; fetal genetic contributions were not significant after correction for multiple testing, with exploratory signals observed for birthweight-related outcomes requiring independent replication. Interpretation A robust causal signal for placenta praevia alongside directionally consistent estimates across the placental disorders domain, suggests that mechanisms related to abnormal implantation and placentation may constitute a major mechanism for how endometriosis liability influences pregnancy. These results suggest that previously reported associations with broader obstetric outcomes may partly reflect confounding or clinical management patterns, and support targeted surveillance for abnormal placentation rather than a generalised elevation of obstetric risk.

Cannabis (marijuana) is the most widely used recreational drug in the USA accounting for about 62 million users in 2024. Among cannabis users, 26% are of prime reproductive age (18-25 years). Delta-9 tetrahydrocannabinol (THC) is the principal psychoactive component of cannabis and has been detected in human seminal fluids. Although abundant evidence indicates adverse effects of THC exposure on spermatogenesis in different species, acute effects of THC on postejaculatory sperm including fertilization potential and subsequent carryover effects on embryo development are largely unknown. The present study was designed to provide missing information on structural and mechanistic effects of THC exposure to postejaculatory sperm function by evaluating sperm indices often overlooked or masked during clinical evaluation. A bovine embryo continuum model was employed to determine effects of THC on sperm structure, kinematics, bioenergetics, and binding mechanisms. Effects of THC on the sperm genomic and epigenomic landscape were determined, complemented by paternal carry over effects on embryo development as a human translational model to elucidate paternal effects on future development, and to mirror sperm exposure during transport within the female reproductive tract. Cryopreserved bovine sperm from three bulls were independently exposed to physiologically relevant concentrations of THC (0 and 32nM, n = 2 individual replicates/bull) for 24 h under non-capacitating conditions at 25{degrees}C followed by quantification of sperm kinematics at 37{degrees}C. Samples of THC-exposed sperm and vehicle-control (0.1% DMSO) were collected in replicate following immediate addition of THC (0 h) and again at 24 h. DNA damage, acrosome integrity, bioenergetics, changes to DNA methylation and embryo development were quantified. Data were analyzed by logistic regression with a generalized linear mixed effect model. Computer-assisted sperm assessment revealed a reduction in progressive motility of THC-exposed sperm after 24 h while other parameters were not affected. Acrosome integrity as determined by flowcytometric analysis with FITC-PSA was severely compromised in THC-exposed sperm (P [&le;] 0.05), despite no detectable difference in capacitation status using merocyanine staining. Similarly, DNA integrity as determined by TUNEL assay was significantly impaired after 24 h of THC exposure (P [&le;] 0.05). Mechanistic effects of THC were explored through characterization of the transmembrane G-protein coupled cannabinoid 1 receptor (CB1). CB1 is expressed in the post-acrosomal region and its abundance decreased as compared to unexposed sperm. Alterations to the methylation landscape of sperm were then determined after 24 h of THC exposure through whole-genome Enzymatic Methyl Sequencing. PCA analysis indicated that sperm from different males formed distinct clusters, implying individual differences among bulls, while the effects of THC exposure produced tighter clusters. Paternal carryover effects on embryos derived by in vitro fertilization from THC exposed sperm had reduced 2-cell cleavage, 8-16 cell morula development, and reduced blastocyst development compared to unexposed sperm (46% vs. 33%). In conclusion, post-ejaculatory mammalian sperm exposure to THC compromises acrosome integrity, induces DNA damage, changes the sperm methylome, and reduces developmental potential. Collectively, these data implicate new considerations for recreational and clinical use of cannabis that impact cellular and molecular mechanisms important for sperm function with detrimental consequences for gamete interaction and embryo development.

Background: Literature on the role of thermal discomfort (heat- and cold-stress) on in-vitro fertilization (IVF) outcomes are scarce and inconclusive. This multi-center research examines association between heat stress and IVF treatment outcomes in Andhra Pradesh, which is prone to year around chronic heat stress. Methods: IVF data were abstracted from clinical chart review of all patients from three IVF from centers 2019 to 2023, which included time-stamped data on each IVF procedure, demographics and pre-existing comorbidities. Weather data were acquired from the National Climatic Data Center (NCDC). IVF outcomes were modelled with respect to time-lagged exposure to ambient temperature stratified by hyper- and hypo-thermic conditions using Poisson and logistic regressions depending on the scale of IVF outcomes adjusting for confounders. Results: Heat stress peaked in June, which corresponded with elevated number of spontaneous abortions/miscarriage (SAM). Under hypo- and hyper-thermic conditions a unit increase ambient temperature was associated with an 11% higher and an 8% lower number of oocytes retrieved, respectively. Adjusting for confounders, a 10 degree F increase in two-day lag heat stress was associated with a 30% higher odds of SAM (odds ratio ~ 1.03; 95% CI = 1.001 to 1.068; p-value < 0.043), and odds of PTB were 3 times higher when three day-lagged heat index (HI) was greater than 35 degree C (odds ratio 1.13 to 7.99; p < 0.05). Conclusion. Our findings warrant strategies to engage IVF patients in mitigating their exposure to thermal discomfort before and during the treatment.

Introduction The aim of this study was to determine the annual age- and sex-specific prevalence of gender-affirming hormone and puberty blocker use among young people with a gender incongruence (GI) diagnosis in Norway. Methods We integrated data from multiple Norwegian national registers to perform a nationwide register-based study of individuals with known sex assigned at birth who were born in the period 1975-2017 and resident in Norway for all or part of the period 2008-2022. We first calculated the annual age- and sex-specific incidence of GI diagnoses in the population. Then, we calculated the annual age- and sex-specific prevalence of androgen, estrogen, and puberty blocker use among individuals with a GI diagnosis who were under age 25 (for androgens and estrogens) or 18 (for puberty blockers) in the year that they collected the prescription. Results The incidence of GI diagnoses has increased among youth in Norway, most notably since 2015 and with the largest increase among teens assigned female at birth. The prevalence of feminizing and masculinizing hormone therapy has increased in this period as well, but mainly among the oldest teens and young adults. The prevalence of puberty suppression is mostly low but has also increased since 2015, especially in recent years among teens assigned male at birth. Conclusion The prevalence of gender-affirming hormone and puberty blocker use has increased among transgender youth in Norway, concurrently with an increase in the incidence of GI diagnoses.

Objective To examine the effects of combined oral contraceptive (OC) use on clinical markers of ovarian reserve by comparing Anti-Muellerian Hormone (AMH), antral follicle count (AFC), and ovarian volume (OV) before and after starting or stopping OC. Methods This analysis is based on data from a prospective cohort study conducted at the University Hospital Tubingen, Germany, as part of the IRTG-2804 project. A total of 54 healthy women were included and categorized into three groups based on their OC use status: OC starters (n = 12), stoppers (n = 16), and long-term OC-users (n = 26). Each participant underwent a transvaginal ultrasound (including AFC and OV) and serum sampling (including AMH) at two time points (S1 and S2), three to six months apart. OC starters were assessed first during the early follicular phase (day 1-7) and then during active OC intake (day 8-21), while stoppers were assessed in the reverse order. Long-term users were assessed twice during active OC intake. Results OC stoppers showed significant within-group increases in all ovarian reserve markers, including AMH ({Delta} = 2.57 ng/mL, p < .001), AFC ({Delta} = 3.88, p = .004), and OV, which almost doubled (1.94-fold increase; 95% CI [1.35, 2.80], p < .001). In contrast, OC starters exhibited a significant decline in AMH ({Delta} = -1.25 ng/mL, p = .013), but no changes in AFC or OV. No significant longitudinal changes were observed among long-term OC users. Conclusion AMH levels decrease after starting OC use whereas AFC and OV are not affected. In contrast, AMH, AFC, and OV recover within three to six months after stopping OC, suggesting a reversible suppression of ovarian reserve markers during OC use. These findings are clinically relevant for fertility counseling and for the interpretation of ovarian reserve markers in women using hormonal contraception.

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.

Background The use of Assisted Reproductive Technology (ART) is increasing worldwide. These treatments involve ovarian stimulation to enable multiple follicle recruitment, hence inducing supraphysiological estrogen levels. While most long-term follow-up of women undergoing ART has concerned cancer incidence, the long-term safety regarding cardiovascular and metabolic diseases remains under-explored. This study was performed to assess the risk of acute myocardial infarction, cerebral ischemic conditions, intracranial hemorrhage, type 2 diabetes mellitus, heart failure, aortic aneurysm or dissection, and chronic kidney disease in women that conceived with ART, and to investigate the role of the underlying infertility and its risk factors. Methods and Findings Swedish national registers allowed us to follow a nationwide cohort of 380,756 women from their first birth between 1992 and 2002 until the end of 2023. The safety of ART was evaluated by comparing women with infertility who conceived with and without ART, while adjusting for baseline differences in age, body mass index, country of origin, socioeconomic factors, pre-existing comorbidity, smoking and year. The role of infertility was additionally explored by comparing all women with and without infertility adjusting for age, as well as the aforementioned baseline characteristics. Cumulative risks were plotted using inverse-probability weighted Kaplan-Meier curves. To facilitate the comparison of groups we also estimated risk differences and ratios at 10-, 20-, and 30-years of follow-up. Use of ART was not associated with cardiovascular disease except for an excess risk of cerebral ischemic conditions, with a 30 year risk ratio of 1.43 (1.09; 1.89). With the exception of cerebral ischemic conditions, intracranial hemorrhage, aortic dissection, and chronic kidney disease, women with a history of infertility exhibited consistently higher risk of all outcomes, adjustment for differences in baseline characteristics explained some but not all of these elevated risks. Conclusions With the exception of ischemic cerebral conditions, the findings provide reassurance regarding the long-term cardiometabolic safety of ART use, while adding to the growing literature suggesting that infertility can act as a marker of womens cardiovascular and metabolic disease.

Background Delineating the cellular origins of extracellular vesicles (EVs) enables the detection of clinically relevant changes in dynamic and complex tissues, such as the endometrium, which are not characterizable through single biomarker assays. Transcriptome deconvolution into cellular composition using deep learning methods provides a means to explore this complexity. However, such computational methods have not been previously applied to EV bulk transcriptomes, and their efficacy in profiling EV population changes and concordance to tissue throughout the menstrual cycle remains unknown. Methods This observational cross-sectional study utilized a deconvolutional generative deep learning algorithm, BulkTrajBlend, trained on a comprehensive human endometrial single-cell RNA sequencing (scRNA-seq) atlas. The model was applied to deconvolve paired bulk transcriptomes from endometrial tissue and uterine fluid EVs (UF-EVs) across the proliferative (P, n=4), early-secretory (ES, n=5), mid-secretory (MS, n=5), and late-secretory (LS, n=5) phases from healthy, fertile women. To validate generalizability, independent UF-EV datasets (ES, n=12; MS, n=12) obtained via different laboratory protocols were included. Deconvolved pseudo-single-cell (pSC) profiles from UF-EV data were subsequently integrated with Visium spatial transcriptomics slides of human endometrium (P, n=2; MS, n=4; ES, n=2). Results We developed a foundation model-based approach utilizing self-supervised learning to determine the cellular origin of EVs from their transcriptomic profiles. By mapping the generated pSC profiles to spatial transcriptomic data, we evaluated spatial origins of EVs. The statistical analysis demonstrated that UF-EV transcriptome deconvolution reflects the dynamic changes in the cellular composition of endometrial tissue across the menstrual cycle phases. The ability to distinguish accurately between proliferative and decidualizing menstrual cycle phases (ROC-AUC = 0.98) using cellular profile of deconvoluted UF-EVs transcriptome enables non-invasive profiling of endometrial tissue. Conclusions Our findings indicate the feasibility of determining endometrial tissue cellular composition using UF-EV transcriptomics. This methodology enables refined, non-invasive endometrial testing, avoiding invasive biopsy procedures. Based on deconvolution results, we are able to correlate UF-EV content to tissue, and distinguish between menstrual cycle phases. These results build toward a multifactorial screening method for abnormalities within the endometrium.

BackgroundMitochondrial dysfunction is a leading contributor to the decline in oocyte quality associated with maternal aging. Prior investigations of mitochondrial function in the ovarian follicle have largely treated the mitochondrial pool as a homogeneous population, reporting aggregate values that may obscure biologically meaningful differences between distinct mitochondrial subpopulations. The present study addresses this limitation by characterizing mitochondrial subpopulation dynamics in oocytes and cumulus granulosa cells at single-organelle resolution using fluorescence-activated mitochondria sorting (FAMS). ResultsAnalysis of the aggregate mitochondrial population in mouse oocytes revealed no significant age-associated differences in mitochondrial DNA copy number or membrane potential, a result that would previously have been interpreted as evidence of minimal age-related mitochondrial change. Subpopulation analysis revealed this conclusion to be incomplete: aged oocytes showed significantly elevated mitochondrial DNA copy number specifically within the high membrane potential and small mitochondrial subpopulations, with no significant differences in the low membrane potential or large subpopulations. NMN supplementation normalized mitochondrial DNA copy number in the high membrane potential and small subpopulations toward young levels while producing an opposing effect in large mitochondria, demonstrating subpopulation-specific rather than uniform rejuvenation. In cumulus cells, significant age-associated changes were detectable at the aggregate level, including a reduction in mitochondrial DNA copy number and an elevation in membrane potential, and subpopulation analysis further resolved these findings. The age-associated reduction in cumulus cell mitochondrial DNA copy number was driven predominantly by the high membrane potential subpopulation. NMN supplementation exerted opposing effects on small and large cumulus cell mitochondrial subpopulations, increasing mitochondrial DNA copy number above both young and aged levels in small mitochondria while further reducing it below aged levels in large mitochondria. ConclusionsViewing the mitochondrial pool as a heterogeneous mixture of functionally distinct subpopulations rather than a uniform population reveals age-associated alterations in oocytes and cumulus cells that are undetectable by aggregate analysis. NMN supplementation exerts subpopulation-specific effects in both cell types, identifying specific mitochondrial subtypes as more precise targets for future mechanistic investigation of age-associated infertility than the mitochondrial pool considered in aggregate.

The human endometrium undergoes cyclic, hormone-driven remodeling that establishes a transient window of receptivity required for embryo implantation, placentation, and maintenance of pregnancy. Decidualization of endometrial stromal cells is a central component of this process and can be induced in vitro using cAMP alone or in combination with ovarian steroid hormones (EPC: estradiol, progesterone, and cAMP). Although cAMP activates the core decidual transcriptional program, whether hormone supplementation induces a more physiologically relevant response remains unclear, particularly in 3D endometrial organoid (Endo-organoid) models which have emerged as a new alternative methodology (NAM). Here, we compared morphological and transcriptomic responses of human endometrial stromal cell-derived Endo-organoids undergoing decidualization induced by cAMP or EPC stimulation. EPC-treated Endo-organoids exhibited enhanced structural remodeling and more advanced morphological transformation compared with cAMP-treated organoids. RNA-seq analysis revealed substantial overlap in canonical decidual gene expression between the two conditions, but EPC induced broader transcriptional and pathway-level changes, including enrichment of metabolic, stress-response, and differentiation-related processes. Together, these findings demonstrate that while cAMP activates the core decidual program, EPC elicits a broader and more physiologically relevant decidualization response in 3D human Endo-organoids, providing guidance for optimizing Endo-organoids to study endometrial receptivity, implantation, and early pregnancy success.

Study question: Does twin status and zygosity (monozygotic vs. dizygotic; same-sex vs. opposite-sex) predict fertility outcomes and intergenerational reproductive patterns compared with singletons? Summary answer: Among females, dizygotic twins had modestly higher completed fertility than singletons and monozygotic twins and were more likely to have a twin birth. Fertility did not differ meaningfully among males. These differences were restricted to the twin generation and did not persist in the next generation, indicating sex-specific and generation-specific effects rather than intergenerational transmission. What is known already: Dizygotic twinning is associated with heritable hyperovulation and higher natural fertility but less is known about whether being a twin or zygosity influences reproductive outcomes across generations. Study design, size, duration: A population-based longitudinal cohort study using part of the Finnish Twin Cohort and national population registers. Participants included monozygotic (MZ; N = 4,068), same-sex dizygotic (SSDZ; N = 8,890), opposite-sex dizygotic (OSDZ; N = 8,474) twins, and singleton controls (N = 1,193,404) born between 1945-1957 (total N =1,254,103; 49.1% female), their mothers, their children, and their grandchildren. Participants/materials, setting, methods: Fertility outcomes (number of biological children, age at first birth, childlessness, multiple births) were derived from Finnish population registers. Analyses followed a preregistered plan (https://osf.io/qbwv3) Main results and the role of chance: Differences in fertility between singletons and twins were modest and varied by sex and zygosity. Differences were observed generally in the mothers of twins and female twins themselves, with limited differences in the offspring of twins as compared to the offspring of singletons. Twins were slightly older at first birth, had fewer total biological offspring, but were more likely to have a twin birth. Dizygotic twins in particular differed from monozygotic twins and singletons. Limitations, reasons for caution: Findings are limited to individuals born in mid-20th-century Finland and thus generalizability to recent populations or non-Nordic contexts may be restricted. Further, analyses are observational, and causal inference is limited due to alternative motivation behind fertility rates like social or cultural reasons. Wider implications of the findings: These findings suggest that zygosity and sex interact to shape reproductive outcomes, offering insight into genetic and environmental contributions to fertility. They highlight the value of large twin cohorts for studying intergenerational reproductive trends and the representativeness of twins in population-based fertility research.

Abstract Purpose: Published clinical outcome data on preconception carrier screening (PCS) in Central Asia are limited. We report the first clinical implementation study from Uzbekistan of a whole-exome sequencing (WES)-based multi-platform PCS program combining exome sequencing with targeted SMA, FMR1, and DMD assays. Methods: We retrospectively analyzed anonymized data from 65 individuals (19 couples, 27 singletons) screened at IMC Genomics, Tashkent, between January 2024 and May 2026. WES covering the protein-coding regions of approximately 20,000 genes was followed by exome-wide bioinformatics filtering and clinical geneticist interpretation. Partly overlapping cohorts underwent SMA carrier screening (n=179), FMR1 CGG-repeat analysis in females (n=155), and DMD deletion/duplication testing in preconception females (n=29). Variants were classified by ACMG/AMP criteria against gnomAD v4.1. Results: Sixty-one of 65 WES-screened individuals (93.8%; 95% CI 85.2 - 97.6%) carried at least one reportable variant (152 instances across 126 genes). Four of 19 couples (21.1%; 95% CI 8.5 - 43.3%) were concordant for pathogenic or likely pathogenic variants in the same autosomal recessive gene; two were referred for preimplantation genetic testing for monogenic disease. SMA screening identified four carriers, including two 2+0 silent carriers; FMR1 analysis identified one intermediate allele; DMD MLPA identified no exonic rearrangements. Conclusion: This first reported WES-based multi-platform PCS program in Uzbekistan was feasible and clinically informative, identifying actionable couple-level reproductive risks and supporting structured implementation of reproductive genetic screening in Central Asia.

Consanguinity is a reproductive union between individuals who share a recent common ancestor. These unions are common in many regions of the world and increase the burden of rare recessive disorders by elevating autozygosity in offspring. Current reproductive genetic screening focuses on a limited set of known pathogenic variants, leaving most recessive risk unaddressed. Here we argue that embryo-level autozygosity, quantified as the fraction of the genome in long runs of homozygosity (FROH), is a potentially actionable genomic biomarker that can be integrated into routine preimplantation genetic testing as a homozygosity-informed embryo-prioritization framework (PGT-H) that can be layered onto existing embryo biopsy workflows when couples are already undergoing IVF with PGT-A or PGT-M. Using forward simulations of first-cousin and double-first-cousin couples, we show that siblings conceived by the same couple span a wide range of FROH; selecting the lowest-FROH candidate from a cohort of five embryos reduces FROH by approximately 40% on average. Combining these reductions with empirical effect-size estimates, we estimate that for first-cousin couples this strategy could reduce risk of intellectual disability by roughly 35-45% (corresponding to an absolute risk reduction of about 1.8-2.2%) and potentially reduce excess recessive disease burden, while also modestly reducing risk of common diseases such as type 2 diabetes. We outline how existing PGT-A and PGT-M workflows could potentially be extended to report embryo-level FROH and discuss ethical and counseling considerations. Autozygosity-based embryo prioritization offers a principled way to address a component of recessive risk that current variant-centric approaches miss.

Background: Placenta accreta spectrum (PAS) is an important cause of severe maternal morbidity. Although prior cesarean delivery is a well-established risk factor, PAS also occurs in women without prior cesarean section (CS), in whom risk may be underestimated. This study evaluated routinely available clinical factors associated with PAS in this population and developed a clinical-history-based prediction model. Methods: We conducted a retrospective cohort study of women without prior CS who delivered at Peking University Third Hospital, China, from January 1, 2022, to December 31, 2023. PAS was diagnosed according to the 2019 International Federation of Gynecology and Obstetrics clinical and/or histopathological criteria. Multivariable logistic regression was used to identify independent risk factors. Model performance was assessed using receiver operating characteristic curves, calibration, decision curve analysis, and stratified 5-fold cross-validation. Analyses were repeated after stratification by placenta previa status. Results: Among 11,148 women without prior CS, 236 had PAS. Independent risk factors in the overall cohort were placenta previa, operative hysteroscopy, uterine curettage, in vitro fertilization, and multifetal pregnancy. The overall clinical prediction model showed an area under the curve of 0.838 (95% confidence interval, 0.81-0.87), with stable performance in internal validation. In stratified analyses, model discrimination was lower among women without placenta previa (area under the curve, 0.734) and those with placenta previa (area under the curve, 0.647). Conclusions: In this single-center cohort, routinely available clinical history was associated with PAS risk among women without prior CS. The proposed model may help identify patients who warrant targeted PAS imaging or specialist assessment, but external validation and integration with imaging features are needed before broad clinical implementation.

Human embryo implantation, occurring approximately one week after fertilization, remains poorly understood due to ethical and technical limitations of in vivo investigation. To overcome these barriers, and model this critical developmental event, encompassing peri- and early post-implantation stages, we used an in vitro embryo attachment model composed of donor-derived endometrial epithelial cells forming an open-faced endometrial layer (OFEL) and human stem cell-derived blastoids recapitulating human day 5 blastocysts in peri-implantation model. Following attachment, developmental progression was further investigated on laminin-coated substrates to capture early post-implantation dynamics. Despite its central role as the primary endocrine signal of early pregnancy, human chorionic gonadotropin (hCG) remains largely uncharacterized in this context. Here, we describe the transcriptomic profile of blastoid-endometrial co-cultures relative to OFEL alone, identifying CGA and CGB3/5/8 as among the most strongly upregulated genes following blastoid attachment to hormonally stimulated OFEL. Consistent with these findings, immunoassays and luteinizing hormone/choriogonadotropin receptor (LHCGR) activation assays of conditioned media confirmed the secretion of heterodimeric, biologically active hCG and its free subunits in co-cultures, but not in endometrial layers alone. Notably, the hyperglycosylated hCG heterodimer was the predominant isoform detected. Co-culture with the endometrial component significantly increased hCG secretion compared with blastoids cultured alone, an effect further enhanced by hormonal priming in the peri-implantation model. Collectively, these findings indicate that a hormonally primed endometrial environment not only promotes blastoid attachment but also amplifies embryonic hCG production and bioactivity, underscoring the importance of maternal endocrine cues in early embryo-endometrium communication. Furthermore, our peri- and early post-implantation models recapitulate key aspects of reciprocal endocrine signaling between embryonic and endometrial tissues, providing a tractable experimental framework to investigate embryo-endometrium crosstalk.

Subfertility and recurrent pregnancy loss (RPL) affect a significant proportion of couples worldwide. Genetic causes can be seen in up to 30% of these individuals but require multiple genetic tests, which often impede a comprehensive work up. Newer genomic technologies, such as PacBio HiFi long read sequencing (LRS) can detect most subclasses of variations (such as structural rearrangement, monogenic disorders) through one single test. In this multicenter study, we enrolled couples with unexplained subfertility and/or RPL and performed HiFi LRS to determine the underlying genetic etiology. Participants were recruited using a standardized inclusion/ exclusion criteria to rule out other known causes of subfertility and/or RPL. 96 individuals were recruited across the 5 sites. Average age of participants was 36 years (range 30-46 years). Among the 84 individuals who completed sequencing, 4.8% were identified with a likely genetic diagnosis and variants of uncertain significance were identified in another 14.2% of individuals. One individual was identified with an ACMG secondary finding, and while multiple carriers for recessive genetic disorders were identified, none of the couples were identified to be at increased risk. This study highlights the utility of performing genomic sequencing in couples with unexplained subfertility and/or RPL, with 1 in 10 couples harboring a clinically significant variant. In addition, use of HiFi LRS allowed for characterization of different subclasses of genomic variations through a single test. Future studies, including exploring the cost effectiveness and resource utilization of LRS as first line test, will help in optimizing care for such couples. TWEETABLE STATEMENTA single long-read genome sequencing test can consolidate multiple genetic investigations and uncover clinically relevant causes in couples with unexplained subfertility and recurrent pregnancy loss. AT A GLANCEO_LIWhy was this study conducted? O_LIMany couples with subfertility and recurrent pregnancy loss remain undiagnosed after multiple conventional genetic tests C_LIO_LIExisting workflows require sequential testing and may miss complex genomic variants C_LI C_LIO_LIWhat are the key findings? O_LILong-read genome sequencing identified clinically relevant variants in [~]1 in 10 couples with unexplained subfertility or recurrent pregnancy loss C_LIO_LIA single assay enabled detection of multiple variant types, including structural and sequence variants C_LI C_LIO_LIWhat does this study add to what is already known? O_LIDemonstrates feasibility of a unified genomic testing approach in a real-world multicenter cohort C_LIO_LISupports a potential shift from fragmented testing toward a single comprehensive genomic workflow C_LI C_LI