Molecular disruptions to the future fetal tissue lineage are associated with delayed euploid blastulation

Objective: To study the underlying molecular activity driving the reduced reproductive potential of Day 7 euploid blastocysts. Design: Euploid inner cell mass and trophectoderm transcriptome analysis examined in association with timing of blastulation. Subjects: Equivalent grade and maternal age-matched Day 5 (n=20) and Day 7 (n=20) surplus cryopreserved euploid blastocysts donated with informed patient consent. Intervention: None Main Outcome Measures: RNA sequencing was used for transcriptome analysis, followed by enriched pathways and gene ontology terms to determine functional relevance. Quantitative real-time PCR was performed for targeted gene expression analysis and validation of differentially expressed genes identified by RNA sequencing. Results: Timing of blastocyst development leads to significant differences in transcription for equivalent grade, maternal age-matched euploid blastocysts. Controlling for cellular lineage, 2880 differentially expressed genes were identified in the Day 7 inner cell mass (padj≤0.05) and 2057 genes in the Day 7 trophectoderm (padj≤0.05), compared to their equivalent grade Day 5 counterparts. However, pathway enrichment was predominantly driven by downregulated gene expression in the inner cell mass of Day 7 blastocysts (padj≤0.05) impacting signal transduction pathways and genes involved in primitive endoderm development. Slower developing blastocysts appear to face metabolic and endoplasmic reticulum stress, leading to increased protein degradation in the inner cell mass, irregular apoptotic activity, as well as compromised trophectoderm epigenetic regulation. Conclusions: The Day 7 inner cell mass transcriptome revealed disproportionate perturbations involving the downregulation of many critical networks and genes required for embryogenesis, providing an array of molecular mechanisms responsible for developmental delay. With no significant impact on Day 7 trophectoderm pathways, reproductive potential of the blastocyst may be primarily driven by the molecular activity of the future fetal tissue lineage, thereby explaining the halving of live birth rates following euploid Day 7 blastocyst transfers. Defining the network of embryonic pathways essential for developmental competence will allow for the future development of culture systems to support optimal extended culture for poor-prognosis patients.