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

Background 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. Similar content being viewed by others Abbreviations - DEGs: - Differentially expressed genes - EMs: - Endometriosis - GnRHa: - Gonadotropin-releasing hormone agonist - scRNA-seq: - Single-cell RNA-sequencing - t-SNE: - T-distributed stochastic neighbour embedding

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We are very grateful to our gynecologic colleagues for patient recruitment. Funding This research was funded by National Natural Science Foundation of China (Grant No. 82271677) Jinwei Miao; Mangzhong Technological Innovation Project of Beijing Obstetrics and Gynecology Hospital, Capital Medical University. (No. FCYYMZB202501) Yanqin Zhang; Beijing Obstetrics and Gynecology Hospital, Capital Medical University. (No. FCYYJC202408) Yanqin Zhang; Laboratory for Clinical Medicine, Capital Medical University, Jinwei Miao; Beijing Hospitals Authority’s Ascent Plan, (Code: DFL20221201) Jinwei Miao. Author information Authors and Affiliations Contributions Yanqin Zhang and Xinyi Zhang were responsible for the data analysis and article writing of this paper. Mengqi Deng were in charge of preliminary data processing, while Yuning Geng and Chunyu Xu were responsible for the acquisition and submission of all samples for this article. Jinwei Miao oversaw the overall conception and quality control of this study. Corresponding author Ethics declarations Consent for Publication Not applicable. Competing Interests The authors declare that they have no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Zhang, Y., Zhang, X., Deng, M. et al. Cell Subtypes and Gene Dysfunction in Ovarian Endometriosis Before and After GnRHa Treatment Revealed by Single-Cell RNA Sequencing. Reprod. Sci. 33, 331–345 (2026). https://doi.org/10.1007/s43032-026-02056-0 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-026-02056-0