Human Reproduction

Study question: Are maternal age and a history of previous pregnancy losses associated with the timing of pregnancy loss for women undergoing ART treatment? Summary answer: Among women who underwent ART treatment, younger women had a higher risk of very early biochemical pregnancy losses. Women with previous pregnancy losses overall experienced pregnancy losses at an earlier gestational age. What is known already: Maternal age and history of pregnancy loss are two of the strongest, independent determinants of pregnancy loss risk. Study design, size, duration: This retrospective cohort study comprises all embryo transfers in Sweden between 2007 and 2022 (n = 202,008). The Swedish Quality Register for Assisted Reproduction (Q-IVF) provides information on confirmed pregnancies from two weeks after embryo transfer. Additional information was retrieved from the Swedish Medical Birth Register and the National Patient Register. Participants/materials, setting, methods: We studied 202,008 embryo transfers resulting in 80,673 biochemically verified pregnancies and 21,240 pregnancy losses. We assessed the associations between two exposures (maternal age and history of pregnancy loss) and four outcomes (implantation failure, pregnancy loss, the proportion of pregnancy losses occurring within the biochemical pregnancy window, and the gestational duration when the pregnancy loss was diagnosed). Fixed and mixed linear and logistic regression models were used, the latter to account for unmeasured confounding shared across pregnancies from the same mothers. Through analyses of subgroups including oocyte recipients (n = 3,558) and women undergoing treatment due to male infertility (n = 41,721), we attempted to tease out the effects of potential unmeasured confounders, such as maternal oocyte aneuploidies. Main results and the role of chance: Mean maternal age was 34 years, 23 % had a history of pregnancy loss, implantation failure occurred after 60 % of embryo transfers and pregnancy loss occurred in 26 % after successful implantation. Higher maternal age was associated with increased risk of implantation failure (OR = 1.02 per year; 95% CI = 1.02, 1.02), and pregnancy loss after successful implantation (OR = 1.05 per year; 95% CI = 1.05, 1.06). We observed no relationship between maternal age and the risk of implantation failure or pregnancy loss among women undergoing ART with donated oocytes. Among those who experienced pregnancy loss, the proportion of pregnancy losses within the biochemical window decreased with increasing maternal age (OR = 0.94 per year; 95% CI = 0.93, 0.94), a result consistent across all subgroups and regardless of oocyte origin. The number of previous pregnancy losses was associated with a lower risk of implantation failure (OR = 0.82; 95% CI = 0.81, 0.84), but not with pregnancy loss in the index pregnancy after accounting for within-women correlations using mixed-effects models (OR = 1.02; 95% CI= 0.99, 1.04). Limitations, reasons for caution: Since all women studied underwent ART treatment, the findings may not apply to naturally conceived pregnancies. The gestational duration of pregnancy losses reflects the time at diagnosis, and thus is an upper bound for the pregnancy loss. Wider implications of the findings: This study confirms (oocyte) maternal age and history of pregnancy loss as influential in risk of implantation failure and pregnancy loss, and suggests that maternal age may delay the timing of the pregnancy loss, particularly among pregnancy losses occurring within the biochemical pregnancy window.

Capacitation is a post-ejaculatory maturation process by which sperm become hyperactive and undergo the acrosome reaction, both of which are required to fertilize an oocyte. Two key features of capacitation are increased intracellular calcium via the calcium channel CatSper and changes in mitochondrial function. In mouse spermatozoa, CatSper-dependent calcium entry promotes mitochondrial calcium uptake and mitochondrial activation, contributing to hyperactivated motility and fertilization ability. Whether this mechanism is conserved in human spermatozoa was unknown. Here, we assessed mitochondrial function and mitochondrial calcium in paired non-capacitated and capacitated human spermatozoa from normozoospermic donors. Capacitation increased mitochondrial respiratory control ratio, coupling efficiency, mitochondrial membrane potential, and mitochondrial calcium. The increase in mitochondrial calcium required extracellular calcium and was prevented by pharmacological inhibition of CatSper or the mitochondrial calcium uniporter (MCU). Inhibition of CatSper or mitochondrial calcium uniporter also attenuated the capacitation-associated increase in mitochondrial membrane potential and reduced hyperactivated motility without affecting progressive or total motility. Pharmacological disruption of mitochondrial function reduced capacitation-associated hyperactivated motility without affecting the acrosome reaction. Together, these findings support a conserved pathway in human and mouse spermatozoa in which CatSper-mediated calcium entry promotes MCU-dependent mitochondrial calcium uptake and mitochondrial activation, contributing to the acquisition of hyperactivated motility during capacitation. These findings identify mitochondrial calcium handling as a potential target for modulating human spermatozoa function.

Objective: To study the prevalence of co-occurring endometriosis and polyendocrine metabolic ovarian syndrome (PMOS), estimate infertility odds, and characterize menstrual symptom profiles associated with this combined phenotype in a population cohort. Design: Prospective digital cohort in the United States (US). Subjects: 90,714 consented participants enrolled between November 2019 and June 2025 who completed relevant surveys; a subset of 25,152 participants with menstrual cycle logs linked to monthly surveys contributed to symptom analyses. Exposure: Participants were classified by self-reported physician diagnosis into four groups: endometriosis alone, PMOS alone, co-occurring endometriosis and PMOS (endometriosis+PMOS), or neither condition (reference group). Main Outcome Measures: (1) Odds of self-reported physician-diagnosed infertility (ever/never, cross-sectional), estimated using multivariable logistic regression adjusted for age, race/ethnicity, and subjective socioeconomic status; (2) number of adverse menstrual symptoms logged per cycle, assessed using negative binomial mixed-effects models adjusted for age, race/ethnicity, and hormonal contraceptive use. Results: Among 90,714 participants, the mean age was 35.1 years (standard deviation: 11.6) and the majority identified as non-Hispanic White (72%). Prevalence estimates were 11% for PMOS alone, 6% for endometriosis alone, and 2% for endometriosis+PMOS. Individuals with endometriosis+PMOS more frequently reported unemployment, low socioeconomic status, active smoking, poorer subjective health, early menarche, current irregular cycles, and a broad range of comorbidities. Infertility odds were highest for endometriosis+PMOS (odds ratio: 12.49 [95% confidence interval: 11.13-13.99]), followed by PMOS alone (6.12 [5.69-6.59]) and endometriosis alone (4.31 [3.93-4.72]), with the combined effect exceeding additive expectations (relative excess risk due to interaction: 3.05 [1.65-4.46]). Individuals with endometriosis+PMOS logged 11% more symptoms per cycle than those with neither condition (incidence rate ratio: 1.11 [1.04-1.19]) and had the highest odds of logging pelvic pain, fatigue, and sleep changes, among other symptoms. Conclusion: In this large US digital cohort, co-occurring endometriosis and PMOS affected 1 in 50 participants and was associated with markedly elevated infertility odds exceeding additive expectations, and a heightened menstrual symptom burden. Several risk factors and health impacts characteristic of each condition appeared amplified in individuals with both conditions, suggesting a subgroup with compounded clinical complexity and greater care needs.

Embryo implantation remains a major limitation of assisted reproductive technology, with failure occurring in approximately 30% of euploid embryo transfers. Implantation requires a synchronized dialogue between the blastocyst and receptive endometrium during the window of implantation (WOI), yet minimally invasive approaches to characterize the structural and molecular features of receptivity remain limited. We analyzed paired sonohysterogram images and uterine lavage samples collected during the proliferative and mid-secretory phases from subjects with regular ovulatory cycles and proven fertility. Endometrial folds were quantified, and lavage samples were analyzed by Luminex multiplex immunoassay. Folds were present in both phases but were significantly more abundant during the mid-secretory WOI, independent of imaging view and endometrial thickness. Folding correlated strongly with circulating estradiol level during the proliferative phase but not the mid-secretory phase, and folding patterns between phases were not correlated, suggesting distinct regulatory mechanisms. Consistent with these structural patterns, uterine lavage demonstrated phase-specific differences in expression of factors associated with endometrial receptivity and implantation, with glandular epithelium, and myeloid-lineage cells emerging as major contributors. Together, these findings identify coordinated structural and secretory processes during the WOI and support further evaluation of endometrial folding and uterine lavage as complementary, minimally invasive markers of endometrial receptivity.

Study question. Are endometrial gene expression profiles reproducible across hormone replacement therapy (HRT) cycles and across different regions of the same endometrial biopsy? Summary answer. Endometrial transcriptomic profiles were highly reproducible across HRT cycles and across non-adjacent and adjacent regions of the same biopsy. What is known already. Endometrial transcriptomic analyses are increasingly used to understand endometrial physiology and to evaluate endometrial reproductive potential. However, this approach requires an endometrial biopsy in a prior endometrial cycle to the embryo transfer cycle in the IVF treatment. Therefore, endometrial transcriptomic analyses rely on the assumption that the gene expression profile is representative of subsequent endometrial cycles and of the whole biopsy specimen. However, inter-cycle and intra-biopsy reproducibility remain insufficiently characterized. Study design, size, duration. This was a prospective, single-center, observational clinical study including 20 participants recruited between June 2024 and March 2025. Participants/materials, setting, methods. Participants underwent two different endometrial preparation cycles under the same HRT conditions at a private fertility clinic, and an endometrial biopsy was collected during both cycles, with the same duration of progesterone exposure, in the mid-secretory phase by the same clinician. RNA was processed for whole endometrial transcriptome analysis using RNA sequencing. Three paired comparisons were performed: biopsies from the same participant collected in different cycles, non-adjacent regions of the same biopsy, and adjacent regions of the same biopsy. Gene expression reproducibility was assessed using Pearson correlation, hierarchical clustering, principal component analysis, and paired differential gene expression analysis. The reproducibility of estimated cell-type proportions was also assessed by deconvolution to observe potential differences in cellular composition. Main results and the role of chance. In the inter-cycle endometrial gene expression comparison, the mean {+/-} standard deviation (SD) gene expression correlation coefficient within participant pairs was 0.974 {+/-} 0.012, significantly higher than that observed between participant pairs (p = 1.56 x 10-6), the mean {+/-} SD Euclidean distance was significantly lower within than between participant pairs (p = 2.24 x 10-6), and no differentially expressed genes were identified within participant pairs. Principal component analysis and hierarchical clustering further supported the similarity of most paired samples. Intra-biopsy comparisons showed even higher reproducibility, with both non-adjacent and adjacent biopsy regions showing mean {+/-} SD correlation coefficients of 0.986 {+/-} 0.003 and 0.986 {+/-} 0.002, respectively. The correlation coefficients were significantly higher than those observed between biopsy pairs (p = 9.91 x 10-12 and p = 2.14 x 10-12, respectively). No differentially expressed genes were detected within intra-biopsy pairs, all paired regions clustered together by hierarchical clustering and principal component analysis, and intra-biopsy pairs showed lower Euclidean distances than inter-biopsy pairs (p = 9.91 x 10-12 and p = 2.14 x 10-12 in non-adjacent and adjacent regions, respectively). Estimated cell-type proportions did not differ significantly between paired samples in any of the three comparisons. Limitations, reasons for caution. The findings are specific to samples analyzed using the AmpliSeq Transcriptome Human Gene Expression Panel protocol. Although other transcriptomic platforms should be tested, we are expecting the same reproducibility due to the consistency of RNA seq platforms. All biopsies were collected by the same clinician, so operator-related variability could not be directly assessed. These findings can be extrapolated only to HRT cycles. Wider implications of the findings. These findings support the reproducibility of endometrial gene expression profiles under controlled HRT conditions from biopsies collected by the same clinician and suggest that a single biopsy region can provide a representative molecular profile of the whole biopsy specimen. These findings strengthen the methodological basis for bulk endometrial RNA sequencing studies and support the reliability of transcriptomic-based tools for endometrial evaluation in an endometrial cycle prior to an embryo transfer cycle for reproductive medicine.

Infertility affects one in six couples worldwide and has a significant genetic component. Genome sequencing efforts have identified increasing numbers of potentially pathogenic variants, including in conserved meiotic genes. However, the lack of functional validation remains a major barrier to establishing variant pathogenicity and clinical diagnoses. Here, we establish an experimental pipeline integrating C. elegans and mouse in vivo models with in silico protein structure predictions to functionally validate potentially pathogenic infertility-associated variants. Using CRISPR-Cas9 we generated worm and mouse models of a homozygous missense variant (P638L) in MSH4, a mismatch repair protein essential for meiotic recombination, identified in a patient with non-obstructive azoospermia (NOA). Homozygous msh-4P546L worms exhibited severe meiotic defects including an absence of chiasmata, accumulation of DNA damage, and loss of crossover markers. Comparable defects were observed in spermatocytes of homozygous Msh4P660L/P660L mice, while Msh4P660L/P660L ovaries lacked oocyte-containing follicles, demonstrating that this variant causes infertility in both sexes. Together, these findings support a model in which the MSH4 P638L substitution disrupts the conversion of early recombination events into crossovers, leading to NOA in men and primary ovarian insufficiency in women. Further worm modelling of human MSH4 genetic variants identified exclusively in heterozygosity (G649R) or also in homozygosity (Y589C) demonstrates the potential of worm models to predict the pathogenicity of missense variants in meiotic proteins found in the human population. We propose that this integrated worm-mouse pipeline provides a powerful framework for accelerating the genetic diagnosis of infertility and informing the clinical management of affected individuals.

Per- and polyfluoroalkyl substances (PFAS) and phthalate metabolites are understudied in follicular fluid of women undergoing assisted reproduction. We characterized these in previously collected follicular fluid from patients at a Nebraska fertility clinic (August 2022 to June 2025) and retrieved demographics and clinical characteristics from fertility records. Spearman correlations and nonparametric tests evaluated PFAS and phthalate correlations and exposure differences by age, race, BMI, and urbanicity. Seven PFAS and four phthalate metabolites were detected in at least half of participants. PFOS and PFHxS concentrations were higher in younger women, and mono-isobutyl phthalate (MiBP) was higher in younger and non-White women. PFAS and phthalate correlations were weak, and exposures did not differ by urbanicity, indicating multiple chemical exposures in follicular fluid with sociodemographic disparities.

BACKGROUND Twin pregnancies have a two- to three-fold higher risk of preeclampsia (PE), but early prediction remains poor because current biomarkers and prediction models are largely derived from singleton pregnancies. Defining the biological changes that precede PE in twins may facilitate the development of more effective risk assessment approaches. OBJECTIVE We aimed to characterize maternal plasma protein changes preceding PE in twin pregnancies and to explore whether early circulating proteins could serve as improved risk stratification for twin PE. STUDY DESIGN We performed a nested case-control study within a prospective twin pregnancy cohort at Peking University Third Hospital. A total of 84 women with twin pregnancies were included, comprising 40 pregnancies complicated by PE and 44 normotensive controls. Longitudinal maternal plasma samples were collected in the first, second, and third trimesters. In the discovery cohort, maternal plasma proteins were profiled by mass spectrometry to identify trimester-specific protein changes associated with PE. Candidate proteins were then validated in an independent validation set. We further assessed the functional relevance of selected proteins in trophoblast and endothelial cell models and constructed prediction models integrating biomarkers with maternal clinical characteristics. RESULTS Twin pregnancies complicated by PE showed distinct trimester-dependent protein alterations, with first- and second-trimester changes mainly involving immune regulation and glycolipid metabolism, and later changes enriched in cardiovascular-related pathways. Several proteins demonstrated early discriminatory patterns, including increased annexin A3, chitotriosidase-1, and ferritin light chain (FTL), together with decreased annexin A2 in the first trimester, whereas the conventional singleton markers (sFlt-1 and PAPP-A) did not differ. ELISA validation of these candidate biomarkers yielded an AUC of 0.93 for twin preeclampsia prediction; integration with standard maternal clinical factors further improved discriminatory performance to an AUC of 0.97. Accordingly, a multivariable model combining annexin A2, annexin A3, chitotriosidase-1, FTL, and maternal clinical factors achieved strong internal performance for early discrimination of PE in twin pregnancies (AUC, 0.97; 95% CI, 0.93-1.00). Moreover, annexin A2 knockdown impaired trophoblast migration and invasion and reduced inflammatory cytokine secretion in endothelial cells, supporting a role in placental development and maternal vascular adaptation. CONCLUSION Twin pregnancies that later develop PE exhibit early and progressive maternal plasma protein alterations that can be detected before clinical disease onset. These findings identify candidate biomarkers and biologically relevant pathways that may help explain why PE in twins differs from singleton disease and may support future twin-specific risk stratification strategies. These results require external validation before clinical application.

Background: Heavy menstrual bleeding (HMB) and menstrual pain (MP) are among the most common menstrual cycle related symptoms with substantial impacts on quality of life. Despite this, population-level evidence on their prevalence across the life course is scarce. Methods: We conducted descriptive analyses of six longitudinal population-based cohorts from the United Kingdom, United States, Norway, and Australia. We estimated cumulative prevalence (across all available timepoints) and age-specific point prevalence of HMB and MP. Cumulative prevalence estimates were stratified by sociodemographic factors, where data permitted. We also examined HMB and MP incidence in adolescent cohorts with repeated measurements. Findings: HMB and MP are commonly reported, with cumulative prevalence across all the cohorts and timepoints (i.e., ever reported) ranging from 36% to 89% for HMB and 46% to 72% for MP. In adolescence, MP (point-prevalence: 33% to 47%) is more commonly reported than HMB (16% to 24%), whereas in later ages (45 years and above), HMB (47% to 70%) is more prevalent than MP (24% to 40%). We found no consistent evidence that symptoms were more likely to co-occur than occur independently; patterns varied across life stages and cohorts. Symptoms were more common in those from lower socioeconomic backgrounds and minoritised ethnic groups. Incidence of both HMP and MP peaked in the years immediately following menarche. Interpretation: Most women experience heavy and/or painful periods at some point. MP is more common in adolescence and HMB in later reproductive life. Generating robust life-course evidence on menstrual symptom patterns is essential to inform clinical guidelines, reduce diagnostic delays, and address inequalities in menstrual health.

Src family kinases (SFKs) constitute a major non-receptor tyrosine kinase class that regulate processes important for pregnancy. We profiled placental SFKs to assess cell-specific expression, tissue localization, and clinical associations in early-onset preeclampsia (PE). Placental SFK transcripts were analyzed using bulk RNA sequencing discovery (GSE203507; n=15 PE, n=15 controls) and external validation (GSE114691; n=40 PE, n=21 controls) cohorts. Single-cell and single-nucleus RNA sequencing atlases mapped cell-specific expression. Computational deconvolution and causal mediation analyses were applied to bulk datasets. Findings were cross-validated by real-time quantitative PCR (qPCR; n=9 early-onset PE, n=7 controls) and immunohistochemistry (IHC). Transcriptomic data showed that LYN was the sole SFK member consistently elevated in early-onset PE placentas across discovery (1.8-fold, FDR<0.01) and validation cohorts (2.2-fold, FDR<0.001). Elevation was verified by qPCR (p<0.0001) and remained significant after adjusting for clinical covariates. Placental LYN abundance correlated positively with maximum antepartum maternal systolic blood pressure (r=0.63, p<0.01). Single-cell transcriptome atlases demonstrated LYN enrichment in myeloid lineages, extravillous trophoblasts, and syncytiotrophoblast (SCT). Deconvolution and mediation analyses indicated that altered tissue composition and increased per-cell expression each accounted for approximately half of the PE-associated LYN increase. IHC localized Lyn protein to Hofbauer macrophages and the apical microvillar SCT membrane in PE placentas. We conclude that placental LYN expression is selectively elevated in early-onset PE and correlates with maternal blood pressure severity. Lyn localization to trophoblasts and placental immune cells suggests potential roles in mediating inflammation and hypoxia-associated signaling in PE pathogenesis.

Female infertility remains a major women's reproductive health challenge, with impaired endometrial receptivity contributing to recurrent implantation failure, early pregnancy loss, and pregnancy complications. Mechanistic studies of human early pregnancy are limited by the lack of physiologically relevant models, and existing in vitro endometrial models rely on exogenous hormone treatment and do not recapitulate the dynamic ovarian control of endometrial growth and differentiation. Here, as a proof of concept, we developed a 3D in vitro ovarian follicle-endometrial organoid co-culture model. Immature murine follicles were cultured in vitro to obtain preovulatory follicles, followed by human chorionic gonadotropin (hCG) stimulation to induce ovulation and luteinization. Luteinized follicles were co-cultured with human endometrial stroma organoids for 6 days. The results showed that luteinized follicles generated a dynamic endocrine environment characterized by progressively increasing progesterone levels and declining estradiol levels. Follicle co-culture induced morphological differentiation and expression of decidualization markers in human endo-organoids. Transcriptomic analysis revealed strong genome-wide concordance between follicle co-culture and conventional decidualization induction using estradiol/progesterone/cAMP (EPC), with significant enrichment of decidualization-associated genes and pathways under both conditions. Nevertheless, the two conditions also exhibited distinct transcriptional states, with EPC preferentially enriched for more mature decidualization-associated programs, whereas luteinized follicle co-culture retained features of the early stage of decidualization. Functional perturbation testing revealed that the progesterone receptor (PR) antagonist RU486 attenuated co-culture-induced endometrial decidualization. Together, these results establish a new in vitro new approach methodology (NAM) that recapitulates ovarian control of endometrial decidualization. This platform provides a human-relevant NAM model to investigate the physiology and pathophysiology of endometrial decidualization, and to evaluate how drugs and environmental chemicals perturb uterine receptivity and early pregnancy.

Background: Both fetal and maternal genomes contribute to birthweight, but the timing and relative magnitude of their effects during gestation remain unclear. Polygenic risk scores (PRSs), constructed from genome-wide association studies (GWAS), capture the cumulative influence of common birthweight-associated variants to quantify genetic predisposition to fetal growth. We constructed fetal and maternal PRSs for birthweight in two UK birth cohorts, the Pregnancy Outcome Prediction Study (POPS) and Born in Bradford (BiB), to evaluate their associations with estimated fetal weight (EFW) throughout pregnancy and with birthweight at delivery. We also investigated whether PRSs could improve risk stratification of infants born below the 10th centile by distinguishing those with pathological intrauterine growth restriction (IUGR) from those who are constitutionally small-for-gestational-age (SGA). Methods: In POPS and BiB, we used LDpred2-auto to estimate PRSs based on the largest available maternal and fetal GWAS for birthweight. We longitudinally assessed EFW associations with PRSs using linear mixed effects models in POPS and BiB. We evaluated birthweight associations with PRSs using linear models, where birthweight was adjusted for gestational age and fetal sex. We estimated R2 and conducted variance decomposition analyses for all models. Genetically-predicted birthweights were estimated from joint models of fetal and maternal PRSs plus ten genetic principal components (PCs). BiB analyses were conducted separately for genetically-inferred European (EUR) and South Asian (SAS) ancestry. Results: While PRSfetus was significantly associated with EFW at baseline ([~]22 weeks gestation) across cohorts, PRSmother was not. Both the interaction of PRSfetus and PRSmother with gestational age (GA) were significantly associated with EFW. The effect of PRSmother on EFW increased more quickly across gestation than the effect of PRSfetus in POPS, but this finding was not replicated in BiB. Jointly, fetal and maternal polygenic risk explained 4% of variance in EFW in POPS and 1% in BiB-EUR. In POPS, fetal and maternal PRSs showed independent contributions to EFW variance of 3% and 0.5%, respectively. Joint and univariable linear models showed significant positive associations of PRSfetus and PRSmother with observed birthweight. In POPS, PRSfetus and PRSmother accounted for [~]10% and [~]4% of variance in birthweight over covariates alone, respectively, which was replicated in BiB-EUR. Maternal and fetal genetic effects jointly explained 11% of birthweight variance in POPS, similar to BiB-EUR, at 12%; however, the variance explained was reduced by half in BiB-SAS, at 6%. Among POPS babies born <10th birthweight centile, the magnitude of the difference between observed and genetically-predicted birthweight was greater in the group judged clinically to be IUGR vs. SGA (p=0.008; point estimate=0.29, 95%CI: 0.12, 0.47). Conclusions: Maternal and fetal PRSs jointly account for [~]11-12% of birthweight variance in European ancestry groups, but much less in a South Asian cohort. In POPS, the effect of birthweight-associated SNPs on estimated fetal weight increases more rapidly across pregnancy for maternal than for fetal genetic effects. Deviation from genetically-predicted birthweight may be a useful metric to help distinguish growth-restricted from constitutionally small babies.

Predicting implantation outcomes in vitro fertilization (IVF) is challenging because clinically relevant information becomes available at different points during treatment. In this study, we developed a stage-aware, leakage-conscious, and explainable machine-learning framework to predict implantation using a public IVF dataset. {beta}-HCG was excluded to avoid potential downstream target leakage. The data were divided into training, validation, and independent test sets. Several machine-learning models, including Logistic Regression, Random Forest (RF), Extra Trees, XGBoost, and Hist Gradient Boosting, were optimized using stratified cross-validation. A fixed RF model was then evaluated at three stages of the IVF process, using baseline couple characteristics, gamete and fertilization variables, and embryological variables, respectively. Model performance improved as additional treatment-related information became available, with the largest improvement observed after the inclusion of embryological variables. The final RF model showed good predictive performance on the independent test set. The number of embryos was consistently identified as the most important predictor across RF, permutation, and SHAP analyses. Overall, the findings suggest that IVF implantation prediction becomes more accurate as later-stage treatment information is incorporated, particularly information related to embryological characteristics. The proposed approach also provides interpretable predictions while reducing the risk of information leakage. Further evaluation using independent clinical datasets is needed before the framework can be considered for clinical use.

Progesterone is essential for the establishment and maintenance of pregnancy, primarily through the classical nuclear progesterone receptor (PGR). However, progesterone also exerts rapid, non-genomic effects through membrane progesterone receptors, including members of the progestin and adipoQ receptor (PAQR) family, whose cellular distribution and potential roles at the human maternal-fetal interface remain poorly understood. We therefore sought to define the cell-type-specific expression landscape of classical and membrane progesterone receptors during early human pregnancy. We analyzed publicly available single-cell RNA-sequencing datasets of the first-trimester human maternal-fetal interface to characterize the expression of PGR and PAQR family members across trophoblast, immune, stromal, and vascular cell populations. Major expression patterns were examined across independent datasets and further assessed using spatial transcriptomic data. PGR expression was predominantly localized to decidual stromal and perivascular populations, whereas individual PAQR family members showed distinct cellular distributions. PAQR6 was enriched in decidual natural killer cells, PAQR7 was broadly expressed in trophoblast populations, PAQR8 was prominently expressed in fetal stromal and macrophage populations, including Hofbauer cells, and PAQR9 showed prominent enrichment in syncytiotrophoblasts. Selected cell-type-specific patterns were further supported by spatial transcriptomic analysis. These findings provide a cell-type-resolved framework for investigating how distinct progesterone receptor systems mediate progesterone signaling across maternal and fetal cellular compartments during early human placentation.

Polyendocrine metabolic ovarian syndrome (PMOS) and early-life irregular cycles (ELIC) carry higher metabolic risks, but real-world glycemic patterns remain inadequately characterized. We analyzed 247,316 continuous glucose monitoring (CGM) days from 716 consented and enrolled participants (11/2019-08/2026) in the Apple Women's Health Study, using cluster-weighted generalized estimating equations to evaluate associations between PMOS, ELIC, and glycemic metrics. Same-day wearable-assessed physical activity, digital dietary logs, and medication health records were assessed as effect modifiers. Compared to participants without PMOS and with regular cycles, those with PMOS had higher mean glucose (6.81 mg/dL; 95% CI: -0.70, 14.72) and %observations above target range (3.77%; 95% CI: 0.04, 8.14). Estimates were smaller-in-magnitude for participants without PMOS but with ELIC, and larger for PMOS with androgen excess. Meeting activity goals, consuming protein-/fiber-rich diets, or using metformin/GLP-1 receptor agonists attenuated these associations. PMOS/ELIC were associated with unfavorable glycemic profiles, suggesting opportunities for risk identification and modification.

The ovary is one of the first organs to age, with declines in oocyte quantity and quality leading to infertility and loss of endocrine function. Why the ovary ages earlier than somatic tissues has remained enigmatic but may relate to its highly dynamic physiology and substantial demand for cellular debris clearance. Each ovulation forms a corpus luteum (CL), a transient endocrine structure which supports pregnancy but regresses in its absence. Because CLs have a short functional lifespan but prolonged structural regression, they represent a substantial source of ovarian debris. We hypothesized that post-ovulatory debris clearance becomes impaired with age and contributes to ovarian fibroinflammation. Using a CL-labeling system, we demonstrated that CLs from reproductively old mice show dysregulated regression, resulting in an accumulation of persistent CLs (pCLs) which appeared concurrently with ovarian multinucleated giant cells (MNGCs). Laser capture microdissection, RNA sequencing, and histological evaluation of CLs, pCLs, and MNGCs demonstrated that pCLs were focal hotspots of proinflammatory and debris-associated signatures enriched in lipids, extracellular matrix, and immune cells. Although pCLs retained luteal features, they were transcriptionally more similar to MNGCs than CLs. Consistent with this, an ex vivo CL model acquired MNGC-associated molecular features during extended culture, suggesting that pCLs may represent sites of MNGC formation. Culture of isolated follicles or ovarian stromal organoids with factors from MNGC-enriched explants induced compartment-specific fibroinflammatory responses. These findings provide novel mechanistic insight into ovarian aging, whereby post-ovulatory debris clearance is compromised with age, leading to pCL accumulation, MNGC formation, and a fibroinflammatory milieu.

Several different mechanisms of regeneration have been proposed to explain repair of the endometrial luminal epithelium (LE) following menstrual/estrous cyclicity, parturition or other damaging insults to the uterine mucosa. These include activity of an as-yet unidentified epithelial stem cell population, mesenchymal transdifferentiation, or integration of bone marrow-derived cells. Here, we used a novel Cag-Flox-STOP-Flox-Pgrmc2-3XFlag (Pgrmc2-3XFlag) reporter mouse coupled with a recently developed GE-specific cre recombinase mouse (Prss29-Cre) to determine if and to what extent the GE contributes to regeneration of the LE following parturition and during aging in nulliparous mice. To initiate regeneration in the endometrium, control (Pgrmc2-3XFlag) and reporter (Prss29-Cre;Pgrmc2-3XFlag) mice were placed with males for one pregnancy. Postpartum (pp) uterine tissues were then collected 5 days (1x5dpp), 3-weeks (1x3wpp) and 7-weeks (1x7wpp) later. A fourth set of uterine tissues was collected 3-weeks after three pregnancies (3x3wpp). The number of glands did not change at any time point. Indelibly marked FLAG+ GE cells were detected by immunohistochemistry and immunofluorescence in GE as expected. As demonstrated in serial uterine sections of uteri from all four treatment groups, GE cells migrated toward the LE and formed junctional points (JP) where GE cells transitioned from cuboidal GE cells to pseudostratified columnar LE cells. Transitioning cells at junctional points expressed markers of both GE (i.e., FOXA2) and LE (i.e., CALB1) tissues. Following transition, GE-derived LE cells expressed PGR and ESR1 consistent with surrounding developmentally-derived LE cells. About 17 FLAG+ LE cell islands were also observed in 1x5dpp 4 mm longitudinal sections at sites distant from GE-LE junctional points. Interestingly, the number of islands did not change from 1x5dpp to 1x7wpp. However, the total number of GE-derived FLAG+ LE cells observed in each island decreased from 1x5dpp to 1x3wpp, but then tended (p=0.07) to increase by 1x7wpp. There was no difference in the number of GE-LE junctional points or GE-derived FLAG+ LE islands between 1x3wpp and 3x3wpp tissues. However, a significant increase in the total (p=0.02) and average (p=0.02) number of FLAG+ LE cells was observed in 3x3wpp LE islands highlighting the proliferative expansion of these cells over the intervening ~80 days. GE-LE junctional points were also observed in 5-month old nulliparous Prss29-Cre;Pgrmc2-3XFlag mice at diestrus, but not in younger 6-week old mice. Collectively, these findings establish the GE as a quantifiable cellular source for LE regeneration during postpartum involution and as part of normal LE cell turnover during the estrous cycle in nulliparous mice. We propose a mechanism whereby GE cells within glandular structures are not terminally differentiated, but rather are stably differentiated to perform glandular function. However, when needed and based on the level of endometrial damage, GE cells can dedifferentiate, become integrated in to LE tissue and then reprogram to take on an LE phenotype as part of the regenerative process following parturition and during estrous cyclicity over time.

Polyendocrine Metabolic Ovarian Syndrome (PMOS, formerly PCOS) is associated with chronic low-grade inflammation. This study aimed to characterize the peripheral cytokine landscape in women with PMOS and identify cytokines associated with disease pathogenesis and phenotypic variation. Plasma concentrations of 48 cytokines were quantified using a multiplex bead-based assay in 102 women with PMOS and 114 healthy controls. We performed receiver operating characteristic (ROC) analysis, odds ratio estimation, correlation analysis, and network analyses to characterise cytokine alterations and identify phenotype-associated inflammatory signatures. Fourteen cytokines were significantly elevated in PMOS compared with healthy controls: IL-18, IL-1Ra, IL-12p70, IL-13, IL-16, MCP-1, MCP-3, CTACK, Eotaxin, RANTES, IP-10, M-CSF, HGF, and {beta}-NGF. ROC analysis identified IL-16, CTACK, and IP-10 as potential discriminators of phenotype A, whereas IL-18, M-CSF, and HGF demonstrated promising discriminatory performance for phenotype B. Odds ratio analysis identified 11 cytokines with odds ratios greater than 1, with IL-16 showing the strongest association with PMOS. Correlation analysis revealed 10 strongly correlated cytokine pairs (r [&ge;] 0.7) in the PMOS group, suggesting coordinated regulation of inflammatory mediators. Gene ontology and network analysis highlighted alterations in chemotaxis and immune cell migration pathways, implicating these processes in PMOS-associated immune dysregulation. These findings reveal a distinct peripheral inflammatory cytokine signature and phenotype-associated differences in PMOS. IL-16 emerged as a prominent candidate for further investigation, given its strong association with PMOS. Peripheral cytokine profiling may provide a minimally invasive approach to characterising immune alterations and identifying candidate biomarkers for disease characterisation and phenotype stratification.

BackgroundPremature ovarian insufficiency (POI) is a condition affecting female fertility e.g., in the context of autoimmune disease. A recent proof-of-concept study showed that B-cell depletion with rituximab (RTX) temporarily restored ovarian function in patients with autoimmune POI. However, the underlying immunological mechanisms behind this response is unknown. ObjectivesTo characterize B- and T-cell phenotypes before and after RTX therapy in the above-mentioned patients and associations with autoantibody levels and treatment response. MethodsCryopreserved cell samples were available from six autoimmune patients with POI, four of whom were positive for autoantibodies against steroidogenic enzymes (21OH, 17OH and/or SCC). Patients received RTX and blood samples were collected at baseline, second infusion (2 weeks), 3, 8 and 12 months. Peripheral B- and T-cell phenotypes were analysed using spectral flow cytometry and correlated with autoantibody levels. ResultsAt baseline, patients displayed signs of B-cell activation by alteration in subset composition compared to matched controls. Upon closer analysis, patients positive for steroidogenic enzyme autoantibodies had significantly increased frequency of CXCR3+switched memory (SWM) B-cells than autoantibody-negative patients, which correlated positively with anti-17OH and-SCC autoantibody levels. In parallel, increased Th1 cell frequencies were also observed in the same patient group. Longitudinal analyses showed expected B-cell depletion followed by repopulation after RTX. Changes in T-cell subsets were observed at 8 months with reduced frequencies of Th1 and Tfh cells. ConclusionAutoimmune POI displayed altered B- and T-cell subsets implicating cell crosstalk, immune cell infiltration to sites of inflammation such as ovaries, and presence of Th1-driven immunopathogenesis. Key messagesO_LIIncreased CXCR3+SWM B cells and Th1 cells were found in patients with autoimmune POI who were positive for autoantibodies against steroidogenic enzymes. C_LIO_LICXCR3 expression on SWM B cells positively associated with anti-17OH and anti-SCC autoantibody levels. C_LIO_LIRTX induced rapid B-cell depletion followed by repopulation and a delayed T-cell effect at 8 months with reduced Th1 and Tfh cell frequencies. C_LI Capsule summaryRTX has recently been shown to restore fertility in autoimmune POI. Lymphocyte profiling identified Th1 bias accompanied by CXCR3+memory B cells that correlate with autoantibody levels. B-cell depletion appears to temporarily alleviate ovarian autoimmunity and ameliorate physiological function.

Background: European Association of Urology (EAU) Guidelines identify urogenital infections as a possible treatable cause of infertility, yet the current recommendation for antibiotic treatment remains weak due to a lack of high-quality evidence. We evaluated the effect of antibiotic treatment of asymptomatic urogenital infections on both conventional and apoptosis-related (flow cytometry sperm apoptosis test, FACS-APO) sperm parameters, as well as on pregnancy outcomes, in infertile couples. Methods: In this retrospective, single-centre study, we analysed 265 couples with an asymptomatic urogenital infection (Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, and Chlamydia trachomatis) confirmed in at least one partner by first-void urine testing, who received simultaneous antibiotic treatment. Paired conventional semen analysis and FACS-APO were performed before and 0-9 months after treatment completion. Partner infection concordance, the effect of follow-up timing on treatment response, and pregnancy outcomes within 12 months were also assessed. Results: Sperm concentration, total sperm count, total and progressive motility, and the proportion of viable sperm increased significantly after treatment, while the proportion of late apoptotic / necrotic sperm and calculated sperm DNA fragmentation decreased significantly (all p<0.05); the proportion of early apoptotic sperm remained unchanged, and semen volume did not change. Among 244 couples with complete bilateral microbiological testing, Ureaplasma urealyticum was by far the most prevalent pathogen (57.8% of men, 87.7% of women), and female partner positivity given male positivity was 83.0%. The improvement in sperm concentration and total sperm count was most pronounced within 3 months after treatment and diminished, or reversed, beyond 6 months. Pregnancy was achieved in 127 of 265 couples (47.9%; per-protocol rate 53.4%) within 12 months, most commonly following in vitro fertilization (72/127) or spontaneously (21/127). Conclusions: Antibiotic treatment of asymptomatic urogenital infections in infertile couples was associated with significant improvement in conventional semen parameters and a reduction in sperm apoptosis and calculated DNA fragmentation. Given the retrospective, uncontrolled design, these findings should be regarded as hypothesis-generating rather than definitive proof of a causal, fertility-enhancing effect, and warrant confirmation in prospective controlled studies.