The aim of this study was to investigate the effects of zona drilling and biopsy on day 3 followed by vitrification on day 5 on the cytoskeleton and development of human embryos, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy in human biopsied and non-biopsied embryos. In total, 98 human blastocysts (50 non-biopsied and 48 following biopsy on day 3) were vitrified on day 5 using either a commercial dimethyl sulphoxide (DMSO)-free vitrification kit or increasing concentrations of DMSO/EG (5%/5–10%/10–20%/20%). Following warming, the blastocysts were allowed to recover in culture for 24 h and were immunostained with α-tubulin, acetylated tubulin, and/or γ-tubulin antibodies in combination with 4′,6-diamidino-2-phenylindole (DAPI). Labelled embryos were examined by both fluorescence and confocal laser scanning microscopy. The survival rates following warming (92% non-biopsied vs 83.3% biopsied) and the incidence of normal spindle chromosome configurations was not statistically different between the two groups (65.2% non-biopsied vs 59.2% biopsied, P>0.05). The incidence of spindle abnormalities including multipolarity, chromosome lagging, congression failure and chromosome bridging were also similar between the two groups (P>0.05). This study is the first to compare the incidence of cytoskeletal abnormalities in biopsied and non-biopsied human embryos following vitrification. We conclude that there was no significant difference in the survival rates and the incidence of spindle abnormalities between the two groups.

Vitrification 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.