Molecular Human Reproduction

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

In vitro fertilization (IVF) is key for genetic improvement programs in bovine. However, embryos produced through IVF have lower developmental competence than those produced under in vivo conditions. Conventional sperm preparation for IVF typically relies on heparin for sperm capacitation but fails to replicate the finely tuned molecular environment of the oviduct, resulting in compromised embryonic competence. Here, we evaluated the effect of HyperBull, a novel capacitation technology, on bovine IVF outcomes using unsorted cryopreserved semen. In a split-sample design, 528 cumulus-oocyte complexes were co-incubated with either control or HyperBull capacitated spermatozoa from the same bull. While overall blastocyst rates were not significantly different between groups (34.21% HyperBull vs. 28.63% control, p=0.148), the proportion of hatched embryos was significantly higher in the HyperBull group (15.82% vs. 9.13%, p=0.016). These findings suggest that modulating capacitation signals prior to insemination enhances embryonic developmental competence, thereby improving readiness for implantation. HyperBull may thus represent a valuable tool to increase the efficiency of IVF programs.

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

Pregnancy results in profound physiological changes driven by dynamic and precisely programmed molecular processes. Maternal peripheral blood is generally the specimen of choice for studying these processes, as it is easily accessible and essential for many aspects of maintaining a healthy pregnancy. Here, we present a high-resolution atlas of the dynamic temporal changes in the transcriptome of maternal peripheral blood in healthy human pregnancy. We generated comprehensive RNA sequencing data in 802 weekly samples from 31 healthy pregnant women from the first trimester until after delivery. Using a strict discovery and replication setup, our longitudinal analysis of gene expression identified 720 genes with robust pregnancy-specific expression patterns. Using weighted graph correlation network analysis, we identified nine pregnancy-associated transcriptional modules that reveal a strong, coordinated enrichment of innate/neutrophil and antiviral immune programs, alongside changes in adaptive immunity (T cell differentiation and signaling), erythropoiesis and hemoglobin metabolism. Cell-type deconvolution revealed that these transcriptomic shifts were accompanied by increased relative neutrophil proportions and reduced naive CD4 and CD8 T cells in pregnancy. We provide a comprehensive characterization of dynamic changes across pregnancy, highlighting maternal blood as a key systemic regulator in healthy gestation. Together, our findings establish a reference atlas of healthy pregnancy, which can be used to identify dysregulated processes and mechanisms in women with pregnancy complications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/715300v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@2a4b28org.highwire.dtl.DTLVardef@ac49d9org.highwire.dtl.DTLVardef@12468c8org.highwire.dtl.DTLVardef@15b282f_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LI720 genes showed robust pregnancy specific expression patterns. C_LIO_LICo-expression analysis clustered the genes into nine modules with distinct dynamics. C_LIO_LIEnrichment in pathways involved in innate and neutrophil-mediated immunity, antiviral responses, T cell differentiation and signaling, erythropoiesis and hemoglobin metabolism. C_LIO_LICell-type deconvolution showed increases in neutrophils and decreases in naive CD4 and CD8 T cells. C_LIO_LIThe atlas of detailed longitudinal transcriptional changes provides a baseline reference for healthy pregnancy. C_LIO_LIResults for all genes and protein-protein interaction networks are made available for interactive exploration. C_LI

BackgroundPolycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and a leading cause of female infertility, with complex genetic, metabolic, and hormonal etiologies. Long non-coding RNAs (lncRNAs) have emerged as important regulators of diverse biological processes, yet their roles in PCOS remain underexplored. Here, we identified and characterized PCOS differentially expressed gene-associated lncRNAs (PDEGAL) with an integrative approach combining expression data, genetic association, and evolutionary analysis. MethodsThirty-three PCOS-associated protein-coding genes were obtained from our prior study, and all their nearby and overlapping lncRNAs were annotated. These candidates were analyzed using UCSC Genome Browser-mapped annotations and datasets, including NCBI RefSeq, GENCODE, GTEx, GWAS SNPs, and conservation, as well as the FANTOM5 cap analysis of gene expression (CAGE) promoter data, to assess their expression, regulatory potential, genetic variant overlaps, and evolutionary conservation. ResultsTwenty-three PDEGALs (18 antisense to, and 5 sharing bidirectional promoters with, known PCOS-associated protein-coding genes) were identified. 17 PDEGALs contained GWAS SNPs with statistically significant disease associations, 9 of which were associated with PCOS-related traits. 5 PDEGALs demonstrated expression in the KGN granulosa cell model of PCOS. Key gene structure element (KGSE) analysis revealed that most PDEGALs are primate-specific. Integrating four criteria--GTEx expression, GWAS SNPs, FANTOM promoterome, and KGSE conservation--highlighted HELLPAR as the only lncRNA fulfilling all four, while five others--PGR-AS1, MTOR-AS1, ENSG00000265179, ENSG00000256218, and LOC105377276--fulfilled three of the four criteria. ConclusionsWe have systematically identified candidate PCOS regulatory lncRNAs with convergent genetic, expression, and evolutionary evidence. These results provide a framework for functional validation and highlight lncRNAs as potential biomarkers and therapeutic targets in PCOS that function by regulating their nearby and overlapping protein-coding genes.

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

Mitochondria undergo significant structural and functional changes during human pre-implantation embryogenesis, yet the transcriptional activity of both nuclear-encoded mitochondria-associated genes and mitochondrially transcribed genes across this developmental window remains poorly characterized. While mitochondria are established as the primary energy source for the early embryo, emerging evidence suggests they may also influence lineage specification through epigenetic regulation and metabolite availability. To investigate this, we reanalyzed two publicly available human single-cell RNA sequencing datasets filtered for mitochondria-associated genes using the MitoCarta 3.0 reference database, with separate analyses conducted on the nuclear-encoded and mitochondrially transcribed subsets. The first dataset spanned individual blastomeres from the oocyte through blastocyst stage, and the second compared trophectoderm and inner cell mass cells isolated from blastocysts. Mitochondria-associated gene expression was sufficient to cluster human blastomeres by developmental stage, with morula and blastocyst stage cells forming well-defined clusters. Mitochondrially transcribed genes were found to be the primary drivers of clustering in earlier developmental stages, while nuclear-encoded mitochondria-associated genes drove clustering at the blastocyst stage. A pronounced shift in the expression of both gene sets was identified at the transition from the 4-cell to the 8-cell stage, with 115 unique differentially expressed genes identified across the two stages immediately following this transition, compared to only 5 across the two prior stages. The timing of this transcriptional upregulation, preceding the known onset of oxidative phosphorylation at approximately the 32-cell stage, suggests a mitochondrial role in early embryogenesis beyond energy production. Analysis of trophectoderm and inner cell mass cells showed that mitochondrial gene expression profiles partially distinguished these two lineages, consistent with known differences in mitochondrial activity between them. These findings suggest that both nuclear-encoded and mitochondrially transcribed gene expression is upregulated prior to the first lineage specification event in the human embryo, potentially contributing to epigenetic regulation and cell fate determination through altered metabolite availability. A limitation of this study is its reliance on transcriptomic data alone; future work incorporating functional metabolite measurements will be needed to establish causality. Nonetheless, these data reframe mitochondria as active participants in early human developmental programming rather than passive energy suppliers.

ABSTRACT/SUMMARYVascular remodeling within the developing fetus and placenta is essential for supporting the growth and function of emerging tissues and organs. Pericytes (PCs) play a central role in stabilizing and maturing microvascular networks by extending along endothelial cells (ECs) and reinforcing vessel integrity. In the placenta, as in other organs, PC-EC communication is mediated in part by platelet-derived growth factor-BB (PDGF-BB) signaling, which governs PC differentiation, proliferation, migration, and survival, ultimately enabling their recruitment and retention along capillaries. In this study, we identified progressive PC investment along feto-placental capillaries in both murine and human tissues across gestation, supported by morphological and molecular evidence. Placental PCs displayed phenotypic heterogeneity comparable to that observed in the brain and heart, suggesting conserved diversity across organ systems. In addition to characterizing PC dynamics, we examined the expression of recently identified soluble PDGF Receptor-{beta} (sPDGFR{beta}) isoforms. These variants were detected at the protein and transcript levels in mouse and human placentas, as well as in a murine trophoblast-embryonic stem cell (TESC) differentiation model that recapitulates aspects of early placental vascular development. Within this model, sPDGFR{beta} expression was independent of ADAM10 activity and exogenous growth factors during early vessel formation but was markedly upregulated during hypoxia. To assess how elevated sPDGFR{beta} might influence PDGF-BB signaling, we exposed TESCl-derived vascular networks to excess PDGF-BB with or without a sPDGFR{beta} mimetic. PDGF-BB alone reduced full-length PDGFR{beta} levels while increasing receptor phosphorylation, consistent with known ligand-induced regulatory mechanisms. Inclusion of the sPDGFR{beta} mimetic shifted these responses toward baseline, suggesting a potential modulatory or feedback role for soluble receptor variants. Together, these findings demonstrate that PCs are progressively recruited to placental capillaries and exhibit diverse phenotypes during development, and that soluble PDGFR{beta} isoforms may modulate PDGF-BB signaling in a manner sensitive to oxygen tension. Understanding these mechanisms provides insight into the regulation of placental vascular maturation and may inform strategies to improve human health by targeting disorders rooted in impaired placental development.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection worldwide and a leading cause of inflammatory reproductive tract disease and infertility in women. Much of the tissue damage associated with genital chlamydial infection arises from host inflammatory responses rather than direct bacterial cytotoxicity. Epithelial cells lining the female reproductive tract represent the primary host cells infected during chlamydial infection and play key roles in initiating innate immune responses. Among the cytokines produced by infected epithelial cells, type-I interferons have emerged as important regulators of host defense and inflammatory signaling; however, the specific contribution of interferon-{beta} (IFN-{beta}) to epithelial transcriptional responses during chlamydial infection remains incompletely defined. In the present study, we investigated the role of IFN-{beta} in coordinating epithelial immune signaling networks during infection with Chlamydia muridarum. Using wild-type murine oviduct epithelial cells (OE-WT) and IFN-{beta}-deficient epithelial cells (OE-IFN{beta}-KO), we performed pathway-focused RT{superscript 2} Profiler PCR array analyses examining transcriptional responses across four biological pathways: (1) innate and adaptive immune responses, (2) type-I interferon signaling, (3) inflammatory and autoimmune responses, and (4) fibrosis-associated pathways. Infection of OE-WT cells resulted in coordinated induction of cytokines, chemokines, and interferon-stimulated genes associated with antimicrobial defense and immune cell recruitment. In contrast, IFN-{beta} deficiency resulted in widespread dysregulation of these transcriptional programs, including reduced induction of interferon-responsive chemokines such as CCL5 and CXCL10, altered inflammatory cytokine expression, and transcriptional signatures consistent with enhanced tissue remodeling responses. Notably, IFN-{beta} deficiency resulted in increased TNF expression accompanied by reduced IL-6 induction, suggesting disruption of balanced inflammatory signaling networks. Pathway analyses further revealed dysregulated expression of fibrosis-associated genes including Serpine1, Ctgf, and Eng in IFN-{beta}-deficient epithelial cells, indicating potential mechanisms linking interferon signaling to tissue remodeling during infection. Collectively, these findings identify IFN-{beta} as a central regulator of epithelial immune networks during chlamydial infection and suggest that disruption of IFN-{beta} signaling may promote inflammatory and fibrotic pathology within the female reproductive tract. Author SummarySexually transmitted infections caused by Chlamydia trachomatis are a major cause of infertility worldwide. Although antibiotic treatment can eliminate the bacteria, damage to the reproductive tract often results from the bodys own immune response to infection. The epithelial cells lining the reproductive tract are the first cells infected and play an important role in initiating immune responses. In this study, we investigated how a specific immune signaling molecule, interferon-{beta} (IFN-{beta}), regulates the gene expression programs activated in epithelial cells during chlamydial infection. Using pathway-focused gene expression arrays, we found that IFN-{beta} coordinates multiple immune pathways, including interferon signaling, inflammatory cytokine networks, and genes associated with tissue remodeling. When IFN-{beta} was absent, many of these pathways became dysregulated, resulting in altered inflammatory signaling and gene expression patterns linked to fibrosis. These findings suggest that IFN-{beta} functions as a key regulator that helps balance protective immune responses with inflammatory processes that can damage reproductive tissues during infection.

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

BackgroundMaternal protein restriction results in a 28% reduction in nephrogenic cells and nephron units in rodent offspring by the 17th day of gestation compared to adequate protein intake. AimsThe present study investigates the association between growth factor expression and some developmental pathways that contribute to nephron reduction during embryonic and fetal development. Experimental DesignPregnant C57BL/6-Tg and C57BL/6J mice were assigned to either normal protein intake (NP-17%) or low protein intake (LP-6%) groups. Body weight of male offspring and kidney growth factor expression were assessed on gestation days (GD) 14 and 18. ResultsOn GD 14, LP pups exhibited a 4% higher body mass (0.1035 g) compared to NP pups (0.0995 g, p = 0.005). By GD 18, LP pups demonstrated a 4% decrease in body mass (0.939 g, p = 0.03) and a 10% increase in the number of cells per metanephric cap area. Three genes (Csf2, Il1b, Il2) were downregulated, while seven genes (Bmp2, Csf3, Fgf8, Gdnf, Bmp7, Fgf3, Ntf3) were upregulated. By GD 14, phagophores and autophagosomes in the ureteric bud increased by 197%, with further increases observed by GD 18. Bcl-2 expression increased significantly in ureteric bud cells, and mTOR activity was elevated by GD 18. ConclusionEarly gestational protein restriction modifies renal growth factor gene expression, influencing cell proliferation and autophagy, and may contribute to reduced nephron numbers by the 18th day of gestation. HIGHLIGHTSO_LIThis study examines the effects of a low-protein diet during pregnancy in mice and demonstrates a significant reduction in embryo-fetal body weight between gestational days 14 and 18. C_LIO_LIProtein restriction induces a distinct cellular pattern in the mesonephros, with a 21% increase in CAP cells at gestational day 14 (GD14), followed by a decrease by gestational day 18 (GD18) compared to offspring from mothers on a normal protein diet. C_LIO_LIAdditionally, increased expression levels of key growth factors essential for kidney development were observed at GD 14, comparing LP with NP intake during pregnancy. C_LIO_LISeven genes were upregulated (Gdnf, Bmp2, Bmp7, Tgf, Fgf8, Fgf3, Csf3, Ntf3), while three genes were downregulated (Csf2, Il1b, Il2). C_LIO_LIOverall, these findings indicate that gene regulation, autophagy, and mTOR signaling mechanisms significantly influence nephron numbers in response to gestational protein restriction beyond the 18th day of gestation. C_LI

PurposeIdiopathic hypogonadotropic hypogonadism (IHH) is characterized by impaired reproductive maturation, and approximately half of all cases lack an identified genetic cause. We investigated the genetic basis of IHH in two large cohorts to identify novel disease-causing genes. MethodsWe analyzed exome and genome sequencing data from 1,822 patients with IHH from two independent cohorts. Rare variants were filtered using pedigree-informed inheritance models. PLEKHA6 expression in the postmortem human hypothalamus were tested at the mRNA and protein level. Functional studies assessed kisspeptin secretion in cell-based assays. ResultsWe identified 18 distinct PLEKHA6 variants in 24 patients from 20 unrelated families (1.3% of cohort). Variants segregated with disease under autosomal recessive and autosomal dominant (with variable penetrance) inheritance patterns. PLEKHA6 was robustly expressed in the hypothalamus and showed clear colocalization with neurokinin B, which served as the marker for the GnRH pulse generator. Functional studies demonstrated that patient variants significantly impaired kisspeptin secretion. ConclusionPLEKHA6 is a novel IHH gene and the first reported regulator of kisspeptin secretion from the kisspeptin-neurokinin B-dynorphin (KNDy) neurons, which have recently been established as the GnRH pulse generator. These findings establish impaired kisspeptin release as a new disease mechanism in IHH and highlight the critical role of neuropeptide trafficking in reproductive function.

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

Purpose: To evaluate the efficacy and safety of oral L-ergothioneine (EGT) in improving ovarian reserve and clinical symptoms in women with diminished ovarian reserve (DOR). As a proof-ofconcept study, we explored correlations between hormonal shifts and symptom amelioration. Methods: This single-center, open-label trial enrolled 40 women (aged 35-45 years) with DOR (baseline AMH: 1.0-3.0 ng/mL) and menstrual disorders. Participants received oral EGT (120 mg/day) for three consecutive menstrual cycles. The primary outcome was the change in serum AMH. Secondary outcomes included sex hormones (FSH, E2), antral follicle count, and validated clinical questionnaires (modified Kupperman Index [KI], PSQI, SF-36, and Menstrual Symptom Score). Results: Thirty-six participants completed the intervention without product-related adverse events. EGT significantly improved core ovarian markers: mean AMH increased from 1.79 {+/-} 0.71 to 2.47 {+/-} 1.52 ng/mL (p = 0.029). Concurrently, basal FSH decreased (8.22 {+/-} 2.93 to 7.05 {+/-} 2.47 mIU/mL, p = 0.032) and E2 increased (46.00 {+/-} 22.70 to 63.46 {+/-} 50.10 pg/mL, p = 0.030). Clinical assessments showed progressive reductions in KI (5.42 {+/-} 3.66 to 1.90 {+/-} 2.16, p < 0.0001) and PSQI scores (6.89 {+/-} 1.82 to 5.50 {+/-} 1.40, p < 0.0001), alongside improved menstrual and SF-36 scores (p < 0.001). Subgroup analysis revealed upward AMH trends across both the 35-39 and 40-45 age cohorts. Crucially, endocrine restoration ({Delta}FSH) significantly correlated with improvements in sleep quality ({Delta}PSQI, r = 0.43, p < 0.05) and E2 increases (r = -0.46, p < 0.05), linking hormonal stabilization directly to systemic relief. Conclusion: Oral EGT safely enhances serum AMH and optimizes the FSH/E2 balance in women with DOR, yielding substantial relief from peri-menopausal and sleep disturbances. This pilot proofof- concept study provides the first clinical evidence supporting EGT's systemic benefits in reproductive aging, laying the groundwork for future placebo-controlled trials. Trial Registration: ChiCTR2500104484; Prospectively registered on 2025-06-18. Keywords: L-Ergothioneine, diminished ovarian reserve, anti-Mullerian hormone (AMH), oxidative stress, clinical trial

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

There is growing interest in understanding how hormonal signaling pathways contribute to the pathophysiology of mood disorders, based on the premise that fluctuations in sex hormones influence mood, a relationship particularly evident in conditions such as premenstrual dysphoric disorder, prenatal depression, postpartum depression, and perimenopausal depression. Estrogen receptor alpha (ER) is predominantly localized in the nucleus, but can also be associated with the cell membrane, thus mediating a broad range of genomic and non-genomic effects through distinct intracellular pathways. By employing a combination of computational simulations and in vitro biochemical and cell-based assays, we systematically evaluated the potential binding and functional interactions of antidepressant compounds with ER. Our results provide compelling evidence that antidepressants may not only affect classical monoaminergic targets but also modulate hormone receptor activity, particularly that of ER. These findings are consistent with the hypothesis that ER plays an important role in mood regulation and highlight it as a potential therapeutic target. Moreover, this work raises the possibility that the clinical efficacy of certain antidepressants may, at least in part, derive from their capacity to influence estrogen receptor-mediated signaling. Significance statementClinical observations suggest a link between female sex hormones and mood, highlighted by the higher prevalence of depression in women and increased vulnerability to depression during hormonal fluctuations. Here, we report that structurally diverse conventional and rapid-acting antidepressants directly interact with estrogen receptor alpha (ER). This interaction is associated with rapid intracellular signaling in cellular models. These findings indicate that, alongside their conventional targets, antidepressants may also engage sex steroid receptor components and signaling. This work broadens our basic understanding of antidepressant pharmacology at the cellular level, offering an additional perspective that may inform future research into the biological mechanisms of mood disorders and suggest a framework for developing targeted therapies for hormone-associated depressive disorders.

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

ObjectiveTo establish a fetal lamb model of complex gastroschisis and characterize the impact on the intestines over time. Summary Background DataGastroschisis is a congenital abdominal wall defect and in its complex form is associated with serious morbidity. Robust large-animal models may help understanding are lacking. MethodsAt gestational day 75, gastroschisis was induced by creating a 1-cm abdominal wall defect reinforced by a silicone ring. Fetuses were assessed either at term or at mid-gestation (13-21 days post-induction). The primary outcome was complex gastroschisis occurrence, defined by bowel stenosis, atresia, volvulus, perforation or necrosis; otherwise classified as simple. At mid-gestation, occurrence was compared between early (13-16 days) and late (17-21 days) intervals. Secondary outcomes included prenatal ultrasound findings, in vivo bowel motility and morphology, ex-vivo bowel contractility, amniotic fluid composition, and histology across complex, simple, and normal groups. ResultsGastroschisis was induced in 32 fetuses. At term (n=14), all survivors (7/14; 50%) had complex gastroschisis, with impaired bowel motility, altered enteric neural contractile responses and smooth muscle remodeling. At mid-gestation (n=18), complex gastroschisis occurred more frequently in the late than in the early group (71% vs. 11%; p=0.035). Mid-gestation gastroschisis fetuses showed greater intra-abdominal bowel dilatation on ultrasound and higher amniotic fluid digestive enzyme levels compared with non-operated littermates, with the greatest dilation observed in complex gastroschisis. ConclusionsThis model consistently reproduces complex gastroschisis in term survivors. After induction, complex gastroschisis occurrence increases with disease duration and is accompanied by structural and functional bowel changes.

Understanding the functions of maternal effect genes during oocyte growth is essential for elucidating the mechanisms of oogenesis and early embryonic development. However, conventional gene knockout and conditional knockout approaches require extensive breeding and are time-consuming. Here, we present a rapid in vitro gene functional analysis system that combines microinjection of mRNA, siRNA and plasmid DNA into mouse secondary follicles with a two-step oocyte growth culture system. Mouse secondary follicles were subjected to microinjection of mCherry mRNA and subsequently cultured for 15 days to produce fully grown oocytes. mCherry fluorescence persisted throughout the oocyte growth period but declined rapidly after fertilization. Despite minor cellular damage occasionally caused by microinjection, injected follicles developed normally and retained developmental competence. To evaluate the efficiency of gene suppression, we introduced siRNA targeting Dnmt3l, which is abundantly expressed during oocyte growth phase. Although Dnmt3l deficiency is known not to affect oocyte growth, we observed that oocyte growth was maintained normally despite a marked reduction in endogenous Dnmt3l mRNA levels in our knockdown model. These results demonstrate that this method enables efficient manipulation of gene expression specifically during oocyte growth while preserving developmental competence, providing a versatile platform for rapid functional screening of maternal effect genes in vitro.

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder marked by numerous renal cysts that impair kidney function, with about half of affected individuals progressing to kidney failure by midlife. Patients exhibit reduced circulating apelin, a ligand of the apelin receptor, known to regulate cardiovascular function including hypertension. We tested whether diminished apelin signaling contributes to cystogenesis and if exogenous apelin receptor activation can improve disease outcomes. MethodsPlasma samples from age- and sex-matched healthy controls and ADPKD participants were analyzed for circulating apelin peptides. To assess direct cystic effects, primary ADPKD renal epithelial cells were grown as 3D collagen-embedded cysts and treated with apelin agonists. Male and female Pkd1RC/RC; Pkd2+/- (PKD) mice were treated for 27 days with apelin agonists, vehicle, or the standard of care drug, Mozavaptan. Kidney and heart weight ratios, BUN, renal cAMP, and kidney transcriptional profiles were evaluated. ResultsCirculating apelin peptides were significantly reduced in ADPKD patients despite normal kidney function (eGFR, BUN, and creatinine). In vitro, both apelin and the small molecule apelin receptor agonist Azelaprag inhibited cyst growth. Apelin and Mozavaptan reduced kidney weight, cystic index, blood urea nitrogen and renal cAMP in PKD mice, whereas Azelaprag did not. Apelin downregulated expression of genes associated with cyst progression, including Lcn2 (Ngal), Postn, and Havcr1 (Kim-1). Mozavaptan, but not apelin, induced diuresis and reduced urinary concentration. ConclusionApelin receptor activation by exogenous apelin inhibited cAMP synthesis and cyst growth and improved kidney function in an orthologous mouse model of ADPKD. We propose that the apelin receptor may be a potential therapeutic target in ADPKD.