Human Reproduction

STUDY QUESTION Is genetic liability to endometriosis associated with iron homeostasis, and is this relationship potentially causal? SUMMARY ANSWER Genetic evidence indicates that reduced systemic iron status is associated with increased risk of endometriosis, with evidence of 8 shared genome-wide significant loci and suggestive but inconsistent evidence for causal bidirectional effects. WHAT IS KNOWN ALREADY Endometriosis is a chronic inflammatory condition associated with local iron accumulation within ectopic lesions and peritoneal cavity, resulting from retrograde menstruation and altered iron homeostasis. Epidemiological studies have suggested that women with endometriosis may exhibit reduced systemic iron stores compared to women without endometriosis, reflected by lower circulating ferritin concentrations, although findings have been inconsistent and may be confounded by menstrual blood loss and inflammation. As observational studies cannot distinguish causal relationships from secondary effects or residual confounding, the potential genetic basis linking iron homeostasis and endometriosis risk remains unclear. STUDY DESIGN, SIZE, DURATION We performed genetic analyses using summary statistics from large-scale genome-wide association studies (GWAS) of endometriosis (overall and stage III/IV disease) and five iron biomarkers (serum iron, ferritin, total iron-binding capacity (TIBC), transferrin saturation, and hepcidin). Analyses included genome-wide genetic correlation using linkage disequilibrium score regression (LDSC), identification of shared genetic variants using multi-trait GWAS (MTAG) and bidirectional Mendelian randomisation to evaluate potential causal relationships. PARTICIPANTS/MATERIALS, SETTING, METHODS Iron biomarker summary statistics came from a six-cohort GWAS meta-analysis (HUNT, MGI, SardiNIA, deCODE, Interval, DBDS; N up to 257,953) of blood-derived serum iron, ferritin, transferrin saturation and TIBC (Moksnes et al., 2022). Endometriosis summary statistics came from a 24-study GWAS meta-analysis (60,674 cases, 701,926 controls; European and East Asian ancestry), 12 of which had surgically confirmed cases (Rahmioglu et al., 2023). Genome-wide genetic correlations between iron biomarkers and endometriosis (overall and stage III/IV disease) were estimated using linkage disequilibrium score regression (LDSC), based on summary statistics aligned to the GRCh37 reference genome and restricted to HapMap3 variants. Multi-trait GWAS (MTAG) was applied to each iron biomarker jointly with endometriosis to enhance discovery of genetic loci. Shared loci were functionally annotated using reproductive and iron related tissues from GTEx v8 and blood from eQTLGen expression quantitative trait loci (eQTL) data. Bidirectional Mendelian randomisation (MR) analyses were performed using genome-wide significant variants across multiple clumping thresholds, with inverse-variance weighting (IVW) as the primary method and sensitivity analyses including weighted median, MR-Egger and MR-PRESSO. MAIN RESULTS AND THE ROLE OF CHANCE Genetic correlation analyses suggested that a genetic predisposition to endometriosis is associated with a profile of lower systemic iron availability. Specifically, genetic liability to endometriosis was associated with higher total iron-binding capacity (TIBC; rg=0.16, p=4x10-4), together with lower transferrin saturation (rg=-0.16, p=0.006) and lower ferritin levels (rg=-0.10, p=0.022), findings that are consistent with reduced iron stores. MTAG identified eight additional genome-wide significant loci for endometriosis and eight loci shared with iron biomarkers, including regions implicating coagulation (F5), reproductive biology (WNT4), and immune and vascular pathways (e.g. ABO, STAT6). Mendelian randomisation analyses provided limited and inconsistent evidence for a causal relationship between iron status and endometriosis. Although the inverse-variance weighted (IVW) model showed nominal associations between higher ferritin levels and a lower risk of endometriosis (OR = 0.85, 95% CI 0.76-0.94; p = 0.002), and between genetic liability to endometriosis and higher TIBC (OR = 1.02, 95% CI 1.01-1.04; p = 0.006), these findings were not consistently supported by sensitivity analyses. MR-PRESSO identified a small number of pleiotropic variants, but their removal did not materially alter the results. LIMITATIONS, REASONS FOR CAUTION Iron biomarker GWAS included males and females, potentially obscuring female-specific effects. Dataset availability restricted analyses to European ancestry, limiting applicability to other populations, and to overall and stage III/IV endometriosis, precluding assessment of other disease subtypes. Heterogeneity across SNP instruments, reflected by Cochran's Q statistics, reduced the precision of Mendelian randomisation estimates. Moreover, the genetic instruments explained only between approximately 1.0% and 18.8% of variance in the iron biomarkers, depending on the clumping threshold, which may have limited power to detect causal effects. WIDER IMPLICATIONS OF THE FINDINGS These findings suggest that endometriosis is genetically associated with reduced systemic iron availability and altered iron homeostasis. Thus, lower systemic iron status observed in women with endometriosis may not be explained solely by menstrual blood loss or dietary factors, but reflect an underlying genetic predisposition. Shared genetic loci implicate coagulation, ABO biology, and immune pathways as potential mechanisms linking iron metabolism and endometriosis. Although Mendelian randomisation did not provide consistent evidence for causality, these findings support a shared genetic architecture and warrant further investigation using female-specific GWAS, refined disease subtypes, and multi-omic approaches. Clinically, these findings suggest that low systemic iron status in women with endometriosis may reflect factors beyond established causes of iron deficiency, including an underlying genetic predisposition.

Menstrual symptoms vary across the cycle, yet most research assumes a normative 28-day cycle with fixed phase durations, obscuring the physiological relevance of natural cycle variation. Using the mcPHASES dataset, we characterised cycle and phase length variation across 96 menstrual cycles from 37 participants, with ovulation timing estimated from daily urinary luteinizing hormone measurements using a Bayesian hierarchical model, and examined associations with daily symptoms in a subset of 64 cycles from 35 participants with complete symptom data. Twelve physical, mental, and behavioural symptom domains were modelled using Bayesian ordinal regression, with posterior uncertainty in phase-length predictors propagated via a measurement error framework. Total cycle length was not associated with daily symptom burden, except sleep disturbances. By contrast, phase length decomposition revealed systematic associations across multiple domains: longer menstrual phase length was broadly associated with greater symptom intensity spanning physical, gastrointestinal, affective, and sleep domains; longer luteal phase duration was associated with greater fatigue and more frequent headaches, but lower sore breast intensity and lower stress; and longer follicular phase duration and later ovulation were each associated with greater sore breast intensity and more frequent mood swings. These associations require knowledge of actual ovulation timing and cannot be recovered from cycle length alone, indicating that the common assumption of a fixed 14-day luteal phase introduces systematic misclassification of hormonal exposure. Daily symptom intensity was also predominantly person-specific, with cycle phase explaining little of the between-person variance across most symptoms. These findings indicate that calendar-based phase assignment is insufficient for research and clinical assessment of hormone-sensitive conditions, and that person-specific baselines, rather than population-level phase averages, are needed for clinically meaningful symptom monitoring.

Background: Inequalities exist for many health outcomes, but there is limited evidence regarding menstrual symptoms despite their importance for health and wellbeing. We aimed to investigate inequalities in menstrual symptoms according to socioeconomic position and childhood adversity. Methods: In two generations (G0 mothers and G1 offspring) from the Avon Longitudinal Study of Parents and Children (ALSPAC), a UK prospective cohort study, we examined associations of multiple indicators of socioeconomic position (SEP) and adverse childhood experiences (ACEs) with menstrual symptoms (pain, abnormal uterine bleeding, and premenstrual syndrome (PMS) measured 3-8-years post-birth in G0 and 17-21-years-old in G1), using multivariable logistic regression. Samples ranged from 4,828 to 9,335 G0 participants and 1,288 to 2,757 G1 participants depending on the exposure-outcome association. Missing data were addressed using multiple imputation and inverse probability weighting. Results: Financial difficulties were associated with greater odds of menstrual pain (G1 OR 1.41; 95% CI 1.07, 1.86: G0 OR 1.55; 95% CI 1.36, 1.76) and irregular cycles (G1 OR 1.60; 95% CI 1.12, 2.29: G0 OR 1.48; 95% CI 1.27, 1.72) in both generations, as well as with short/long cycle lengths in G0 only. Lower education and manual social class were also associated with these three menstrual symptoms in at least one generation. Conversely, higher SEP was associated with PMS in both generations. Higher cumulative ACEs were consistently associated with menstrual pain (4+ compared to none: G1 OR 2.15; 95% CI 1.48, 3.11: G0 OR 1.52; 95% CI 1.29, 1.80) and irregular cycles (G1 OR 1.92; 95% CI 1.20, 3.09: G0 OR 1.54; 95% CI 1.26, 1.87) but not cycle length. Lower parental education, financial difficulties, and cumulative ACEs were associated with heavy bleeding in G1 offspring only, whereas financial difficulties, own manual social class, and cumulative ACEs were associated with prolonged bleeding in G0 mothers only. Higher cumulative ACEs were also associated with PMS in G1 offspring only. Conclusions: We found evidence of inequalities according to socioeconomic disadvantage and childhood adversity for multiple menstrual symptoms, although some associations were only observed in one generation. Findings suggest that menstrual symptoms are disproportionately experienced by socially and socioeconomically disadvantaged women.

Objective: To perform internal and external validation of a gradient-boosted decision tree (GBDT) fusion model that integrates zygote morphokinetic parameters with conventional embryo assessment features for blastocyst prediction, and to compare its discriminative performance against senior embryologists. Methods: This retrospective cohort study included 631 normally fertilized zygotes from 218 treatment cycles. A GBDT fusion model integrating 84 zygote morphokinetic parameters and 8 conventional assessment features was evaluated internally (5-fold cross-validation) and externally on a public dataset of 523 embryos with blastocyst outcomes. Model performance was assessed using area under the ROC curve (AUC), area under the precision-recall curve (AUPRC), F1 score, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Discrimination was compared with embryologist consensus using the DeLong test; agreement was assessed with Cohen's kappa. Results: The model achieved an internal AUC of 0.78 (95% CI 0.74-0.82), AUPRC 0.72, F1 0.73, sensitivity 0.74, specificity 0.77, PPV 0.72, and NPV 0.79. External validation on the public dataset demonstrated acceptable generalizability (AUC 0.76, 95% CI 0.71-0.81). The model significantly outperformed embryologist consensus (AUC 0.70, P<0.001) with moderate agreement (kappa=0.56). Decision curve analysis confirmed clinical net benefit at threshold probabilities of 0.15-0.55. Conclusions: The GBDT fusion model integrating zygote morphokinetics with conventional assessment demonstrates good discrimination and external generalizability for blastocyst prediction, providing an interpretable decision-support tool for embryo selection in IVF practice.

Background: Early access to pregnancy assessment units now detects many tubal ectopic pregnancies (TEP) at a stage when they could resolve spontaneously, creating a management dilemma. Methods: We performed a hypothesis-generating exploratory analysis in a retrospective study to assess whether serum progesterone (P4) levels in women with TEP are associated with management outcome. Results: Ninety-one cases of TEP managed in a single centre over three years were analysed. Receiver operating characteristic (ROC) curve analysis was used to explore serum levels of progesterone (P4), first human chorionic gonadotropin (hCG) and peak hCG (alone and in combination) in relation with successful completion of expectant management. Decision-tree analysis using first hCG and P4 was additionally performed to explore clinical sequential risk stratification. 23% (n=21) successfully completed expectant management. P4 concentrations in the expectant management group (median 3 nmol/L, IQR 2.00 to 8.50) were significantly lower than in those requiring surgical or medical management (median 17 nmol/L, IQR 5.75 to 29.25; p=0.0002). Area under the ROC curve (AUC) values for P4, log10 first hCG, log10 peak hCG and P4 with log10 first hCG were 0.766, 0.814, 0.811 and 0.835, respectively, for predicting successful expectant management. However, hCG was not significantly outperformed. Nonetheless, Youden optimised thresholds for hCG and P4 are reported, alongside decision-tree analysis that identified sequential first hCG and P4 thresholds associated with successful expectant management. Conclusion: Lower P4 levels are associated with successful expectant management of TEP but they do not outperform hCG either alone or as an adjunctive marker.

Background: Endometriosis is a chronic pain condition in which hormonal therapies form the cornerstone of long-term management. Treatment tolerability is critical for adherence and therapeutic success, but most comparative studies and reviews have focused on their ability to reduce menstrual pain, while their impact on non-menstrual pelvic pain (NMPP), bleeding patterns, adverse events (AEs), treatment discontinuation and quality of life (QoL) remain poorly characterised. This systematic review and meta-analysis evaluate these outcomes across currently available hormonal therapies, providing practical evidence for clinical decision-making. Methods: PubMed/MEDLINE, Scopus, and Embase were searched up to November 2025 for randomised controlled trials comparing at least two active first- or second-line hormonal treatments for endometriosis. Studies without confirmed endometriosis, treatment duration less than three months and comparing therapies to placebo only were excluded. Data were extracted by two reviewers from reports. Pain outcomes were pooled as mean differences (MD, 95% CI), with bleeding patterns, AEs, and discontinuations as proportions. Analyses were performed in R. PROSPERO: CRD420251137785. Findings: Of 1892 records screened, 48 trials (5583 women) met our inclusion criteria. Overall pelvic pain (0-10 scale) was significantly reduced across all treatment categories (p<0.001): combined oral contraceptives (COCs) (MD 3.17), oral and long-acting progestogens (MD 3.83; MD 4.29), and GnRH-analogues (MD 3.81). Sensitivity analyses restricted to studies reporting NMPP yielded comparable results. GnRH-agonists showed the most favourable bleeding profile, followed by continuous COCs. However, all regimens reported class-specific AEs, including mood changes, nausea, headache, weight gain, and decreased libido (pooled proportions >10%). Overall discontinuation due to AEs was 7.7%, and vaginal bleeding was the leading cause. Heterogeneity across meta-analyses was high. Risk of bias (RoB2) was moderate to high. Interpretation: Given similar reductions in overall pelvic pain across hormonal therapies, treatment decisions should prioritise differences in bleeding profiles, therapy-specific AEs, and QoL. Funding: None.

Background and aimsAssisted reproductive technologies (ART), including in vitro fertilisation (IVF), account for over 10 million births worldwide. ART-conceived young offspring show altered cardiovascular phenotypes, including cardiac remodelling and raised blood pressure, but the mechanisms remain unclear. Mitochondrial disturbance during preimplantation development may link early ART exposure to later cardiac dysfunction. However, to our knowledge, no one has assessed mitochondrial function in adult offspring from IVF pregnancies. In this study, investigated the effects of IVF and embryo vitrification on blastocyst mitochondrial redox balance and metabolism, and determined whether these effects persisted into the adult heart. Methods and ResultsIGS-CD1 mouse blastocysts from naturally mated donors or IVF were transferred fresh or after vitrification-warming. IVF reduced blastocyst total, trophectoderm and inner cell mass cell number, while vitrification lowered the inner cell mass proportion and increased apoptosis. Both exposures depolarised mitochondrial membrane potential and depleted glutathione; reactive oxygen species rose with an interaction, being highest in vitrified IVF embryos. IVF reduced live birth rate and litter size. In the adult offspring, high-resolution respirometry of isolated mitochondria from left ventricle revealed reduced oxidative phosphorylation capacity with an increased H2O2 production, altered OXPHOS subunit abundance and reduced complex I, III and IV activities. ConclusionsIVF and vitrification impose distinct disturbance on preimplantation embryo redox states and bioenergetics, and this early disturbance is followed into adulthood with a reduced mitochondrial aerobic capacity and increased basal ROS production. These results have important implications for IVF practices and suggest that mitochondria may be permanently programmed by this procedure. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/744765v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1cd1bd7org.highwire.dtl.DTLVardef@ded6b8org.highwire.dtl.DTLVardef@1e2ddf7org.highwire.dtl.DTLVardef@15abc84_HPS_FORMAT_FIGEXP M_FIG C_FIG IVF and vitrification impose distinct and partly independent effects on the preimplantation embryo that persist into the adult offspring heart. At the blastocyst stage, IVF reduced cell number and vitrification altered lineage allocation, while both exposures lowered mitochondrial membrane potential ({Delta}{Psi}m) and glutathione (GSH) and raised reactive oxygen species (ROS); vitrification additionally increased apoptosis. After embryo transfer, IVF reduced live birth rate and litter size, whereas vitrification altered postnatal growth trajectory. In adult offspring, ventricular mitochondria, vitrification reduced OXPHOS capacity and IVF reduced LEAK respiration, while both exposures increased H2O2/ O2 flux, reduced respiratory chain enzyme activities and altered OXPHOS subunit abundance.

Background. In Europe, first-trimester combined screening with the Fetal Medicine Foundation (FMF) algorithm identifies women at increased risk of preeclampsia who may benefit from personalized aspirin prophylaxis. However, a substantial proportion of early-onset preeclampsia (EOPE) remains undetected at clinically acceptable specificity. Objective. To evaluate the first-trimester performance of MaiRa for early-onset preeclampsia (EOPE) risk stratification by benchmarking it against FMF screening in the same women, characterizing discordant patient-level classification profiles and exploring potential implementation strategies. Study Design. This secondary case-control analysis was nested within the prospective, multicentre PREMOM cohort [NCT04990141], which enrolled women with singleton pregnancies across 14 tertiary hospitals in Spain. First-trimester MaiRa and FMF risk estimates were evaluated in the same 126 pregnant women, comprising 99 uncomplicated controls and 27 EOPE cases, defined by disease onset before 34 weeks. Discrimination was compared using a stratified paired bootstrap analysis of the areas under the receiver-operating-characteristic curves. Performance was assessed at prespecified clinical thresholds, and detection rates were evaluated at fixed false-positive rates. Universal and contingent MaiRa implementation strategies were also evaluated. Results. MaiRa showed greater first-trimester discrimination for EOPE than FMF combined screening (AUC, 0.974 vs 0.900; P=.040) and consistently achieved higher detection rates across fixed false-positive rates. At false-positive rates of 5% and 10%, MaiRa detected 85.2% and 92.6% of EOPE cases, compared with 44.4% and 70.4% for FMF, respectively. Patient-level analysis demonstrated that MaiRa identified 12 of 27 EOPE cases (44.4%) classified as low risk by FMF; these pregnancies generally exhibited less abnormal conventional first-trimester profiles, including fewer maternal risk factors, lower mean arterial pressure and lower uterine artery pulsatility index, yet 8 of 12 (66.7%) subsequently developed severe EOPE. Exploratory implementation analyses showed that universal MaiRa screening achieved the highest EOPE detection, whereas a contingent strategy using FMF for triage and reflex MaiRa testing reduced molecular testing to 35.7% of pregnancies while maintaining 77.8% sensitivity and 97.0% specificity. Conclusion. MaiRa provided greater first-trimester discrimination for EOPE than conventional combined screening and detected additional pregnancies that later developed severe disease despite less abnormal conventional screening profiles. The findings suggest that maternal plasma cfRNA profiling captures biological alterations not fully reflected by combined first-trimester screening and support further prospective evaluation in an independent, unselected obstetric population. Key words: early-onset preeclampsia; first-trimester screening; cell-free RNA; liquid biopsy; Fetal Medicine Foundation algorithm; combined screening; risk stratification; aspirin prophylaxis.

The distribution of fat-soluble compounds (including antioxidants) in follicular fluid (FF) remains sparsely documented in relation to in vitro fertilization (IVF) outcomes and existing studies have reported diverging associations. This study aimed to describe plasma and FF concentrations of fat-soluble micronutrients in women undergoing IVF and to analyze their adjusted associations with ovarian function, embryo development and pregnancy outcomes. In 2021-2022, plasma and FF samples were collected from 82 women (first IVF cycle) at oocyte puncture, along with lifestyle data covering the three preceding months. Eleven compounds (two tocopherols, three xanthophylls, five carotenes and retinol) were quantified. All compounds were detected in both compartments (lowest in FF) except phytoene, undetectable in FF. Plasma and FF -tocopherol concentrations were positively associated with plasma estradiol levels before oocyte puncture (both p<0.01) while FF -carotene and lycopene were inversely associated with plasma progesterone concentrations (p=0.01 and 0.02, respectively). Plasma phytofluene and phytoene were positively associated with mature oocyte rate (p=0.03 and p=0.01, respectively), while FF retinol was negatively associated (p=0.03). Carotenes, tocopherols and retinol were inversely associated with later IVF outcomes: fertilization rate (p<0.001 for plasma g-tocopherol, 0.02 for FF retinol), top-quality embryo (p=0.02 for plasma phytofluene), biochemical pregnancy at day 7 post-embryo transfer (p=0.05 for plasma -tocopherol, 0.02 for plasma -carotene), clinical pregnancy (p=0.03 for plasma -tocopherol, 0.01 for plasma phytoene) and live birth (p=0.04 for plasma -tocopherol, 0.02 for plasma phytoene). Plasma and FF g-tocopherol were positively associated with embryo fragmentation (both p<0.05). Finally, among xanthophylls, only plasma {beta}-cryptoxanthin was positively associated with plasma progesterone concentrations (p=0.02). Our findings of heterogeneous associations between tocopherols, carotenes, retinol and IVF outcomes across the stages of IVF suggest a beneficial effect limited to early outcomes and support a complex and context-dependent role of these compounds in female reproduction. This manuscript has been submitted to PlosOne on August 19, 2026.

Background: Endometriosis affects approximately 10% of reproductive-age women and is associated with substantial diagnostic delay and heterogeneous symptom presentation. Prior machine-learning prediction models have relied on comorbidity data alone or on small candidate-variant genetic scores, with inconsistent or incompletely reported performance. No study has combined a well-powered, multi-ancestry polygenic risk score (PRS) with environmental, reproductive, and symptom data in a single hybrid model. We developed and evaluated hybrid risk-prediction models integrating a genome-wide, multi-ancestry PRS with clinical and symptom data for endometriosis in the US-based All of Us Research Program. Methods: Among 69,376 participants (15,382 endometriosis cases, 53,994 controls) across six genetically inferred ancestry groups, we computed individual-level PRS values using PRS-CS weights derived from an independent, multi-ancestry GWAS. Five nested logistic regression, random forest, and XGBoost models progressively added age, ancestry, and within-ancestry genetic principal components (Model 1), environmental and reproductive factors (Model 2), symptom and comorbidity indicators (Model 3), all covariates combined (Model 4), and PRS x environment interactions (Model 5). Performance was assessed by AUROC in a held-out test set and 5-fold cross-validation, with class-weighted, Youden-optimized thresholds used for sensitivity, specificity, and predictive values; permutation importance identified top contributors. Pairwise AUROC differences were tested with a Holm-corrected DeLong-type test. Results: Discrimination improved from AUROC 0.63 (PRS, age, ancestry, principal components) to 0.72 for the full model, driven mainly by symptom and comorbidity data. XGBoost consistently outperformed logistic regression and random forest. The PRS ranked among the top individual predictors by permutation importance in nearly every model, alongside age, while genetic and demographic information alone gave only modest discrimination, and PRS x environment interactions did not improve on environmental factors alone. Threshold optimization yielded balanced sensitivity and specificity (~0.67/0.65) versus near-zero sensitivity at a default threshold. Conclusions: Combining the PRS with symptom and comorbidity data gave the best discrimination compared to solely a well-powered, multi-ancestry PRS as a predictor of endometriosis. This study clarifies both the promise and current limits of hybrid genetic-clinical prediction for endometriosis and points to symptom-based phenotyping, molecular subtyping, and external validation as priorities.

In vitro fertilization (IVF) laboratories equipped with timelapse incubators generate vast quantities of sequential embryo images, yet the absence of standardized, annotated databases impedes the development of reproducible computational tools for embryo assessment. Here we describe the construction of a standardized time-lapse imaging database comprising 631 normally fertilized zygotes from 218 treatment cycles, integrating timelapse image sequences, patient clinical records, and embryo developmental outcomes. We further present a gradient boosting decision tree (GBDT) ensemble framework that integrates zygote morphokinetic parameters-continuous time-series features extracted via a validated CNN-based segmentation algorithm (US Patent US11210494B2)-with conventional embryo assessment grades (categorical features per the Istanbul consensus). The fusion framework employs equal-weight initialization followed by iterative residual-decreasing training to optimally combine heterogeneous feature types. Ablation analysis demonstrated that the integrated model achieved an AUC of 0.78, significantly outperforming morphokinetics-only (AUC 0.71) and conventional-only (AUC 0.65) models, confirming the complementary value of the two data modalities. The database and fusion framework provide a reproducible foundation for embryo development assessment and are generalizable to other multimodal data integration tasks in reproductive medicine.

Reproduction is one of the most fundamental biological processes in the human body, yet the molecules governing it remain incompletely understood. Here, we have characterized the role of PKHD1L1 and its globally relatively common splice donor variant rs17368310 in female fertility. We demonstrate estrogen-responsive expression of PKHD1L1 in the human endometrial and Fallopian tube epithelium, identify the change in the rs17368310 mRNA sequence in endometrial tissue, and assess the possible effects of the variant on the PKHD1L1 protein through structural modeling. We reveal that women homozygous for rs17368310 have a persistently lower child count compared to other genotypes not only among all women but also among women who have undergone medical treatments for infertility in the Finnish population. We further show that rs17368310 associates with female infertility-related traits also in the Mexican population. These findings elucidate the effects of rs17368310 on fertility in millions of reproductive-age women across different populations.

Direct experimental analysis of the mammalian oviduct is constrained by limited tissue access and the short lifespan of ex vivo preparations. Extracellular matrix-embedded three-dimensional epithelial organoids provide longer-term in vitro models. However, their inward-facing apical surface and the absence of supporting stromal cells limit physiological studies of the oviduct, including ciliary activity and maternal-embryonic interactions. Here, we provide a step-wise protocol detailing the generation of mouse and human oviductal assembloids in which epithelial cells form an outward-facing (apical-out) layer around a stromal core. Epithelial and stromal cells from adult mouse oviducts or human Fallopian tubes are isolated, expanded separately, and subsequently aggregated in a rotational culture system. The protocol also outlines morphological and immunostaining criteria for confirming cellular organization, whole-mount detection of external cilia, measurement of ciliary beat frequency, and co-culture of mouse assembloids with preimplantation embryos. Mouse and human assembloids retained epithelial and stromal identity and displayed cilia at the accessible outer surface. In a proof-of-concept experiment, embryos co-cultured with the assembloids developed to blastocysts at a rate similar to that of in vivo-derived blastocysts. This reductionist system provides a straightforward and tractable model to investigate oviduct physiology and embryo-maternal communication while allowing direct manipulation and observation of the epithelial interface. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/743297v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1917a7borg.highwire.dtl.DTLVardef@41d7org.highwire.dtl.DTLVardef@e2bf98org.highwire.dtl.DTLVardef@90c9f3_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryThe protocol for generating mouse and human oviductal assembloids by combining epithelial and stromal cells for studying oviductal function in an in vitro setting.

Background Oral contraceptive (OC) use, particularly during adolescence, may increase depression risk in some individuals, but it remains unclear who is susceptible to mood-related side effects and who is not. We aimed to detect single nucleotide polymorphisms (SNPs) and genes that moderate the effect of OC use on depression in young adulthood using data from the UK Biobank. Methods N=202,243 participants were followed from birth to age 23.29 (SD: 2.58) years. We used Cox models for counting processes to test the association between OC use and incident depression in young adulthood, and conducted a genome-wide-by-drug-interaction study (GWDIS) of SNP by OC use interactions alongside a standard genome-wide association study (GWAS) on incident depression in young adulthood. Results Over half of all participants (57.5%) initiated OC and 1.0% received a depression diagnosis during follow up. OC initiators had a 20% higher hazard of incident depression than non-initiators (HR=1.20, 95% CI=1.04-1.37). No SNPs reached genome-wide significance in the GWDIS, though eight showed suggestive interaction signals (p<1e-5). At the gene level, FSIP1 (p=5.90e-5) and EHBP1 (p=6.47e-5) showed suggestive signals, but none passed the genome-wide threshold. No SNPs reached genome-wide significance in the GWAS. Conclusions We did not find evidence for genetic variants that moderate the association between OC initiation and depression. If such effects exist, they are likely to be small and polygenic, suggesting there is currently no solid basis for using genetic data for individualised contraception counselling concerning mood-based side effects.

Healthy ovarian follicle development and ovulation require coordinated communication between oocytes and surrounding somatic cells. Although oocyte-secreted factors (OSFs), such as GDF-9 and BMP-15, are established regulators of this communication, the non-OSF maternal factors that support OSF delivery and signaling during late-stage follicle development remain poorly understood. Here, using an oocyte-specific Dhx36 knockout mouse model, we identify the G-quadruplex (G4) helicase DHX36 as a non-OSF maternal factor required for antral follicle development and hormone-induced ovulation. Dhx36 deficiency caused severe defects in granulosa cell proliferation and cumulus expansion, accompanied by impaired activation of SMAD2/3 and SMAD1/5/8, while ERK1/2 activation remained intact. Although the expression of major OSFs was largely unchanged, Dhx36-deficient oocytes exhibited disrupted microvilli and transzonal projections (TZPs), resulting in defective OSF delivery and impaired oocyte-cumulus communication. Proteomic, lipidomic, and ultrastructural analyses further revealed dysregulated phospholipid metabolism, membrane organization, autophagy, and organelle homeostasis, including abnormal lysosomal, mitochondrial, and endoplasmic reticulum structures. Integrative transcriptomic and proteomic analyses identified concordant downregulation of genes involved in these processes, whose promoters were enriched in potential G4 motifs. Consistently, Dhx36 deficiency was associated with reduced RNA polymerase II activity, while pharmacological G4 stabilization impaired transcription of selected genes. Together, these findings establish DHX36 as a maternal regulator that links oocyte intrinsic homeostasis to intercellular communication, suggesting that DHX36-dependent maintenance of membrane and organelle integrity is essential for OSF delivery, cumulus cell function, antral follicle development, and ovulation.

Background. DNA methylation patterns in cord blood are robustly associated with birthweight in the general population. However, it remains unknown whether these associations extend to clinically relevant populations, such as preterm neonates or those born small for gestational age, and whether they directly reflect birthweight or are driven indirectly by genetic, familial, maternal, and obstetric factors. Methods. We calculated a birthweight methylation profile score (MPSBW) using weights of 835 CpGs previously associated with birthweight in the general population and evaluated its association with birthweight in 67 monochorionic (MC) twin pairs including 134 neonates (97% born preterm) from the Twinlife study. MC twin pairs are identical twins sharing a single placenta, often unequally, which can result in unequal resource distribution and differential fetal growth. Results. We examined the association between within-pair differences in birthweight and MPSBW, thereby estimating the association independent of factors shared equally by co-twins. A 500-gram increase in birthweight was associated with a 0.256 SD increase in MPSBW (p<0.005) in this population of preterm neonates. Adjustment for polygenic score for birthweight (PGSBW) confirmed that the observed epigenetic associations were not driven by common genetic variation underlying birthweight. Interestingly, a similar effect size (0.226 SD per 500 g birthweight increase; p<0.05) was observed in the within-pair analysis, which controls for all shared influences within a twin pair. Conclusion DNA methylation is associated with individual differences in birthweight in a high-risk clinical population of MC twins, independent of shared genetic, familial or maternal influences.

Sperm motility and function are central to mammalian fertilization and are tightly regulated by intracellular calcium (CaCa2+) signaling. This signaling is primarily orchestrated by the CatSper Ca2+; channel complex located in the flagella of spermatozoa. However, the natural ligands that activate CatSper remain largely unknown in many species, despite the conservation of CatSper in mammals. Here, we present a signaling role for soluble N-terminal ZP2 fragments in regulating CatSper activity and sperm physiology in mice and humans. ZP2 has been implicated in mediating sperm binding and recognition at the oocyte surface interface; however, new evidence is starting to unveil the molecular mechanisms and function of ZP2 during fertilization transition. Here, we show that the during fertilization, cleaved ZP2 N-terminal fragment triggers a rapid and robust increase in intracellular CaCa2+ levels in sperm. This increase depends strictly on CatSper function, as demonstrated through pharmacological analysis and CatSper1 knockout mice. This calcium influx is sufficient to induce acrosomal exocytosis in a subset of human and mouse sperm. AlphaFold-based structural modeling suggests a potentially conserved extracellular interaction site between the soluble N-terminal ZP2 fragments and the CatSper complex. In human sperm, ZP2 treatment significantly modulates motility parameters, including flagellar movement and velocity, while inducing a CatSper-dependent increase in intracellular CaCa2+ similar in magnitude to that evoked by progesterone. Species-matched ZP2 stimulation elicits the stronger calcium response, underscoring evolutionary adaptations in ligand-channel protein pairs. Taken together, our findings reveal a conserved signaling pathway from ZP2 to CatSper that integrates oocyte-derived signals into the regulation of sperm motility and acrosomal exocytosis. This pathway provides new mechanistic insights into fertilization and highlights potential targets.

Objectives: Menopausal symptoms are heterogeneous and commonly assessed by questionnaires. We explored serum two-dimensional gel electrophoresis (2-DE) protein spots associated with menopausal symptom burden. Methods: This exploratory cross-sectional study included 27 women aged 45-55 years. A total of 550 matched serum 2-DE spots were quantified. A frequency-adjusted symptom burden score was calculated as the sum of severity x frequency products across 10 symptoms. Spots were screened using Spearman rank correlation with Benjamini-Hochberg false discovery rate (FDR) adjustment, followed by qualitative image review. Spots #285 and #636 were prioritized for vasomotor and psychological domain analyses. Results: The median age was 51.0 years; 13 participants were menstruating and 14 were amenorrheic. The median overall symptom burden score was 45.0 (interquartile range, 6.5-58.5) and was inversely correlated with spots #285 and #636. Spot #285 was inversely correlated with vasomotor symptom score, including inverse correlations in both menstrual-status groups. Spot #636 was inversely correlated with psychological symptom score overall, with a stronger descriptive correlation among menstruating participants. Neither candidate remained significant after FDR adjustment. Conclusions: Spots #285 and #636 are hypothesis-generating candidates requiring molecular identification, analytical validation, multiplicity-aware confirmation, and independent replication.

The major events of female germline establishment, from primordial germ cell (PGC) specification to assembly of primordial follicles, occur during embryonic/fetal development. This study presents a single-cell RNA-sequencing atlas of the bovine fetal ovary at four gestational timepoints: estimated day 50, and timed pregnancies at days 70, 90, and 120, capturing the progression of PGCs through commitment, meiotic entry, and early oocyte growth. Fourteen transcriptionally distinct cell populations were identified, including stromal, epithelial, endothelial, immune, somatic support cell, and germ cell lineages. Sub-clustering of the germ cell population resolved six developmental stages (PGCs, transitioning oogonia, proliferative oogonia, committed oogonia, meiotic prophase I oogonia, and oocytes), while that of the somatic support cell compartment revealed five granulosa cell subtypes (steroidogenic, pre-granulosa 1, pre-granulosa 2, pre-granulosa 3, and epithelial cells). Trajectory analysis reconstructed the developmental path of PGCs to oocytes, with sequential activation of meiotic and oocyte-specific gene programs. Representation of all six germ cell stages at day 120 pointed to asynchronous oogenesis in the fetal ovary, which was validated and shown to be region-specific by protein immunolocalization. Intercellular signaling networks between germ cells and the somatic niche were mapped, revealing strong interactions through BMP, KIT, IGF, IGFBP, WNT, and MDK pathways with temporal specificity across gestational ages. The bovine germ cell and pre-granulosa cell subtypes demonstrate significant transcriptional parallels with similarly-staged cells from human fetal ovaries, establishing the cow as a reliable model for human germ cell and ovarian development. Collectively, these data provide a developmental roadmap for bovine oogenesis at the single-cell resolution that advances fundamental understanding of gametogenesis and informs strategies for advanced assisted reproduction.

Importance The menopausal transition has been associated with an increased risk of depression, although findings are inconsistent. While most research has focused on menopausal stage, some studies suggest that later age at menopause may be associated with lower depression risk. Objective To examine the association between age at menopause and depression risk during perimenopause and early postmenopause using multivariable regression and genetic approaches. Design Prospective cohort study using data from the mothers of the Avon Longitudinal Study of Parents and Children (ALSPAC), a UK birth cohort that recruited pregnant women in 1991-1992. Setting UK community-based cohort study. Participants Up to 3,307 women with repeated measures of depressive symptoms across the perimenopausal and postmenopausal periods and data on observed or genetically predicted age at menopause. Exposure Observed age at menopause, a polygenic risk score (PRS) for age at menopause, and genetically predicted age at menopause. Main Outcome(s) and Measure(s) Depressive symptoms during the perimenopausal and early postmenopausal periods were assessed using the Edinburgh Postnatal Depression Scale (EPDS), with depression defined as a score >= 13. Results Effect estimates across multivariable regression and genetic analyses were small and directionally consistent with lower odds of depression with older age at menopause, although most confidence intervals included the null. In analyses using observed age at menopause, there was little evidence of an association with depression during perimenopause (Odds ratio (OR) per year increase in age at menopause 0.98, 95%CI 0.89-1.08) or postmenopause (OR 1.00, 95%CI 0.89-1.13). Results were similar when using a PRS as a genetic proxy for age at menopause during perimenopause (OR per standard deviation (SD) increase in PRS 0.98, 95%CI 0.89-1.09) but suggested lower odds of depression during postmenopause (OR 0.92, 95%CI 0.86-0.99). Mendelian randomization analyses did not support a causal effect (OR per year increase 1.00, 95%CI 0.89-1.13 for perimenopause, and OR 0.97, 95%CI 0.86-1.09 for postmenopause). Conclusions and Relevance Age at menopause is unlikely to be a major driver of midlife depression risk. However, consistent effect directions across approaches suggest a small association may exist, but further research in larger samples is needed to confirm this.