123 Identification of transcriptome markers for bovine embryo quality

A molecular diagnostic for embryo quality is not available and visual/microscopic diagnostics are limited. It is inferred that a molecular diagnostic for embryo quality will improve success rate for assisted reproductive technologies (ART) with human and livestock applications. Most losses in ART happen early in pregnancy during critical embryo development. Current technology to identify blastocysts with greatest chance of pregnancy success relies on visual inspection of the embryos before transfer, contributing to variation in the success of pregnancy from embryo transfer. In a previous study, 3 transcripts of interest (TOIs) were discovered to be different between embryo qualities 1 and 2 in Day 7, stage 6/7 embryos. These identified TOIs were further investigated to aid in the identification of the best quality embryos before embryo transfer. In experiment 1, for transcription profiling by reverse transcription quantitative polymerase chain reaction (RT-qPCR), bovine oocytes were collected, fertilized, and cultured. The whole bovine Day 6 embryos (Morula, stage 4, n = 29) were visually graded, and each individual embryo from different grades (1, 2, and 3) was subjected to RNA extraction. In experiment 2, whole bovine Day 6 embryos (Morula, stage 4, n = 22) were graded and then, a 5% to 20% biopsy was taken for RNA extraction. RNA was converted to cDNA and pre-amplified with specific primers for RT-qPCR for the 3 TOIs. Luciferase RNA was added to each RNA extraction from an embryo or biopsy as an extraction control. A housekeeping gene (HKG) was used as an internal control. The RT-qPCR analysis from a whole embryo or biopsy yielded a Cq value for luciferase (extraction control), a Cq value for HKG, and a Cq value for a particular marker transcript for each embryo. These data were used to generate a normalized ratio of expression (Rnor) for each embryo which represented the ratio of expression level of a marker gene to the expression level of HKG in each embryo. The luciferase data were used to adjust for possible extraction differences among samples. Three TOIs were determined to be either lowly expressed or not expressed at all in good-quality embryos. Analysis of these 3 TOIs revealed that these genes were not detected or were expressed at very low levels in grade 1 (good-quality embryos) compared to greater concentrations in grade 2 and 3 embryos. Similar profiles were observed between whole embryos of Day 6 and Day 6 embryo biopsies. This study validated an RNA extraction and RT-qPCR method for studying gene expression in a single embryo and a biopsy sample. Further research is warranted to investigate the roles of the defined marker genes in the early embryonic development and their impact on the quality of embryos and the relationship between the quality assessment based on these marker gene expressions and after embryo transfer success in establishment of pregnancy. Analysis of these TOI in addition to other embryo evaluations may help identify the best candidates for embryo transfer.

This research was funded by OEDIT grant 008724–00002 and CSU Ventures grant 008677–00002.