Abstract Study question Do human blastocyst embryos affected by mitochondrial disorders have gene expression disruption? Summary answer A global transcriptional repression was found in embryos carrying mutations in mitochondrial genes, primarily affecting oxidative phosphorylation and cell survival pathways. What is known already Mitochondria are thought to play a critical role for embryo development by supplying adequate energy levels. Whether a metabolic rescue through mitochondrial metabolism modifications is taking place during the blastocyst stage, remained to be elucidated. For instance, abnormally elevated mtDNA levels were detected in human blastocyst embryos carrying the m.3243>G pathogenic variant, suggestive of a compensatory response, while this was not the case for embryos carrying another mtDNA mutation (m.8344A>G). Study design, size, duration To investigate if mitochondrial mutations affect gene expression of human blastocysts, transcriptome profiling between 33 control and 9 mitochondrial embryos was performed and analyzed by RNA-Sequencing. Participants/materials, setting, methods In total, 42 blastocyst embryos (Day-5/6/7) from 27 unrelated couples were collected after a preimplantation genetic testing analysis, concluding in an affected status. Among them, 33 were affected by a non-metabolic, non-mitochondrial genetic disorder (control group), and 9 were affected by a mitochondrial disorder (mitochondrial group). Transcriptomic analyses were performed on whole blastocyst embryos, by RNA-Sequencing. Main results and the role of chance Gene expression profiling of human blastocyst embryos revealed a global transcriptional repression in mitochondrial embryos, with a total of 566 genes being down-regulated, while only 52 genes were up-regulated (p ≤ 0.05; fold-change=2). A similar pattern was observed among all mitochondrial embryos, affecting a significant proportion of differentiation factors (such as KLF4, p = 1.88x10-2; OXT2, p = 3.32x10-3 and POU5F1, p = 8.03x10-3), as well as nuclear genes encoding mitochondrial proteins (n = 59). If oxidative phosphorylation was at the top of the most significant deregulated pathways (p = 6.32x10-14), cell survival (p = 2.19x10−10) and autophagy (p = 4.56x10-9) were found to be significantly decreased in these embryos, questioning their viability. Limitations, reasons for caution The number of mitochondrial embryos was limited due to the rarity of the material, however similar molecular profiles were detected among them. The control group included embryos affected by genetic disorders, although the resulting potential transcriptional biases were neutralized by selecting embryos affected by various and distinct genetic disorders. Wider implications of the findings The differentially expressed genes identified in this study represent biomarkers predictive of mitochondrial dysfunction, which will be useful for the establishment of therapeutic or mitochondrial replacement trials. Because of the role of mitochondria, they are also interesting to test in the context of in-vitro fertilization, as biomarkers of preimplantation development. Trial registration number Not applicable