Abstract The placenta plays a vital role in supporting fetal growth and development by facilitating nutrient exchange, hormone signaling, and immune regulation. Environmental toxicants such as perfluoroalkyl and polyfluoroalkyl substances (PFAS) can disrupt placental homeostasis, leading to increased risk of disease later in life. Paternal environmental exposures may influence offspring health by inducing epigenetic changes in sperm, which may affect sex-dependent placental programming. Here, male mice were exposed to a PFAS mixture (PFOS, PFOA, PFNA, PFHxS, GenX; 20 μg/l each) for 10 weeks and mated with unexposed females. We identified 6656 and 3242 differentially methylated regions (DMRs) in E12.5 placentas of female and male offspring, respectively. Female placentas showed predominantly hypermethylation (78.5%), whereas male placentas were mostly hypomethylated (62%). Gene ontology analyses linked female placental DMRs to cell and embryonic morphogenesis, and male DMRs to anatomical size regulation and hemostasis. Bulk RNA sequencing revealed 168 and 148 differentially expressed genes (DEGs) in female and male placentas, respectively. Female DEGs were related to immune regulation, while male DEGs were involved in muscle and mesenchyme development. Sex-by-PFAS interaction models identified distinct methylation and transcription biological pathways. Overlap analysis between F0 sperm and F1 placentas identified 265 shared genes, including 289 female-specific and 124 male-specific genes. Paternal exposure to the PFAS mixture increased glycogen concentrations in male, but not female, placentas. No effects were observed at imprinted loci. These findings demonstrate that paternal preconception exposure to a PFAS mixture alter placental epigenomic and transcriptomic landscapes, which may contribute to sex-dependent trajectories of fetal health and development and offspring phenotypes.
Abstract The development of in vitro systems capable of supporting early human folliculogenesis holds significant promise for fertility preservation, particularly for patients who cannot undergo ovarian tissue transplantation. Mitochondria-targeted antioxidants have shown beneficial effects in later stages of oocyte maturation, but their impact during early folliculogenesis remains unclear. This study aimed to investigate the effects of the mitochondrial-targeted antioxidant Mito-Q on early-stage human follicles. Using human ovarian tissue and isolated pre-antral follicles obtained from women undergoing fertility preservation, two complementary in vitro models were established. Ovarian cortex fragments were cultured for up to 14 days, and isolated pre-antral follicles were maintained in three-dimensional culture for 8 days. Following an initial comparison of the oxidised and reduced forms, the reduced form was selected for further experiments. Outcomes included follicle density and morphology, apoptosis, cytotoxicity, growth, survival, and gene expression. Mito-Q did not improve follicle survival or overall follicle density. Instead, increasing concentrations were associated with a significant increase in apoptosis in granulosa cells within ovarian tissue and reduced growth of isolated pre-antral follicles. These effects were accompanied by changes in the expression of mitochondrial stress-related genes, suggesting activation of cellular stress responses without functional benefit for follicle development. The findings indicate that mitochondrial antioxidant supplementation does not universally benefit early folliculogenesis and may be detrimental at higher concentrations. These results highlight the importance of stage-specific approaches to redox modulation and suggest that careful optimisation of antioxidant strategies is required to improve in vitro follicle development and fertility preservation outcomes.
Abstract The ovarian stroma surrounds ovarian follicles and provides a supportive microenvironment during follicle quiescence and subsequent growth. In this study, we investigated the effect of testosterone exposure on human ovarian stromal composition, specifically collagen and elastin content and stromal cell density (SCD), and its relationship with non-growing follicle density (NGFD). Whole ovaries were obtained from transgender men receiving long-term testosterone therapy (mean age 27.6 ± 1.7 years, n = 8) at oophorectomy and compared with ovarian cortical biopsies from age-matched healthy women obtained at caesarean section (mean age 31.8 ± 1.5 years, n = 8). Testosterone-exposed ovarian tissue demonstrated a more fibrotic stromal composition, characterised by increased collagen and reduced elastin, resulting in a higher collagen–elastin ratio indicative of increased intrinsic tissue rigidity. SCD was significantly higher in testosterone-exposed tissue, consistent with stromal cell hyperplasia, whereas NGFD was comparable to controls. These findings indicate that elevated circulating testosterone alters ovarian stromal composition and cellularity without reducing the non-growing follicle pool. Although interpretation is limited by the use of pregnancy-derived control tissue, these findings provide insight into the effects of testosterone on the ovarian microenvironment and warrant further investigation of their functional significance for transgender men considering pregnancy or fertility preservation.
Abstract The female reproductive tract is a multi-tissue system in which epithelial, stromal, immune, and vascular cells coordinate responses to hormonal signals. Single-tissue organoids have advanced epithelial modeling in vitro but cannot capture the inter-tissue communication underlying key reproductive processes. Assembloids, which recombine cells from distinct tissue compartments in three-dimensional space, offer a means to restore these missing interactions. This review examines reproductive assembloid research across three biology-driven contexts. In menstrual cycle modeling, organoid-based systems have recapitulated epithelial breakdown and regeneration, yet stromal, immune, and vascular integration remain incomplete. In implantation modeling, embryo-endometrial co-cultures and receptive assembloids have begun to capture maternal–fetal crosstalk, though immune and vascular dimensions are largely missing. In endometriosis and adenomyosis modeling, assembloids have started to reconstruct pathogenic microenvironments that single organoids cannot replicate. Three issues are examined in depth. The first is the gap between what current models can and cannot reveal about reproductive biology. The second is the risk that added complexity may only confirm findings already established in vivo. The third is the point at which an additional tissue component stops contributing new information. Progress in this field is likely to depend not on greater structural complexity but on questions that only multi-tissue models can address.
Endometriosis is a multifaceted disease, causing debilitating pelvic pain in some patients, while being completely asymptomatic in others. The pathophysiology of endometriosis pain is not well understood and poorly correlated to stage. We hypothesized that this clinical heterogeneity may be explained by examining differences in gene expression. We performed RNA sequencing of 27 formalin-fixed, paraffin-embedded peritoneal biopsies from 9 symptomatic (Sx) and 10 asymptomatic (ASx) subjects. 890 genes were differentially expressed between Sx and ASx samples. Of these, the most significant including genes involved inflammatory signaling (IL16, IL17RA, JAK3, SMPD3, RELT), cell adhesion (OLFML1, CDON, VCAN), and neuromodulation (SEMA6D, ADRA2C, SLC7A5). Gene Set Enrichment Analysis identified functional enrichment in 22 gene ontology (GO) pathways, of which 7 represented immunologic pathways. Weighted Gene Correlation Network Analysis (WGCNA) identified 11 co-expression modules significantly correlated with symptomaticity, including one module strongly enriched for immunologic/inflammatory functions. Of all molecules identified as associated with pain, IL16 expression also correlated with symptom severity when cases were stratified into mild vs. severe pain based on clinical criteria. These findings suggest that endometriotic lesions of symptomatic subjects are characterized by distinct molecular signatures, including altered expression of inflammatory pathways, highlighting potential mechanisms underlying symptom variability and identifying candidate pathways for future therapeutic interventions.
