Mitochondrial and Ribosomal Stress Underlying Pronuclear Envelope Breakdown Failure: Insights From Single‐Cell Transcriptomics in Human Zygotes

Pronuclear Envelope Breakdown (PNEB) failure is a critical factor contributing to the early developmental arrest of intracytoplasmic sperm injection (ICSI) embryos; however, its molecular mechanism remains inadequately understood. This study aimed to elucidate the core regulatory network underlying PNEB failure occurrence and its impact on embryonic development. Single-cell sequencing was used to identify differentially expressed genes (DEGs) between PNEB‑type two pronuclei (2PN) zygotes and 3PN control zygotes, and weighted gene coexpression network analysis (WGCNA) was performed to identify module genes associated with PNEB failure. The least absolute shrinkage and selection operator was applied to model disease types and screen core genes, thereby establishing a multiomics integration strategy. Reactive oxygen species (ROS) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) fluorescence staining were conducted to analyze oxidative stress and mitochondrial function. A total of 1294 DEGs were identified, including genes involved in the oxidative phosphorylation pathway. Mitochondrially encoded reduced nicotinamide adenine dinucleotide dehydrogenase 1 (MT-ND1) and mitochondrially encoded cytochrome c oxidase III (MT-CO3) were significantly upregulated, whereas genes associated with the DNA repair pathway were downregulated. WGCNA revealed a light-green module strongly associated with PNEB failure (r = 0.89, P = 1.2e - 5). Hub genes, ribosomal protein L10a (RPL10A) and ribosomal protein L38 (RPL38), within this module were implicated in ribosome biogenesis. The PPI network confirmed functional interactions between MT-ND1 and RPL10A, suggesting that dysregulated mitochondrial function and ribosomal assembly are central to PNEB failure. ROS and JC-1 fluorescence staining showed a significant decrease in the JC-1 red/green fluorescence intensity ratio in the PNEB failure group (p < 0.05), while ROS levels were significantly elevated (p < 0.01). This study reveals that mitochondrial metabolic dysfunction and ribosomal assembly abnormalities contribute to PNEB failure, thereby disrupting nuclear envelope stability. Furthermore, it identifies the MT-ND1-RPL10A/RPL38 axis as a potential novel molecular marker for assessing ICSI embryo quality.