115 Detecting embryo developmental potential by single blastomere RNA-seq

Selecting high quality in vitro-produced (IVP) bovine embryos for transfer is a difficult task when using only visual embryo observation. We aimed to develop a single blastomere biopsy technique at morula stage to allow for the detection of differences in gene expression profiles without compromising embryo development. The further goal was to reveal whether any of these differences would make it possible to distinguish between embryos developing into blastocysts and embryos with arrested development in morula stage. In vitro-produced bovine embryos were cultured in groups until Day 5, when 65 morulae were biopsied with a microneedle and one blastomere was aspirated. The biopsied morulae were further individually cultured in culture media droplets under mineral oil until Day 8, when blastocyst formation was recorded. For whole transcriptome sequencing, six biopsy samples from embryos arrested in morula stage and six biopsy samples from embryos developing to the blastocyst stage were chosen. The samples were sequenced using the SOLiD 5500 Wildfire platform together with paired-end sequencing chemistry (50 bp forward and 50 bp reverse). Raw data was mapped to the bovine reference genome bosTau7 using Lifescope software (Thermo Fisher Scientific). The quality threshold was set to 10; the mapping confidence was more than 90. Reads with a score lower than 10 were filtered out and average mapping quality was 30. Analysis of the RNA content and gene-based annotation was done within whole transcriptome workflow. For statistical and functional analysis, Bioconductor packages DeSEqn 2 and ReactomePA implemented in R were used. DeSEqn 2 package allows testing for differential expression by use of the negative binomial distribution and a shrinkage estimator for the distribution’s variance (Anders et al. 2010). The log₂FC cutoff was set at 2. Package performs sample comparison, adjusts P-value to overcome multiple testing problems and controls for false discovery rate (FDR) (Benjamini et al. 1995). Out of 65 biopsied morulae, 32 developed to blastocysts (49.2%). Out of 108,760 successfully mapped genes, 1,204 showed a difference in mRNA expression level. Out of these, 155 genes had a log₂FC value over 2 and were expressed in embryos developing to blastocysts after biopsying. Among other most expressed genes were, for example, HSBP1 and ATP5G3. HSBP1 is responsible for maintaining normal cell development and ATP5G3 in mitochondrial function. The pathway enrichment analysis of embryos developing to blastocysts revealed significant enrichment in organelle biogenesis and maintenance, mRNA splicing, and mitochondrial translation pathways. These findings suggest principal differences in gene expression patterns and functional networks of embryos able to reach the blastocyst stage compared with embryos arrested in development. Our preliminary data suggest that single blastomere biopsy and selected gene expression profiles at morula stage offer additional possibilities for the early embryo selection.

This study was supported by Enterprise Estonia grant EU30020, institutional research funding IUT 8-1, and Horizon 2020 Project SEARMET 692299.