Biology of Reproduction

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.

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.

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.

Progesterone governs the establishment and maintenance of pregnancy in ruminants, acting through the nuclear progesterone receptor (PGR) and, potentially, through membrane progesterone receptors (mPRs) of the PAQR family. The cellular distribution of mPRs in ruminant reproductive tissues, however, remains undefined. We generated and validated rabbit polyclonal antibodies against the intracellular C-terminal peptides of bovine PAQR6 (mPR{delta}) and PAQR8 (mPR{beta}), and used them, with quantitative PCR of the uterus and conceptus, to map these receptors in the ovine uterus and conceptus and in the bovine ovary. Both antisera showed high ELISA titer against their immunizing peptides, negligible preimmune reactivity, and complete loss of tissue signal after peptide pre-absorption. By qPCR, PAQR6, PAQR8 and PGR transcript levels did not change significantly across the peri-implantation period in either the conceptus or the endometrium; in contrast, the progesterone-responsive gene ZBTB16 increased markedly in the conceptus (up to ~45-fold by day 21, p < 0.05 at days 17, 19 and 21), whereas FKBP5 showed a numerical increase without reaching statistical significance. Both genes remained relatively stable in the endometrium, indicating conceptus-specific changes in progesterone-responsive gene expression. At the protein level, PAQR6 and PAQR8 localized to the luminal and glandular epithelium of the endometrium across the cycle and early pregnancy, and to follicular (granulosa) and luteal cells of the ovary. Dual immunofluorescence in the ovine endometrium showed that both PAQR6 and PAQR8 are enriched at the apical surface of the luminal epithelium and are spatially distinct from the basolateral adherens-junction marker E-cadherin. These results provide a protein-level map of PAQR6 and PAQR8 in the ruminant reproductive tract, identify the apical epithelial surface as a site of receptor enrichment, and show that the endometrial epithelium retains membrane progesterone receptors while progesterone-responsive gene expression changes dynamically in the conceptus during the peri-implantation period. The validated ruminant-reactive antibodies provide a resource for future functional studies.

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.

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.

Epithelial cells of the female reproductive tract (FRT) form a protective barrier and serve as the first line of defense against sexually transmitted infections (STIs). They recognize pathogens, release cytokines and initiate innate immune responses. Sex hormones 17{beta}-estradiol (E2) and progesterone (P4) fluctuate throughout the menstrual cycle and influence epithelial cell functions. Generally, E2 confers protection against viral STIs like HIV-1 and HSV-2, whereas P4 increases susceptibility. However, the underlying mechanisms remain incompletely understood. MicroRNAs, small non-coding RNAs that regulate gene expression by inhibiting translation of target mRNAs, significantly influence viral infections, by promoting or restricting viral pathogenesis. Here, we examined the effect of sex hormones on microRNA expression in vaginal epithelial cells (VECs), the first responders to pathogens in the FRT. Using next generation sequencing analysis, the effect of physiological concentrations of E2 or P4 on microRNA transcription profile of VECs grown in air-liquid interface cultures was characterized. Thirteen miRNAs were uniquely regulated by E2 and 102 by P4, with 29 miRNAs that overlapped between both. The top four differentially expressed miRNAs from both treatments were validated by RT-qPCR. The mRNA targets of unique E2-miRNAs were involved in adherens junction, tight junction and chemokine signaling pathways; whereas the mRNA targets of unique P4-miRNAs were involved in MAPK signaling pathway. Interestingly, several miRNAs regulated by E2- (eg. hsa-miR-9-3p, hsa-miR-200c- 5p, hsa-miR-548c-5p) and by P4 (eg. hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-27b-3p) targeted genes related to cell cycle pathway. Together, these findings indicate that microRNAs regulated by E2 and P4 influence mRNAs in the pathways associated with cell adhesion, cell growth and development and innate immune responses, critical for maintenance of epithelial barrier integrity and regulating pathogen susceptibility in the vaginal tract. These hormone-regulated miRNAs may serve as potential targets for therapeutic interventions.

NANOG is a conserved regulator of epiblast identity and is essential for hypoblast development and embryo survival after the blastocyst stage in mice. However, its role during post-blastocyst development remains poorly understood in non-rodent mammals. Here, we investigated the function of NANOG during ovine embryogenesis using CRISPR/Cas9-mediated gene ablation. NANOG ablation did not affect blastocyst formation or the initial development of the epiblast. However, NANOG-deficient blastocysts contained significantly fewer SOX17+ hypoblast cells by day (D) 8, followed by reduced hypoblast migration during early post-hatching stages in D12 in vitro embryos. These defects were transient, as hypoblast development was comparable between NANOG-deficient and wild-type conceptuses during subsequent in vivo conceptus elongation from embryonic day (E) 11 onwards. In contrast, although extraembryonic membranes development and conceptus elongation proceeded normally, NANOG-deficient conceptuses lacked SOX2+ epiblast cells and failed to form an embryonic disc (ED) from E11 onwards. These findings demonstrate that NANOG is dispensable for hypoblast development but is required for epiblast maintenance and ED formation during conceptus elongation in sheep.

Introduction: With global warming, heatwaves are increasing in severity, affecting species worldwide. Recent studies show associations between heat exposure in pregnancy and adverse birth outcomes such as preterm birth, low birthweight and stillbirth across multiple species. This suggests potentially conserved mechanisms such as maternal inflammation, dehydration and changes in placental physiology in response to heat exposure. Moreover, the gestational timing of antenatal heat likely impacts the type and severity of outcome, yet this is not fully understood. Methods: We conducted a scoping review of studies on heat exposure and pregnancy outcomes in humans and other animals, to identify common mechanisms and temporal dynamics of effects. Temperature-, trimester- and species- specific patterns were reviewed across 197 studies and ten species (human, cow, sheep, pig, goat, rat, mouse, guinea pig, lizard and rabbit). Results: Qualitative trends indicate that high temperatures are associated with adverse birth outcomes. In particular, third trimester exposure increases risk of preterm birth and low birthweight. Many studies emphasise placental involvement in adverse outcomes and point to potential aberrations in placental physiology or cortisol signaling pathways. Conclusions: Overall, literature presents that heat exposure increases risk of adverse birth outcomes across studies, and taxa. Understanding the relationship between heat exposure and adverse birth outcomes, as well as the mechanisms and timing, is vital for informing public health policies and creating mitigating strategies for expecting mothers.

Uterine leiomyomas, commonly known as fibroids, are benign tumors of the female reproductive tract. They are the most common tumor of the female reproductive system and the leading cause of hysterectomy in the United States. Progesterone, estrogen, and their receptors are important drivers for the development and growth of leiomyomas. How estrogen drives leiomyoma growth remains unclear. We have previously shown that simvastatin can inhibit leiomyoma growth by modulating intracellular calcium. This suggests that calcium signaling is important for leiomyoma growth. We therefore hypothesized that estrogen may drive leiomyoma growth by stimulating calcium release through the membrane estrogen receptor GPR30 (also known as GPER). We found that estrogen dose-dependently induced calcium release in leiomyoma cells. The GPR30-specific agonist G-1 also induced calcium release, indicating that GPR30 mediates estrogen-mediated calcium release. The GPR30 antagonist G-15 blocked estrogen-induced calcium release and significantly reduced leiomyoma proliferation. Our results demonstrate that GPR30 is a major driver of estrogen-dependent growth in leiomyoma cells by modulating intracellular calcium. These findings may have high therapeutic relevance to leiomyomas and other estrogen-dependent tumors.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

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.

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.

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.

During embryogenesis, the cortical and medullary compartments of the adrenal gland emerge from two distinct sources: intermediate mesoderm-derived steroidogenic progenitors that comprise the cortex, and migratory neural crest-derived catecholaminergic progenitors that give rise to the medulla. The distinct cellular origins and paracrine signaling environments that foster induction, differentiation, and/or migration of cortical and medullary layers present challenges to recapitulating their co-emergence and incorporation in a human pluripotent stem cell (hPSC) based system. To address this, we developed an approach for modeling the adreno-gonadal primordium (AGP) in compound organoids that are organized along a gradient of NR5A1 expression and which unexpectedly incorporate SOX10-expressing Schwann cell precursors that ultimately generate chromaffin cells. Deconstruction of paracrine signaling within AGP-like organoids (AGPLOs) identified a fibroblast growth factor 9 (FGF9)-driven mechanism that expands bridge cell-like chromaffin progenitors and correlates to paracrine signaling cues encountered by physiological correlates along their migration corridor to the cortex in vivo. Combination of NR5A1-expressing cells with chromaffin cell clusters generated corticomedullar-like assembloids (CMLAs) that generated steroids and catecholamines and were responsive to adrenocorticotropic hormone. This work establishes a platform for modeling the reciprocal signaling relationships that drive adrenal gland development and function in health and disease and provides an approach for generating hPSC-derived adrenal cells/tissues that can be applied therapeutically.

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.

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.

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.

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.

Alterations in the female urinary microbiome have been linked to several several common urinary conditions that increase in prevalence after menopause. Here we use sequencing and metabolomic data to compare the urinary bladder microenvironments between women who are premenopausal, postmenopausal, and postmenopausal using vaginal estrogen. While the urinary microbiome differs between pre- and postmenopausal women, at least 6 weeks of vaginal estrogen is associated with higher urinary lactobacilli and fewer opportunistic pathogens in the urinary postmenopausal microbiome, which closer approximates what is seen in the premenopausal context. Changes in urinary Lactobacillus species are correlated with vaginal pH and the same Lactobacillus species found in the vaginal microbiome tend to also be identified within the urinary bladder microbiome. Urinary metabolites differ by context (e.g., pre-, post-, and post-menopausal using vaginal estrogen), but the majority of metabolites that drive these differences are unannotated. Of the metabolites with shared associations among different Lactobacillus species, L. iners have consistently inverse relationships compared to L. crispatus, L. gasseri, or L. jensenii.