Single-cell RNA and DNA methylome profiling reveal liquid helium vitrification enhances porcine parthenogenetically activated blastocyst viability

Cryopreservation of porcine blastocysts remains a significant challenge due to their high lipid content, which can lead to cryoinjury and reduced developmental competence. While liquid nitrogen (LN) vitrification causes significant molecular damage, LHe vitrification enhances oocyte viability, reduces cryoinjury, and better preserves ultrastructure and gene expression. Despite these advantages, the specific effects of LHe vitrification on porcine blastocysts are still underexplored. LHe vitrification significantly improved post-thaw survival rates (96.53 ± 4.27%) compared to conventional LN (71.20 ± 6.82%) and modified LN protocols (90.77 ± 7.88%). scRNA-seq and scWGBS achieved mapping rates of 83.70% and 33.69%, respectively, and a bisulfite conversion efficiency of 99.27%, ensuring high-quality data for gene expression and methylation analysis. Vitrification significantly altered gene expression and epigenetic regulation, with hypermethylation and downregulation of genes associated with energy metabolism (ARG2, AUH, IDH3A, NDUFV1), immune function (CD53), and protein processing (B4GALT5, OSTC, DPM3, EXT1), resulting in mitochondrial and immune dysfunction, as well as protein instability. KEGG pathway analysis revealed disruptions in the TCA cycle, pentose phosphate pathway, homologous recombination, and fatty acid biosynthesis, which impaired blastocyst viability, ATP availability, immune responses, and cellular communication. Conversely, hypomethylation and upregulation of genes regulating energy metabolism (GPAM, AICDA), oxidative stress response (ITGAV), and cell adhesion (PLAU, MRAP2) suggest an adaptive response to mitigate cellular stress and damage. However, excessive upregulation following vitrification may lead to metabolic strain, oxidative stress accumulation, lipotoxicity, and an increased risk of apoptosis. Our findings demonstrate that LHe offers superior metabolic and epigenetic preservation compared to LN, thereby reducing molecular damage and enhancing post-thaw development. These results establish LHe vitrification as an effective method that increases blastocyst viability and reproductive biotechnology applications.