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Endometrial organoids have recently been developed, but they consist of only epithelial cells, and their apical surface faces inward, preventing blastocyst attachment. We developed an apical-out mouse endometrial organoid incorporating epithelial and stromal cells, and examined its ability to recapitulate implantation with mouse blastocysts. Mouse uteri were digested with collagenase and cultured in monolayers. The resulting aggregates were then transferred to low-attachment plates for 3D culture. After 7 days, self-organized aggregates contained E-cadherin-positive epithelial cells outside and vimentin-positive stromal cells inside. Mucin 1 signals were observed on the apical side of epithelial cells, confirming the apical-out orientation. Organoids were stimulated with sex steroid hormones and co-cultured with blastocysts. Time-lapse imaging revealed the four implantation steps: blastocyst attachment, epithelial invagination, entosis and invasion. Invaded cells expressed proliferin while surrounding stromal cells expressed cyclooxygenase 2, indicating trophoblast differentiation and decidualization. This novel organoid closely recapitulates the mouse endometrium and implantation process in vitro.<\/jats:p>","DOI":"10.1242\/dev.204461","type":"journal-article","created":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T10:01:56Z","timestamp":1747216916000},"source":"Crossref","is-referenced-by-count":13,"title":["Establishment of an <i>in vitro<\/i> implantation model using a newly developed mouse endometrial organoid"],"prefix":"10.1242","volume":"152","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-7337-524X","authenticated-orcid":false,"given":"Taishi","family":"Fujimura","sequence":"first","affiliation":[{"name":"Yamaguchi University Graduate School of Medicine 1 Department of Obstetrics and Gynecology , , Minamikogushi 1-1-1, Ube 755-8505 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0737-5556","authenticated-orcid":false,"given":"Isao","family":"Tamura","sequence":"additional","affiliation":[{"name":"Yamaguchi University Graduate School of Medicine 1 Department of Obstetrics and Gynecology , , Minamikogushi 1-1-1, Ube 755-8505 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Azumi","family":"Yoshimura","sequence":"additional","affiliation":[{"name":"Yamaguchi University Graduate School of Medicine 2 , Minamikogushi 1-1-1, Ube 755-8505 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshihide","family":"Yoneda","sequence":"additional","affiliation":[{"name":"Yamaguchi University Graduate School of Medicine 1 Department of Obstetrics and Gynecology , , Minamikogushi 1-1-1, Ube 755-8505 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hitomi","family":"Takasaki","sequence":"additional","affiliation":[{"name":"Yamaguchi University Graduate School of Medicine 1 Department of Obstetrics and Gynecology , , Minamikogushi 1-1-1, Ube 755-8505 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amon","family":"Shiroshita","sequence":"additional","affiliation":[{"name":"Yamaguchi University Graduate School of Medicine 1 Department of Obstetrics and Gynecology , , Minamikogushi 1-1-1, Ube 755-8505 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuichiro","family":"Shirafuta","sequence":"additional","affiliation":[{"name":"Yamaguchi University Graduate School of Medicine 1 Department of Obstetrics and Gynecology , , Minamikogushi 1-1-1, Ube 755-8505 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shun","family":"Sato","sequence":"additional","affiliation":[{"name":"Yamaguchi University Graduate School of Medicine 1 Department of Obstetrics and Gynecology , , Minamikogushi 1-1-1, Ube 755-8505 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Norihiro","family":"Sugino","sequence":"additional","affiliation":[{"name":"Yamaguchi University Graduate School of Medicine 1 Department of Obstetrics and Gynecology , , Minamikogushi 1-1-1, Ube 755-8505 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"237","published-online":{"date-parts":[[2025,5,14]]},"reference":[{"key":"2025051406015282900_DEV204461C1","doi-asserted-by":"publisher","first-page":"e230478","DOI":"10.1530\/REP-23-0478","article-title":"Development of polarity-reversed endometrial epithelial organoids","volume":"167","author":"Ahmad","year":"2024","journal-title":"Reproduction"},{"key":"2025051406015282900_DEV204461C2","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1002\/rmb2.12435","article-title":"A gene network of uterine luminal epithelium organizes mouse blastocyst implantation","volume":"21","author":"Aikawa","year":"2022","journal-title":"Reprod. 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