ANGPTL4 induces granulosa cells proliferation dysfunction in PCOS by regulating the immune microenvironment and WNT signaling pathway.

backgroundPolycystic ovary syndrome (PCOS) is a common disorder affecting the reproductive, endocrine, and metabolic systems in women. Dysfunction of granulosa cells (GCs) and imbalance of the immune microenvironment are key pathological mechanisms underlying PCOS. Previous studies have shown that angiopoietin-like protein 4 (ANGPTL4) is significantly involved in the development of PCOS. However, the precise effect of ANGPTL4 on the pregnancy outcomes of PCOS patients has not been fully clarified.

methodsThis study enrolled 168 PCOS patients and 175 controls who received their first IVF treatment during 2023–2024. GCs transcriptome datasets from GEO were analyzed using least absolute shrinkage and selection operator (LASSO) regression and support vector machine-random forest (SVM-RF) algorithms to screen for hub genes. SVM-RF integrates support vector machine (SVM) and random forest (RF) for synergistic feature selection, enhancing hub gene screening accuracy, implemented in R 4.4.1 with the caret package (v6.0-94), using default parameters for SVM (kernel = “radial”) and RF (ntree = 500, mtry = sqrt(number of features). A miRNA-TF-hub gene regulatory network was constructed. Immune infiltration and functional enrichment analyses were conducted to explore hub gene features. The expression levels of ANGPTL4, WNT2, and CTNNB1(β-CATENIN) were detected in ovarian GCs and follicular fluid of patients. We used adenovirus to construct ANGPTL4 overexpressing cell lines and verified the impact on cell proliferation with CCK8 assay.

resultsThis study found upregulated ANGPTL4 expression and downregulated WNT2 and β-CATENIN expression in the ovarian GCs of PCOS patients. Follicular fluid ANGPTL4 levels were significantly higher in 168 PCOS patients, correlating with poorer IVF outcomes, such as fewer oocytes, lower quality embryos, and higher biochemical miscarriage and abortion rates. Bioinformatics analysis of GSE155489 identified 4,818 differentially expressed genes, with ANGPTL4 identified as hub gene via LASSO and SVM-RF. ANGPTL4 expression was negatively associated with WNT-inhibitory pathways. The vitro experiments showed that ANGPTL4 overexpressing cells exhibited reduced proliferation, and this phenomenon could be partially reversed by the WNT agonist (SKL2001).

conclusionThis study confirms that ANGPTL4 is overexpressed in PCOS patients, which is closely associated with reproductive outcomes, immune regulation, and metabolic disorders. ANGPTL4 affects ovarian granulosa cell function by inhibiting the WNT signaling pathway. ANGPTL4 is expected to become a novel biomarker for PCOS diagnosis and prognosis evaluation. Meanwhile, targeting the ANGPTL4/WNT signaling pathway provides new insights for the treatment of PCOS.