{"paper_id":"45940617-6b1b-4ce1-9c8b-b51a4bd94d22","body_text":"Abstract\nBackground\nFibrosis, 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).\nMethods\nWe 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.\nResults\nWe 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.\nConclusion\nCellular 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.\nSimilar content being viewed by others\nAbbreviations\n- DEGs:\n-\nDifferentially expressed genes\n- EMs:\n-\nEndometriosis\n- GnRHa:\n-\nGonadotropin-releasing hormone agonist\n- scRNA-seq:\n-\nSingle-cell RNA-sequencing\n- t-SNE:\n-\nT-distributed stochastic neighbour embedding\nReferences\nPhilippa TKS, Andrew WH. Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell. 2021;184(11):2807–2824.\nTaylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet. 2021;397(10276):839–52.\nGuo M, Bafigil C, Tapmeier T, Hubbard C, Manek S, Shang C, et al. Mass cytometry analysis reveals a distinct immune environment in peritoneal fuid in endometriosis: a characterisation study. BMC Med. 2020;18:3.\nPractice Committee of the American Society for Reproductive Medicine. Endometriosis and infertility. Fertil Steril. 2004;81:1441–6.\nZondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Viganò P. Endometriosis. Nat Rev Dis Primers. 2018. https://doi.org/10.1038/s41572-018-0008-5.\nNirgianakis K, Ma L, McKinnon B, Mueller MD. Recurrence patterns after surgery in patients with different endometriosis subtypes: a long-term hospital-based cohort study. J Clin Med. 2020;9(2):496.\nBulun SE. Endometriosis. N Engl J Med. 2009;360(3):268–79.\nVallve-Juanico J, Houshdaran S, Giudice LC. The endometrial immune environment of women with endometriosis. Hum Reprod Update. 2019;25(5):564–91.\nVigano P, Ottolina J, Bartiromo L, Bonavina G, Schimberni M, Villanacci R, et al. Cellular components contributing to fibrosis in endometriosis: a literature review. J Minim Invasive Gynecol. 2020;27(2):287–95.\nSaare M, Krigul KL, Laisk-Podar T, Ponandai-Srinivasan S, Rahmioglu N, Lalit Kumar PG, et al. DNA methylation alterations-potential cause of endometriosis pathogenesis or a refection of tissue heterogeneity? Biol Reprod. 2018;99:273–82.\nPaul F, Arkin Y, Giladi A, Jaitin DA, Kenigsberg E, Keren-Shaul H, et al. Transcriptional heterogeneity and lineage commitment in myeloid progenitors. Cell. 2016;164:325.\nWang W, Vilella F, Alama P, Moreno I, Mignardi M, Isakova A, et al. Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle. Nat Med. 2020;26(10):1644–53.\nLv H, Zhao G, Jiang P, Wang H, Wang Z, Yao S, et al. Deciphering the endometrial niche of human thin endometrium at single-cell resolution. Proc Natl Acad Sci USA. 2022;119(8):e2115912119.\nFerrero H, Corachán A, Aguilar A, Quiñonero A, Carbajo-García MC, Alamá P, et al. Single-cell RNA sequencing of oocytes from ovarian endometriosis patients reveals a differential transcriptomic profile associated with lower quality. Hum Reprod. 2019;34(7):1302–12.\nZhang Y, Chang X, Wu D, Deng M, Miao J, Jin Z. Down-regulation of exosomal miR-214-3p targeting CCN2 contributes to endometriosis fibrosis and the role of exosomes in the horizontal transfer of miR-214-3p. Reprod Sci. 2021;28(3):715–27.\nMa J, Zhang L, Zhan H, Mo Y, Ren Z, Shao A, et al. Single-cell transcriptomic analysis of endometriosis provides insights into fibroblast fates and immune cell heterogeneity. Cell Biosci. 2021;11(1):125.\nFonseca MAS, Haro M, Wright KN, Lin X, Abbasi F, Sun J, et al. Single-cell transcriptomic analysis of endometriosis. Nat Genet. 2023;55(2):255–67.\nHu C, Li T, Xu Y, Zhang X, Li F, Bai J, et al. Cell Marker 2.0: an updated database of manually curated cell markers in human/mouse and web tools based on scRNA-seq data. Nucleic Acids Res. 2023;51(1):870–6.\nQueckbörner S, von Grothusen C, Boggavarapu NR, Francis RM, Davies LC, Gemzell-Danielsson K. Stromal heterogeneity in the human proliferative endometrium-a single-cell RNA sequencing study. J Pers Med. 2021;11(6):448.\nSchwab KE, Gargett CE. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium. Hum Reprod. 2007;22:2903–11.\nMasuda H, Anwar SS, Buhring HJ, Rao JR, Gargett CE. A novel marker of human endometrial mesenchymal stem-like cells. Cell Transplant. 2012;21:2201–14.\nChan RW, Schwab KE, Gargett CE. Clonogenicity of human endometrial epithelial and stromal cells. Biol Reprod. 2004;70:1738–50.\nKonrad L, Kortum J, Nabham R, Gronbach J, Dietze R, Oehmke F, et al. Composition of the stroma in the human endometrium and endometriosis. Reprod Sci. 2018;25(7):1106–15.\nJabbour HN, Kelly RW, Fraser HM, Critchley HO. Endocrine regulation of menstruation. Endocr Rev. 2006;27(1):17–46.\nTan Y, Flynn WF, Sivajothi S, Luo D, Bozal SB, Davé M, et al. Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Nat Cell Biol. 2022;24(8):1306–18.\nZhu S, Wang A, Xu W, Hu L, Sun J, Wang X. The heterogeneity of fibrosis and angiogenesis in endometriosis revealed by single-cell RNA-sequencing. Biochimica et Biophysica Acta (BBA). 2023;1869(2):166602.\nLv C, Ren C, Yu Y, Yin H, Huang C, Yang G, et al. Wentilactone A reverses the NF-κB/ECM1 signaling-induced cisplatin resistance through inhibition of IKK/IκB in ovarian cancer cells. Nutrients. 2022;14(18):3790.\nYin H, Wang J, Li H, Yu Y, Wang X, Lu L, et al. Extracellular matrix protein-1 secretory isoform promotes ovarian cancer through increasing alternative mRNA splicing and stemness. Nat Commun. 2021;12(1):4230.\nFan W, Liu T, Chen W, Hammad S, Longerich T, Hausser I, et al. Ecm1 prevents activation of transforming growth factor β, hepatic stellate cells, and fibrogenesis in mice. Gastroenterology. 2019;157(5):1352-1367.e13.\nZhang C, Cheng H, Ye X, Cui H, Li Y, Zhu H, et al. Ecm1 promotes migration and invasion in endometriosis. Reprod Biol. 2024;24(1):100826.\nDing D, Wang X, Chen Y, Benagiano G, Liu X, Guo SW. Evidence in support for the progressive nature of ovarian endometriomas. J Clin Endocrinol Metab. 2020;105(7):dgaa189.\nde Barcena Arellano ML, Gericke J, Reichelt U, Okuducu AF, Ebert AD, Chiantera V, et al. Immunohistochemical characterization of endometriosis-associated smooth muscle cells in human peritoneal endometriotic lesions. Hum Reprod. 2011;26(10):2721–30.\nGargett CE. Uterine stem cells: what is the evidence? Hum Reprod Update. 2007;13(1):87–101.\nDucommun S, Jannig PR, Cervenka I, Murgia M, Mittenbühler MJ, Chernogubova E, et al. Mustn1 is a smooth muscle cell-secreted microprotein that modulates skeletal muscle extracellular matrix composition. Mol Metab. 2024;82:101912.\nBerger M, Bergers G, Arnold B, Hämmerling GJ, Ganss R. Regulator of G-protein signaling-5 induction in pericytes coincides with active vessel remodeling during neovascularization. Blood. 2005;105(3):1094–101.\nDe Vries L, Zheng B, Fischer T, Elenko E, Farquhar MG. The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol. 2000;40:235–71.\nPatel M. Recent trends in medical management of endometriosis. J Obstet Gynaecol India. 2024;74(6):479–83.\nKiba A, Banno K, Yanokura M, Asada M, Nakayama Y, Aoki D. Differential mRNA expression profiling in ovarian endometriotic tissue with versus without leuprolide acetate treatment. J Obstet Gynaecol Res. 2015;41(10):1598–606.\nLin X, Dai Y, Tong X, Xu W, Huang Q, Jin X. Excessive oxidative stress in cumulus granulosa cells induced cell senescence contributes to endometriosis-associated infertility. Redox Biol. 2020;30:101431.\nYu CX, Song JH, Li YF, Tuo Y, Zheng JJ, Miao RJ. Correlation between replicative senescence of endometrial gland epithelial cells in shedding and non-shedding endometria and endometriosis cyst during menstruation. Gynecol Endocrinol. 2018;34(11):981–6.\nCicinelli E, Trojano G, Mastromauro M, Vimercati A, Marinaccio M, Mitola PC, et al. Higher prevalence of chronic endometritis in women with endometriosis: a possible etiopathogenetic link. Fertil Steril. 2017;108(2):289-295.e1.\nPoli-Neto OB, Carlos D, Favaretto A, Rosa-E-Silva JC, Meola J, Tiezzi D. Eutopic endometrium from women with endometriosis and chlamydial endometritis share immunological cell types and DNA repair imbalance: a transcriptome meta-analytical perspective. J Reprod Immunol. 2021;145:103307.\nHasan A, Rahim A, Afzal M, Naveed AK, Ayub S, Jahan S. Serum albumin and C3 complement levels in endometriosis. J Coll Physicians Surg Pak. 2019;29(8):702–5.\nAcknowledgements\nWe are very grateful to our gynecologic colleagues for patient recruitment.\nFunding\nThis 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.\nAuthor information\nAuthors and Affiliations\nContributions\nYanqin 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.\nCorresponding author\nEthics declarations\nConsent for Publication\nNot applicable.\nCompeting Interests\nThe authors declare that they have no competing interests.\nAdditional information\nPublisher's Note\nSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nRights and permissions\nSpringer 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.\nAbout this article\nCite this article\nZhang, 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\nReceived:\nAccepted:\nPublished:\nVersion of record:\nIssue date:\nDOI: https://doi.org/10.1007/s43032-026-02056-0","source_license":"public-domain-us","license_restricted":false}