84 Characterization and effect of endometriosomes on the development and quality of pre-implantation bovine embryos

The efficiency of IVF in cattle is low. Small extracellular vesicles (sEVs) mediate communication between maternal cells and the embryo, generating changes at different levels in the embryo. The sEVs secreted by endometrial cells (endometriosomes) have had beneficial effects on the development and quality of embryos produced in vitro in different species. This study evaluated the effect of sEVs secreted in vitro by bovine endometrial stromal (BESC) and epithelial (BEEC) cells on the development and quality of bovine IVF embryos. From endometrial tissue in the secretory phase, BESC, and BEEC were isolated and cultured for 2 days in Dulbecco’s Modified Eagle Medium supplemented 1% with insulin-transferrin-selenium. From the obtained conditioned culture medium, endometriosomes were isolated from BESC (BESC-E) and BEEC (BEEC-E) by polymer-based precipitation and purified by ultrafiltration. The endometriosomes were then characterised by western blot, scanning transmission electron microscopy (STEM), and dynamic light scattering (DLS). Embryos were produced by IVF. One group was cultured in KSOM containing BESC-E (80 ng/μL), and one in KSOM containing BEEC-E (80 ng/μL). An endometriosome-free control cultured under exactly the same conditions as the treatments, was included. Each group contained 125 presumed zygotes in total. Cleavage (Day 3) and blastocyst development (Day 7) rates were assessed. The results were analysed using the chi-squared test and Fisher’s exact test (P < 0.05). Endometriosomes from both cell types were successfully isolated; STEM revealed a vesicular morphology consistent with sEVs reported in the literature. The sEV-specific proteins CD9, CD63, and TSG101 were identified by western blot, validating the microphotographs obtained by STEM. The DLS revealed an average diameter of 200 nm for BEEC-E and BESC-E, approximately. The BEEC-E significantly increased the blastocyst formation rate (43.5%), compared to the control (28.8%), in contrast to BESC-E (34.4%). In addition, immunofluorescence showed a larger inner cell mass in embryos cultured with BESC-E and BEEC-E compared to the control. However, further replicates are needed to validate these results. Supplementing the embryo culture medium with BEEC-E may improve the development and quality of bovine embryos generated by IVF. However, further studies are needed to elucidate the mechanisms underlying this improvement.