109 Evaluating bovine embryo developmental competence to establish pregnancy using noninvasive RNA-seq and morphological analysis

In vitro embryo production is widely used in biomedical research and livestock production. Despite decades of optimization in assisted reproductive technology procedures, the efficiency of blastocyst production in cattle remains <40%, and half of the blastocysts fail to establish pregnancies upon transfer to recipients. Although studies have implicated the role of several genes important for blastocyst development, the molecular understanding of early embryo developmental failure in cattle has been largely unknown. In the present study, we hypothesized that the transcriptomic signature of a 1-cell (1C) stage bovine embryo and its developmental kinetics in vitro could determine the embryo’s competence to form a blastocyst and establish a pregnancy. Our study had two objectives. First, we aimed to analyze the transcriptomic signature of the 1C embryo and its association with developmental phenotypes including embryonic arrest (early, 1–4C; mid, 4–8C; and late, 16C-Morula), cleavage time (early, <30 h; late, >30 h post-insemination), abnormal cleavage (skipped and reversed), blastocyst formation (Day 7), and pregnancy (Day 90). Second, we investigated the association of embryo developmental kinetics in vitro with the competence to form a blastocyst and establish pregnancy. For live embryo RNA-seq, ~65–150 pL of cytoplasm from IVF-produced zygotes was used to make a cDNA library using Takara SMART-Seq® v4Ultra® Low Input RNA kit and sequenced at Hi-seq (20–35 million reads/samples). Zygotes (n = 115) were biopsied for RNA sequencing and then cultured in an EmbryoScope (VitroLife) to track developmental kinetics, followed by embryo transfers on Day 7. Pregnancy data were collected until Day 90. Data analysis was performed to evaluate the association between the transcriptomic signature of individual zygotes with their developmental indices in vitro and pregnancy upon transfer. Results demonstrated abnormal expression of mitochondrial function genes in early arrest embryos compared with others. Differential gene expression of zygotes with arrested and blastocyst phenotypes showed β-estradiol as a common upstream regulator for genes essential for blastocyst formation. However, 1C embryos (n = 13 pairs) with identical transcriptomic signatures showed different development phenotypes in culture (arrested and blastocyst), suggesting no association between their transcriptome and developmental outcome. EmbryoScope image analysis revealed that 73.9% of late-cleaved embryos (n = 23) and 86.1% of abnormally cleaved embryos (n = 36) failed to make transferable quality blastocysts, suggesting that such characteristics could be used to predict embryo quality. Our molecular analysis suggested that the 1C zygote transcriptome may not be able to predict its developmental outcome; however, estradiol and mitochondrial pathway status can affect blastocyst development and quality.