Identification of transcriptome markers associated with in vitro bovine embryo quality in single embryos and biopsies

Current technology to identify blastocysts with the greatest chance of pregnancy success relies on visual inspection of the embryos before transfer, which contributes to variation in embryo transfer pregnancy rates. It was hypothesized that ribonucleic acid (RNA)-sequencing (RNA-Seq) could be employed to discover differentially expressed transcripts between Grade 1 (G1, excellent/good) and Grade 3 (G3, poor) quality day 7 in vitro produced (IVP) embryos. Bovine oocytes were collected, fertilized, cultured, and then graded on day 7. Using RNA-Seq of pooled early and expanded blastocysts by quality grade, three messenger RNA transcripts were found to be upregulated in G3 vs G1 embryos. These transcripts were studied in G3 vs G1 whole or biopsied morula-stage embryos in three independent trials. A pre-amplified complementary deoxyribonucleic acid method using primers for all three transcripts and housekeeping genes was developed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). These transcripts were either lowly expressed or not expressed at all in G1 quality morulae and biopsies. Expression of these transcripts in G3 embryos and biopsies had significantly greater variation (Levene's test). Some of the G3 embryos had transcript levels that were consistent with G1 embryos, potentially indicating a higher quality compared to subjective visual methods of evaluation. In conclusion, an RNA extraction and RT-qPCR method for studying marker transcript expression in a single IVP embryo and biopsy sample was validated. Three transcript markers were identified that differ in expression based on embryo grade in both blastocysts and morulae. These transcripts in addition to other embryo evaluations may identify the best candidates for embryo transfer.