81 Maternal extracellular vesicles from in vitro and in vivo system have different impact in pre-implantation bovine embryos development

In vitro production system affects embryonic development, quality and competence. The early embryo-maternal interaction is absent during in vitro culture, which could explain the lower development of in vitro produced embryos. Embryo-maternal communication could occur through the extracellular vesicles (EVs). The EV characteristics and cargo vary according to tissue and cellular origin, affecting the interaction with target cells. The aim of this work was to determine how EVs from maternal cells could affect the pre-implantation embryonic development and whether there are differences between endometrial EVs produced in vivo or in an in vitro culture system. For this purpose, EVs released by in vitro-cultured endometrial cells (EVC) and from uterine fluid (UF) collected from cows at Day 5 after ovulation, were added to the culture medium of bovine embryos produced in vitro. Three replicates of three groups of 10 embryos each were used per treatment; EVC, EV-UF, and negative control (NC). A total of 2.4 × 10⁸ particles in 20 µL of phosphate-buffered saline (PBS) were added twice to 500 µL of culture medium at Day 5 and 7 of development. Similar volume of PBS was added to NC. The EVs were marked with PKH67 dye (Sigma Aldrich) to evaluate internalization by the embryonic cells. The effect of the EVs was assessed in terms of expanded and hatched blastocyst rates, embryo diameter and expression level of crucial genes. Then RT-quantitative polymerase chain reation was performed to evaluated 10 genes related to oxidative stress (SOD1, GPX1), apoptosis (BAX, BCL2), pluripotency genes (NANOG, SOX2, OCT4), implantation-related gene (IFNT), pregnancy-associated glycoprotein (PAG1) and glucose metabolism (G6PD). The embryo diameter was measured using the Micrometrics software at Day 5, 7, and 9 of development. Kruskal–Wallis test was performed for all parameters except for embryo diameter were an ANOVA test was carried out. Data among groups were considered statistically different with P < 0.05. The EVs from EVC and UF were internalized by embryonic cells. Maternal EVs had a positive effect on embryo development and quality, inducing an increase of IFNT and SOX2 expression and a tendency to increase NANOG while decreased the expression of genes related to apoptosis (BAX) and oxidative stress (SOD1). However, the expanded and hatched blastocyst rates and IFNT expression were higher in the group with EVs from UF. There also was a sustained increase (from Day 5 to 9) in diameter of the embryos cultured with EV-UF. It was concluded that the positive effect of maternal EVs on embryonic development differs according to the origin of EVs.

This research was supported by Fondecyt 1210334 and ANID 21201060.