Location: Livestock and Range Research LaboratoryTitle: Circulating microRNAs as biomarkers of early embryonic viability in cattle Author
|Pohler, K - University Of Missouri|
|Green, J - University Of Missouri|
|Moley, L - University Of Missouri|
|Hung, W - University Of Kansas Medical School|
|Hong, X - University Of Kansas Medical School|
|Christenson, L - University Of Kansas Medical School|
|Smith, M - University Of Missouri|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/19/2015
Publication Date: 6/18/2015
Citation: Pohler, K.G., Green, J.A., Moley, L.A., Hung, W.T., Hong, X., Christenson, L.K., Geary, T.W., Smith, M.F. 2015. Circulating microRNAs as biomarkers of early embryonic viability in cattle. Society for the Study of Reproduction Meeting Abstract. CD.
Technical Abstract: Embryonic mortality (EM) is considered to be the primary factor limiting pregnancy success in cattle and occurs early (< day 28) or late (= day 28) during gestation. The incidence of early EM in cattle is approximately 25% while late EM is approximately 3.2 to 42.7%. In cattle, real time ultrasonography can efficiently be used to diagnosis pregnancy starting as early as d 28 of gestation. Most EM occurs before d 28. In recent years there has been a research focus on microRNAs (miRNAs) and their potential as novel biomarkers. MicroRNAs represent an attractive biomarker for earlier pregnancy diagnosis because they appear in exosomes in the maternal circulation. Exosomes, which are about 30 to 90 nm in size, have been shown to be secreted by various cell types and are known vehicles of miRNA transport. We hypothesized that specific miRNAs would differ in the maternal circulation of non-pregnant and pregnant cows. The objective of this study was to determine if circulating exosomal-derived miRNAs could serve as a biomarker of embryonic presence and/or viability in cattle. In this study, cows were randomly assigned to be artificially inseminated (AI) with high fertility (live) semen (n= 36; treatment group) or heat-treated (dead) semen (n=8; control group) on d 0 (day of estrus). Blood was collected from all animals on d 17 and 24 followed by pregnancy diagnosis via ultrasound on d 30. Following diagnosis of pregnancy on d 30, cows were retrospectively classified within the AI assignment as pregnant (n=17), non-pregnant (n=19). In addition, expression of IFN-stimulated genes (ISG-15, Mx2 and OAS-1) were measured in each group to select cows that had low IFN-stimulated gene expression at d 0 followed by either high message at d 17 (pregnant and non pregnant) or low message (control). Cows with increased IFN-stimulated gene expression on d 17 but not pregnant on day 30 were considered to have experienced EM. Exosomes were isolated via high-speed ultra centrifugation, quantified via nanoparticle tracking analysis and western blot analysis (CD 81 Positive). Exosomal miRNAs were extracted from the d 17 and d 24 samples and miRNA sequencing was performed on the Illumina HiSeq2500 Sequencing System (pregnant n=4/day; non-pregnant n=4/day; control n=4/day). Sequence reads were mapped to a reference transcriptome using Nextgene software. In total, 194 miRNAs were identified from d 17 and 211 miRNAs from d 24 samples. Differential abundance of miRNA was characterized as a statistically significant difference (P < 0.05) with a fold change greater than 2. At d 17, there were 15 miRNA that were significantly greater in abundance incontrol versus pregnant cows and no no miRNA were elevated in pregnant versus control. Comparison of non-pregnant and pregnant cows indicated differential abundance of 28 miRNA 25 miRNA had greater levels in non-pregnant and 3 had greater levels in pregnant cows. On d 24 there were 18 differentially abundant miRNAs between control and pregnant cows (2 elevated in control; 16 elevated in pregnant) along with 10 differentially abundant miRNA in non-pregnant compared to pregnant cows (3 elevated in non-pregnant; 7 elevated in pregnant). In summary, preliminary data suggest that circulating exosomal-derived miRNAs may provide a novel biomarker to assess embryonic presence and viability in cattle.