Biology of Reproduction

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.

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.

Equine endometrosis is a major cause of subfertility in mares characterized by fibrotic remodeling of the endometrium. Although transforming growth factor beta 1 (TGF-{beta}1) is implicated in fibrogenesis, the relationship between endometrosis severity and transcripts associated with tissue maintenance and proliferation remains incompletely defined. Present study evaluated endometrial mRNA expression of IGF1, MKI67, TGFB1, and ACTA2 in relation to endometrosis severity and defined histopathological features. Forty-seven endometrial samples were graded according to the modified Kenney and Doig (KD) categories. Relative mRNA expression was quantified by RT-qPCR and histopathology was extended using a standardized feature-based assessment. TGFB1 mRNA expression was higher in category I+ than in categories I and III (p = 0.041) and in samples with glandular basal lamina disruption (p = 0.020). MKI67 mRNA expression was lower in samples with luminal epithelial erosion (p = 0.049). IGF1 mRNA expression correlated negatively with KD category ({rho} = -0.401, p = 0.015), glandular degeneration ({rho} = -0.340, p = 0.043), overall inflammatory infiltration ({rho} = -0.387, p = 0.020), lymphocytic infiltration ({rho} = -0.426, p = 0.010), and neutrophilic infiltration ({rho} = -0.448, p = 0.006). MKI67 correlated positively with ESR1 ({rho} = 0.887, p < 0.001). These findings indicate that early endometrosis-compatible lesions are associated with increased TGFB1 transcription and that epithelial damage is accompanied by reduced MKI67 expression. The inverse associations between IGF1 expression and both lesion severity and inflammatory infiltration support a link between progressive histopathological changes and reduced expression of a growth factor involved in tissue maintenance.

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.

Polycystic ovary syndrome (PCOS), a common cause of infertility, is marked by persistently high luteinizing hormone (LH)-pulse frequency, presumably driven by high-frequency GnRH pulses. Prenatally androgenized (PNA) mice mimic neuroendocrine PCOS symptoms including high LH-pulse frequency. GnRH neurons from adult PNA mice have a higher firing rate than those from vehicle (VEH) mice; this is reversed in prepubertal mice despite more excitatory inputs at both ages. We hypothesized voltage-gated Ca2+ currents (ICa) help set intrinsic excitability of GnRH neurons and are altered by development and/or PNA treatment. Whole-cell patch-clamp recordings were used to measure GnRH neuron ICa in 3wk-old and adult VEH and PNA mice. PNA treatment increased ICa density and depolarized the ICa-half inactivation potential at both ages. In VEH but not PNA mice, the Ca2+-half activation potential was depolarized in adults versus 3wks. Age decreased the inactivation rate of a fast ICa regardless of PNA treatment. GnRH neuron firing rate during current injections was higher at 3wks than in adulthood in VEH mice only. Blocking small-conductance Ca{superscript 2}-activated K current with apamin increased GnRH neuron firing rate except in adult PNA mice. Apamin changed the post-spike-train membrane response from hyperpolarization to depolarization; during development, this net effect of apamin became smaller in PNA mice. In summary, while GnRH neurons from PNA mice have increased ICa, they lack some developmental changes in ICa kinetics and intrinsic excitability observed in VEH mice. Ca{superscript 2}-activated K currents are less prominent in GnRH neurons from adult PNA mice, perhaps contributing to increased spontaneous firing. Significance statementHyperactivation of GnRH neurons, which control reproductive endocrine function, can lead to increased LH-pulse frequency and is a hallmark of hyperandrogenemia polycystic ovary syndrome (PCOS). We used a mouse model of prenatal androgenization (PNA) that recapitulates the neuroendocrine aspects of PCOS to test the role of calcium currents (ICa) in the PNA phenotype and the typical pubertal process. PNA treatment increased ICa in GnRH neurons both before and after puberty. Calcium plays a crucial role in neurosecretion thus this may enhance GnRH release. Another role of calcium is activation of calcium-sensitive potassium currents, which tend to decrease action potential firing rate. Despite increased ICa, calcium-activated potassium currents are less effective in adult PNA mice, perhaps contributing to GnRH neuron hyperactivation.

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 [&le;] 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 [&le;] 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.

BackgroundUnderstanding the causes of the exceptional rate of evolution of the mammalian placenta is likely to aid the understanding of placental development and the aetiology of the human-specific pregnancy disorder pre-eclampsia (PE). As retroelements (REs) are often lineage-specific and known to be co-opted for placental functioning, here we consider the RE binding of GATA3 and DLX5, these transcription factors being dysregulated in PE, and their downstream consequences. MethodsMultiomics analyses identified the retroviral regulatory sequence LTR8B in the PSG gene array, as a contributor to expression diversification in the placenta. To characterize this genomic domain, we performed copy number variation analysis and whole-genome sequencing. Multiomics data was employed to identify loci that might act as an active chromatin loop boundary around the PSG region. CRISPR-Cas9 knockouts with aligned RNAseq and epigenetic mark data tested for trophoblast-specific cis-regulatory elements (CREs-enhancer and/or promoter sequences) of resulting loci. Functional assays were employed to characterize the phenotypic effects of a candidate locus. Structural analysis of PSG family members also identified an additional RE, MER65-int. RNA-seq and antibody staining was employed to consider polyadenylation and functional diversification. ResultsThe LTR8B CRE facilitates the binding of transcription factors (e.g., GATA3, DLX5, TFAP2A/C), resulting in a diversified PSG gene expression pattern within a primate-specific genomic region that exhibits high intraspecies variability. The LTR8B/PSG9 regulatory element influences other PSG family members. PSG9, unique among PSGs, produces both secreted and membrane-anchored isoforms, MER65-int providing alternative polyA signals, enabling the evolution of secreted PSG variants through the truncation of the ancestral CEACAM proteins transmembrane domain. The LTR8B/PSG9 locus regulates the differentiation of multinucleated trophoblasts (syncytialization) and, like chorionic gonadotropin and syncytin1, determines the identity of syncytiotrophoblasts. Notably, PSG9 is the most upregulated PSG in PE, with levels correlated with GATA3 and DLX5 levels. ConclusionsREs contribute to the structural and expression evolution of PSG genes, facilitating lineage-specific placental evolution. The LTR8B/PSG9 regulatory network plays a central role in syncytiotrophoblast differentiation. Given the association between DLX5/GATA3 dysregulation and elevated PSG9 levels, along with PSG9s expression in the first trimester, PSG9 shows potential as a predictive biomarker for PE.

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.

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.

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.

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.

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.

The signaling pathways that control embryonic development, migration, and differentiation of gonadotropin-releasing hormone (GnRH) neurons, as well as the postnatal fate, function, and survival of differentiated cells, are the subject of ongoing research. Here, we examined the role of phosphoinositides in this complex multistep process by generating GnRH neuron-specific phosphatidylinositol 4-kinase alpha knockout mice. These mice were healthy and indistinguishable from their control littermates in size. However, adult knockout females and males were infertile due to underdeveloped gonads and reproductive organs. Furthermore, hypothalamic GnRH immunoreactivity was absent, and expression of the hypothalamic Gnrh1 gene and pituitary gonadotroph-specific genes was reduced. In contrast, hypothalamic kisspeptin immunoreactivity was preserved, and Kiss1 expression was modified in a nuclei specific-manner, consistent with the loss of circulating sex steroid hormones. Embryonic neurogenesis and migration of GnRH neurons were not impaired, as evidenced by normal Gnrh1 expression in the hypothalamus of neonatal animals and the presence of immunoreactive GnRH neurons in infantile mice in comparable number and distribution to age-matched controls. However, their cellular degeneration was evident, accompanied by reduced Gnrh1 expression. GnRH neuron-specific tdTomato expression confirmed their postnatal degeneration and death, whereas ectopic tdTomato cells located in the lateral septum remained unaffected. Together, these findings indicate that phosphoinositides dependent on phosphatidylinositol 4-kinase alpha activity are not critical for embryonic steps in the development of the GnRH neuronal network, but are essential for the postnatal function and survival of these cells. Significance StatementDifferentiation of neuroendocrine GnRH cells involves neurogenesis in the olfactory placodes, migration to the hypothalamus, projection to the median eminence, and connections with upstream neurons, including kisspeptin neurons. Here we show that knockout of phosphatidylinositol 4-kinase alpha in GnRH neurons does not affect these strps of embryonic development. However, the activity of this enzyme is essential for postnatal survival of GnRH neurons; in the absence of this gene, the neurons die, causing infertility in both female and male mice.

BackgroundLow-dose aspirin (LDA) reduces preeclampsia (PE) risk by up to 40%, yet its molecular effects on chorion trophoblast cells (CTCs) a fetal membrane lineage at the feto-maternal interface remain obscure. CTCs form a structural and immunoregulatory barrier whose dysfunction drives inflammation-associated membrane pathology in PE. Extracellular vesicles (EVs) released by CTCs may encode cellular stress and adaptation states, offering a molecular window into aspirins timing-dependent effects on PE risk modification. MethodsHuman CTCs were challenged with cigarette smoke extract (CSE) to model oxidative stress-driven PE pathology. Two paradigms were tested: (1) prophylactic aspirin (4 and 40 {micro}g/ml) before and/or flanking CSE, and (2) therapeutic aspirin after CSE challenge. EVs were isolated via ultracentrifugation and size exclusion chromatography, characterized by nanoparticle tracking and immunoblotting, and profiled by quantitative mass spectrometry. Network pathway analysis and machine-learning biomarker selection defined EV-encoded molecular states. ResultsCTC-derived EVs from CSE-exposed cells carried a PE-like proteomic signature marked by suppressed VEGF/ECM remodeling, activated TNF-p53 apoptotic signaling, and heightened inflammation. Prophylactic low-dose aspirin shifted EV cargo toward preserved angiogenic capacity (VEGFA, COL1A1, MMP14) with attenuated apoptotic and NF-{kappa}B signatures. High-dose aspirin produced broad transcriptional suppression without vascular benefit in EVs. Therapeutic aspirin partially rescued injury-associated EV cargo but failed to restore angiogenic signatures. Machine-learning analysis of EV proteomes identified a prophylactic biomarker panel anchored by HSPA8, SERPINF2, COL4A1, and PLOD1, linked to angiogenic recovery and redox balance. ConclusionsCTC-derived EV proteomic signatures capture dose-and timing-dependent aspirin effects, positioning the chorion as a pharmacological "secondary responder" favoring cellular resilience over classical anti-inflammatory suppression. EV-based molecular profiling might offer a framework for stratifying aspirin responders from non-responders toward personalized PE prevention.

The human fallopian tube plays a critical role in reproductive biology, yet the structural organization and immune repertoire of this tissue remain incompletely characterized. Here, we performed an in-depth analysis of human fallopian tube tissue from women of reproductive age across three distinct anatomical regions (isthmus, ampulla, and fimbriae) across the menstrual cycle. Using antibody-based imaging for EPCAM, CD8A, and CD20 together with automated image analysis, the epithelial thickness and spatial distribution of T and B lymphocytes was assessed. No significant differences in epithelial thickness were observed between proliferative and secretory phases within any tubal region. In contrast, significant regional differences were identified, with the epithelium being thickest in the isthmus and thinnest in the ampulla. Both CD8A+ T lymphocytes and CD20+ B lymphocytes were detected throughout the fallopian tube, and a strong correlation between T and B lymphocyte abundance was observed across patients. Spatial analysis further revealed that both lymphocyte populations were preferentially localized within the mucosal compartment adjacent to the lumen. Notably, intraepithelial B lymphocytes were identified throughout the fallopian tube. Together, these findings provide new insight into epithelial organization and immune cell distribution in the human fallopian tube, highlighting the complexity of the tubal immune microenvironment and its potential relevance for reproductive biology.

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.

BackgroundPlacental growth and function are imperative for healthy fetal growth; data on placentas can inform research and clinical care. Measuring placental size after delivery should be easy, but current methods are hard to standardize and error prone. We developed PlacentaVision using artificial intelligence (AI)-based models, to automatically, accurately, and precisely measure placentas from digital photographs. ObjectiveWe aimed to compare placental disc morphology between gross pathology examination (human measurements) and our automated PlacentaVision model (AI measurements). MethodsPlacentaVision is a multi-site study to assess placental morphology, features, and pathologies from digital photographs. We built a large dataset of digital placenta photographs and clinical data from singleton births at three large hospitals: Northwestern Memorial (Chicago; n=24,933), UPMC Magee-Womens (Pittsburgh; n=1198) and Mbarara Regional Referral (Uganda, n=1715). Data and images were from the medical record for Northwestern, part of a biobank study for Magee, and from our prospective studies for Mbarara. We compared long and short disc axis length (defined by Amsterdam criteria) between human and AI-based PlacentaVision measurements by calculating the difference and using Bland-Altman; we stratified by site, disc shape, infant sex, and term/preterm birth. ResultsMean (SD) disc length was 19.2 (3.1) and 18.6 (3.1) cm from PlacentaVision and human measurement, respectively, with a difference of 0.57 (2.19) cm. Disc width was 16.3 (2.3) cm and 16.1 (2.4) cm from PlacentaVision and human measurement, respectively, with a difference of 0.25 (1.85) cm. Bland-Altman limits of agreement were -3.7 to 4.9 cm for length and -3.4 to 3.9 cm for width. Irregularly-shaped placentas had a greater difference between PlacentaVision and human measurements compared to those with round/oval shapes (length differences of 1.53 and 0.45 cm respectively). Further, there were length differences by site (Northwestern 0.6, Magee 0.0, and Mbarara 0.4) and gestational age at birth (preterm 0.71, term 0.53 cm), but similar results for male and female placentas. Results for width were similar to length. ConclusionsAI-based measurements were less than a cm from human measurements overall. Our findings of larger differences for irregular shapes and preterm may indicate it is difficult for humans to measure irregular or small placentas according to protocol. PlacentaVision can automate and standardize the process.

IntroductionNon-obstructive azoospermia (NOA) represents the most severe form of male infertility. Current clinical tools have limited ability to predict sperm production or guide surgical sperm retrieval. Conventional B-mode ultrasound provides qualitative grayscale images and cannot characterize testicular microstructure relevant to spermatogenesis. Quantitative ultrasound (QUS) provides objective parameters from raw radiofrequency data, which quantitatively measure tissue heterogeneity. We hypothesize that men with spermatogenesis will have different QUS features compared to men without spermatogenesis (measured by total motile count, TMC, on semen analysis), with the goal of identifying imaging biomarkers for prognosis and intraoperative guidance. MethodsWe prospectively analyzed men presenting for infertility evaluation who underwent high-frequency ultrasound imaging and semen analysis. Imaging was performed using a 36-MHz transducer with fixed acquisition parameters. Ninety-two QUS features were extracted from manually annotated testicular regions of interest, including Nakagami distribution parameters (m, {omega}, k), envelope statistics, and texture features. Univariate associations between each QUS feature and TMC were assessed using Spearman correlation with Bonferroni correction. Top-performing features were evaluated using logistic regression and receiver operating characteristic (ROC) analysis to discriminate sperm presence or absence (TMC>0 vs TMC=0). ResultsThirty-seven men (18 azoospermic, 19 with sperm present in the ejaculate) contributed 135 regions of interest. Seventeen of 92 QUS features significantly correlated with TMC after correction. The coefficient of variation of the Nakagami k-factor within the superficial testicular parenchyma (K_Zone1_Cv) demonstrated the strongest correlation ({rho}=0.51, corrected p<0.001), suggesting that greater spatial heterogeneity in the superficial parenchyma was associated with higher sperm counts. K_Zone1_Cv discriminated sperm presence with an AUC of 0.77 (95% CI 0.60-0.92), sensitivity 73.7%, and specificity 83.3%. QUS features with the highest univariate association were highly intercorrelated, suggesting a shared biological signal. ConclusionQuantitative ultrasound-derived measures of testicular microstructure heterogeneity correlate with sperm production and demonstrate moderate discrimination of sperm presence. These findings suggest QUS may provide a non-invasive imaging biomarker of spermatogenesis. Study findings warrant further assessment and validation in male infertility for sperm retrieval prognosis and the potential for intra-operative surgical guidance.

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.

The population of kisspeptin neurons located in the rostral periventricular area of the third ventricle (RP3V) is thought to have a key role in generating the GnRH surge that triggers ovulation. Using a modified GCaMP fibre photometry procedure, we have been able to record the in vivo population activity of RP3VKISS neurons across the estrous cycle of female mice. A marked increase in GCaMP activity was detected beginning on the afternoon of proestrus that lasted in total for 13{+/-}1 hours. This was comprised of slow baseline oscillations with a period of 91{+/-}4 min and associated with high frequency rapid transients. Very little oscillating baseline or transient activity was detected at other stages of the estrous cycle. Concurrent blood sampling showed that the peak of the LH surge occurred 3.5{+/-}1.1 h after the first baseline RP3VKISS neuron baseline oscillation on the afternoon of proestrus. The time of onset of RP3VKISS neuron oscillations varied between mice and across subsequent proestrous stages in the same mice. To assess the impact of estradiol on RP3VKISS neuron activity, mice were ovariectomized and given an incremental estradiol replacement regimen. Minimal patterned GCaMP activity was found in OVX mice, and this was not changed acutely by any of the estradiol treatments. However, on the afternoon of the expected LH surge, the same oscillating baseline activity with associated transients occurred for 7.1{+/-}0.5 h. These observations reveal an unexpected prolonged oscillatory pattern of RP3VKISS neuron activity that is dependent on estrogen and underlies the preovulatory LH surge as well as potentially other facets of reproductive behavior.