The Identification of Possible Microrna Biomarkers of Successful Pregnancy in Timed Embryo Transfer Recipients

Abstract The objective of the current study was to look for potential microRNA (miRNA) biomarkers indicative of a successful pregnancy in timed embryo transfer (ET) recipients. Commercial beef cows were synchronized using a 7-day CIDR co-synch protocol and GnRH injection given 66 hours following CIDR removal. At 6.5 days post GnRH administration, cows (n = 78) had a frozen thawed embryo delivered into the uterine horn ipsilateral to the corpus luteum detected by rectal palpation. A blood sample was collected by coccygeal venipuncture and serum isolated and frozen at -80°C 24 days post embryo transfer. At 38 days post embryo transfer pregnancy was determined by rectal ultrasound by a trained individual and confirmed by Biopryn assay. A total of 42 recipients were determined to be pregnant and 36 were determined to be non-pregnant. Of the 42 pregnant recipients, 10 did not calve and 32 delivered a calf at full term. Day 24 post ET serum samples from pregnant cows that calved normally (n = 4), pregnant cows that did not calve (n = 4), and pregnant cows that had an abnormal pregnancy at time of ultrasound and did not calve (n = 4) were selected for further analysis. These samples had RNA extracted with the microRNA fraction enriched and were then subjected to RNA sequencing (LC Sciences). Differential miRNA abundance was determined using ANOVA adjusted for false discovery rate using the Benjamini-Hochberg method with significance set at P < 0.05. A total of 747 miRNAs were detected and annotated to the bovine genome. A total of 36 miRNA showed differential abundance (P < 0.05) between groups of recipients. Of them, bta-mir-185-p3_1ss21CT, bta-miR-224_R-1, bta-mir-125b-1-p3, bta-miR-363_L+1, and bta-miR-494_R+1 had increased (P < 0.01) abundance in animals that calved to ET compared with cattle that did not calve to ET. MicroRNA PC-5p-120366_31 and PC-5p-89234_47 had increased abundance (P < 0.01) in recipients that had an abnormal appearing pregnancy compared with the other two recipient groups. MicroRNA bta-mir-1306-p3 had increased abundance (P < 0.01) in the two groups of recipients that had normal appearing pregnancies compared with the groups whose pregnancies were abnormal at time of ultrasound. These microRNAs could be used as biomarkers for an insufficient pregnancy that will not go to full term and therefore could be used to increase the efficiency of large scale ET.