Reproduction

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.

Study questionWhich proteins underpin oocyte developmental competence, as modelled by oocytes of variable competence matured in vivo, or matured in vitro under different conditions (capacitation in vitro maturation (CAPA) or standard in vitro maturation (IVM))? Summary answerSignificant differences in the global proteome were observed in both oocytes and their corresponding cumulus cells depending on the mode of oocyte maturation, with key variations in eukaryotic translation, autophagy and endocytosis pathways within oocytes, and changes in reactive oxygen species detoxification and serine biosynthesis in cumulus cells. What is known alreadyWithin the ovarian follicle, mammalian oocytes must acquire the necessary molecular machinery to support successful fertilisation and embryonic development. Close contact with the surrounding cumulus cells ensures coordinated nuclear and cytoplasmic maturation of the oocyte, along with the accumulation of proteins stored within the oocyte in cytoplasmic lattices and endo-lysosomal vesicular assemblies. Study design, size, durationThis basic science study utilised a mouse model to assess proteomic changes across three oocyte competence models. Key proteins identified in mouse oocytes were also assessed in discarded immature human germinal vesicle (GV) oocytes and MII oocytes following rescue-IVM. Three oocyte maturation methods were tested: i) in vivo maturation, (ii) CAPA and (iii) standard IVM. In vivo maturation served as a positive control group, whereby metaphase II (MII) mature oocytes were collected from mice stimulated with pregnant mare serum gonadotropin (PMSG) and triggered with human chorionic gonadotropin (hCG), simulating full ovarian stimulation. For the in vitro maturation groups, immature cumulus oocyte complexes (COCs) were collected from mildly stimulated (23 hr PMSG) mice. For the standard IVM group, immature COCs were matured in media containing amphiregulin and epiregulin for 18 hours. For the CAPA group, COCs were held for 24 hours in pre-IVM conditions in the presence of c-type natriuretic peptide (CNP), oestradiol, insulin and follicle stimulating hormone (FSH), and then matured via IVM in media containing FSH, amphiregulin and epiregulin. Four biological replicates were performed for mouse proteomics experiments, three biological replicates performed for mouse immunocytochemistry experiments and six replicates were performed for embryology experiments. Participants/materials, settings, methodsFour to six-week-old C57BL/6JAusb mice were used for all mouse experiments. Embryology outcomes were used to confirm the variation in oocyte developmental competence between the three maturation groups. For the in vivo, CAPA and IVM groups, mature MII COCs were collected and separated into oocytes and cumulus cells. Oocytes and cumulus cells were subjected to mass spectrometry and bioinformatic analysis was performed using Proteome Discoverer and Ingenuity Pathway Analysis, with data validated by immunofluorescence. To assess conservation of proteins in human oocytes, 49 oocytes were collected from 36 patients following assisted reproduction technology (ART) cycles and subject to immunofluorescence. Rescue-IVM was also performed with half of the human oocyte cohort to obtain MII oocytes. Main results and the role of chanceProteomic profiling identified around 1600 proteins in mouse oocytes and 3100 in mouse cumulus cells across all three treatment groups (at least 2 peptides per protein). Differential expression analysis and pattern analysis collectively revealed a signature of proteins that were consistently differentially expressed between in vivo and in vitro oocyte maturation systems (log2FC of {+/-} 1 and a p-value [≤] 0.05). These subsets of proteins were mapped to biological processes including eukaryotic translation, autophagy and endocytosis pathways within oocytes. Orthogonal validation of clathrin, ribosomal protein L24 and eukaryotic initiation factor 2A supported the proteomic findings and expression was conserved in human oocytes. Changes in reactive oxygen species detoxification and serine biosynthesis were observed in mouse cumulus cells, with fluorescence intensity changes in ferredoxin-1 and phosphoglycerate dehydrogenase supporting the dysregulation of cumulus cell processes during in vitro maturation. Large scale dataThe mass spectrometry data are available via ProteomeXchange with identifier PXD073269. Limitations, reasons for cautionThe foundational mechanisms of oocyte developmental competence remain elusive, particularly in humans where MII oocytes are heterogenous in quality within the same stimulation cycle and patient. In this study, C57Bl6/J mice were used as the model species, allowing precise control over differing models of oocyte quality and capacity to analyse large numbers of oocytes. However, care is required when interpreting the significance of these findings in mice to mechanisms regulating human oocyte quality. Nonetheless, the in vivo stimulation and both IVM protocols used in this study are clinically relevant and developmentally matched. This study has also not addressed oocyte developmental competence in gonadotropin-free IVM oocytes, which is now a clinical reality. Wider implications of the findingsThis study confirms that mouse oocytes, matured in vitro in two clinically relevant systems, show reduced developmental competence when compared to in vivo matured oocytes. Through examination of the global proteome in oocytes, molecular pathways including eukaryotic translation, autophagy and endocytosis were dysregulated in in vitro oocytes. Recent findings have revealed the critical role of these pathways to developmental competence in the context of in vivo development. In cumulus cells, changes in reactive oxygen species detoxification and serine biosynthesis were observed, adding to the extensive knowledge around metabolic activity in cumulus cells as a critical facet of oocyte quality. Combined, this data suggests that the necessary processes of protein storage and degradation in oocytes and metabolism in cumulus cells constitute important components of oocyte quality. These processes appear suboptimal in current IVM systems, providing a future research direction to optimise IVM protocols with consideration to these protein pathways. Study funding/competing interestsThis study was funded by a National Health and Medical Research Council Investigator Fellowship (APP1023210) awarded to R.B.G. and by a gift from Open Philanthropy. The following competing interests are declared: R.B.G.is a consultant to Dioseve Inc.. L.E.W is a co-founder, shareholder, director and advisor of Jumpstart Fertility Inc.. L.E.W. is also an advisor and shareholder in EdenRoc Sciences, the parent company of Metro Biotech NSW and Metro Biotech, and in Life Biosciences LLC and its daughter companies. His UNSW Industry Scientia position is partly funded by Proto Axiom. All other authors have no competing interests to disclose.

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.

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.

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.

Corncob bedding is commonly used for housing rodents in research, but previous work has linked corncob to altered reproductive behavior, disrupted estrous cycling, aggression, and welfare impacts across non-murine rodents. Furthermore, corncob is used as a licensed commercial rodenticide. Corncob contains endocrine-disrupting compounds (EDCs) that interfere with aromatase activity and estrogen signaling, processes critical for normal sexual behavior and development, yet effects on reproductive outcomes in mice remain unexplored. We conducted two experiments to test whether corncob bedding influences breeding performance and male sexual development. In Experiment 1, we analyzed breeding records to compare breeding performance of NSG mice housed on corncob versus cellulose bedding across two 3-month phases (N = 488 litters). Pairs housed on corncob produced significantly fewer pups than pairs housed on cellulose. To understand this effect, in Experiment 2, hormonal and morphological effects of corncob were assessed in male mice from four genetic backgrounds (C57BL/6, BALB/c, FVB, and CD1; N = 32 cages). Mice were bred and born on aspen or corncob, with half switched at weaning and half unchanged. Corncob produced timing-dependent effects in male reproductive physiology and development. Early-life corncob exposure altered baculum morphology and reduced testosterone, estradiol, and anogenital distance. In contrast, post-weaning corncob exposure resulted in hyper-masculinization, indicated by increased anogenital distance. Alongside prior evidence that corncob contains EDCs, our results raise serious concerns about its suitability as bedding in animal research. Continued use of corncob introduces uncontrolled variation that compromises animal welfare, reproduction, experimental validity, and reproducibility.

The developing female reproductive tract is highly sensitive to external hormonal stimulation, which can result in infertility and gynecologic diseases. To determine the underlying mechanisms, we used a mouse model to test the immediate, cell type-specific effects of neonatal exposure to the estrogenic chemical, diethylstilbestrol (DES), on the developing uterus. We found that control uterine epithelium is in a partial epithelial-mesenchymal transition state that is lost following DES exposure. This is accompanied by evidence of premature differentiation including altered apical-basal cell polarity and absence of the Lgr5+ epithelial stem cell population required for uterine gland formation. Cell-cell communication between epithelial and mesenchymal cells is restructured, and Wnt signaling is persistently reduced. The DES-exposed uterine mesenchyme has early signs of fibrosis through increased deposition of extracellular matrix collagen.Mechanistically, DES exposure causes cell type-specific changes in chromatin accessibility and gene expression, most prominently in epithelial cells. These changes can be explained in part by cell-specific alterations in chromatin looping at enhancer regions in concert with alterations in ER binding. These findings suggest that reprogramming cell type-specific differentiation trajectories and extracellular matrix characteristics underlie the long-term phenotypic effects of developmental exposure to estrogenic endocrine disrupting chemicals. These changes lead to functional impairment of adult tissues and increased cancer risk. Significance StatementUterine development is strongly impacted by brief exposure to estrogenic endocrine disruptors, but it is unclear why development is such a sensitive time point. This study employed multiomic analysis to identify cell type-specific uterine developmental trajectories in neonatal mice exposed to the estrogenic chemical, diethylstilbestrol, and compared these to controls. Control epithelium was under the influence of carefully orchestrated Wnt/{beta}-catenin signaling and was in a partial epithelial-to-mesenchymal transition state. DES exposure repressed Wnt/{beta}-catenin signaling and drove the epithelium toward full differentiation, resulting in the loss of both epithelial stem cells and normal apical-basal polarity. These changes provide an explanation for how endocrine disruptors can divert intrinsically programmed developmental trajectories to alter adult organ function.

Menopausal hormone therapy (MHT) is prescribed for climacteric symptoms including hot flushes and weight gain and contains estrogens such as 17 beta-estradiol (17{beta}E2). However, estrogen receptor activation by MHT may increase reproductive cancers and cardiovascular event risk in some people. As the protective metabolic effects of 17{beta}E2 are partly mediated through the arcuate nucleus of the hypothalamus, restricting 17{beta}E2 actions to the brain could serve as a safer mechanism of MHT. 10{beta},17{beta}-Dihydroxyestra-1,4-dien-3-one (DHED) is a prodrug of 17{beta}E2 which is enzymatically converted to the parent hormone exclusively within the brain. DHED has demonstrated positive benefit in rodent models of centrally-mediated maladies including hot flushes, depression and cognitive decline, without peripheral hormonal burden. Therefore, we hypothesized that DHED treatment in obese female mice would act within the hypothalamus to provide the same beneficial metabolic effects as 17{beta}E2. Female mice were ovariectomized, placed on a high fat diet and split into either control, DHED, or 17{beta}E2 treatment groups. Body weight, uterus weight and glucose tolerance were recorded along with gonadal hormone receptor expression in the brain. Delivery of DHED at a similar dose as 17{beta}E2 failed to improve metabolic parameters or recapitulate the hypothalamic responses induced by 17{beta}E2. Delivery of DHED at higher doses, which elicited estrogen-like actions within the brain, still failed to improve metabolic health. Our findings suggest that peripheral actions, in addition to hypothalamic targets, may be required to mediate 17{beta}E2s protective effects on metabolism and that brain-targeted MHT may be unsuitable for improving metabolic health during menopause.

The oviduct provides the dynamic microenvironment that supports fertilization and early embryo development yet replicating its hormonally regulated secretory activity in vitro remains a major challenge. Here, we established bovine oviductal epithelial organoids that reproduce the structural polarity and endocrine responsiveness of the native oviduct. Exposure to either estradiol or progesterone resulted in distinct transcriptomic and proteomic landscapes that were characteristic of the follicular and luteal phases, respectively. This included the upregulation of canonical phase-specific markers, such as OVGP1, NTS, HP and TGM2. Proteomic profiling of organoid-derived secretions (ODS) revealed extensive overlap with in vivo oviductal fluid. Integration of transcriptomic and proteomic datasets by multi-omics factor analysis identified coherent biological signatures defining each hormonal state. Functionally, ODS obtained from estradiol-treated organoids enhanced sperm capacitation and acrosome reaction, recapitulating the activity of follicular-phase oviductal fluid. These findings demonstrate that hormonally responsive oviductal organoids generate bioactive secretions that emulate the molecular and functional features of the native oviductal environment, providing a sustainable and physiologically relevant platform for studying gamete-maternal communication and improving assisted reproduction technologies.

The fallopian tube serves as a sperm reservoir, and it is the site where the oocytes become fertilized. Here, we describe development of an organ-on-a-chip microfluidic model of the fallopian tube (FT Chip) lined by primary human epithelial cells and stromal fibroblasts derived from the FT ampulla. Abundant tissue folds lined by hormone-responsive, epithelial cells resembling those seen in vivo formed on-chip, but not in epithelial organoids cultured in gel cultures. Comparative time-resolved analysis of human sperm versus oocyte-sized microparticles introduced into the epithelial channel in the presence of estradiol revealed that sperm movement was significantly reduced, while the oocyte-sized particles increased, relative to movements in acellular chips. When the non-hormonal contraceptive TDI-11861 was administered to the chip, dose-dependent inhibition of human sperm motility was detected. Thus, this FT Chip may offer a human preclinical tool to study FT physiology and assess the efficacy and mechanism of action of contraceptives.

Ferroptosis is linked to various diseases, but the role of transferrin (TF) in endometriosis (EM) remains unclear. Expression levels of ferroptosis-related proteins, including transferrin (TF), transferrin receptor (TFRC), and glutathione peroxidase 4 (GPX4), were analyzed by western blotting. Compared to normal endometrial stromal cells, eutopic and ectopic endometrial stromal cells from EM patients exhibited significantly enhanced proliferative and migratory abilities, accompanied by a marked reduction in glutathione (GSH) levels in both eutopic and ectopic tissues. TF and TFRC expression was upregulated in ectopic endometrium relative to normal controls, while GPX4 expression was downregulated. To evaluate the functional role of TF, siRNA-mediated knockdown was performed in endometrial stromal cells, with knockdown efficiency confirmed by western blotting. Functional assays demonstrated that TF knockdown not only suppressed cell proliferation (CCK-8 and clonogenic assays) and migration (wound healing assay) but also significantly increased apoptosis rate (flow cytometry with Annexin V-FITC/PI staining).These findings implicate TF in the pathogenesis and progression of endometriosis, likely through modulating endometrial stromal cell proliferation, migration, and apoptosis.

Polycystic ovary syndrome (PCOS) is a heterogenous reproductive disorder that is often associated with metabolic dysfunction, as well as comorbidities such as pregnancy complications. Although metabolic traits like hyperinsulinemia (i.e., elevated insulin without hypoglycemia) likely exacerbate the reproductive and metabolic features of PCOS, the precise impacts of specific metabolic traits on PCOS pathogenesis, symptom severity, and comorbidity incidence are not known. The aim of our study was to investigate the relationships between insulin levels, PCOS-like traits, and pregnancy complications by limiting endogenous insulin production in a mouse model of PCOS. Using Ins1-null mice with modulated Ins2 gene dosage (Ins1-/-:Ins2+/- versus Ins1-/-:Ins2+/+ littermates), we longitudinally assessed metabolic and reproductive phenotypes in PCOS-like mice generated via prenatal anti-Mullerian hormone (PAMH) exposure. We observed mild reproductive characteristics of PCOS in PAMH mice of both genotypes, including increased anogenital distances, delayed puberty, and disrupted estrous cycling, but did not detect robust PAMH-induced metabolic changes across six months. In the absence of PAMH-aggravated metabolic dysfunction or hyperinsulinemia--even in mice fed a high-fat, high-sucrose diet--reducing Ins2 gene dosage did not notably change most measured traits. However, high-fat, high-sucrose-fed PAMH pregnant dams exhibited a diminished pregnancy-induced insulinogenic response and a trend for reduced {beta}-cell mass compared to control mice, together with superior blood glucose homeostasis despite the physiological challenges of pregnancy. Therefore, while Ins1-null PAMH mice did not manifest pronounced PCOS-like metabolic features, prenatal AMH exposure can cause shifts in metabolic homeostasis during pregnancy.

Early human pregnancy is a critical period characterized by rapid growth and extensive maternal-fetal communication that influence maternal and fetal outcomes. Circulating extracellular vesicles (EVs) have the capacity to capture cargo that reflect these processes in real-time; however, signatures of EV subtypes during early pregnancy are poorly defined. Here we quantified mitochondrial DNA (mtDNA) and performed transcriptomic and proteomic profiling of small ([~]100 nm) and large ([~]200 nm) plasma EVs from n=10 normal pregnancies (11-15 weeks) to define subtype-specific molecular signatures. mtDNA and mitochondrial protein content were more abundant in large EVs (lEVs). lEVs also contained a more complex set of long RNAs enriched for placental, immune, and mitochondrial-related transcripts compared with small EVs (sEVs). Proteomic profiling showed enrichment of canonical EV markers and extracellular matrix proteins in sEVs, whereas lEVs were preferentially associated with pregnancy-specific proteins, including proteins related to placental hormone production. MicroRNAs (miRNAs) accounted for [~]25% of small RNAs in both EV subtypes with miR-223 and miR-16 enriched in lEVs and miR-639 enriched in sEVs. These data together, support a model where small and large plasma EVs have distinct, yet complementary signatures reporting systemic adaptations during the critical 11-15 week transition period. This work establishes a foundational framework for future studies linking EV signatures to placental dysfunction and adverse outcomes.

Despite the importance of inflammation to menstruation, we lack detailed understanding of how immune dynamics contribute to endometrial repair. We performed detailed phenotypic characterisation of uterine immune cells using single cell RNA sequencing, flow cytometry and multiplex immunohistochemistry in a mouse model of simulated menstruation. Uterine tissues were collected from age-matched controls or from key phases of menstruation, including tissue breakdown, repair and remodelling. Our findings reveal distinct compositional changes across different phases of menstruation and highlight predominant roles for monocytes, macrophages, and neutrophils in endometrial repair. Immunohistochemistry revealed the spatial association of these myeloid cell subsets with areas of tissue repair and remodelling. Bioinformatic analysis highlighted key roles for monocyte, macrophage and neutrophil signalling during endometrial repair with thrombospondin 1 and secreted phosphoprotein 1 emerging as key signalling pathways. These data significantly advance our understanding of menstrual physiology and identify potential therapeutic targets for menstrual disorders. summaryThis study investigates immune cell dynamics in a mouse model of menstruation, highlighting roles for monocytes, macrophages, and neutrophils in non-fibrotic endometrial repair, and identifies key signalling pathways as potential therapeutic targets for menstrual disorders.

BACKGROUNDDuring spermiogenesis, histones are exchanged by protamines (PRMs) in spermatids, which results in DNA hypercondensation and protection. Rodents and primates express two PRMs (PRM1 and PRM2) in a species-specific ratio. Maintaining this ratio is necessary for functional chromatin reorganization and alteration is associated with sub- or infertility in mice and humans. Prm1 and Prm2 deficient mice are infertile, while Prm1+/- males are subfertile showing a severely altered PRM ratio. Prm2+/- males are fertile and display a protamine ratio comparable to WT. OBJECTIVESHere, we addressed the question whether loss of one allele of Prm1 and one allele of Prm2 affects fertility. MATERIAL AND METHODSDouble heterozygous (dHET) mice lacking one allele of Prm1 and one allele of Prm2 were generated and analyzed RESULTSdHET males were infertile with sperm showing retention of histones and TNPs, high levels of PRM2 precursor and decreased levels of mature PRM2. In mature sperm the PRM ratio and the total PRM content was not altered. However, CMA3 staining revealed incomplete protamination and sperm nuclei appeared more rounded and slightly bigger, suggesting impaired DNA-hypercondensation. In dHET sperm, DNA degradation was apparent, but to a lower level compared to sperm from Prm1 and Prm2 deficient males. Increased 8-OHdG levels suggested oxidative stress in the epididymis of dHET mice. However, a fraction of dHET sperm were capable of fertilization, with embryonic development up to 8-cell stage. DISCUSSION AND CONCLUSIONThese results suggest, that male factor infertility might not be reliably detected by measuring PRM1/PRM2 ratio but rather by determining the level of protamination by e.g. CMA3 analysis and pre-PRM2 retention.

Artificial ovarian scaffolds represent a promising therapeutic strategy for preserving reproductive health in patients. However, current in vitro approaches are limited by inadequate biomimicry of the native tissue microenvironment, leading to poor development of in vitro ovarian models. In this study, we developed region-specific hydrogel scaffolds incorporating solubilized decellularized ovarian extracellular matrix (dECM) with mechanically tuned properties to enhance the functionality of engineered 3D ovarian models. Ovine ovarian dECM was isolated by mechanical and chemical decellularization methods and subsequently solubilized and incorporated in varying concentrations in homogenous alginate (0.5%) and a composite mixture of 1% gelatin with 0.5% alginate (1:1). The synthesized hydrogels were characterized for rheological properties, including Youngs modulus, pore size, and viscosity, and cytocompatibility assays were conducted using Chinese hamster ovary (CHO) cells. The study demonstrated that both 0.5% alginate and the composite gelatin-alginate hydrogels successfully replicated the mechanical properties of native human ovarian cortical and medullary tissue, with Youngs modulus of 0.84 {+/-} 0.16 kPa, pore size (60-150 nm), and toughness of 0.4Pa, respectively. Zonal hydrogel scaffolds incorporating ovarian dECM demonstrated significantly enhanced cell viability compared to hydrogels supplemented with dECM. The study emphasises the critical role of integrating both mechanical and biochemical attributes while developing functional artificial ovarian constructs for transplantation and regenerative medicine applications. This work contributes to advancing strategies for creating physiologically relevant in vitro models of ovarian tissue.

The association between sub-optimal paternal diet and offspring well-being is becoming established. However, the underlying mechanisms are yet to be fully defined. The aim of this study was to establish the impact of over- and under-nutrition, with or without macronutrient supplementation, on male reproductive fitness and post-fertilisation development. Male C57/BL6J mice were fed either control diet (CD), isocaloric low protein diet (LPD), high fat/sugar Western diet (WD) or LPD or WD supplemented with methyl-donors and carriers (MD-LPD or MD-WD respectively) for 8 weeks before mating with virgin C57/BL6J females. Placental tissue was collected at embryonic day (E)8.5, to assess early placental (ectoplacental cone) morphology and metabolism and E17.5 for sex-specific transcriptomic profiling. Post-mating, stud male tissues were harvested for assessment of testicular morphology and gene expression, gut microbiota composition and metabolic status. WD and MD-WD males displayed increased adiposity, hepatic cholesterol and free fatty acids and gut microbiota dysbiosis when compared to CD fed males. In the testes, WD and MD-WD perturbed the expression of genes associated with metabolism and transcription regulation. Additionally, we observed differential expression of multiple genes within the Wnt signalling pathway, central in the regulation of cellular proliferation, migration, survival, and cell fate determination during development. Despite no impact on fundamental male fertility, significant changes in ectoplacental cone metabolism, fetal growth, and placental gene expression were observed in response to specific dietary regimens. Interestingly, while CD male and female placentas displayed 301 genome-wide, sexually-dimorphic genes, LPD, MD-LPD, WD and MD-WD male and female placentas possessed only 13, 0, 14 and 15 sexually-dimorphic genes respectively. Our data show that while sub-optimal paternal diet has minimal impact on male fertility, fetal and placental development are perturbed in a sex-specific manner.

The endometrium, the inner lining of the uterus, is a dynamic tissue that undergoes precise molecular and structural changes to achieve a receptive state capable of supporting embryo implantation. Although the uterine environment was long considered sterile, molecular studies have detected microbial signals and bioactive compounds that may influence endometrial function. Endometrial epithelial organoids (EEOs) provide a three-dimensional in vitro model that recapitulates the architecture, polarity, and hormonal responsiveness of native endometrial tissue. This study aimed to elucidate how bacterial-derived compounds, including D-lactate (D-lac), commonly associated with Lactobacillus communities, and lipopolysaccharides (LPS), a component of Gram-negative bacteria, affect the transcriptomic profile of the endometrial epithelium under a hormonally induced receptive state. EEOs were exposed to different concentrations of these compounds, and relative metabolic activity was monitored through resazurin-based assays, revealing no significant alterations across the conditions tested. Transcriptomics analysis of hormonally stimulated EEOs, mimicking the mid-secretory phase, revealed that D-lac modulated genes related to epithelial development, tissue remodelling and growth regulation, whereas LPS influenced genes associated with inflammatory signalling and immune response. While key markers of receptivity remained largely stable, small transcriptional changes suggest that microbial signals may modulate the functional balance of the receptive endometrium. These findings highlight a modulatory role of microbial signals on endometrial epithelial function and demonstrate that EEOs are a robust platform for exploring host-microbe interactions in the uterus, offering new insights into the mechanisms underlying uterine receptivity.

1.BackgroundHuman infertility affects approximately 17.5% of the global population, with male factors accounting for nearly half of all cases. The identification of reliable molecular biomarkers is crucial for improving the diagnosis and assessment of male fertility. In this study, we developed and optimized an untargeted high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) workflow for comprehensive lipidomic and metabolomic profiling of human spermatozoa using only 1.25 million cells per sample. ResultsCompared to previous reports, our optimized method achieved unprecedented analytical depth, identifying 473 lipid species and 955 structurally annotated metabolites, corresponding to nearly 7.600-fold improvements in detection efficiency per cell over published approaches. Lipidomic analysis revealed cholesterol, fatty acids, phosphatidylcholines, and phosphatidylethanolamine plasmalogens as the most abundant lipid classes, consistent with the structural complexity of the sperm plasma membrane. Metabolomic profiling showed strong enrichment of lipid-related and steroidogenic pathways, including phospholipid biosynthesis, glycerolipid metabolism and androgen and estrogen metabolism. The integration of lipidomic and metabolomic data highlighted functionally interconnected pathways related to membrane dynamics, energy metabolism, and hormone biosynthesis. ConclusionsOverall, this work establishes a robust, sensitive, and scalable analytical framework enabling high-coverage molecular characterization of spermatozoa from limited sample material, laying the groundwork for future biomarker discovery and clinical applications in male infertility research. One Sentence SummaryDevelopment of a highly sensitive untargeted HPLC-ESI-MS/MS lipidomic and metabolomic workflow that achieves unprecedented molecular coverage from only 1.25 million human spermatozoa, revealing interconnected lipid and metabolic pathways and providing a robust foundation for biomarker discovery in male infertility. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/703749v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@10b3132org.highwire.dtl.DTLVardef@1caf850org.highwire.dtl.DTLVardef@746adborg.highwire.dtl.DTLVardef@1135539_HPS_FORMAT_FIGEXP M_FIG C_FIG

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