The morula stage transcriptome is characterized by marked upregulation of genes that mediate key mitochondrial functions

RNA sequencing may uncover the transcriptional programs mediating embryogenesis. Compaction involves cellular polarization/differentiation, in preparation for blastulation. Genes involved in energy mobilization and waste disposal have been shown to be key at this transition1. Blastocoel cavity formation depends on ATPase activity, with peak mitochondrial oxygen consumption occurring at the morula stage2. The study aimed to characterize differential gene expression across preimplantation embryo development. Prospective cohort Fresh embryos from IVF cycles between January-June, 2016 were collected from day 3 to 7. Embryos had ∼2-4 cells were removed for aneuploidy screening by next generation sequencing (NGS), prior to RNA Sequencing. Differential gene expression between embryo cohorts was calculated using DESeq2 (adjusted significance, p<0.05). A likelihood ratio test was used to account for heterogeneity due to patient, batch, and ploidy and growth status (arrested/ongoing). Pathways were grouped according to cellular functions (Table 1). Of the 20,553 protein-coding transcripts, 3 were found to have significant differential expression (POLR1D, SULT2A1, CSAG3) between cleavage (n=29), morula (n=9), and blastocyst stage (n=43). Transcriptional activity peaked at the morula stage. The morula stage was characterized by marked upregulation, including genes mediating mitochondrial function: CISD1 (outer membrane protein protective against mitochondrial cell death); ISCA1 (iron-sulfur cluster machinery component in mitochondrial respiration); MFN2 (determinant of mitochondrial morphology); MRPL42 (encodes mitochondrial ribosomes for protein synthesis); and SUCLG1 (conversion of succinyl CoA and ADP/GDP to ATP/GTP). Transcripts mediating major mitochondrial functions play an important role in compaction prior to blastulation. Enrichment in processes related to mitochondrial function may reflect the switch from anaerobic glycolysis to aerobic metabolism at the morula stage. A deeper understanding of the molecular pathways that drive the cleavage-to-blastocyst transformation may facilitate future advances in embryo culture technique to optimize energy generation, minimize stress, and ultimately improve blastulation rates.Tabled 1Differentially expressed genes in morula vs.cleavage/blastocyst stage with p<0.05PathwaysDownregulatedUpregulatedGenesLog2 Fold ChangeGenesLog2 Fold ChangeMitochondrial functionCISD1//ISCA1//MFN2//MRPL42//SUCLG13.3//3.3//4.4//3.1//4.4Anti-apoptoticFBXL2-5.4MAST26.3Lysosomal functionCLN56.8Transcription mediatingPOLR1D//POLR2L3.2//3.7Post-translational modificationNMT2-3.9Uncategorized functionC4orf34.0 Open table in a new tab