59 Effect of amniotic progenitor cell microvesicles on freezing of in vitro-produced bovine embryos and on pregnancy rate after embryo transfer

Abstract

Microvesicles (MV) are involved in communication mechanisms based on microRNAs active in the regulation of gene decisive in biological processes such as gametogenesis, fertilization, implantation and embryo development. In this context, in our previous study (Perrini et al. 2018 Reprod. Fertil. Dev. 30, 658-671), amniotic derived MV, and to a lesser extent MV derived from endometrial cells, were effective on stimulating embryo development, demonstrating that MV-exposed embryos show a larger number of cells constituting the inner cell mass, greater viability, higher expression of GPX1 gene (protective against lipid peroxidation) and lower expression of BAX gene (involved in apoptosis) compared with the control. In this study, further effects of amniotic-derived MV on bovine embryo were evaluated. Bovine embryo survival rate after cryopreservation and on pregnancy rate after embryo transfer of fresh or cryopreserved MV-exposed embryos were studied. Embryos were produced from 3782 oocytes. Basing on our previously published protocol (Perrini et al. 2018 Reprod. Fertil. Dev. 30, 658-671), presumptive zygotes were randomly transferred in SOFaa (control, CTR) or cultured by adding 50 × 10⁶ of amniotic-derived MV mL⁻¹ in the SOFaa on Day 5 post-fertilization (Perrini et al. 2018 Reprod. Fertil. Dev. 30, 658-671). The embryo developmental rate was evaluated at Day 7 (blastocyst stage), and after that, part of the embryos were fresh transferred, whereas part of them were cryopreserved in 1.5 ethylene glycol using a standard slow freezing curve. One fresh or cryopreserved embryo was transferred in each recipient. Statistical analyses were performed by ANOVA with nonparametric Kruskal-Wallis test and for pregnancy rate by chi-square test. Differences were considered statistically significant at P < 0.05. Results showed that blastocyst rate was 34.59 ± 1.32% (709/2050) in CTR and 34.24 ± 1.71% (593/1732) with MV. After cryopreservation, embryo survival was statistically different (P < 0.05) between CTR (32.71 ± 6.26%; 87/266) and MV (43.09 ± 5.73%; 78/181), respectively. The pregnancy rate was statistically different (P < 0.05) after embryo transfer of fresh embryos, with 66.67% (20/30 cows) of pregnancies for MV and 36.67% (11/30) for CTR. The transfer of cryopreserved embryos again provided a statistically (P < 0.05) different result: 36.67% (11/30) for MV embryos versus 10% (3/30) for CTR. These data show that the pregnancy rate of fresh or cryopreserved MV embryos is higher compared with CTR. Based on our previous data demonstrating higher expression of GPX1 gene and lower expression of BAX gene in presence of MV, these results indicate that the higher expression of the GPX1 gene probably limits the damage of lipid peroxidation by improving the survival rate following cryopreservation, whereas the less expressed BAX gene limits damages due to apoptosis. In conclusion, amniotic-derived MV probably play a biological role in the interaction between the embryo and the endometrium, providing a more resource-rich environment.