Sugars exert a major influence on the vitrification properties of ethyleneglycol based solutions and have low toxicity to embryos and oocytes

A systematic approach was taken to assess the vitrification properties of e thylene glycol-based solutions supplemented with carbohydrates. Solutions w ere prepared by weight (gravimetrically) using ethylene glycol as the cryop rotectant, 0.9% NaCl in water, and six different sugars: D-glucose, D(-)-fr uctose, D-sorbitol, sucrose, D(+)-trehalose, and raffinose. Sugars were add ed on a molal basis (0.1, 0.5, and 1 m). Characteristics of the solutions w ere measured during warming by differential scanning calorimetry using a co oling rate of 100 degrees C/min and a warming rate of 10 degrees C/min. In the absence of carbohydrates a 59 wt% EG-saline solution formed a stable gl ass. When EG was replaced by an equimolal concentration of glucose, fructos e, or sorbitol (monosaccharides) at 0.1, 0.5, or 1.0 m there was no change in the total solute concentration at which vitrification occurred, but the glass transition (Tg) occurred at a higher temperature than in EG-saline al one. When EG was replaced by an equimolal concentration of sucrose or treha lose (disaccharides) both the Tg and the lowest total solute concentration required for vitrification became progressively higher as the molecular wei ght, or the ratio of sugar to EG in the solutions, increased. At the highes t tested disaccharide concentration (1 m) vitrification was achieved at a t otal solute concentration of 65 wt% (sucrose) and 67 wt% (trehalose). The p olysaccharide raffinose significantly modified the vitrification properties of ethylene glycol solutions. When 0.5 or 0.1 m raffinose replaced EG on a n equimolal basis the glass transition point was raised more than with eith er the monosaccharides or the disaccharides. Raffinose allowed vitrificatio n at a total solute concentration of 67 wt% (0.5 in) and 63 wt% (0.1 m). Th e maturation of immature mouse oocytes, and the development of embryos in m edia containing 5-7 mM of any sugar was comparable to controls, indicating that they are not toxic. Exposure of freshly collected GV or MII oocytes to sugar concentrations between 0.5 and 1.0 M, for up to 10 min had no signif icant effect on the proportion which subsequently formed two cells. We conc lude that added sugars do contribute to a solutions overall vitrification p roperties, and their properties should be taken into consideration when vit rification solutions are being designed or modified. (C) 1999 Academic Pres s.