Hyperhomocysteinemia reduces the high-quality embryo rate in PCOS patients undergoing IVF/ICSI: clinical evidence and a preliminary exploration of mechanisms in KGN cells

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.