Effects of vitrification on blastomere viability and cytoskeletal integrity in mouse embryos

Abstract

SummaryVitrification is widely used to cryopreserve supernumerary embryos following in vitro fertilization (IVF). The mouse model was used to investigate the effects of vitrification on blastomere viability, using viability markers, and on the cytoskeleton, by analysing spindle/chromosome configurations, using confocal scanning microscopy. Ninety cleavage and morula stage dimethyl sulphoxide (DMSO)/EG vitrified mouse embryos were either processed immediately following warming for viability assessment by labelling with the fluorescent markers carboxyfluorescein-diacetate succinimidylester (CFSE) and propidium iodide (PI) or were cultured to the blastocyst stage and immunostained with α-tubulin antibody to visualize microtubules and DAPI or PI to visualize DNA. Sixty-five fresh embryos were also used as the control. Vitrified embryos showed high survival rates following warming, but they had a higher incidence of damaged blastomeres compared with fresh embryos. Most mitotic spindles examined in all groups were normal, but multivariable analysis revealed that the proportion of abnormal spindles was significantly higher in vitrified/warmed embryos (P < 0.05). This study is the first to examine the immediate effects of vitrification on blastomere viability, using fluorescent markers and shows that although vitrification results in a higher incidence of damaged blastomeres, vitrified embryos may compensate for this limited number of damaged/abnormal cells, as development to the blastocyst stage was not compromised.