BACKGROUND: The process of epithelial-mesenchymal transition (EMT) may promote fibrosis in ovarian tissue related to polycystic ovary syndrome (PCOS), thus affecting ovarian function and hormonal balance. OBJECTIVE: This study aimed to explore key genes associated with EMT in PCOS and their potential molecular regulatory mechanisms, exclusively from the perspective of transcriptomics and single-cell RNA sequencing (scRNA-seq), combined with Mendelian Randomization (MR) analysis. METHODS: The dataset for PCOS and EMT-related genes (EMT-RGs) were sourced from public databases. The key genes in this study were identified via differential expression analysis, MR, and evaluation of expression levels. Enrichment analysis and a series of functional analyses were conducted on these genes to further elucidate their potential mechanisms. Subsequently, using scRNA-seq data and validation of the expression of key genes, key cell group in PCOS were identified, followed by pseudo-time and cell communication analyses to provide deeper insights. RESULTS: Three key genes, NUCB2 [odds ratio (OR) = 0.8634, 95% confidence interval (CI): 0.8145-0.9152, P <
0.0001], PGF (OR = 0.8393, 95% CI: 0.7185-0.9805, P <
0.05), and CRIM1 (OR = 0.7539, 95% CI: 0.6556-0.670, P <
0.0001), were identified as having a unidirectional causal association with PCOS and were associated with a reduced risk of PCOS. In public datasets, NUCB2 exhibited significantly increased expression in PCOS samples, while PGF and CRIM1 showed the opposite trends. These three genes were enriched in pathways related to cellular functions, metabolic processes, and the operation of the nervous system, and they were co-expressed in smooth muscle. Additionally, five cell clusters were annotated, among which fibroblasts were identified as key cells due to their highest expression of all three key genes. Further analysis revealed a bifurcation event occurring during the mid-development stage of fibroblasts, with PCOS samples displaying a higher abundance of fibroblasts. In PCOS samples, fibroblasts exhibited more extensive communication with secretory epithelial cells, indicating a more complex intercellular interaction within this condition. CONCLUSION: This study identified three EMT-RGs: NUCB2, PGF, and CRIM1, which were associated with a reduced risk of PCOS, with fibroblast identified as a key cell group in the disease's pathology. This provides new insights for PCOS research.