Xuehong Zhang

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive and metabolic disorder. Whether gut microbiota-associated metabolites contribute to ovarian dysfunction under metabolically adverse conditions remains unclear. We investigated microbiota-associated metabolic alterations in women with PCOS and overweight/obesity and explored the functional relevance of indole-3-acetic acid (IAA). Methods: Women with PCOS and overweight/obesity and non-PCOS infertility controls were enrolled for clinical microbiome and metabolomic profiling. The association between microbial features and IAA metabolism was investigated using integrated multi-omics analysis. The IAA-producing capacity of Bacteroides fragilis was examined in vitro. The effects of B. fragilis and IAA were further evaluated in mice under basal and high-fat diet (HFD)-induced metabolic stress conditions, and IAA-associated AHR signaling responses were examined in KGN granulosa cells with or without palmitic acid (PA)-induced lipotoxic stress. Results: Exploratory clinical profiling identified altered gut microbial and indole-related metabolic features in women with PCOS and overweight/obesity, including increased B. fragilis abundance and IAA levels, which were positively correlated. IAA levels were also increased in serum and follicular fluid. The B. fragilis strain used in this study produced IAA in vitro. In mice, B. fragilis or IAA exposure alone did not produce detectable overt ovarian abnormalities under basal dietary conditions, whereas selected ovarian, endocrine, and estrous-cycle abnormalities were observed when either intervention was combined with HFD-induced metabolic stress. In KGN cells, IAA reduced proliferation and altered G1/S cell-cycle progression, accompanied by increased AHR expression and nuclear translocation. AHR inhibition or knockdown partially attenuated PA + IAA-associated cell-cycle alterations. PA-induced lipotoxic stress further enhanced IAA-associated cellular responses. Conclusions: These findings support a context-dependent relationship among microbiota-associated IAA signaling, granulosa-cell dysfunction, and selected PCOS-like abnormalities under metabolic stress, with AHR contributing to the cellular response. The clinical microbiota–metabolite associations were identified in women with PCOS and overweight/obesity and require validation in lean PCOS populations and broader PCOS phenotypes.

Background:Itaconic acid and its isomers citraconic acid and mesaconic acid are a recently identified class of metabolites with anti-inflammatory and antioxidant effects. This study investigates their roles in ischemia-reperfusion-induced acute kidney injury.Methods:In this study, a rat model of renal ischemia-reperfusion injury (IRI) was established, and itaconic acid, citraconic acid and mesaconic acid were administered as a preoperative intervention. After the operation, renal injury was evaluated by contrast-enhanced ultrasound, biochemical analyses and histopathological staining.Results:The intervention with itaconic acid and its isomers reduced the production of the inflammatory cytokines interleukin-18 (IL-18) (p < 0.05), inhibited activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome (p < 0.05), and reduced reactive oxygen species levels (p < 0.05). The intervention with itaconic acid and mesaconic acid reduced the production of the inflammatory cytokine interleukin-1β (IL-1β) (p < 0.05). The expression of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) spot protein in the intervention group was significantly lower than that in the renal ischemia-reperfusion injury (IRI) group (p < 0.01). Conclusions:These findings suggest that itaconic acid, citraconic acid, and mesaconic acid may be potential therapeutic agents for renal ischemia-reperfusion through their anti-inflammatory and antioxidant effects.

In female animals, the functional status of the ovary is closely associated with ovarian hemodynamics. Contrast‐enhanced ultrasonography (CEUS) is a noninvasive imaging technology that allows for the visualization of microvascular blood flow. However, there is a lack of research on the hemodynamics of rat ovaries. This study conducted CEUS dynamic monitoring of ovarian hemodynamics in rats during infancy and pregnancy, revealing significant variations in time–intensity curve (TIC) parameters between the two periods. The findings demonstrate the ability of CEUS to quantitatively assess the hemodynamic characteristics of rat ovaries, indirectly indicating their functional status. Moreover, these results offer a technical method for the noninvasive monitoring of ovarian functional diseases by CEUS.

Objective: This study aims to investigate the role of thrombospondin-1 (THBS1) in polycystic ovary syndrome (PCOS) pathogenesis and its mechanism in regulating granulosa cell (GC) function. Methods: Follicular fluid and granulosa cells from 21 PCOS patients and 21 age-matched non-PCOS controls were analysed for THBS1 expression and clinical correlations. A dehydroepiandrosterone (DHEA)-induced PCOS rat model with adeno-associated virus serotype 9 (AAV9)-mediated THBS1 knockdown was used to assess phenotypic changes. The KGN human granulosa-like cell line was employed to evaluate THBS1 overexpression effects on proliferation, apoptosis, and steroidogenesis. Mechanistic studies included RNA sequencing with Gene Set Enrichment Analysis (GSEA), co-immunoprecipitation, molecular docking against the latent TGF-β1 crystal structure (PDB 9VJJ), molecular dynamics simulation, an active/total TGF-β1 ELISA, and pharmacological TGF-β receptor inhibition. Results: THBS1 was elevated in PCOS follicular fluid and granulosa cells and correlated positively with serum AMH and LH after Benjamini–Hochberg FDR correction. AAV9-mediated ovarian THBS1 knockdown (37.4% protein reduction, p = 0.006) ameliorated cystic morphology, restored estrous cyclicity, and normalised serum AMH/LH/T. In KGN cells, THBS1 overexpression suppressed proliferation, induced apoptosis and inflammatory cytokines, and dysregulated steroidogenic enzymes. Transcriptome analysis revealed upregulation of canonical TGF-β/Smad pathway components (SERPINE1, SMAD7, TGFB2, INHBA, CCN2, COL1A1/2). Molecular docking and 100-ns dynamics simulation supported a stable interaction between THBS1 and latent TGF-β1 (ΔG_TOTAL ≈ −120 kcal·mol⁻¹). Co-immunoprecipitation confirmed physical association in cells, and ELISA showed elevated TGF-β1 in PCOS follicular fluid and rat serum, both attenuated by THBS1 knockdown. Pharmacological TGF-β receptor inhibition with SB-431542 rescued THBS1-induced cellular dysfunction. Conclusions: THBS1 is associated with PCOS-related granulosa cell dysfunction through the TGF-β/Smad pathway and represents a candidate biomarker and exploratory therapeutic target that warrants validation in independent multicentre cohorts.

Background: Homocysteine (Hcy) is a sulfur-containing intermediate metabolite during methionine metabolism. Studies indicate that individuals with polycystic ovary syndrome (PCOS) have higher circulating Hcy levels relative to unaffected populations. Hyperhomocysteinemia (HHcy) has been implicated in multisystem cytotoxicity. This study was designed to evaluate the association between Hcy levels and embryo quality and pregnancy outcomes in PCOS patients undergoing assisted reproductive technologies (ART). Mechanistic insights were explored through in vitro assays and combined network pharmacology analyses. Methods: A total of 599 patients with PCOS who were undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) were retrospectively analyzed. Patients were stratified into three groups based on Hcy levels (low, intermediate, and high). Comparative analyses of clinical and laboratory parameters were performed across strata. Multivariable logistic regression models were developed to evaluate the independent association between Hcy levels and reproductive outcomes after adjustment for potential confounders. In vitro assays were conducted using KGN cells, a human granulosa-like tumor-derived cell line. Cellular viability after exposure to graded concentrations of Hcy was quantified using the Cell Counting Kit-8 (CCK-8) assay, while cell apoptosis was observed via flow cytometry. For systems-level analysis, Hcy-associated targets were retrieved from SwissTargetPrediction, TargetNet, BATMAN-TCM, and PharmMapper databases. PCOS-related targets were curated from OMIM, GeneCards, DrugBank, and DisGeNET repositories. Overlapping targets were analyzed for protein-protein interaction (PPI) network construction using the STRING platform, followed by Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Results: Comparative univariate analysis showed a significant difference in the high-quality embryos among the three Hcy strata, whereas other clinical parameters did not reach statistical significance. Multivariable regression analysis identified a robust inverse association between Hcy level and high-quality embryo rate (β = -1.04, 95% CI: -1.36 to -0.72; p < 0.0001), which remained stable after adjustment for confounding variables, including creatinine clearance rate (β = -1.06, 95% CI: -1.40 to -0.72; p < 0.0001). In vitro results showed that exposure to Hcy (80–160 µM, 24 h) significantly reduced KGN cell viability and increased apoptotic rates in a concentration-dependent manner. Network pharmacology analysis identified 102 intersecting targets that may mediate Hcy-associated effects in PCOS. KEGG pathway enrichment revealed significant involvement in lipid metabolism and atherosclerosis, viral infections (i.e., hepatitis B and cytomegalovirus), the AGE-RAGE signaling pathways in diabetic complications, and the TNF signaling cascade. Conclusion: Higher Hcy levels were inversely correlated with high-quality embryo formation in PCOS patients, with each increase in Hcy associated with a measurable decline (1.4%) in embryo quality. Experimental validation indicated that Hcy exerts cytotoxic and pro-apoptotic effects on KGN cells. Combined network pharmacology further suggested that Hcy may influence PCOS. These results support a contributory role of HHcy in impaired PCOS and provide a preliminary mechanistic understanding for future investigation.

Introduction: This study aimed to investigate the association between 25-hydroxyvitamin D [25(OH)D] levels in serum (VD-S) and follicular fluid (VD-FF) and the laboratory outcomes of in vitro fertilization (IVF) in patients with diminished ovarian reserve (DOR). Methods: This prospective cohort study enrolled 145 women undergoing IVF, comprising 74 patients with DOR and 71 with normal ovarian reserve (NOR) as controls. VD-S and VD-FF levels were measured. The DOR group was further stratified into vitamin D (VD) non-deficient (VDH) and VD deficient (VDL) subgroups. Baseline characteristics and IVF laboratory outcomes were compared between the groups and subgroups. Results: A significant positive correlation was observed between VD-S and VD-FF levels (r = 0.769, P < 0.001). Patients in the DOR group had significantly lower 25(OH)D levels in both serum ( P = 0.048) and follicular fluid ( P = 0.015) compared to the NOR group. Within the DOR cohort, the VDH subgroup demonstrated significantly higher oocyte retrieval rates (95.0% vs . 84.6%, P = 0.021) and normal fertilization rates (72.9% vs . 56.3%, P = 0.016) than the VDL subgroup when stratified by VD-S. Similar results were found when stratifying by follicular fluid levels. Furthermore, the VDH subgroup exhibited significantly higher high-density lipoprotein (HDL) levels ( P < 0.05). Conclusion: In patients with diminished ovarian reserve, higher 25(OH)D levels in serum and follicular fluid are positively associated with higher oocyte retrieval and normal fertilization rates during IVF cycles. These findings suggest that VD deficiency may be a modifiable factor associated with IVF outcomes in this specific population.

Introduction: Improving pregnancy outcomes for women with diminished ovarian reserve (DOR) is a key area of focus in assisted reproductive technology (ART). Objective: This study aims to investigate the association between 25-hydroxyvitamin D (25-[OH]D) levels in follicular fluid (FF) of women with diminished ovarian reserve (DOR) and embryo quality during in vitro fertilization (IVF), and to perform a metabolomics analysis. Methods: A total of 54 women with DOR and 62 women with normal ovarian reserve (control) were enrolled. Based on FF 25-(OH)D levels, both DOR and control cases were stratified into two subgroups: the vitamin D non-deficient (VDH) and vitamin D deficient (VDL). Baseline characteristics and IVF laboratory outcomes were compared among groups and subgroups. Untargeted metabolomic profiling of FF was then performed to identify differences between subgroups. Results: FF vitamin D levels were significantly lower in the DOR group than in controls. In both cohorts, the normal fertilization rate was significantly higher in the VDH subgroup than in the VDL subgroup. Metabolites significantly upregulated in the VDH subgroup included phospholipids, hydroxychloroquine, and serinyltryptophan. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed significant enrichment in glycerophospholipid metabolism in the VDH subgroup. In the DOR group, the DOR-VDH subgroup exhibited upregulation of steroid hormone biosynthesis and vitamin metabolism pathways, particularly those mediated by cytochrome P450, relative to the DOR-VDL subgroup. Conclusion: Vitamin D may enhance the follicular microenvironment in DOR patients primarily by regulating lipid metabolism, thereby improving oocyte and embryo quality and developmental potential.