Background: Embryo implantation is a complex process regulated by interactions between endometrial epithelial and stromal cells. The endometrium plays a critical role in this process, providing a supportive environment for embryo attachment. However, conventional 2D cell culture models fail to fully replicate the complex 3D structure and cellular interactions of the endometrium. To overcome these limitations, 3D organoid models have been developed to better mimic the in vivo endometrial environment. Methods: In this study, a multicellular uterine organoid model was developed using porcine endometrial epithelial cells (pEECs) and porcine endometrial stromal cells (pESCs) to evaluate the effects of the endometrial environment on embryo implantation. First, single-cell endometrial organoids (pEOs) were formed by culturing pEECs in Matrigel, and their basic cellular characteristics were assessed. Then, a multicellular uterine organoid model was established by combining pEOs with pESCs. Finally, porcine embryos were co-cultured with this model to examine its effect on embryo attachment. Results: The multicellular uterine organoid model facilitated embryo attachment, demonstrating that the 3D structure and cellular interactions of the endometrium play a significant role in embryo implantation. The presence of both epithelial and stromal cells contributed to a more physiologically relevant environment that supported embryo adhesion. Conclusions: This study demonstrates that a multicellular uterine organoid model can serve as a useful in vitro system for porcine embryo implantation research. This model may contribute to a better understanding of embryo development and implantation mechanisms, with potential applications in regenerative medicine and biotechnology.

Background: Porcine embryonic development is widely utilized in the medical industry. However, the blastocyst development rate in vitro is lower compared to in vivo . To address this issue, various supplements are employed. Extracellular vesicles (EVs) play the role of communicators that carry many bioactive cargoes. Additionally, the contents of EVs can vary on the estrous cycle. Methods: We compared the effects of adding EVs derived from porcine uterine fluid (UF), categorized as non-EV (G1), EVs in estrus (G2) and EVs in diestrus (G3). After in vitro culture (IVC) was performed in three different groups, cleavage rate and blastocyst development rate were examined. In addition, glutathione (GSH) and reactive oxygen species (ROS) levels were measured 2 days after activation to assess oxidative stress. Results: Using NTA and cryo-TEM, we confirmed the presence of EVs with sizes ranging from 30 nm to 200 nm, that the particles were suitable for analysis for analysis. In IVC data, the highest cleavage rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Similarly, the highest blastocyst development rate was observed in G2, which was significantly different from G1 but not significantly different from the next highest, G3. Conclusions: These results indicate that estrus derived EVs contain biofactors beneficial for early blastocyst development, including GSH which protects the blastocyst from oxidative stress. Additionally, although diestrus-derived EVs are expected to have some effect on blastocyst development, it appeared to be less effective than estrus-derived EVs.