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

In mammals, during the pre-implantation phase, the exchange of signals between the mother and the embryo is essential for the development and implantation of the embryo [1]. In cattle, it was considered that the absence of communication between a receptive endometrium and a viable embryo could be an important cause of the low pregnancy rates [2]. These considerations led to the hypothesis that there should be a paracrine communication between the mother and the embryo. Microvesicles (MVs) are involved in the communication mechanism based on microRNAs active in the regulation of gene expression at the transcriptional and post transcriptional level in the most critical biological processes such as gametogenesis, fertilization, implantation and embryonic development [3]. In this context, we evaluated the efficacy of MVs derived from amniotic cells on embryo development and demonstrated that these embryos show a greater number of cells constituting the internal cellular mass, a greater viability, a higher expression of GPX1 gene (protective against lipid peroxidation) and a lower expression of BAX gene (involved in apoptosis processes) compared to control [4]. After these in vitro results, we aimed to evaluate the effect of MVs on embryo survival after cryopreservation and on pregnancy rate after embryo transfer of fresh or cryopreserved MV-embryos in the bovine species. Embryos were produced in 18 replicates from 3.812 oocytes using routine protocol. Presumptive zygotes were randomly transferred in SOFaa (control, CTR) or cultured by adding 50x106 MVs/ml in the SOFaa on day 5 post fertilization. The embryo rate was evaluated at day 7 (blastocyst stage) and, after that, the embryos were transfer fresh, in cows synchronized by Cloprostenol, or cryopreserved. Results showed that blastocyst rate was 34.62±1.32% in CTR and 34.27±1.71% with MVs. After cryopreservation, embryo survival was statistically different (P<0.05) between CTR and MVs (32.70±6.26 and 43.51±5.73, respectively). The pregancy rate was statistically different (P<0.05) after embryo transfer of fresh embryos, with 66.67% (20/30 cows) of pregnancies for MVs and 36.67 (11/30) for CTR. The transfer of cryopreserved embryos provided again a statistically (P<0.05) different result: 36.67% (11/30) for MV-embryos and only 10% (3/30) for CTR. Our data show that pregnancy rate of fresh and cryopreserved MV-embryo is higher compared to CTR. These results indicate that the higher expression of GPX1 gene, due to the presence of MVs, probably limits the damage of lipid peroxidation by improving the survival of cryopreservation, while the less expressed BAX gene limits damages from apoptosis. Moreover, amniotic derived MVs probably have a biological role in the interaction between the embryo and the endometrium providing a more resourceful environment. [1] Fazeli A. Maternal communication with gametes and embryos, Theriogenology, 70:1182–1187, 2008. [2] Thatcher WW, Guzeloglu A, Mattos R, Binelli M, Hansen TR, Pru JK. Uterine-conceptus interactions and reproductive failure in cattle, Theriogenology, 56:1435–1450, 2001. [3] Machtinger R, Laurent LC, Baccarelli AA. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation, Human Reproduction Update, 22:182–193, 2016. [4] Perrini C, Esposti P, Cremonesi F, Lange Consiglio A. Secretome derived from different cell lines in bovine in vitro embryo production, Reproduction Fertility and Development, 30:658–671, 2018.