Exosomal micrornas as a biological marker of maternal recognition of pregnancy in the mare

Abstract

Pregnancy maintenance mandates a signal from the embryo or by-product of the presence of the embryo in the uterus of the mare. This signal must occur between days 12 and 16 post-ovulation. A mobile conceptus prior to day 16 is obligatory for maintenance of pregnancy. However other communication between the embryo and suppression of endometrial prostaglandin F2&alpha; has been thoroughly researched yet remains enigmatic. Exosomes, cell secreted vesicles of endocytic origin, have been associated in a wide variety of important physiological cell-to-cell communication. Exosomes are secreted from numerous cell types and have been isolated from a variety of biological samples including; urine, breast milk, serum, plasma, and semen. Exosome presence in peripheral fluids makes them attractive, non-invasive biomarkers. Exosomes contain specific cargo including messengerRNA (mRNA), microRNA (miRNA), and proteins. Exosomes containing miRNA have been implicated in normal and complicated pregnancies as well as signature miRNAs associated with pregnancy status in women. We hypothesized that exosomal miRNA profiles between pregnant and non-pregnant mares would differ due to the changing uterine environment during maternal recognition of pregnancy. To test this hypothesis exosomes were isolated from serum samples in a simple cross-over design before (n=8) during (n=5) and after (n=3) maternal recognition of pregnancy. Pregnancy was confirmed using trans-rectal ultrasonography and embryo collection. Exosome isolation was performed using Exoquick™ (System Biosciences, Inc.) and total RNA was isolated using TriReagent BD (Molecular Research Center, Inc.). The first experiment profiled 380 human miRNAs to identify differences by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) between pregnant and non-pregnant mares after maternal recognition of pregnancy (n=3) on day 16 post-ovulation. For the second experiment, we designed 340 equine miRNAs primers to assess differences with qRT-PCR between pregnancy status. Samples from day 9 and 11 (n=8) and day 13 (n=5) were profiled to represent time points before and during maternal recognition of pregnancy in the mare. Pathway analysis of significantly different (P<0.05) miRNAs was performed employing Diana mirPath software. qRT-PCR identified six miRNAs differentially expressed on day 16 post-ovulation; two only in pregnant, two only in non-pregnant, and two significantly up-regulated in non-pregnant mares. qRT-PCR analysis revealed one, four, and seven miRNAs differentially expressed on days 9, 11, and 13 respectively with only day 9 samples present at higher levels in samples from pregnant mares. miRNAs significantly different in day 16 samples were not found to be significant in earlier time points. Pathway analysis indicated focal adhesion molecules as the primary pathway of miRNAs that were expressed higher in non-pregnant samples on days 11 and 13. Therefore we conclude that exosomal miRNAs from serum do demonstrate differential expression, and can be used as biomarkers for pregnancy and maternal recognition of pregnancy. Predicted targets of these miRNAs may be biologically relevant for determining the signal for maternal recognition of pregnancy in the mare.