玻璃化冷凍對於胚胎細胞骨架之影響

玻璃化冷凍對於囊胚期胚胎細胞骨架之影響
未冷凍: 85% 正常細胞骨架/紡錘體
冷凍: 68% 正常細胞骨架/紡錘體

但玻璃化冷凍對囊胚期胚胎存活及著床率影響並不明顯差異



http://www.ncbi.nlm.nih.gov/pubmed/22028018

Hum Reprod. 2011 Oct 25. [Epub ahead of print]

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

Chatzimeletiou K, Morrison EE, Panagiotidis Y, Vanderzwalmen P, Prapas N, Prapas Y, Tarlatzis BC, Handyside AH.

Source

Section of Reproductive Medicine, First Department of Obstetrics & Gynaecology, Aristotle University Medical School, Papageorgiou General Hospital, Thessaloniki 56403, Greece.

Abstract

BACKGROUNDVitrification 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.METHODSA 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.RESULTSIn 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).CONCLUSIONSThe 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.