58 Derivation and characterization of porcine endometrial organoids and their effects on the embryonic development

Endometrial organoids (EOs) play a pivotal role in advancing our understanding of the complex biology of maternal-embryonic crosstalk and offer a remarkable tool for investigating various aspects of reproductive processes involved in embryo implantation. This work was carried out to generate EOs and optimize their cryopreservation and co-culture with embryos in pigs. Uteri (n = 6) of diestrus sows were harvested after slaughtering, and endometrial tissues were aseptically collected. The tissues were then digested with collagenase for 2 h, purified using a 70-µm cell strainer, and suspended in cooled reduced growth factors Matrigel. Domes of Matrigel containing the endometrial cells were formed in Petri dishes and covered with organoid formation medium and was kept in a humid atmosphere of 5% CO₂ at 38.5°C. The medium comprised an advanced Dulbecco’s Modified Eagle Medium with a specific cocktail of supplements, growth factors, and hormones. To improve the long-term storage of EOs, a rock inhibitor (RI, Y-27632, 10 μM) was added to the cryopreservation medium. Real-time quantitative (q)PCR was used to compare gene expressions in control and RI-cryopreserved EOs. Day 7 parthenogenetically activated embryos (n = 90, 6 replicates) were co-cultured with EOs, and embryonic cell numbers and attachment to polystyrene-coated cell culture dishes were monitored in comparison to the control group. Ratios of embryo attachment were analysed with chi-squared test, while data of embryo cell number and qPCR were analysed with Student’s t-test. Significant differences were considered when P < 0.05. Results indicated that EOs with a typical spherical shape and sizes of 100–300 µm can be retrieved on Day 7 of culture and EOs can be maintained in the Matrigel dome for successive 20 days with a proportional increase in size, reaching around 1 mm in diameter. Individual EOs can be harvested and cultured on tissue culture dishes for colony and outgrowth formation. Immunofluorescence analysis revealed the cytoplasmic expression of endometrial markers (mucin-1, pan-cytokeratin, and vimentin), and the nuclear expression of Ki67, indicating that more than 80% of the organoid nuclei are proliferative. The expression of these proteins was maintained after EOs culture and outgrowth formation, with a marked reduction in the expression of Ki67 to 18% of EOs nuclei after 7 days of EOs culture. The RI increased the yield and quality of organoids after freezing/thawing, and qPCR results showed that RI increased the expression of anti-apoptotic genes BCL2 and BCLXL, as well as miR-21, while reducing the expression of apoptotic genes BAK and BAX, in addition to reducing miR-155 and miR-100 (P < 0.05). Co-cultured embryos showed a 5-fold increase in embryonic cell numbers (P < 0.05), and the proportion of trophoblast outgrowths increased 7-fold compared to the control embryos (P < 0.05). Embryos that were co-cultured over the EOs outgrowths showed embryonic attachment after 24 h. Immunofluorescence staining of the attached embryos showed nuclear expression of trophoblast marker CDX2 in the periphery of the EOs outgrowths, which would act as a model for embryo implantation. The current work optimizes the derivation and cryopreservation of EOs and provides a valuable platform to study the intricate processes involved in porcine embryo implantation.