Cytoskeletal analysis of human blastocysts by confocal laser scanning microscopy following vitrification

BACKGROUND Vitrification of human blastocysts is being used increasingly to cryopreserve supernumerary embryos following IVF. In this study, we investigate the effects of aseptic vitrification on the cytoskeleton and development of human blastocysts, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy. METHODS A total of 55 fresh blastocysts and 55 day 5 dimethylsulphoxide/ethylene glycol vitrified blastocysts, which were allowed to remain in culture for 24 h post-warming, were rapidly fixed in ice cold methanol, and immunostained with an a-tubulin antibody to visualize microtubules in combination with antibodies against acetylated tubulin (to visualize spindles, poles and mid bodies), gamma tubulin (to identify spindle poles) and 4(6-diamidino-2-phenylindole) to visualize DNA. RESULTS In total, 213 spindles were analysed in the control (fresh) group of which 183/213 (85.9%) were normal, 20/213 (9.4%) were abnormally shaped, 9/213 (4.2%) were multipolar and 1/213 (0.5%) was monopolar. A total of 175 spindles were analysed in the vitrified group, of which 120/175 (68.6%) were normal, 39/175 (22.3%) were abnormally shaped, 10/175 (5.7%) were multipolar and 6/175 (3.4%) were monopolar. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often associated with chromosome lagging, or congression failure, was significantly higher in the vitrified group compared with the fresh group (P< 0.05). CONCLUSIONS The high survival rate following thawing and the large proportion of normal spindle/chromosome configurations suggests that vitrification at the blastocyst stage on Day 5 does not adversely affect the development of human embryos and the ability of spindles to form and continue normal cell divisions. However, there was a significantly higher incidence of abnormal spindles in the vitrified group compared with the fresh group, notably of spindles with a focused and an unfocused pole as well as chromosome bridging and disorganized middle spindle fibres at telophase. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to damage during vitrification, the fate of the abnormal spindles and any potential effects that may be reflected on the chromosomal constitution of the developing blastocysts.