Ice-free vitrification of embryos and its impact on post-warming outcomes.

Vitrification protocols are widely used for mammalian embryo cryopreservation. Post-warming outcomes are impaired by cryodamage from crystalline intracellular ice, whose formation depends on both the cooling and warming rates. Here, time-resolved X-ray diffraction measurements on bovine embryos indicated that current standard tools and protocols achieved ice-free cooling, but that intracellular ice always formed during warming. Increasing cooling rates by a factor of ~ 30 over current standard rates allowed ice-free cooling and warming, even when cryoprotectant concentrations were reduced by 30%. Embryos cooled at ultrafast rates exhibited survival and developmental competence rates close to those of unvitrified controls and significantly better than those cooled at standard rates. Increased global H3K9me3 intensity was found with both standard and ultrafast cooling relative to controls, while DNA methylation (5mC and 5hmC) was comparable for all groups. Transcriptomic analysis indicated adhesion and cell-junction assembly genes were downregulated while DNA damage repair pathway genes were upregulated with standard cooling relative to controls, and cell-development and differentiation pathway genes were downregulated with standard cooling relative to ultrafast cooling. Embryos vitrified with the ultrafast cool protocol retain full developmental potential and can establish successful pregnancies. Overall, increased cooling rates improved post-cryopreservation outcomes in part by minimizing ice formation, minimizing DNA damage, and preserving gene expression related to embryonic developmental functions.