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While myofibroblast activation is central to fibrogenesis, the cellular origins and regulatory mechanisms remain incompletely understood. This study demonstrates that the macrophage\u2013myofibroblast transition (MMT) is a novel source of myofibroblasts in endometriosis and is regulated by the TGFB1\/SMAD3 signaling pathway. Using single-cell RNA sequencing, we identified a distinct subpopulation of CD68+ macrophages co-expressing ACTA2 and extracellular matrix (ECM)-related genes in the human endometrium, which exhibited a myofibroblast-like transcriptional profile and were predominantly located at a fibrotic terminal state along the pseudotime trajectory. Histological and ultrastructural analyses revealed varying degrees of fibrosis and elevated TGFB1 expression in eutopic and ectopic endometrium in endometriosis patients and mouse models. Immunofluorescence confirmed that MMT-positive cells, co-expressing CD68 and \u03b1-SMA, were enriched in endometriotic tissues and primarily derived from M2 macrophages. In mouse models of endometriosis, pharmacological inhibition of TGFB1\/SMAD3 signaling significantly reduced the number of MMT-positive cells and attenuated collagen deposition, particularly in the eutopic endometrium. Furthermore, reduced ectopic lesion volume and epithelial ultrastructural damage following pathway inhibition suggested impaired ectopic lesion survival. These results demonstrate that the TGFB1\/SMAD3 pathway-driven MMT might be a novel contributor to endometrial fibrosis in endometriosis. Targeting the TGFB1\/SMAD3 signaling axis may provide a dual antifibrotic and anti-lesion strategy, offering therapeutic potential to intervene in the progression of endometriosis.<\/jats:p>","DOI":"10.1093\/molehr\/gaaf051","type":"journal-article","created":{"date-parts":[[2025,10,3]],"date-time":"2025-10-03T11:58:02Z","timestamp":1759492682000},"source":"Crossref","is-referenced-by-count":11,"title":["TGFB1\/SMAD3-driven macrophage\u2013myofibroblast transition promotes fibrosis progression in endometriosis"],"prefix":"10.1093","volume":"31","author":[{"given":"Yuchan","family":"Zhong","sequence":"first","affiliation":[{"name":"Division of Reproductive Medicine, West China Second University Hospital of Sichuan University , Chengdu, Sichuan,","place":["China"]},{"name":"Key Laboratory of Birth Defects and Related Diseases of Women and Children of Ministry of Education , Sichuan University, Chengdu, Sichuan,","place":["China"]},{"name":"NHC Key Laboratory of Chronobiology, Sichuan University , Chengdu, Sichuan,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Huang","sequence":"additional","affiliation":[{"name":"Division of Reproductive Medicine, West China Second University Hospital of Sichuan University , Chengdu, Sichuan,","place":["China"]},{"name":"Key Laboratory of Birth Defects and Related Diseases of Women and Children of Ministry of Education , Sichuan University, Chengdu, Sichuan,","place":["China"]},{"name":"NHC Key Laboratory of Chronobiology, Sichuan University , Chengdu, Sichuan,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujing","family":"Li","sequence":"additional","affiliation":[{"name":"Division of Reproductive Medicine, West China Second University Hospital of Sichuan University , Chengdu, Sichuan,","place":["China"]},{"name":"Key Laboratory of Birth Defects and Related Diseases of Women and Children of Ministry of Education , Sichuan University, Chengdu, 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Chengdu, Sichuan,","place":["China"]},{"name":"Key Laboratory of Birth Defects and Related Diseases of Women and Children of Ministry of Education , Sichuan University, Chengdu, Sichuan,","place":["China"]},{"name":"NHC Key Laboratory of Chronobiology, Sichuan University , Chengdu, Sichuan,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chang","family":"Liu","sequence":"additional","affiliation":[{"name":"Division of Reproductive Medicine, West China Second University Hospital of Sichuan University , Chengdu, Sichuan,","place":["China"]},{"name":"Key Laboratory of Birth Defects and Related Diseases of Women and Children of Ministry of Education , Sichuan University, Chengdu, Sichuan,","place":["China"]},{"name":"NHC Key Laboratory of Chronobiology, Sichuan University , Chengdu, Sichuan,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yixian","family":"Han","sequence":"additional","affiliation":[{"name":"Division of 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