Experiments were conducted to find a suitable cryoprotectant and suitable procedure for vitrification of 8-cell mouse embryos. The method was then applied clinically to the cryopreservation of human embryos in our assisted reproduction programme. Mouse embryos were vitrified with 30 or 40% 1,2-propanediol (PROH), dimethylsulphoxide (DMSO), ethylene glycol, glycerol, or acetamide, each diluted with a solution containing 30% Ficoll plus 0.5 M sucrose. Embryos were exposed to the solutions for 0.5 or 2 min at 20 or 25 degrees C, cooled in liquid nitrogen and warmed rapidly. Embryo survival was assessed by in-vitro development. In PROH-, DMSO- and acetamide-based solutions, higher survival rates (29-82%) were obtained with less permeating conditions, suggesting that these cryoprotectants are considerably toxic. In glycerol- and ethylene glycol-based solutions, however, higher survival rates (74 and 92% respectively) were obtained with more permeating conditions, suggesting that these cryoprotectants are less toxic. Human embryos on days 2-3 were vitrified in an ethylene glycol-based solution (EFS40). Survival, assessed by the morphology, was higher in 4-cell embryos on day 2 and 8-cell embryos on day 3 than in 2-3-cell embryos on day 2 or 2-7-cell embryos on day 3. From 18 transfers, one ended with the delivery of healthy twin babies.

Abstract.To assess the permeability of mouse oocytes and embryos, matured oocytes and embryos at various stages of development were placed in five cryoprotectant solutions at 25 C for 25 min. From the cross-sectional areas of the oocytes/embryos, the relative change in volume was analyzed. In oocytes, shrinkage was least extensive and recovery was quickest in the propylene glycol solution, showing that propylene glycol permeates the oocytes most rapidly. Dimethyl sulfoxide, acetamide, and ethylene glycol permeated the oocytes slightly more slowly than propylene glycol. The oocytes in glycerol shrunk extensively and then expanded marginally, indicating slow permeation. The volume changes of 1-cell and 2-cell embryos were similar to those of oocytes, showing little change in permeability. In 8-cell embryos, the volume recovered much faster than in the earlier stages especially in glycerol and acetamide. In morulae, the volume recovery was much faster in glycerol and in ethylene glycol; in ethylene glycol, the extent of shrinkage was small and the recovery was fast, indicating an extremely rapid permeation. Although the permeability of oocytes/embryos generally increased as embryo development proceeded, the degree of increase varied greatly among the cryoprotectants. Interestingly, the volume change in propylene glycol was virtually unaffected by the stage of development. Such information will be valuable for determining a suitable protocol for the cryopreservation of oocytes/embryos at different stages of development. Key words: Mouse, Embryo, Oocyte, Permeability, Cryoprotectant (J. Reprod. Dev. 51: 235-246 , 2005) he survival of mammalian oocytes and embryos after cryopreservation varies with the stage of maturation and development. In general, oocytes/embryos at earlier stages appear to be more sensitive to cryopreservation. In the mouse, the rate of survival after cryopreservation is lower for unfertilized oocytes than embryos and increases as development proceeds up to the 8-cell or morula stage, although it may decrease as the blastocoel enlarges Cryoprotectants for mammalian oocytes and embryos are virtually limited to these five agents both in slow freezing and in vitrification [8].Although the protective action of cryoprotectants is considered colligative [9], each agent has its own specific properties. Among them, the permeating p r o p e r t y i s o f g r e a t i m p o r t a n c e , b e c a u s e permeation of the cell with a cryoprotectant is critical for the successful cryopreservation of

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