We recently developed and validated a methodology based on early morphokinetics to predict 6 categories of bovine embryos, including 4 categories of blastocysts: early hatching blastocysts (EHB), hatching blastocysts (HB), low hatching blastocysts (LHB), and arrhythmic blastocysts (AB) (Reis et al. 2018 Anim. Reprod. 15, 601). We hypothesised that different early morphokinetic pathways could (1) be accompanied by different biological patterns (EGA, etc.) and (2) influence interaction with environmental constraints (cryopreservation, etc.). The objective of this study was (1) to investigate whether transcriptomic differences between the predicted EHB, HB, LHB, or AB could be observed immediately after the fourth embryonic cycle (just after EGA); (2) to assess cryotolerance of these morphokinetic categories. Time lapses were produced during 4 days post invitro insemination (dpi) (Reis et al. 2018). Study 1: 128 embryos having finished the fourth embryonic cycle were individually dry frozen at 4.7 dpi, classified as EHB, HB, LHB, or AB, and pooled into four batches of eight embryos/category (total 16 samples). Total RNA was extracted, amplified using the SMARTseq V4 ultralow input kit (Clontech), libraries were prepared using the Nextera XT Illumina library preparation kit, and sequenced (paired-end 50-34bp) on an Illumina NextSEqn 500 instrument. Identification of differentially expressed genes was achieved using Limma package (R). The P-values were adjusted using the Benjamini and Hochberg false discovery rate (Saenz-de-Juano et al. 2014). Fold change >2 or <0.5 and adjusted P<0.05 were considered significant. Enrichment analysis was performed using the Web server enrichR. Study 2: 90 embryos showing signs of expansion (diameter >135µm and −2µm at zona pellucida) were slow frozen at 6.2 or 7 dpi, classified as EHB (n=8), HB (n=29), LHB (n=32), or AB (n=21), and further thawed and cultured invitro for 72h. Cryotolerance was evaluated at 24 and 72h post-thawing. Statistical analysis was performed using chi-square or Fisher test. Study 1: the AB category presented 258, 699, and 899 upregulated genes and 40, 62, and 60 downregulated in comparison to LHB, HB, and EHB, respectively. The functional comparison of the two extremes (EHB vs. AB), at 4.7 dpi, showed that AB embryos present intense transcription regulation activity. The monocarboxylic acid binding pathway was upregulated in EHB. Study 2: the post-thawing invitro survival was similar between EHB, HB, LHB, and AB (50%, 34.5%, 31.3%, and 42.9%, respectively). The hatching/alive ratio in EHB category tended to be lower than AB (1/4 vs. 8/9; P=0.052). Frozen/thawed EHB and HB presented reduced hatching rates, whereas LHB and AB significantly increased the hatching potential after thawing compared with their not-frozen counterparts of the learning database (25% vs. 100%; 50% vs. 100%; 70% vs. 11%; 88.9% vs. 18%, respectively). Morphokinetic prediction highlighted a new classification of bovine embryos with different biological features. This could contribute to improve practice on IVF embryo transfer taking into account these differences.
