Small RNA-seq analysis of extracellular vesicles from porcine uterine flushing fluids during peri-implantation

Abstract Background: The extracellular vesicles (EVs) of uterine flushing fluids (UFs) mediate intrauterine communication between conceptus and uterus in pigs. The small RNAs of UFs-EVs are widely recognized as important factors that influence embryonic implantation in humans, mice, cattle and sheep. Therefore, it can be hypothesized that small RNAs of UFs-EVs are essential for implantation in pigs. Results: Cup-shaped EVs of porcine UFs were isolated and characterized, and then PKH67 stained extracellular vesicles were taken up by endometrial epithelial cells and porcine trophectoderm cells in a time-dependent manner. The expression of small RNAs in these EVs on days 10 (D10), 13 (D13) and 18 (D18) of pregnancy was comprehensively profiled through sequencing. From nine libraries, a total of 152 known microRNAs (miRNAs), 43 novel miRNAs and 6248 known Piwi-interacting RNAs (piRNAs) and 110 novel piRNAs were identified. Hierarchical cluster analysis and principal component analysis suggested that the expression profiles of small RNAs in D13 group were different from those in D10 and D18 groups. Bioinformatics analysis showed that the miRNAs differentially expressed in the comparisons between D10 and D13, D13 and D18, and D10 and D18 groups were involved in important processes and pathways related to immunization, proliferation, angiogenesis, apoptosis and development, which play important roles in embryonic implantation. Conclusions: Our results reveal that EVs from porcine UFs contain various small RNAs with potentially vital effects on implantation. This research also provides resources for studies of miRNAs and piRNAs in the cross-talk between embryo and endometrium.