A novel rat model of ovarian endometrioma: single-cell RNA sequencing reveals its impact on ovarian function

Background Ovarian endometrioma (OEM) is a leading cause of infertility in women of reproductive age; however, the cellular and molecular mechanisms by which it impairs ovarian function remain poorly understood. Direct investigation of OEM-induced ovarian dysfunction is hampered by the difficulty in obtaining clinical ovarian tissue samples, and the lack of stable animal models that faithfully simulate the pathological features of human OEM represents a critical bottleneck in this field. Methods A rat model of OEM was established by suturing homologous rat endometrial tissue onto the ovarian surface. The impact of OEM on ovarian function was evaluated through the estrous cycle, serum hormone levels, and histopathological staining. Single-cell RNA sequencing (scRNA-seq) was employed to analyse ovarian tissue from the OEM group and the control group, with the aim of mapping cellular landscapes, identifying the composition of cell subpopulations, conducting differential gene enrichment analyses, and characterising changes in differentiation trajectories and intercellular communication networks. Key phenotypic findings were further validated through histopathological staining, immunofluorescence, Real-time Reverse Transcription PCR, and Western blot. Results A rat model of OEM was successfully established by suturing homologous rat endometrial tissue onto the ovarian surface. Estrous cycle monitoring revealed disrupted estrous cycles in OEM model rats. ELISA demonstrated significantly decreased serum AMH and E2 levels, accompanied by elevated FSH levels in the OEM group. CD10 immunostaining confirmed the positive expression of endometrial tissue, and Prussian blue staining revealed markedly increased iron deposition in the OEM group ovaries. The scRNA-seq identified a total of 70,041 cells, revealing seven major cell types. OEM significantly altered the cellular composition of the ovary, with decreased proportions of granulosa and endothelial cells and an increased overall proportion of immune cells; within the immune-cell compartment, the proportions of macrophages and B cells decreased, whereas those of T cells and neutrophils increased. Granulosa cell analysis revealed disrupted differentiation trajectories, attenuated intercellular AMH and Estradiol signaling, and significantly downregulated mRNA and protein expression of AMH, FSHR, and ESR2 in the OEM group. Stromal cell analysis demonstrated significant enrichment of TGF-β and collagen signaling pathways, with upregulated mRNA and protein expression of COL1A1, TGFB1, and ACTA2 in the OEM group. Masson’s trichrome and Sirius Red staining confirmed significantly increased fibrotic areas in the OEM group ovaries. Immune cell analysis revealed significant enrichment of chemokine signaling and IL-17 signaling pathways, enhanced CCL and CXCL chemokine communication networks, and upregulated mRNA and protein expression of inflammatory mediators, including CXCL1, CCL2, and IL1b in the OEM group. Conclusions This study successfully established a rat model that simulates the key pathological features of OEM. Utilizing scRNA-seq, we constructed, for the first time, a comprehensive cellular landscape of the OEM-affected ovary, revealing the synergistic effects of granulosa cell dysfunction, ovarian stromal fibrosis, and immune dysregulation in OEM-induced ovarian functional decline.