Generation of porcine endometrial organoids and their use as a model for enhancing embryonic attachment and elongation.

Endometrial organoids (EOs) are acceptable models for understanding maternal-embryonic crosstalk. This study was conducted to generate EOs and optimize their cryopreservation and provide co-culture modeling with embryos. The endometrial tissues were used for culturing the organoids inside domes of Matrigel®. To improve the long-term storage of EOs, 10 µM ROCK inhibitor (RI) was added to the cryopreservation medium. Day-7 parthenogenetically activated embryos were co-cultured with EOs or EOs outgrowths, and embryonic cell numbers and embryo attachment were monitored. Spherical EOs 100-300 µm in size can be retrieved on Day 7 of culture, and larger EOs, approximately 1.5 mm in diameter, can be maintained in the Matrigel® dome for 21 days. The nuclear expression of Ki67 indicates that more than 80% of EOs nuclei were proliferative. EOs exhibit unique novel characters such as formation of extracellular matrix and ability for fusion. RI increased the yield and quality of organoids after freezing or thawing. The cell number of co-cultured embryos increased five-fold, and the proportion of trophoblast outgrowths increased seven-fold compared with those of control embryos. The embryos cultured with EO-conditioned medium showed a better attachment rate than the other models and -for the first time- we reported embryonic elongation. Immunofluorescence staining of the attached embryos showed CDX2 in the periphery of EOs outgrowths. The 3D assembly and cryopreservation of EOs was optimized, and EO coculture supported embryo attachment, trophoblast outgrowth, and elongation which provides a valuable tool for studying the intricate processes involved in porcine embryo implantation.