Biology of 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.

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.

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.

BackgroundPolycystic ovary syndrome (PCOS) is a prevalent endocrine disorder with complex pathophysiology and limited therapeutic options. Identifying key molecular drivers and potential drug candidates is critical for improving clinical outcomes. MethodsWe integrated multi-cohort transcriptomics (GSE155489, GSE138518, GSE226146) with weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) network analysis, and drug repurposing. Differential expression analysis identified 1,039 DEGs, and WGCNA identified 10 PCOS-associated modules. Intersection of DEGs with module genes yielded 498 core candidate genes, which were subjected to functional enrichment, PPI network analysis, and connectivity map-based drug repurposing (CLUE/LINCS). Candidate drugs were further evaluated by molecular docking and ADMET prediction using a triple intersection strategy (hub genes, high differential expression, drug-target evidence). ResultsFunctional enrichment revealed significant enrichment in cell adhesion and TGF-beta signaling. PPI network analysis identified CD44 as the top hub gene (degree=42). Drug repurposing identified 106 candidate drugs, including troglitazone and enzalutamide. Using the triple intersection strategy, five genes (ID2, NR4A1, GJA5, ID1, MYH11) were prioritized for molecular docking. GJA5 showed strong predicted binding affinity with flufenamic acid (-7.88 kcal/mol), and cytosporone B exhibited favorable druglikeness (0 Lipinski violations). ConclusionThis study systematically characterizes PCOS-associated gene networks and provides a prioritized set of candidate targets and drugs through a purely computational framework. CD44 emerges as a key network node with potential relevance in PCOS pathophysiology. These findings offer testable hypotheses for future mechanistic studies and drug discovery efforts in PCOS.

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.

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.

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.

ObjectivesCD4+ T cells play key roles in regulating immune responses during pregnancy, therefore we aimed to understand the CD4+ T cell surface proteome and transcriptome during pregnancy. MethodsCD4+ T cells were analysed in blood and decidua from term-pregnancies (>37 weeks), and non-pregnant blood. >350 surface proteins were screened via flow cytometry, and transcriptomes were analysed using single-cell RNA sequencing with >130 CITE-seq barcoded antibodies. ResultsSurface protein screening identified changes to ILT4/CD85d, CD9, IFN-{gamma} receptor {beta}-chain, CX3CR1 and CCR5 in the pregnant blood and decidual CD4+ T cells. CX3CR1 and CCR5 had the highest expression on the effector-memory T cell (TEM) subset in the blood, with expression consistent across subsets in decidua. CD126/IL-6R was lower in pregnant blood and decidual CD4+ T cells, while scRNAseq identified enrichment in the IL-6R signalling pathway in naive CD4+ T cells in pregnant blood. Both sIL-6R and IL-6 concentrations were increased in plasma during pregnancy, suggesting perturbations to the IL-6/IL-6R signalling axis. Meanwhile, decidual CD4+ T cells had increased expression of transcription factor RUNX3 in the CD69+ tissue-resident-like subset. ConclusionsOur findings demonstrate altered molecular expression in CD4+ T cells during pregnancy. This provides important mechanistic insight of their adaptation and regulation during placental development, which may drive placental dysfunction or pregnancy complications including preeclampsia, fetal growth restriction and stillbirth. These new data may inform future studies that focus on determining the significance of differentially- expressed immune features in pregnancy to identify potential targets for immune modulation to treat pregnancy complications and infections.

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.

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.

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.

BackgroundHormonal transition phases represent windows of increased neuroplasticity across the female lifespan. In this study, we aim to investigate the brain anatomical architecture of hormonal transition phases by directly comparing menarche, as a period of rising levels of steroid hormones, and menopause, as a time of declining levels. MethodsWe fit linear models on cross-sectional and linear mixed-effect models on longitudinal magnetic resonance imaging (MRI) datasets, to explore the effects of menarche onset (ABCD study data, Ncross-sectional=1274, Nlongitudinal=611) and transition into menopause (UK Biobank data, Ncross-sectional=1614, Nlongitudinal=212) on 66 cortical and 135 subcortical brain volumes, and to identify brain structures with opposing but regional overlapping effects in both periods. Models were adjusted for age and corrected for multiple comparison (P <.05; FDR-corrected). ResultsCross-sectionally, using a between-subject design, 83 brain volumes showed effects of menarche-onset and 17 volumes showed effects of menopause-transition. Of these, seven brain volumes were significantly affected by both transitional periods, showing opposing directional volume changes. Longitudinally, using a within-subject design, 56 brain volumes exhibited menarche effects, of which 46 replicated cross-sectionally. No menopause effect survived correction for multiple comparison, likely due to limited longitudinal sample size. ConclusionOur findings confirm regionally overlapping brain structural alteration between the two hormonal phases - menarche and menopause - showing the hypothesized opposite effect directions. Additionally, our results show the robustness of menarche effects, which converged across cross-sectional and longitudinal study designs. Taken together, our results contribute to a better understanding of hormone related neuroplasticity, emphasizing the importance of not only understanding individual phases, but understanding the overarching patterns across the female reproductive lifespan.

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.

Maternal pancreatic {beta}-cells undergo functional and structural changes to adapt to increased metabolic demands during pregnancy. Lactogen signaling via the prolactin receptor (PRLR) contributes to these adaptations by increasing {beta}-cell mass, insulin transcription and glucose-stimulated insulin secretion[1-4]. In other lactogen-responsive tissues such as the mammary glands and specific hypothalamic nuclei, gestation induces epigenetic changes, some of which persist long after birth[5, 6]. We have previously found that prolactin treatment in islets regulates the expression of epigenetic modifiers[7, 8]. However, whether lactogen signaling in {beta}-cells mediates epigenetic changes to regulate chromatin accessibility has not been examined. Therefore, our objective was to determine whether PRLR signaling alters chromatin accessibility of {beta}-cells to facilitate transcriptional regulation. Using single-cell ATAC-sequencing, we identified differentially accessible regions (DARs) in {beta}-cells which had 718 overrepresented motifs following prolactin treatment of murine islets. Validating this approach, these included motifs bound by established PRLR signaling effectors such as the STAT family of transcription factors (TFs). Using RNA-sequencing we identified transcriptional changes in 41 TFs whose motifs were overrepresented in DARs, including several previously linked to PRLR signaling within {beta}-cells, including Myc, Mafb and Esr1. Importantly, we also identified TFs not previously associated with PRLR signaling, including OVOL2 an established regulator of epigenetic landscape within cells. OVOL2 is a transcription factor involved in EMT inhibition and energy homeostasis with unknown roles in pancreatic {beta}-cells. Here, we establish that OVOL2 acts as a negative regulator of lactogen-dependent effects on {beta}-cell proliferation, establishing a novel regulator of PRLR signaling.

AimsThe Mediterranean diet is associated with reduced cardiometabolic risk, yet its physiological effects during pregnancy and its impact on placental metabolism remain incompletely understood. This study aimed to determine whether maternal adherence to a Mediterranean diet during pregnancy influences placental lipid metabolism and signalling pathways involved in nutrient handling, tissue remodelling, and inflammation, and to assess their relationship with pregnancy outcomes. MethodsPlacental samples and clinical outcome data were analysed from pregnant women participating in an unblinded randomized clinical trial of a Mediterranean diet intervention. Placental lipid composition was quantified and the expression of genes and signalling pathways involved in lipid metabolism, nutrient transport, inflammation, and tissue remodelling was evaluated. ResultsMaternal adherence to a Mediterranean diet during pregnancy was associated with significant alterations in placental lipid composition, including reduced C18:0 and C24:0 and increased C18:1n9c, C20:3n6, and C22:0, with lower total short-chain fatty acids and higher monounsaturated fatty acids. Placental expression of lipid metabolism regulators ALOX15 and PPAR{gamma} was reduced, alongside downregulation of AKT and p38 MAPK signalling pathways. Placentas from mothers adhering to the Mediterranean diet also showed lower expression of amino acid and glucose transporters SLC3A2 and SLC2A1, as well as altered inflammatory and extracellular matrix remodelling markers, including decreased SOCS3 and GHR and increased PAI1 and MMP3. ConclusionsMaternal adherence to a Mediterranean diet during pregnancy modifies placental lipid composition and regulates pathways involved in lipid handling, nutrient transport, inflammation, and tissue remodelling, providing insight into mechanisms linking maternal diet with placental metabolic function.

Preeclampsia (PE), or gestational hypertension, affects around 5% of pregnancies and leads to approximately 70,000 maternal and 500,000 fetal deaths per year worldwide, with increased cardiovascular and metabolic disease in survivors. PE is associated with elevated circulating levels of the alternative splice isoform of VEGF receptor 1 (sFlt1), defects in placental vasculature, kidney damage and, in severe disease, fetal growth restriction. Current mouse models induce PE via direct expression of sFlt1 or elevation of blood pressure, which bypass the natural risk factors for human disease, such as age, obesity, hypertension and diabetes. These risk factors have in common reduced expression of Kruppel-like factors 2 and 4 (KLF2/4), the endothelial transcription factors that protect against cardiovascular disease. We now report that inducible deletion of KLF4 in maternal endothelium (KLF4iECKO) results in gestational hypertension, elevated sFlt1, defective placental vasculature, kidney damage and fetal growth restriction. KLF4iECKO may thus serve as a mouse PE model suitable for mechanistic analysis and screening of treatments that address upstream risk factors.

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

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

Transcriptomic analyses are widely used to elucidate the molecular mechanisms driving gametogenesis and reproduction in fish, yet their accuracy depends heavily on appropriate normalization of gene expression data. Conventional approaches that rely on single or multiple reference genes are problematic during teleost oogenesis, as profound structural and physiological remodeling of the ovary challenges the assumption that commonly used reference transcripts remain stable. In this study, we assessed by qPCR the transcriptional variability of four widely used reference genes (actb, ef-1, gapdh, and 18S rRNA) throughout the oogenic cycle of the thicklip grey mullet (Chelon labrosus), using geNorm and NormFinder analyses, and we additionally evaluated total cDNA concentration as an alternative normalization factor. To examine the performance and interpretive consequences of each normalization strategy, we compared expression patterns of key steroidogenic genes (star, cyp19a1a, and cyp11b) normalized by individual reference genes, combinations of reference genes, or total cDNA concentration. All evaluated reference genes displayed notable transcriptional variability across oogenesis, confirming their limited suitability as sole internal controls. In contrast, normalization approaches integrating multiple reference genes and/or total cDNA concentration consistently provided greater stability and more reliable biological interpretation. These results support a refined and more robust normalization framework for transcriptional analyses in fish ovaries, particularly during stages of extensive tissue remodeling. Our findings demonstrate cDNA-based normalization is straightforward, rapid, and easy to implement across laboratories, providing a practical alternative for achieving accurate, reproducible transcript quantification in fish ovary studies.

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