Hypermethylation of Klotho and Peroxisome Proliferator-Activated Receptor γ Concomitant with Overexpression of DNA Methyltransferase 1 in Adenomyosis

Cellular senescence is known to be involved in tissue repair, but its role in adenomyosis remains unclear. This study was tasked to evaluate the expression of Klotho, a well-known aging-suppressing protein, as well as PPARγ and DNMT1 in adenomyotic lesions (AD) in comparison with that of control endometrium (CT). We performed immunohistochemistry analysis of markers of cellular senescence p16 and p21, along with Klotho, PPARγ and DNMT1 in CT and AD samples, followed by the quantification of gene expression of Klotho, PPARγ and DNMT1 in epithelial organoids derived from AD and CT samples and methylation-specific PCR to evaluate promoter methylation status. The effect of forced expression and knockdown of DNMT1 on Klotho and PPARγ expression in ectopic endometrial epithelial cells was evaluated. We found that both p16 and p21 immunoreactivity in AD was significantly higher while that of Klotho and PPARγ was significantly lower than CT samples, which was concomitant with elevated immunoexpression of DNMT1. The results were confirmed by transcriptional analysis using epithelial organoids derived from AD and CT samples. In addition, the promoter regions of both Klotho and PPARγ genes were hypermethylated in AD as compared with CT, and treatment with HDAC and DNMT inhibitors reactivated the expression of both Klotho and PPARγ. Forced expression of DNMT1 resulted in downregulation of both Klotho and PPARγ but its knockdown increased their expression. Thus, overexpression of DNMT1 seems to facilitate the promoter hypermethylation of both Klotho and PPARγ in AD, resulting in their reduced expression that is suggestive of the role of senescence in adenomyosis. Similar content being viewed by others Data Availability (data transparency) The data presented in this study are available upon written request from the corresponding author explaining the use and purposes. Code Availability Not applicable.

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This research was supported in part by grant 82071623 (SWG) from the National Natural Science Foundation of China, and grant SHDC2020CR2062B (SWG) from Shanghai Shenkang Center for Hospital Development. Author information Authors and Affiliations Corresponding author Ethics declarations Ethical Approval All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients for being included in the study. The study was approved by the institutional ethics review board of Shanghai OB/GYN Hospital (No. 2021-35), Fudan University. Each patient enrolled in this study signed an informed consent for all the procedures and to allow data collection and analysis for research purposes. Consent for Publication All authors approved this manuscript and consented for publication. Consent to Participate All subjects enrolled in this study signed an informed consent for all the procedures and to allow data collection and analysis for research purposes. Conflict of Interest J.F. and X.L. have no conflict of interest. S.W.G. is a member of the Scientific Advisory Board of Heranova BioSciences and of FimmCyte A.G., and has provided consultancy advice to these companies, as well as to Sound Bioventures, but these activities had no bearing on this work. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Electronic Supplementary Material Below is the link to the electronic supplementary material. 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 Fan, J., Liu, X. & Guo, SW. Hypermethylation of Klotho and Peroxisome Proliferator-Activated Receptor γ Concomitant with Overexpression of DNA Methyltransferase 1 in Adenomyosis. Reprod. Sci. 32, 668–683 (2025). https://doi.org/10.1007/s43032-024-01599-4 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-024-01599-4