Profiling circulating microRNAs in the serum of pregnant and non-pregnant pigs reveals a plethora of reproductive status-dependent microRNAs

Abstract

Circulating, non-coding RNAs, such as microRNAs (miRNAs) have been proposed to be powerful pathophysiological indicators of pregnancy in animals and humans. Since their discovery, it is known that miRNAs can take part in numerous biological processes, including cell proliferation and differentiation during early embryonic development and establishment of pregnancy. Our recent studies have indicated that maternal blood can carry miRNAs reported previously at the embryo–maternal interface in pigs. To expand the scope of our research, we tested the hypothesis that miRNAs previously identified in conceptuses, trophoblasts, endometrium and uterine lumen-derived extracellular vesicles (EVs) collected before Day 20 of pregnancy can show reproductive status-dependent profiles in the serum of cyclic and pregnant crossbred pigs. Custom-designed TaqMan arrays, multiplex real-time reverse transcription (RT)-PCR and real-time RT-PCR allowed us to identify a number of reproductive status-dependent miRNAs in serum samples collected from pigs during the estrous cycle or pregnancy (Days 16 and 20). We found that serum samples were enriched with miRNAs involved in processes important during the estrous cycle and early pregnancy, e.g. cell sensitivity and viability, angiogenesis, embryonic cell proliferation and differentiation. Further validation revealed different abundance of ssc-miR-143-3p and ssc-miR-125b in pregnant and non-pregnant animals and correlation of ssc-miR-125b levels with litter size. In addition, analyzed serum samples contained both EVs and Argonaute2 proteins, which are known to be involved in miRNA transportation and intercellular communication. In summary, we identified several circulating miRNAs that differ in abundance between cyclic and pregnant animals and could serve as potential indicators of reproductive status in pigs during breeding management.