Gene expression analysis under normal and stress conditions during in-vitro embryo development and subanalysis of gene expression in the blastocyst stage according to its Mitoscore®.

We analyzed the differences in the gene expression profile, especially pathways related to mitochondrial activity, between human embryos of different developmental stages, different Mitoscore® values and under stress conditions such as embryo arrest and the vitrification/warming process. In total, 44 vitrified specimens were collected for RNA-seq analysis, including 11 MII oocytes, 10 non-arrested cleavage-stage embryos, 5 arrested cleavage-stage embryos and 18 blastocysts (10 blastocysts cultured for 4-5 hours after warming and 8 blastocysts cultured for 0 hours after warming). All specimens were warmed and whole sampled in PCR tubes with 2 µL of 10× Reaction Buffer (SMART-Seq v4 Ultra-Low Input RNA kit for Sequencing, Takara Bio, USA). Amplified cDNA obtained from mRNA was purified using AMPure XP magnetic beads (Illumina, CA, USA). Libraries were constructed using NexteraXT DNA sample preparation (Illumina, CA, USA) and sequencing was done in duplicate in a total of four runs using an Illumina NextSeq 500 (Illumina, CA, USA) with a 300-nucleotide read length in a paired-end design (150-bp fragments). FastQC was used for checking the quality of the raw sequence data. Those fragments that did not meet quality requirements were trimmed using Trimmomatic. Alignment and quantification were performed using the Salmon algorithm (reference genome GRCh38). We observed that disruption of normal in-vitro culture and high Mitoscore® values are related to an upregulation of cellular stress pathways in human embryos. Despite the crucial role of mitochondria in cellullar stress, an upregulation of mitochondrial activity pathways is only observed during embryo arrest. Overall design: Analysis of general and mitochondrial-related differences in gene expression between different embryo stages and conditions: normal in-vitro culture (MII oocytes> non-arrested cleavage-stage embryos> blastocysts), embryo arrest (arrested cleavage-stage embryos vs non-arrested cleavage-stage embryos), vitrification/warming recovery (blastocysts cultured for 4-5 hours after warming vs blastocysts cultured for 0 hours after warming) and blastocysts Mitoscore® level (high Mitoscore® blastocysts vs low Mitoscore® blastocysts).