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Current in vitro models fail to replicate the complexity of the human endometrium throughout the entire menstrual cycle. This study aimed to assess the physiological response of human endometrial organoids (hEOs) to in vitro hormonal treatments designed to mimic the hormonal fluctuations of the menstrual cycle. Endometrial biopsies from three healthy women were used to develop hEOs, which were treated over 28 days with three hormonal stimulation strategies: (1) estrogen only (E) to mimic the proliferative phase, (2) the addition of progesterone (EP) to simulate the secretory phase, and (3) the further addition of cAMP (EPC) to enhance the secretory functions of hEOs. Gene and protein expression were analyzed using qPCR, IHC, and ELISA. The hEOs exhibited proliferation, gland formation, and appropriate expression of markers such as E-cadherin and Ki67. The hormonal treatments induced significant changes in PR, HSD17B1, PAEP, SPP1, and other genes relevant to endometrial function, closely mirroring in vivo physiological responses. The prominent changes were observed in EPC-treated hEOs (week 4) with significantly high expression of uterine milk components such as glycodelin (PAEP) and osteopontin (SPP1), reflecting mid- to late-secretory phase physiology. This model successfully recapitulates human menstrual cycle dynamics and offers a promising platform for studying endometrial disorders and advancing personalized treatments in gynecology.<\/jats:p>","DOI":"10.3390\/cells13211811","type":"journal-article","created":{"date-parts":[[2024,11,4]],"date-time":"2024-11-04T03:57:34Z","timestamp":1730692654000},"page":"1811","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Proof-of-Concept for Long-Term Human Endometrial Epithelial Organoids in Modeling Menstrual Cycle Responses"],"prefix":"10.3390","volume":"13","author":[{"given":"Yanyu","family":"Jiang","sequence":"first","affiliation":[{"name":"Laboratory of Translational Fertility Preservation, Department of Oncology and Pathology, Karolinska Institutet, 171 76 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5820-3490","authenticated-orcid":false,"given":"Arturo Reyes","family":"Palomares","sequence":"additional","affiliation":[{"name":"Laboratory of Translational Fertility Preservation, Department of Oncology and Pathology, Karolinska Institutet, 171 76 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6596-9451","authenticated-orcid":false,"given":"Patricia","family":"Munoz","sequence":"additional","affiliation":[{"name":"Department of Biosciences and Nutrition, Karolinska Institute, 141 52 Huddinge, Sweden"},{"name":"Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Karolinska Institute, 171 76 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6828-2583","authenticated-orcid":false,"given":"Ivan","family":"Nalvarte","sequence":"additional","affiliation":[{"name":"Department of Biosciences and Nutrition, Karolinska Institute, 141 52 Huddinge, Sweden"},{"name":"Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Karolinska Institute, 171 76 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1997-3107","authenticated-orcid":false,"given":"Ganesh","family":"Acharya","sequence":"additional","affiliation":[{"name":"Department of Clinical Science, Intervention and Technology-CLINTEC, Karolinska Institute, 141 52 Huddinge, Sweden"},{"name":"Center for Fetal Medicine, Karolinska University Hospital, 171 76 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose","family":"Inzunza","sequence":"additional","affiliation":[{"name":"Department of Biosciences and Nutrition, Karolinska Institute, 141 52 Huddinge, Sweden"},{"name":"Department of Laboratory Medicine, Karolinska Institute, 141 52 Huddinge, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2165-0152","authenticated-orcid":false,"given":"Mukesh","family":"Varshney","sequence":"additional","affiliation":[{"name":"Department of Biosciences and Nutrition, Karolinska Institute, 141 52 Huddinge, Sweden"},{"name":"Department of Laboratory Medicine, Karolinska Institute, 141 52 Huddinge, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4378-6181","authenticated-orcid":false,"given":"Kenny Alexandra","family":"Rodriguez-Wallberg","sequence":"additional","affiliation":[{"name":"Laboratory of Translational Fertility Preservation, Department of Oncology and Pathology, Karolinska Institutet, 171 76 Stockholm, Sweden"},{"name":"Department of Reproductive Medicine, Division of Gynecology and Reproduction, Karolinska University Hospital, 171 76 Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.mce.2008.02.026","article-title":"Hormone and growth factor signaling in endometrial renewal: Role of stem\/progenitor cells","volume":"288","author":"Gargett","year":"2008","journal-title":"Mol. 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