An association between chromosomal abnormalities in rapidly frozen 2-cell mouse embryos and the ice-forming properties of the cryoprotective solution

摘要

Summary. This paper investigates the effect of straw handling on the viability of 2-cell mouse embryos rapidly frozen in dimethyl sulphoxide (DMSO) solutions. During the brief (3 min) equilibration step, straws were either rotated periodically to keep the embryos in suspension, or kept still to allow the embryos to settle onto the inner surface of the straw. The effects of these straw movements were tested with cryoprotectant solutions containing 1路5, 3路0 or 4路5 m-DMSO. Rapidly cooled straws containing 4路5 m-DMSO vitrify throughout on cooling, but ice forms on warming. The survival and normality of embryos frozen in 4路5 m-DMSO was not influenced by straw handling as 91鈥?2% formed blastocysts in vitro, 77鈥?8% formed normal fetuses, and no chromosomal rearrangements were observed. In solutions containing <4.5 m-DMSO ice formation occurred throughout (1路5 m-DMSO), or in parts (3路0 m-DMSO) of the cryoprotectant during cooling. The viability of embryos frozen in 3路0 or 1路5 m-DMSO solutions was reduced both in vitro and in vivo and structural chromosome aberrations, predominantly tri- and quadri-radial rearrangements, were observed. The reduction in embryo viability, and the chromosomal damage was particularly pronounced in embryos frozen in 3路0 m-DMSO in straws which were rotated during the equilibration step (47% blastocysts, 15% fetuses, 77% chromosome rearrangements). The results indicate that rapid freezing of 2-cell mouse embryos in 4路5 m-DMSO is safe and efficient, whereas freezing at lower DMSO concentrations is associated with severe chromosome damage, and reduced viability in vitro and in vivo.