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

Abstract

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\m=.\5,3\m=.\0 or 4\m=.\5m-DMSO. Rapidly cooled straws containing 4\m=.\5m\ x=r eq-\ DMSO vitrify throughout on cooling, but ice forms on warming. The survival and normality of embryos frozen in 4\m=.\5m-DMSO was not influenced by straw handling as 91\p=n-\92%formed blastocysts in vitro, 77\p=n-\78%formed normal fetuses, and no chromosomal rearrangements were observed. In solutions containing < m-DMSO ice formation occurred throughout (1\m=.\5m-DMSO), or in parts (3\m=.\0m-DMSO) of the cryoprotectant during cooling. The viability of embryos frozen in 3\ m=. \ 0 or 1\m=.\5m-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\ m=. \ 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\m=.\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.

Effect of the Polyvinylpyrrolidone Concentration of Cryoprotectant on Mouse Embryo Development and Production of Pups: 7.5% of PVP is Beneficial for In Vitro and In Vivo Development of Frozen-Thawed Mouse Embryos

“…Both of these mechanisms can lead to membrane damage, disruption of the zona pellucida and subsequent cell death. Rapid vitrification of early stage mouse embryos can result in chromosomal aberrations, and this is associated with the freezing properties of the cryoprotectants [32]. However, vitrification procedures need much higher concentrations of cryoprotectants than conventional slow freezing.…”

Human oocyte and ovarian tissue cryopreservation and its application

“…It has been reported that chilling injury affects the membrane, the microtubules [41], the cytoskeletal organization [42] and the zona pellucida [25]. Chromosome abnormalities also were observed after cryopreservation of human and mouse oocytes [43][44][45]. Most striking sample is the effects of freezing on the second meiotic spindle where microtubles are disrupted or disassembled because of tubulin depolimerization [46].…”

A high degree of aneuploidy in frozen-thawed human preimplantation embryos

“…Environmental factors or in vitro conditions may cause mosaicism and multinucleation in preimplantation embryos Munne et al 1997;Pickering et al 1995) and studies has indicated an association between embryo cryopreservation and chromosomal abnormalities (Munne et al 1997;Shaw et al 1991).…”