BACKGROUND: Despite recent technical improvements, many human preimplantation embryos fail to develop to the blastocyst stage or implant after transfer to the uterus. A possible cause for this developmental arrest is the high incidence of nuclear and postzygotic chromosomal abnormalities observed during cleavage, including chaotic chromosome complements, suggestive of defects in mitotic chromosomal segregation. The underlying mechanisms are largely unknown, but similarities with chromosome instability in human cancers led to the proposal that cell cycle checkpoints may not operate at these early stages. METHODS: To investigate this and to examine whether spindle abnormalities contribute to chromosome malsegregation, we have used fluorescence and confocal laser scanning microscopy, following immunolabelling with antibodies specific for α-tubulin, γ-tubulin, or acetylated tubulin, combined with a DNA fluorochrome to visualize nuclei, spindle and chromosome configurations in normal and arrested human embryos, from cleavage to blastocyst stages. RESULTS: In addition to frequent interphase nuclear abnormalities, we identify for the first time various spindle abnormalities including abnormal shape and chromosome loss and multipolar spindles at cleavage and blastocyst stages. CONCLUSIONS: We propose that a major pathway leading to postzygotic chromosomal abnormalities is the formation of binucleate blastomeres with two centrosomes which result either in a bipolar spindle and division to two tetraploid blastomeres, or in a multipolar spindle, chromosome malsegregation and chromosomal chaos.

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. 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. 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). 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.

SUMMARY: Oligoasthenoteratozoospermia (OAT) is defined by a combined low count < 20 × 10⁶ sperm/ml, poor motility < 50 % forward progression or < 25 % rapid linear progression and abnormal morphology (5–8 % normal using Kruger strict criteria) and has been associated with increased levels of sperm aneuploidy. Here we report on the cytogenetic findings from three ‘spare’ embryos from a couple that were referred for ICSI because of OAT. The embryos were processed for sequential FISH in three hybridization rounds using probes for chromosomes 3, 7, 9, 13, 17, 18, 21, X and Y. Molecular cytogenetic analysis of nine chromosomes revealed that all three embryos were female polyploid. One of them was uniformly tetraploid for all chromosomes tested, while the remaining two embryos showed evidence of abnormal postzygotic segregation of chromosomes, causing the derivative blastomeres to have uneven chromosomal constitution. In one of them in particular, the non-disjoining chromosomes showed preferential segregation to the same pole, rather than randomly moving towards either pole, suggesting an abnormal spindle and causing the derivative blastomeres to have significantly uneven chromosomal constitutions. The possible scenarios leading to polyploidy and chromosomal imbalance through cytokinetic failure and subsequent abnormal centrosomal distribution are outlined.