Endometrial Mesenchymal Stem Cell-Derived Exosomal miR-4669 Promotes EMT in Adenomyosis by Inducing M2 Macrophage Polarization via the DUSP6/ERK Pathway

Adenomyosis (AM), a gynecological disorder that severely affects female reproductive health. AM-associated macrophage (AAM) polarization-induced epithelial–mesenchymal transition (EMT) is a key driver of AM progression. In this study, we investigated the role and underlying mechanisms of endometrial mesenchymal stem cell (eMSC)-derived exosomes in regulating AAM polarization and the subsequent EMT of endometrial epithelial cells (EECs). In vitro coculture studies revealed that AM eutopic eMSCs markedly induced M2 macrophage polarization via exosomes and promoted EMT of EECs. Differentially expressed microRNAs (DE-miRNAs) between exosomes derived from normal eMSCs (N-eMSCs) and AM eutopic eMSCs (A-eMSCs) were identified using miRNA sequencing and miR-4669 was found to be the most significantly upregulated miRNA. Internalization of exosomal miR-4669 by macrophages induced their polarization toward the M2 phenotype and promoted the EMT of EECs. Mechanistic analysis using luciferase assay, mRNA sequencing, and rescue experiments revealed that miR-4669 induced M2 macrophage polarization via downregulation of DUSP6 and activation of MAPK/ERK signaling. The polarized M2 macrophages promoted the EMT of ISK cells via TGF-β1 secretion. In an AM xenograft mouse model, miR-4669 depletion inhibited AM progression by targeting the DUSP6/ERK1/2 pathway in macrophages. Overall, AM A-eMSC-derived exosomal miR-4669 facilitates M2 macrophage polarization by targeting the DUSP6/ERK signaling pathway, thereby promoting EMT of EECs via TGF-β1 secretion. These findings open avenues for developing novel preventive and therapeutic strategies for AM. Graphical Abstract Similar content being viewed by others Data Availability The raw miR-seq and mRNA-seq data have been uploaded to the Gene Expression Omnibus (GEO) database (accession numbers: GSE260626 and GSE287900). Uncropped original Western blot and relevant data are provided in the Supplementary files. The data generated in this study are available upon reasonable request from the corresponding author.

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This work was supported by the National Natural Science Foundation of China (Grant No. 81403321), Science and Technology Project of Jiangsu Provincial Administration of Traditional Chinese Medicine (Grant No. MS2023039, MS2023079, and ZD202419), and Development Fund Project of the Affiliated Hospital of Xuzhou Medical University (Grant No. XYFY202322). Funding National Natural Science Foundation of China (Grant No. 81403321), Science and Technology Project of Jiangsu Provincial Administration of Traditional Chinese Medicine (Grant No. MS2023039, MS2023079, and ZD202419), and Development Fund Project of the Affiliated Hospital of Xuzhou Medical University (Grant No. XYFY202322). Author information Authors and Affiliations Contributions Y.Q. and X.W. conducted the experiments, wrote the original draft, and performed formal analysis and visualization; J.C. contributed to investigation, resources, and writing-review & editing; J.W., Q.D., F.Z., and X.C. were responsible for data curation, validation, and software; Y.L. and G.W. provided funding acquisition, resources, supervision, and writing-review & editing; T.G., M.H., and Z.L. conceptualized the study, acquired funding, administered the project, provided resources, supervised the research, and contributed to writing-review & editing. All authors have read and agreed to the published version of the manuscript. Corresponding author Ethics declarations Ethics Approval This study was approved by the Medical Ethics Committee of Jiangsu Province Hospital with Integration of Chinese and Western Medicine (Approval No. 2023-LWKYZ-050). All patients included in the study provided informed consent. Consent for Publication All authors provided consent for publication. Consent to Participate Not applicable. Competing Interests The authors declare no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Meihua Huang, Zhenli Li, and Tao Gui are also the co-corresponding authors of this work. Supplementary Information Below is the link to the electronic supplementary material. ESM 1 (download PNG ) (PNG 727 KB) ESM 2 (download PNG ) (PNG 975 KB) ESM 3 (download PNG ) (PNG 1.96 MB) ESM 4 (download PNG ) (PNG 2.23 MB) 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 Qiu, Y., Wei, X., Cao, J. et al. Endometrial Mesenchymal Stem Cell-Derived Exosomal miR-4669 Promotes EMT in Adenomyosis by Inducing M2 Macrophage Polarization via the DUSP6/ERK Pathway. Reprod. Sci. 32, 2922–2945 (2025). https://doi.org/10.1007/s43032-025-01944-1 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-025-01944-1