Polycystic ovary syndrome (PCOS) is frequently associated with alterations in gut microbiota composition, although the specific microbial metabolites that influence ovarian function remain incompletely understood. Myricetin has been reported to improve reproductive and metabolic features in experimental PCOS models and to activate brown adipose tissue (BAT); however, whether these effects are mediated by the gut microbiota has not been clarified. In dehydroepiandrosterone (DHEA)-induced PCOS mice, myricetin treatment was associated with improved estrous cyclicity, ovarian morphology, fertility, and insulin sensitivity. These changes coincided with notable shifts in gut microbial composition and the serum metabolome. Integrated multi-omics analysis suggested that methyl indole-3-acetate (MIA), a microbiota-derived tryptophan metabolite, was among the metabolites most consistently elevated following myricetin administration. Fecal microbiota transplantation from myricetin-treated donors partially improved ovarian function and glucose homeostasis in recipient mice. Supplementation with MIA enhanced BAT thermogenesis and the expression of thermogenic genes, whereas surgical removal of interscapular BAT attenuated the metabolic improvements observed with MIA. Together, these results suggest that myricetin may improve PCOS-like reproductive and metabolic disturbances through microbiota remodeling and elevated production of MIA, which supports BAT activity. This study identifies MIA as a previously unrecognized microbiota-derived metabolite that links gut microbial metabolism to BAT function and reproductive outcomes, providing new mechanistic insight into how dietary compounds may influence ovarian function in PCOS.
Diminished Ovarian Reserve (DOR) is a significant manifestation of female reproductive aging that profoundly impacts fertility and quality of life. In recent years, the emergence of aging reprogramming technologies has provided new insights into the mechanisms underlying DOR and potential therapeutic approaches. This review synthesizes current knowledge on the role of aging reprogramming in DOR, focusing on key molecular events such as telomere shortening, mitochondrial dysfunction, and epigenetic alterations. Furthermore, we summarize clinical diagnostic criteria and emerging biomarkers for DOR, while exploring the limitations of existing treatment strategies and the potential interventions based on aging reprogramming. By addressing these issues, this review aims to highlight the interplay between aging processes and ovarian function, paving the way for innovative therapeutic avenues to improve reproductive health in women experiencing DOR.
The koala (Phascolarctos cinereus), recently listed as endangered, faces significant reproductive challenges compounded by the high prevalence of Chlamydia pecorum infection, which may cause severe urogenital pathology and infertility. Spermatogonial stem cell (SSC) biobanking represents a promising conservation strategy, but its implementation requires detailed knowledge of spermatogenic organisation and SSC identity in this species. Here, we provide the first integrated histological, immunofluorescent, and ultrastructural characterisation of the koala testis, with a focus on spermatogonia and their somatic support cells. Quantitative morphometric analysis of H&E-stained sections revealed that seminiferous tubules occupy a lower proportion of the testicular parenchyma in the koala (∼62%) than reported for other mammalian species, and that urogenital disease status is associated with graded impairment of spermatogenic epithelial integrity. Transmission electron microscopy confirmed the ultrastructural features of koala Sertoli, Leydig, and peritubular myoid cells, and enabled identification of Type A, intermediate, and Type B spermatogonia based on nuclear morphology and chromatin organisation as well as intercellular cytoplasmic bridges consistent with clonal spermatogonial development. Using PNA-lectin acrosomal labelling, an eight-stage classification system for the koala seminiferous epithelial cycle was adapted and refined, with stage frequencies quantified. Immunofluorescent screening of seventeen antibody targets identified PLZF and STRA8 as conserved markers of undifferentiated and differentiating spermatogonial populations, respectively, enabling their abundance, proliferative activity, and distribution across the seminiferous epithelial cycle to be mapped. Together, these findings establish the first molecular and morphological framework for the koala spermatogonial compartment, providing a foundation for future SSC identification, isolation, and biobanking.
Probiotics have attracted significant attention as a potential strategy to improved testicular molecular signatures relevant to reproduction in ruminants. In this study, Bohai Black cattle were used as a model to systematically elucidate the mechanisms by which Bacillus maritimus strain N2 positively modulates testicular transcriptomic and metabolic signatures through multi-omics techniques. The study found that feeding N2 to the cattle specifically enriched the rumen with Adlercreutzia bacteria, which have anti-inflammatory potential, and markedly reduced levels of pro-oxidant toxins, such as phenolic derivatives, in the host circulatory system, accompanied by an increase in the antioxidant metabolite quercetin. Integrated analysis indicated that these metabolic changes were closely associated with a testicular gene co-expression module enriched for oxidative phosphorylation. Ultimately, N2 intervention significantly enhanced the expression of key protein markers involved in steroid synthesis, germ cell development, and blood-testis barrier integrity in the testes. This study is the first to depict a cross-organ regulatory axis of probiotics through 'enriching rumen Adlercreutzia-remodeling host metabolic profile-activating testicular energy and barrier programs,' providing an important mechanistic example for a deeper understanding of gut-testis interactions.
In brief: Successful implantation requires coordinated decidual differentiation of endometrial stromal cells. This study shows that anandamide treatment of decidualizing cells is associated with reduced neurogenic locus notch homolog protein 1 (NOTCH1) activation and selective alterations in decidual, inflammatory, and structural programs. Abstract: Decidualization of endometrial stromal cells is essential for human embryo implantation and pregnancy maintenance. The endocannabinoid system (ECS), particularly anandamide (AEA), has been implicated in endometrial receptivity, but the mechanisms through which AEA influences decidualization have been only partially elucidated. This study investigates the effects of AEA during decidualization of immortalized human endometrial stromal cells (HESCs), focusing on the neurogenic locus notch homolog protein 1 (NOTCH1) pathway, inflammatory mediators, cell-cycle arrest/senescence-associated markers, and cytoskeletal remodelling. Expression was analysed by quantitative real-time PCR, western blot, and immunofluorescence. Anandamide treatment during decidualization selectively reduced forkhead box O1 (FOXO1) and insulin-like growth factor binding protein 1 (IGFBP1), without affecting prolactin (PRL); homeobox A10 (HOXA10) was reduced during decidualization but was not further modified by AEA. Anandamide also attenuated NOTCH1 activation and reduced HES-related family bHLH transcription factor with YRPW motif 1 (HEY1) expression. Given the role of NOTCH signalling in cellular plasticity and cytoskeletal organization, NOTCH1/HEY1 attenuation after AEA exposure was accompanied by changes in snail family transcriptional repressor 1 (SNAI1), vimentin organization, and N-cadherin distribution. Consistent with the inflammatory component of stromal decidualization, decidualized HESCs showed increased interleukin-1 beta (IL1B), IL-8/C-X-C motif chemokine ligand 8 (CXCL8/IL-8), and prostaglandin-endoperoxide synthase 2/cyclooxygenase-2 (PTGS2/COX-2), whereas AEA reduced selected inflammatory mediators. Analysis of cyclin-dependent kinase inhibitor 1A (CDKN1A/p21) and cyclin-dependent kinase inhibitor 2A (CDKN2A/p16) suggested a short-term senescence-associated response, with AEA attenuating p21 expression. These findings suggest that AEA interferes with transcriptional and structural pathways critical for decidualization, highlighting a potential role of ECS-NOTCH1 crosstalk in implantation failure and endometrial dysfunction.
In brief: Maternal progesterone modulates the uterine environment that governs conceptus elongation, signaling, and implantation in cattle, yet supplementation strategies inconsistently improve pregnancy success. This review synthesizes current understanding of progesterone-regulated conceptus-maternal interactions and highlights the use of longitudinal biomarkers (IFNT, ISG, and PAG) to better resolve how progesterone influences early pregnancy establishment. Abstract: Progesterone is the key maternal hormone regulating uterine function during early pregnancy to support the developing conceptus and promote uterine receptivity. In turn, the conceptus acquires the capacity to signal its presence and further modulates a maternal response to allow for attachment and initiation of placentation. Supplementation of progesterone is proposed to optimize conceptus-maternal interactions and favor pregnancy establishment due to advanced temporal changes in the uterine milieu allowing for hastened conceptus elongation. Nevertheless, supplemental progesterone has not consistently increased pregnancy per service when applied in beef or dairy cattle, indicating that further research is required. Beyond increasing conceptus elongation, the extent to which supplemental progesterone impacts conceptus development and signaling to favor pregnancy establishment remains poorly understood. Evaluation of interferon tau, interferon-stimulated genes, and pregnancy-associated glycoproteins can be used to infer about the progression of the conceptus through key developmental milestones but has not been widely applied in the context of progesterone supplementation. Importantly, assessment of these conceptus- and pregnancy-specific biomarkers may be performed in vivo and can provide more adequate insight into the timing of conceptus-maternal interactions in response to supplementation of progesterone, especially when examined temporally.
CoRSIVs are genomic regions with CpG-methylation patterns that differ between individuals, yet are consistent between tissues, within the same individual; therefore, their methylation can be profiled in bodily fluids that are easily obtained, like blood and semen. Bearing in mind the simple epigenetic profiling of CoRSIVs, we tested whether this type of Differentially-Methylated Regions (DMRs) is associated with bovine fertility. Sequence-Read-Archive (SRA) meth BLAST was used to estimate CoRSIVs methylation status in 18 healthy, representative, and age-matched Holstein bulls, among which nine had high (H) sire-conception rate (SCR), and the other nine had low (L) SCR (group averages of SCR: 3.3±0.6 and -3.8±1.8, respectively). This method was also applied to morula and trophoblast SRA methylomes. Analysis with meth BLAST was effective for most (80%) CoRSIVs and showed that CoRSIVs are reprogrammed during blastocyst formation, although this method was incapable of specifically determining the methylation level in CoRSIVs with retrotransposons. In sperm, the effect of global methylation was evident in a common (25%) type of CoRSIVs that is highly (94.5%±4.3%) methylated in sperm. Specifically, a failure to retain hyper-methylation in the sperm plus strand was significantly (p < 0.00025) indicative of low SCR. Comparing global DNA methylation using the latter type of CoRSIVs between sperm and blood can be used as a better biomarker for fertility than using other DMRs with more complex epigenetics.
In brief: Daughters of women with endometriosis are known to be at increased risk of developing this disease. Using a mouse model, we demonstrate that uterine dysfunction reminiscent of that in endometriosis patients can also be passed from the father to the daughter, potentially via an epigenetic pathway amenable to therapeutic intervention.
In brief: Ovine preantral follicle diameter positively affected survival, antrum formation, and early growth during culture, although these effects may be modulated in the presence of leukemia inhibitory factor. Follicle culture sustained oocyte growth and formation of transzonal projections, albeit less effectively than in vivo. Abstract: Ovine follicle culture recapitulates several features of the preantral-to-antral transition, including morphological changes and granulosa cells proliferation and differentiation. Although the influence of initial follicle diameter on in-vitro development has long been suspected, it had not been investigated systematically. To date, only a few studies in other species have compared the in-vitro progression of preantral follicles from distinct size classes. In the present study, we finely characterized the effect of initial follicle diameter (ranging from 199 to 340 µm) on subsequent in-vitro development, by incorporating this factor as a continuous variable in statistical analysis. Regardless of the presence of ovine FSH, increasing initial follicle diameter was associated with enhanced survival and antrum formation, and larger final follicle and oocyte diameters. Initial follicle diameter also impacted growth dynamics, with a positive effect during early culture (days 0-6) but a negative effect during late culture (days 13-20). The presence of leukemia inhibitory factor modulated some of these effects. Follicle culture sustained oocyte growth, albeit to a lesser extent than in-vivo development. Among oocytes retrieved from in-vitro grown follicles measuring 500-900 µm, 33% had reached 110 µm (excluding the zona pellucida), a threshold commonly associated with meiotic competence. Furthermore, using high-resolution confocal microscopy, we demonstrate for the first time that follicle culture supports the formation of actin-rich transzonal projections.
In brief: SNX9 is first shown to be essential for normal decidualization. Its downregulation in decidual tissue impairs trophoblast invasion and may underlie severe preeclampsia, revealing a new molecular pathway in this poorly understood dangerous pregnancy complication. Abstract: Preeclampsia (PE) is a gestational hypertension disorder emerging after 20 weeks of pregnancy, complicating 5%-8% of pregnancies and representing a leading cause of maternal-fetal morbidity and mortality. Despite its clinical significance, the etiology and pathogenesis of PE remain obscure. Sorting nexin 9 (SNX9), a key regulator of intracellular trafficking and endomembrane dynamics, has been poorly explored in reproductive physiology. This study investigates the role of SNX9 in PE, demonstrating significant downregulation of SNX9 in decidual tissues from preeclamptic patients. In vitro decidualization models showed that SNX9 expression correlated with decidualization progression, as evidenced by upregulation of decidual markers (IGFBP1, PRL), while SNX9 knockdown impaired decidualization. Transwell assays revealed that aberrant SNX9 expression restricted trophoblast invasion into endometrial stromal cells. In pregnant mice, SNX9 expression in decidual tissues positively correlated with decidualization regulators (Wnt4, Bmp2) and markers (Prl8a2, Dtprp). Consistent expression patterns were observed in pseudopregnant mice after artificial decidualization induction, excluding embryonic influences. Collectively, these findings establish SNX9 as essential for normal decidualization, with dysregulated SNX9 potentially contributing to PE pathogenesis. This study uncovers a novel link between endomembrane dynamics and PE, providing insights into its molecular mechanisms.
In brief: In brief Decidualization of endometrial stromal cells is essential for embryo implantation and modulates endometrial immunity, -especially natural killer cells, while the potential of menstrual blood stromal cells is unclear. This study shows that decidualization reshapes the immunomodulatory effects of endometrial stromal cells and menstrual blood stromal cells, shifting peripheral natural killer cells to a pregnancy-supportive phenotype, advancing knowledge in reproductive immunology. Abstract: Decidualization of endometrial stromal cells (EnSCs) is essential for implantation in part by modulating the endometrial -immune network. Although menstrual blood-derived stromal cells (MenSCs) are considered as EnSC surrogates, little is known about their impact on regulation of the endometrial immune system. Here, we compared how decidualization affects the ability of EnSCs and MenSCs to modulate natural killer (NK) cells. EnSCs and MenSCs were isolated and those cell sources with good decidualization capacity were selected. The expression of HLA-ABC and HLA-DR before and after decidualization was assessed. The impact of EnSCs and MenSCs, either in the presence or absence of the decidualization cocktail, on NK cell markers, and NK cell-mediated cytotoxicity against EnSCs, MenSCs, and K562 cells, was evaluated. IFN-γ pretreatment and decidualization increased HLA-ABC expression in MenSCs. Decidualization in both EnSCs and MenSCs led to a marked reduction in the expression CD16 and NKG2D while increasing CD9+ and CD56bright NK cell frequency. Decidualization, caused a significant decrease in the perforin and IFN-γ levels of NK cells and significantly diminished NK cell cytotoxicity toward K562 cells. We observed that certain aspects of NK cells are modulated solely by the decidualization cocktail, while other aspects are actively regulated by stromal cells beyond the cocktail's effects. This study demonstrates that the immunoregulatory functions of EnSCs and MenSCs are profoundly altered by the decidualization milieu, with certain aspects being further modulated through direct involvement of the stromal cells themselves.
In brief: In brief Sperm motility and male fertility depend on the proper assembly and function of the mitochondrial sheath during late spermiogenesis. This study shows that sperm mitochondria-associated cysteine-rich protein (SMCP) maintains reactive oxygen species homeostasis during mitochondrial sheath assembly, thereby supporting mitochondrial sheath function, sperm motility, and male fertility. Abstract: Abnormal morphology or function of the sperm mitochondrial sheath is a major cause of asthenozoospermia, yet the mechanisms governing its assembly and functional maturation remain poorly understood. Sperm mitochondria-associated cysteine-rich protein (SMCP) is a mitochondrial sheath-associated protein, and its regulation at the protein level during late spermiogenesis has attracted substantial interest. Here, we show that SMCP is expressed during mitochondrial sheath assembly in late spermiogenesis. SMCP is initially detected in the cytoplasm of elongating spermatids and, following cytoplasm removal, a fraction is retained in the mitochondrial sheath of mature spermatozoa. SMCP deficiency leads to markedly reduced sperm motility and male infertility without overt alterations in mitochondrial sheath morphology. Integrative proteomic and functional analyses further reveal that SMCP loss impairs mitochondrial respiratory chain function and reduces ATP production. Mechanistically, SMCP associates with glutaredoxin-1 (GLRX), and SMCP deficiency disrupts GLRX localization, accompanied by elevated reactive oxygen species levels during mitochondrial sheath assembly and in mature sperm. These redox defects likely contribute to compromised mitochondrial sheath function. Collectively, our findings identify SMCP as a regulator of functional module assembly within the sperm mitochondrial sheath and support the concept that mitochondrial sheath assembly represents not only a morphogenetic process but also a developmental window for functional maturation and redox programming, with the SMCP-GLRX complex serving as a localized antioxidant module during this period.
In brief: In brief The establishment of the primordial follicle pool governs female reproductive lifespan but is poorly understood in ruminants. This study provides the first single-cell atlas of goat ovarian development, revealing key cellular interactions and identifying LUZP2 as a candidate oocyte-associated regulator potentially involved in primordial follicle formation. Abstract: Mammalian primordial follicles, essential for female reproduction, form a finite nonrenewable pool perinatally, with species-specific developmental variations. This study employed single-cell RNA sequencing to map the transcriptional landscape of primordial follicle formation in goats, focusing on fetal (embryonic day 75 [E75], E140) and postnatal (postpartum day 0) stages using over 37,000 ovarian cells. These stages encompassed the midfetal stage (after syncytial rupture and the beginning of primordial follicle formation) to the formation of the primordial follicle pool in goats. Seven ovarian cell types were identified, with oocyte and granulosa cell subtypes analyzed in depth. Key interactions between oocytes, proliferative granulosa cells, and granulosa cells-1 via tight junctions, ECM-receptor signaling, and endocytosis were implicated in primordial follicle assembly. Transcription factor analysis identified LUZP2 as a novel oocyte-enriched transcriptional regulator potentially involved in primordial follicle formation, and its expression pattern was validated by real-time quantitative PCR and immunofluorescence. Overall, this study provides the first single-cell transcriptomic atlas of goat ovarian development, delineating dynamic gene expression patterns and cellular interactions during primordial follicle pool establishment. These findings advance understanding of conserved and species-specific mechanisms governing female reproductive lifespan in mammals.
In brief: The preimplantation embryonic development progress is accompanied by dynamic changes of transcriptome, and many transcription factors have been found to responsible for this progress. This study shows that SP1 is necessary for proper zygotic genome activation and morula-to-blastocyst transition by regulating transcriptome establishment and histone modifications in mouse preimplantation embryos. Abstract: Specificity protein 1 (SP1) is the most active member of the specificity protein and Krüppel-like factor (Sp/KLF) family and is widely expressed across all mammalian cell types. However, more detailed studies on the role of SP1 in preimplantation embryonic development are needed. Here, we analyzed the role of SP1 in the development of preimplantation mouse embryos through supplementation with a small molecular inhibitor (plicamycin) and microinjection of Sp1 siRNA. We found that SP1 was indispensable for zygotic genome activation (ZGA) and the morula-to-blastocyst transition. Plicamycin supplementation arrested embryo development at the 2-cell stage and resulted in aberrant RNA polymerase II preconfiguration. Sp1 expression knockdown by Sp1 siRNA microinjection caused most embryos to arrest at the morula stage, and the expression of NANOG, POU5F1, and CDX2 significantly decreased. Both plicamycin supplementation and Sp1 siRNA microinjection decreased embryonic H3K4me3 levels and increased H3K9me3 levels. Moreover, when Sp1 was overexpressed, the embryos were arrested at the 2-cell stage, the H3K4me3 level increased, and the H3K9me3 level decreased. In conclusion, our findings demonstrate that SP1 is crucial for mouse preimplantation embryonic development through the regulation of gene expression and histone modifications.
In brief: Noninvasive embryo quality assessment beyond morphology remains an urgent need. By identifying exRNA in spent embryonic culture medium as a potential noninvasive biomarker, this study provides a foundation for noninvasive strategies based on exRNA in spent culture medium, ranging from embryo quality assessment to applications in reproductive medicine, developmental biology. Abstract: The extracellular RNA (exRNA) transcriptome of the preimplantation embryo culture medium constitutes a comprehensive profile of embryonic RNAs, offering a noninvasive resource for elucidating developmental status. To delineate its relationship with the maternal-to-zygotic transition (MZT) and evaluate its predictive potential for developmental outcomes, we performed systematic exRNA transcriptome profiling of individually cultured cleavage-stage embryos. This study included 34 spent embryo culture medium (SECM) samples for day 3 in-vitro preimplantation cleavage embryos from 30 patients undergoing in-vitro fertilization (IVF)/intracytoplasmic sperm injection. Twenty-four morphologically high-grade samples (grade I, n = 12; grade II, n = 12) and 10 low-grade samples (grade III, n = 6; abnormal pronuclei, n = 2; arrested embryos, n = 2) were included. Embryo transfer after the SECM collection was traced for subsequent clinical pregnancy outcome. ExRNA transcriptome from cleavage SECM showed 81.78% concordance with embryonic gene expression profiles. A total of 1,058 differentially expressed exRNA markers were identified across embryo morphological grading groups, revealing MZT-related molecular dynamics prior to blastocyst formation. A 300-gene signature significantly correlated with embryonic developmental potential was established. The accuracy and robustness of the models were validated using clinical samples. The LogitBoost model built on exRNA profile yielded an area under the curve value above 0.95, indicating encouraging performance for embryo assessment. This study establishes SECM exRNA profiling as a noninvasive method for capturing key molecular events during early embryo development, particularly MZT activation. The identified exRNA biomarkers and machine learning models provide a promising framework for the objective assessment of embryo developmental potential, warranting further validation to overcome the limitations of conventional morphology-based selection in IVF.
In brief: Redox imbalance compromises granulosa cell survival and follicle fate. This study identifies the mitochondrial-derived peptide Humanin (HN) as a cytoprotective factor that protects granulosa cells under oxidative condItions. Abstract: Granulosa cell function is essential for proper ovarian physiology. Redox imbalance compromises granulosa cell survival, thereby impacting follicle fate within the ovary. Humanin (HN), a small mitochondrial-derived peptide, exerts cytoprotective effects in several tissues under pro-oxidant conditions. The present study aimed to evaluate the cytoprotective effects of HN under oxidative conditions in granulosa cells, using a human granulosa cell line (KGN) and an in-vitro rat ovary culture to assess its action within the ovarian microenvironment. KGN cells showed a significant increase in endogenous HN mRNA expression in response to oxidative conditions induced by H2O2. Upon oxidative insult, exogenous HN enhanced cellular antioxidant capacity by significantly increasing catalase activity levels, without modifying superoxide dismutase expression or activity, or redox status in KGN cells. Importantly, HN significantly decreased H2O2-induced granulosa cell apoptosis in KGN cells, as determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. This protective effect was associated with modulation of key apoptosis-related genes, including reduced expression of BAX and the caspase-3 precursor (CASP3), while BCL2 expression remained unchanged. Consistently, in rat ovaries, HN protected granulosa cells of antral follicles under oxidative conditions. In conclusion, our findings support a cytoprotective role of HN in granulosa cells under oxidative conditions, which may contribute to the maintenance of antral follicle survival within the ovarian microenvironment.
In brief: This review synthesizes the technical landscape of current spatial transcriptomic platforms, addresses computational challenges unique to ovarian tissue, and surveys biological discoveries across ovarian development, aging, follicle dynamics, and cancer, providing a practical framework to guide platform selection and analytical strategy in reproductive biology. Abstract: The ovary is a structurally complex organ whose function depends on precisely coordinated interactions among multiple cell types. Spatially resolved transcriptomics (ST) has emerged as a powerful complement to single-cell RNA sequencing (scRNA-seq), enabling gene expression profiling within intact tissue and preserving the spatial context that dissociation-based methods inherently lack. This review provides a comprehensive overview of the major ST platforms, including sequencing-based technologies (Visium, Visium HD, Stereo-seq, and GeoMx) and imaging-based technologies (Xenium, MERSCOPE, and CosMx), with a focus on their distinct technical features, resolution trade-offs, and suitability for ovarian research. We survey 40 published studies applying ST to ovarian biology, spanning atlases, ovarian aging, follicle development and ovulation, and ovarian cancer. We also discuss typical computational analyses as well as their challenges specific to ovary, including cell segmentation of morphologically diverse cell populations, deconvolution of mixed-cell capture spots in sequencing-based platforms, quality control, batch correction, and spatially aware downstream analyses encompassing trajectory inference, cell-cell interaction modeling, gene regulatory network reconstruction, and more. Across these biological contexts, multimodal integration, pairing ST with scRNA-seq, spatial proteomics, or chromatin accessibility profiling, has proven increasingly valuable for resolving the full molecular complexity of ovarian biology. Nevertheless, some challenges persist, and no single platform is universally optimal for all research questions. Thoughtful alignment between biological objectives, tissue scale, and platform capability will be critical for advancing ST from descriptive mapping toward mechanistic and clinically translatable discovery.
In brief: Polyendocrine metabolic ovarian syndrome (PMOS) is associated with metabolic, reproductive, and immune dysfunction, yet the mechanisms driving these abnormalities remain unclear. This study identifies Toll-like receptor 4 (TLR4) as a key contributor of PMOS-like pathology in mice, linking gut barrier disruption and endotoxin-associated immune activation to impaired metabolic and reproductive function. Abstract: Polyendocrine metabolic ovarian syndrome (PMOS), formerly termed polycystic ovary syndrome (PCOS), is a reproductive disorder with heterogeneous symptoms and severity. Despite extensive research documenting chronic immune dysfunction as a hallmark of PMOS, the specific mechanisms of immune activation remain poorly understood. Emerging evidence suggests that gut-derived bacterial endotoxins, particularly lipopolysaccharide (LPS), can breach intestinal barriers and trigger systemic inflammation via Toll-like receptor 4 (TLR4). This study examined the role of TLR4 in PMOS-like pathology using a letrozole (LET)-induced mouse model. In LET-treated wild-type female mice, serum LPS and its carrier protein lipopolysaccharide-binding protein (LBP) were elevated compared with LET-treated TLR4-/- mice. Additionally, TLR4 deficiency attenuated multiple PMOS-like features, including elevated luteinizing hormone, anovulation, and metabolic dysfunction. The LET-treated TLR4-/- mice also preserved estrous cycling and fertility, maintained gut barrier integrity, and reduced inflammatory markers. These findings support TLR4 as an important contributor to multiple features of PMOS-like pathology. This novel work highlights TLR4-mediated inflammation as a potential target for anti-inflammatory treatments in women with PMOS.
Evidence suggest that lipotoxicity can cause hyperandrogenesis, but little is known about the intra-ovarian environment of women with polycystic ovary syndrome (PCOS) and whether it displays features associated with lipotoxicity. The objective of this secondary analysis was to compare follicular fluid concentrations of testosterone, markers of lipid, lipid metabolism and inflammation between women with PCOS and without PCOS. We conducted a controlled cross-sectional study on 80 participants. Medical records were used to determine PCOS diagnosis: 15 women were identified as having PCOS, while 65 had a negative diagnosis. Inclusion criteria were 18 and 40 years old with a body mass index raging between 18 and 40 kg/m2. Follicular fluid was analysed for total testosterone, non-esterified fatty acids (NEFA), triglycerides, NEFA metabolites (acylcarnitines and C16/C13 ratio) and inflammatory cytokines. Compared to women without PCOS, women with PCOS had increased follicular fluid levels of testosterone (7.08 nM vs 0.29 nM), triglycerides (0.30 nM vs 0.17 nM), palmitoylcarnitine (43.7 nM vs 28.4 nM) IL-6 (13.04 pg/mL vs 8.9 pg/mL), while TNF-α remained similar. These differences remained statistically different after adjustment for BMI, except for IL-6. These group differences were also confirmed in paired analyses of 13 BMI-matched pairs of PCOS vs non-PCOS women, exception made for IL-6. In conclusion, intra-ovarian inflammation, but more importantly, lipid overexposure, may play a role in the pathogenesis of PCOS, probably through lipotoxic effects.
