MicroRNA profile of in vitro bovine embryo cultured in presence of amniotic extracellular vesicles shifts toward in vivo collected blastocysts

The absence of maternal-embryo signals could be an important cause of the poor pregnancy rates of in vitro produced embryos, compared to those collected in vivo. In the context of paracrine communication, co-culture of embryo with amniotic-derived EVs improved their quality compared to control (CTR) [1] and, after cryopreservation, provided higher in vitro embryo hatcting and recipient pregnancy rate [2]. After these results, the aim of this study was to evaluate miRNA profiling of in vitro produced blastocysts with or without EV supplementation, using in vivo produced blastocysts as CTR. In vitro embryos were produced based on our protocol [1] with or without 100x106 EVs/ml in the SOFaa on day 5 post fertilization [1]. Grade 1 blastocyst (B7) were immediately snap frozen in liquid nitrogen for genomic study. These embryos were obtained from three replicates. In vivo embryos were obtained from three cows superovulated by Folltropin and inseminated by the same cryopreserved semen. After flushing, only B7 were snap frozen for genomic study. Samples for RNA isolation were obtained from 3 pools of 10 embryos each for each condition (vivo, vitro-CTR and vitro+EVs). Total RNA was isolated by NucleoSpin1 miRNA kit. Concentration and quality of RNA were determined by Agilent 2100 Bioanalyzer. Libraries were prepared using TruSeq Small RNA Library Preparation kits (Illumina). Differential expression analyses between samples were run with the Bioconductor edgeR package (false discovery rate [FDR] < 0.05). MicroRNA cluster analysis was performed with Genesis. The average quantity of total RNA extracted from each pool was of 3.5 ng. Our results show that the miRNAs identified were 1.74E5, 2.3E5 and 3.6E5 for vivo, vitro-CTR and vitro+EVs respectively. PCA calculated on DE-miRNAs showed a separation of the three groups with a distinctive miRNA trait. The miRNAs differentially expressed among three comparisons (vivo vs vitro-CTR, vivo vs vitro+EVs and vitro-CTR vs vitro+EVs) were 20, 15, and 2 respectively. PC1, which explains 62.4% of the variance, clearly separates in vivo and in vitro produced embryo even if EV addition seems to ameliorate the effect of in vitro production and this agrees with the embryo quality and the pregnancy rate after EV supplementation [1,2]. Indeed, vitro-CTR and vitro+EVs embryos differ significantly for two miRNAs (miR 130a, miR-181b) that are found to be higher in our vitro-CTR embryos compared to vitro+EV ones. The miR-181b was found to be higher in degenerate bovine embryos compared to good blastocyst too [3]. In conclusion, this is the first study reporting the complete miRNAs profiling of in vitro blastocysts compared to those obtained in vivo. Addition of EVs during in vitro production seems to influence the expression of specific miRNAs involved in the success of embryo implantation. [1] Perrini C & Lange Consiglio A. Reproduction Fertility and Development, 30:658-671, 2018. [2] Lange-Consiglio A et al. Reproduction Fertility and Development, 31:155, 2019. [3] Kropp J et al. Front Genetic, 24:91, 2014.