Androgen receptor (AR) signalling is central to male sexual differentiation. Notably, however, fetal testis formation and androgen-dependent masculinization are temporally and mechanistically distinct processes that are not always clearly differentiated in developmental and toxicological interpretations. Evidence from human androgen insensitivity syndromes (AIS) and rodent genetic models demonstrate fetal testis formation proceeds independently of AR, whereas downstream masculinization is AR dependent. Across mammals, early testis differentiation is driven by intrinsic programs that establish Sertoli cells, organize testis cords, and specify fetal Leydig cells in the absence of AR activity. Consistently, mouse Ar-knockout models show persistent fetal testis formation, and individuals with complete androgen insensitivity syndrome (CAIS) develop structurally intact testes that produce normal or elevated testosterone despite absent virilization. These conserved outcomes reflect shared developmental timing, as key morphogenetic events occur before AR is expressed in Sertoli or fetal Leydig lineages in both rodents and humans. By contrast, AR-mediated androgen signalling becomes essential once androgens from the fetal testes begin to act on peripheral target tissues, governing masculinization of external genitalia, testis descent, and postnatal maturation of somatic testis compartments, as demonstrated by graded AIS phenotypes and cell-specific Ar ablation in mice. This developmental uncoupling explains why disruption of androgen signalling still allows establishment of early testis architecture yet predictably impairs downstream male reproductive development. Recognizing this distinction is crucial for interpreting human genetic disorders, aligning rodent models with human biology, but also to refine toxicological frameworks to target androgen-dependent windows of vulnerability.

The regulation of gene expression by microRNAs (miRNAs) is essential during development. However, it is necessary to move beyond individual miRNAs to understand their functions and the intricate interplay as a coordinated system. To address this, we developed and implemented a network approach named miRNA-gene synergic co-regulatory networks. This method builds networks identifying pairs of genes jointly regulated (co-regulation) by multiple miRNAs (synergism). Using these networks, focusing on placental genes, we evaluated the coordinated regulation of miRNAs from different origins (maternal circulating extracellular vesicles or placental tissue) and at different times (increased expression in the first or third trimester of pregnancy) using both public and original data. We observed that synergic co-regulatory networks displayed properties compatible with scale-free topology. When comparing the co-regulation networks produced by miRNAs differentially expressed at distinct gestational periods, we identified that miRNAs from the maternal circulation were more coordinated at the first trimester, while the miRNAs from the placental tissue were more coordinated at the third trimester. Regarding maternal circulating miRNAs in the first trimester, we identified a co-expression module that was negatively associated with birth weight. This module, together with other miRNAs expressed at higher levels in the first trimester, regulates TGF-β signaling pathways that are important for uteroplacental circulatory development. Overall, the results of this study reinforce the idea that miRNAs, whether associated with extracellular vesicles in maternal circulation or derived from placental tissue, constitute a regulatory system with dynamic coordination throughout pregnancy.

The formation of N6-methyladenosine (m6A) is the most prevalent internal RNA modification in eukaryotic transcripts and plays an essential role in spermatogenesis. PRRC2A is a m6A reader that is indispensable for meiotic progression and is essential for maintaining spermatocyte identity during mouse spermatogenesis. However, its contribution to human spermatogenesis remains unknown. In this study, we investigated a man diagnosed with non-obstructive azoospermia (NOA) and two with severe oligozoospermia, all carrying biallelic PRRC2A variants. All patients exhibited impaired spermatogenesis. the patient with NOA showed marked arrest at meiotic metaphase I or delayed entry into metaphase. RNA-seq and m6A-seq data demonstrated that his PRRC2A deficiency was accompanied by altered chromatoid body-associated RNA regulatory architecture and affected histone-to-transition protein exchange during spermiogenesis. Despite the presence of PRM2-positive spermatids, testicular spermatozoa retrieved from the NOA patient failed to achieve fertilization via TESE-ICSI, possibly due to impaired DNA integrity, arising from a perturbed histone-to-protamine transition. In the remaining cases, repeated ART attempts resulted in fertilization failure or early embryonic arrest. The successful pregnancy was ultimately achieved only through donor sperm IVF-ET. The concordance between molecular defects and consistently poor fertilization outcomes across independent individuals supports the pathogenic role for PRRC2A deficiency. We suggest that the disruption of an m6A reader compromises meiotic progression, as well as sperm functional competence and fertilization capacity, although the limited sample size precludes formal statistical inference. By linking epitranscriptomic dysregulation to adverse ART outcomes, this study expands the molecular basis of severe male factor infertility and has direct implications for genetic diagnosis, prognosis of fertilization potential, and reproductive counselling.

Infertility represents a major global health challenge and has driven increasing reliance on ART. However, fertilization success in vitro is often limited when physiological egg-sperm interaction is preserved, largely due to premature hardening of the zona pellucida (ZP), the extracellular matrix surrounding the oocyte that controls sperm access. ZP hardening is physiologically triggered after fertilization by ovastacin-mediated cleavage of ZP protein 2 (ZP2) to protect the early embryo. However, if not inhibited prematurely, this cleavage reduces egg competence. In vivo, ovastacin is restrained by the plasma protein fetuin-B, largely absent in IVF media. Whether this process can be pharmacologically targeted has yet to be explored. Here, we identify and functionally characterize small-molecule hydroxamate inhibitors that transiently inhibit ovastacin in murine oocytes and evaluate their ability to control fertilization competence. A lead compound preserved ZP2 integrity and fertilization rates at 86% (±1%) after 8 h of in vitro culture, compared to 57% (±7%) in controls. Importantly, inhibitor-treated embryos developed to the blastocyst stage without abnormalities. These findings establish transient ovastacin inhibition as simple, non-invasive pharmacological strategy to ensure IVF outcomes while preserving physiological egg-sperm interaction mechanisms.

Preeclampsia is a pregnancy complication associated with de novo gestational hypertension, proteinuria, and dysregulations in the coagulation and fibrinolytic systems. Platelet-derived factors include various growth and differentiation factors, such as transforming growth factor (TGF)-β. The TGF-β type III receptor (TGFBR3), also known as betaglycan, is a co-receptor for the TGF-β superfamily and an important regulator of reproduction and fetal development. Here, we test the hypothesis whether or not TGFBR3 is involved in platelet-mediated upregulation of placental plasminogen activator inhibitor-1 (PAI-1). Perivillous fibrin deposits and PAI-1 levels were analyzed via immunohistochemistry, gene expression as well as protein expression in healthy and preeclamptic placental tissue. Placental villi and trophoblast cell line BeWo were incubated in presence or absence of washed platelets from pregnant women, platelet-derived soluble factors, or platelet-derived extracellular vesicles. Additionally, BeWo cells were treated with TGF-β-receptor inhibitors and silenced for TGFBR3 expression. PAI-1 and its encoding gene SERPINE1, as well as perivillous fibrin deposits, were significantly increased towards term of gestation and showed a significant upregulation in placental villi from preeclamptic pregnancies. Furthermore, SERPINE1 and PAI-1 were substantially upregulated in response to washed platelets and platelet-derived factors, but not to platelet-derived extracellular vesicles. The upregulation of SERPINE1 was blocked to control levels with the TGF-β inhibitor P144, whereas silencing of TGFBR3 had no effect. Our results suggest that platelet-derived factors induce placental PAI-1 expression independently of TGF-β receptor 3. Increased maternal platelet activation may contribute to significantly upregulated placental PAI-1 and perivillous fibrin deposits in preeclampsia.

The inaugural European Ovary Workshop (EOW 2025), that was held on 17-20 February 2025, in Lagos, Portugal, brought together 165 scientists, clinicians, and early career researchers from 28 countries to advance ovarian biology from fundamental mechanisms to clinical translation. The program featured 12 scientific sessions, 17 keynote lectures, 15 selected short talks, 89 flash talks, and >90 posters spanning meiosis, folliculogenesis, ovarian reserve, aging, microenvironment, bioengineering, fertility preservation, environmental toxicology, wildlife conservation, patient-public involvement in science, and ethics. A comprehensive networking program combined formal and informal activities to stimulate scientific exchange and interaction across disciplines and career stages. Post-workshop feedback (n = 86) reflected high satisfaction with scientific content (rated 4.6/5), speaker quality (4.7/5), and networking (4.4/5), while identifying opportunities to expand clinical topics, diversify model organisms, and improve poster logistics. EOW 2025 established a unique community and a biennial platform to accelerate discovery and translation in ovarian research.

Decidualization of endometrial stromal cells is a critical process for establishing uterine receptivity and successful embryo implantation, involving coordinated biochemical, cellular, and biomechanical remodeling. Emerging evidence indicates that this process is associated with activation of endoplasmic reticulum stress (ERS) and the development of distinct stromal subpopulations, including mature and senescent decidual cells. However, how these pathways integrate to regulate the mechanical properties of the endometrium remains unclear. Here, we investigated the impact of decidualization dysregulation on stromal cell nanomechanics and its contribution to implantation failure. Our first approach used an in vitro decidualization model of human endometrial stromal cells (cell line), in which an excessive ERS was induced by thapsigargine. Then, decidualization, senescence and unfolded protein response were evaluated by molecular analyses, cellular nanomechanical properties by atomic force microscopy, and decidual functionality with a blastocyst-like spheroid implantation assay (using Swan71 cells). Decidualization induced a significant decrease in cellular stiffness (Young's modulus), consistent with a more permissive phenotype for embryo implantation. In contrast, exacerbated ERS-induced prior to decidualization-disrupted the balance between mature and senescent decidual cells and prevented this biomechanical softening, restoring stiffness to levels comparable to those on nondecidualized cells. Functionally, these alterations impaired trophoblast adhesion and expansion in vitro. Finally, to provide a physiologically relevant view of how decidualization operates in vivo, we studied endometrial samples from fertile women (N = 13) and from women with recurrent implantation failure (N = 11). These patients exhibited reduced expression of key decidualization and senescence markers evaluated by qPCR, similar to our results with exacerbated ERS, supporting the clinical relevance of our findings. Together, our results demonstrate that successful decidualization requires tight coordination between ERS, cellular senescence, and biomechanical remodeling. Disruption of this integrated program leads to a mechanically nonpermissive endometrium, providing a novel mechanistic framework for implantation failure.

Reproductive aging in females is characterized by the irreversible depletion of ovarian follicles, yet the structure and function of the post-reproductive ovary remain poorly defined. Using paired histological and bulk transcriptomic analyses of ovaries from reproductively young (2 m), reproductively old (18 m), and post-reproductive (24 m) mice, we mapped how ovarian identity evolves beyond follicle exhaustion. As expected, follicle loss, stromal remodeling, and increased collagen deposition were observed in the reproductively old and post-reproductive cohorts. Transcriptomic analyses revealed a shift from reproductive functionality to an immune-dominant signature with age. Correspondingly, post-reproductive ovaries exhibited increased infiltration of T cells, macrophages, and multinucleated giant cells. Although old and post-reproductive ovaries diverged substantially from young ovaries, they also showed discrete transcriptomic differences, indicating that the ovary continues to undergo molecular changes after reproductive senescence. Lastly, age-dependent changes in ovarian factors that are predicted to be secreted suggest that the post-reproductive ovary could be a source of pro-inflammatory signaling mediators with the potential to modulate extra-ovarian tissues. These findings challenge the assumption that the post-reproductive ovary is inert, instead indicating that it acquires an immune identity with potential endocrine and paracrine influence on whole-body aging.

Particulate matter (PM) exposure has been increasingly linked to metabolic and reproductive disorders, yet the mechanisms through which PM-related metabolic alterations influence endometrial function remain incompletely understood. In this study, female C57BL/6 mice were fed a normocaloric diet or high-fat diet (HFD) with or without PM exposure for 12 weeks. Body weight, fertility outcomes, and molecular alterations were assessed in visceral adipose tissue (VAT) and endometrial tissue (EMT). In parallel, 3T3-L1 adipocytes were co-cultured with primary human endometrial cells to examine adipocyte-endometrial interactions in vitro. PM exposure was associated with attenuated overall weight gain but increased visceral adiposity and reduced fertility, particularly under HFD conditions. In both VAT and EMT, PM exposure was accompanied by increased expression of pro-inflammatory cytokines and elevated levels of p-NF-κB and p-c-jun. Alterations in adipokine profiles were observed, characterized by increased leptin and decreased adiponectin expression with PM exposure, particularly after the HFD. Hormone receptor expression displayed tissue-specific patterns, with higher AR and ER-α/ER-β ratios in VAT and opposite trends in EMT. Markers of cellular stress and apoptosis were also elevated in PM-exposed groups. In the co-culture model, PM exposure was associated with increased levels of p-Nrf2, p-ERK, and p-p38, together with altered leptin and adiponectin expression levels. Taken together, these findings suggest that PM exposure, particularly in the context of diet-induced obesity, may be associated with dysregulation of the adipose-endometrial crosstalk involving inflammation, oxidative stress-related signaling, and hormonal imbalance, which could contribute to adverse reproductive outcomes.

In regenerative medicine, several attempts have been made to produce functional de novo gametes from mouse embryonic stem cells by utilizing three-dimensional (3D) culture systems. We attempted to perform neogametogensis in a novel 3D niche to generate neo-gametes ready to be used for insemination. Mouse embryonic stem cells were initially cultured on a gelatin-coated 6-well plate with a monolayer of fibroblasts before being encapsulated in sodium alginate spheres. These spheres were then immersed in specially formulated epiblast-like cell followed by primordial germ cell-like cell medium to promote differentiation into germ-like cells. Over the course of differentiation, immunofluorescence analysis revealed consistent expression patterns of spermatogenic markers. Cells were assessed for DAZL (early germ cell marker) VASA (spermatocyte), BOULE (post-meiotic) and acrosin (spermatid). The differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored via time-lapse microscopy. Spherified neogametes on D(day)22, 29, and 36 achieved fertilization rates of 61.1%, 82.7%, and 80.0%, respectively, and blastulation rates of 20.5%, 36.0%, 26.3%, respectively. Controls exhibited fertilization rates of 89.1% and blastulation rates of 76.3%. When embryo morphokinetics were considered, D29 embryos reached compaction at 64 h and blastulation at 77 h, mirroring closely the control's timing of 63 and 77 h, respectively. D22 embryos displayed the most delayed embryo development, of which compaction occurred at 68 h and blastulation at 106 h. Despite normal fertilization and successful blastulation, the efficiency rate remained below optimal levels. Nevertheless, our system could produce viable offspring, demonstrating that replicating the 3D seminiferous tubule environment is crucial for generating artificial gametes. This eliminates the need for allogenic/xenogenic transplantation in experimental animals.

Male fertility depends on sperm motility, which enables sperm to traverse the female reproductive tract to the site of fertilization. Soluble adenylyl cyclase (sAC) is a key regulator of sperm motility, defining it as a promising non-hormonal contraceptive target for women and men. To evaluate the efficacy of sAC inhibitors under conditions mimicking the female reproductive tract, we employed a capillary assay using cervical mucus isolated from humans or cows as well as methylcellulose, a viscoelastic liquid that simulates cervical mucus. Addition of a sAC inhibitor, either directly to semen, modeling an on-demand male contraceptive, or to the cervical mucus, approximating a female contraceptive, effectively blocked sperm migration. These studies provide proof-of-concept for the efficacy of sAC inhibitors under physiologically relevant conditions.

Recurrent pregnancy loss (RPL), defined as two or more consecutive miscarriages, affects 1-5% of couples attempting to conceive and most frequently occurs between 6 and 8 weeks of gestation, coinciding with critical stages of placental development. Despite this temporal association, placental contributions to RPL pathogenesis remain poorly understood. We hypothesized that trophoblast lineage development, including extravillous trophoblast (EVT) and syncytiotrophoblast (STB) differentiation, are disrupted in RPL. To test this hypothesis, we established and functionally characterized human trophoblast stem (TS) cell lines derived from products of conception (POC) obtained from consenting idiopathic RPL cases. Four TS cell lines were successfully derived from five POC samples, representing both normal and abnormal karyotypes. All established TS cell lines exhibited canonical stem-state trophoblast features, including epithelial morphology, ELF5 promoter hypomethylation, expression of trophoblast-specific microRNAs, and trophoblast marker expression. Despite this preserved identity, TS cell lines displayed marked heterogeneity in proliferation and differentiation capacity. TS cells derived from POC with abnormal karyotypes exhibited prolonged doubling times and impaired differentiation, while those from normal karyotypes more closely resembled cytotrophoblast-derived control TS cells. Although all TS cells formed morphologically comparable STB spheroids, functional deficits in human chorionic gonadotropin β secretion were observed in a cell-line-specific manner. EVT differentiation capacity varied substantially across POC-derived TS cell lines. Transcriptomic analyses revealed karyotype-associated transcriptional heterogeneity, including enrichment of senescence- and cell cycle-associated pathways across subsets of POC-derived TS cell lines. Together, these findings demonstrate that patient-specific TS cell lines retain trophoblast identity yet exhibit meaningful functional heterogeneity that becomes evident upon differentiation, underscoring the importance of functional validation in newly established in vitro placental cell lines. Overall, POC-derived TS cells are a powerful platform for investigating molecular mechanisms underlying RPL.

The testis-expressed (TEX/Tex) genes are highly expressed in the testes of various species. Several members of the TEX catalog have been suggested to play a role in spermatogenesis, but the functions of most members remain unknown. Here, we initially confirmed that TEX2 is significantly upregulated in spermatids and further successfully generated a Tex2 knockout (KO) mouse model using CRISPR/Cas9 technology. However, we found that the Tex2 KO male mice were fertile. Interestingly, Papanicolaou staining and scanning electron microscopy showed slight abnormalities in sperm morphology and reduced sperm motility as assessed by computer-assisted sperm analysis in Tex2 KO mice. Collectively, despite Tex2 having enriched expression in mouse testis, our genetic KO studies revealed that Tex2 is not essential for spermatogenesis or fertility in male mice, although its absence in sperm causes subtle abnormalities in morphology and motility.

Endometriosis is a chronic systemic disease affecting ∼10-15% of women of reproductive age, often resulting in chronic pelvic pain and infertility. Despite its high prevalence, diagnosis is frequently delayed. tRNA-derived fragments (tRFs) are an emerging class of small non-coding RNAs that have been implicated in gene regulation and various pathological processes but remain understudied in endometriosis. This study aimed to characterize temporal changes in circulating tRF abundance after experimentally induced endometriosis in a baboon model. Endometriosis was induced in eight female baboons, and peripheral blood samples were collected at five time points post-inoculation. RNA was extracted from serum samples and sequenced to profile tRF abundance. Differential expression analysis was performed. Significant changes in tRFs were observed as early as 3 months after induction. A ranking based on log fold change and adjusted P-values revealed the 10 most significantly altered tRFs. These tRFs demonstrated consistent differential abundance throughout all time points. This is the first study to investigate circulating tRFs in endometriosis. Our longitudinal findings suggest that tRFs may serve as promising candidate biomarkers for endometriosis.

The establishment of a functional utero-placental interface depends on the precise temporal and spatial coordination of blastocyst attachment, decidual transformation, trophoblast invasion, and uterine spiral artery remodeling (SAR). Disruption of these tightly regulated processes underlies major placental pathologies, including preeclampsia, fetal growth restriction, and stillbirth. Although significant advances have been made in defining the molecular networks governing placental development, the contribution of cellular prion protein (PRNP) remains poorly characterized, warranting a focused review. Classically studied in the context of neurobiology and prion-associated neurodegenerative diseases, PRNP is now emerging as an important regulator of reproductive and vascular physiology. This review provides an overview of current evidence supporting non-neuronal roles of PRNP during early pregnancy, with particular emphasis on decidualization and utero-placental vascular adaptation. PRNP displays dynamic expression in the uterine glands, decidua, and vascular smooth muscle cells (VSMCs), where it contributes to tight-junction integrity, decidual cell survival, and autophagic homeostasis. Within the vascular compartment, PRNP regulates trophoblast-directed VSMC phenotypic switching by limiting proliferation, promoting platelet-derived growth factor-β-focal adhesion kinase-dependent migration, and protecting VSMCs from TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, processes essential for effective SAR. Notably, PRNP expression is transcriptionally regulated by odd-skipped related transcription factor 1 (OSR1), which is itself negatively modulated by trophoblast cells. Collectively, these findings position PRNP beyond its traditional neurobiological role as a sensitive molecular indicator of decidual competence and vascular remodeling status, highlighting its potential as a biomarker and therapeutic target in placental disorders.