Human Endometriosis Scaffold Can Enhance Cell Seeding and Engraftment; an In Vitro and In Vivo Study

The extracellular matrix (ECM) critically influences cell behavior, yet its properties in human endometrial lesions (HEL) and human uterine fibromas (HUF) are not well characterized. This study aimed to characterize their ECM and evaluate its impact on cell engraftment and proliferation while optimizing a decellularization protocol. HEL and HUF tissues, collected during laparoscopic surgeries, were decellularized using a novel protocol. Complete cell removal and preserved ECM microstructure were confirmed by histology, DAPI, Masson’s trichrome staining and scanning electron microscopy. The decellularized scaffolds were used as a platform for three-dimensional culture of human endometrial-derived mesenchymal stem cells (hEMSCs), with HUF serving as a fibrotic control originated from the same organ system. The biological impact of the ECM was assessed via immunohistochemistry for engraftment marker matrix metalloproteinase-9 and proliferation marker antigen Kiel-67. The in vivo recellularization potential of HEL scaffolds was further evaluated in a rat model, with HEL scaffold group at two timepoints (n = 6/group) and a sham control (n = 3). Results confirmed complete decellularization with maintained ECM integrity in both HEL and HUF. In vitro evaluation indicated that hEMSCs seeded more efficiently onto HEL scaffolds (51.16 ± 28.84) compared to HUF scaffolds (6.16 ± 7.29) (p = 0.012). The in vivo peritoneal implanted HEL scaffolds demonstrated significant time-dependent host cell recruitment and remodeling compared to the sham control. In conclusion, the decellularized HEL scaffold provides a superior ECM platform for cell seeding and engraftment compared to HUF, making it a promising platform for modeling endometriosis in both in vitro and in vivo settings. Similar content being viewed by others Data Availability The datasets used and/or analyzed during the current study are available from the co-corresponding authors on reasonable request. Abbreviations - dHEL: - Decellularized human endometrial lesion - dHUF: - Decellularized human uterine fibroma - DAPI: - 4’ ,6 -diamidino-2-phenylindole - DMSO: - Dimethyl sulfoxide - ECM: - Extracellular matrix - EDTA: - Ethylenediamine tetra-acetic acid - HEL: - Human Endometrial Lesion - H&E: - Hematoxylin & eosin staining - hEMSCs: - Human endometrial derived mesenchymal stem cells - HUF: - Human uterine fibroma - IHC: - Immunohistochemistry staining - Ki-67: - Antigen Kiel-67 - MMP-9: - Matrix metalloproteinase-9 - MTT: - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - OD: - Optical density - PBS: - Phosphate buffered sodium - SDS: - Sodium dodecyl sulfate - SEM: - Scanning electron microscopy

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Acknowledgments We express our profound gratitude to Dr. Masoomeh Shiravi Khoozani for her invaluable contribution in providing histopathological details of our study's in vitro and in vivo explants. Her insights and expertise were instrumental in enhancing the quality of our study, and we are deeply thankful for her dedicated involvement. Fig. 1 is “Created with BioRender.com”. Funding No funding was received for conducting this study. Author information Authors and Affiliations Contributions All authors contributed to the study conception and design. Conceptualization and Methodology were designed by Abdol-Mohammad Kajbafzadeh and Masoumeh Majidi Zolbin. Material preparation, data collection and analysis were performed by Ashkan Azimzadeh, Roxana Sahmani, Bahareh Mohammadi, Saman Behboodi Tanourlouee, Masoumeh Ekhtiari, Negar Mohammadi Ganjaroudi, Parmida Sadat Pezeshki, and Ali Mohebbi. The first draft of the manuscript was written by Negar Mohammadi Ganjaroudi and all authors commented on previous versions of the manuscript. All authors read and approved of the final manuscript. Corresponding authors Ethics declarations Ethics Approval All procedures were performed in accordance with the Tehran University of Medical Sciences’ Guidelines and approved by the university’s Ethics Committee (IR.TUMS.VCR.REC.1398.406). The study was conducted in accordance with the principles outlined in the Declaration of Helsinki. Informed consent achieved from all premenopausal patient cases undergoing laparoscopy or laparotomy regarding treatment of endometriosis or uterine fibroma, describing the procedure and aims of the study in compliance with the regulations concerning the use of human tissues. All the animal procedures were approved by the animal ethics committee of the Tehran University of Medical Sciences, School of Medicine and Education Section of Basic Sciences and undergone surgery in accordance with the animal welfare Act and Guide for The Care and Use of Laboratory Animals and ARRIVE guidelines. Competing Interests The authors have no competing interests to declare relevant to this article’s content. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ashkan Azimzadeh and Roxana Sahmani are equally contributed as co-first authors. 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 Azimzadeh, A., Sahmani, R., Mohammadi Ganjaroudi, N. et al. Human Endometriosis Scaffold Can Enhance Cell Seeding and Engraftment; an In Vitro and In Vivo Study. Reprod. Sci. 33, 84–93 (2026). https://doi.org/10.1007/s43032-025-02032-0 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-025-02032-0