Cell Subtypes and Gene Dysfunction in Ovarian Endometriosis Before and After GnRHa Treatment Revealed by Single-Cell RNA Sequencing.

Fibrosis, angiogenesis and chronic inflammation are the intrinsic characteristics of endometriosis. It is accompanied by significant changes in the cell composition of both ectopic and eutopic endometrial tissues, occurring both before and after treatment with a gonadotropin-releasing hormone agonist (GnRHa). To further understand the pathological characteristics of fibrosis associated with endometriosis, it is worthwhile to explore the cellular heterogeneity of both ectopic and eutopic endometrium, as well as the changes before and after GnRHa treatment, using single-cell RNA sequencing (scRNA-seq). We performed scRNA-seq on a total of six samples (eutopic endometrium and ectopic lesions) from three patients with confirmed endometriosis. The patients comprised two untreated groups and one group that had undergone three months of GnRHa treatment. We profiled the transcriptomes of approximately 73,531 single cells from these samples. To this end, we aimed to characterize the changes in both the eutopic endometrium and the ectopic lesions during treatment. We observed a significant difference in the cellular composition between ectopic and eutopic endometrium in endometriosis patients. We divided stromal cells into five main subgroups, named ACTA2 + cluster, ECM1 + cluster, PDGFRB + cluster, Stromal cluster 4, and Stromal cluster 5 respectively. The ACTA2 + cluster is found predominantly in ectopic tissues and ECM1 + cluster mainly exists in eutopic endometrial tissues. In samples from GnRHa-treated patients, we observed a significant reduction in the number of cells within the ECM1 + cluster compared to untreated patients. This cluster represents the most critical cell subpopulation susceptible to hormonal influence, and the initiation of fibrosis may be triggered by decreased ECM1 expression in stromal cells. Analysis of immune cell composition revealed that CD8 + T-cells were significantly increased in ectopic endometrium compared to both eutopic endometrium and, furthermore, to post-GnRHa treatment tissues. The ratio of NK1 cells was significantly decreased, and the percentage of macrophages was increased in ectopic lesions compared to eutopic endometrium. Furthermore, GnRHa treatment induced a marked elevation in the percentage of neutrophils in ectopic endometrium. Cellular heterogeneity exists between ectopic and eutopic endometrium in ovarian endometriosis. Our analysis revealed significant stromal cell differences between these tissues and identified an ECM1-high subpopulation as the key cellular group in the non-fibrotic state. Furthermore, reduced ECM1 protein expression appears to initiate the fibrotic process in endometriosis.