Timing matters: molecular signatures linked to developmental and implantation processes in day 5 and day 6...

To determine whether human blastocysts requiring an additional day of in vitro culture to achieve expansion exhibit differences in trophectoderm transcriptomic profiles associated with developmental and implantation-related processes.

Prospective in vitro experimental cohort study including ribonucleic acid sequencing of mural trophectoderm biopsies from day 5 (D5) and day 6 (D6) (euploid) blastocysts.

Twenty-one blastocysts donated by 16 couples undergoing intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A). Transcriptomic comparison focused on 21 blastocysts, classified as D5 (n = 13) vs. D6 (n = 8) embryos, and euploid D5 (n = 6) vs. euploid D6 (n = 4) embryos.

Blastocyst developmental timing (D5 vs. D6) and embryo ploidy status.

Identification of differentially expressed genes (DEGs) and deregulated molecular pathways between D5 and D6 (euploid) embryos, including functional enrichment analysis to explore their potential roles in embryo development and implantation.

Global transcriptomic analysis revealed partial overlap between D5 and D6 blastocysts, indicating a largely shared gene expression landscape with underlying biological variability. Differential expression analysis identified 111 DEGs between D5 and D6 embryos, with functional enrichment analyses highlighting pathways related to translation, metabolism, and cellular organization. An exploratory euploid-only subanalysis yielded a larger number of genes meeting the selected differential expression thresholds, potentially reflecting reduced biological variability within the subset, together with enrichment of pathways associated with oxidative phosphorylation, ribosomal function, and energy metabolism, although these findings should be interpreted cautiously given the limited sample size.

Human D5 blastocysts exhibit transcriptomic profiles enriched in pathways related to translation, metabolism, and cell adhesion compared with D6 blastocysts. However, these differences are subtle and occur within a largely shared global transcriptomic landscape, suggesting that they reflect variations in developmental timing rather than clearly distinct functional states. Although these findings are consistent with biological processes relevant to implantation, they should be interpreted within the broader context of embryo development, where additional molecular and cellular mechanisms also contribute to implantation competence.