STUDIES ON CRYOPRESERVATION OF EMBRYO AND OOCYTES IN IN VITRO FRETILIZATION AND EMBRYO TRANSFER

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Cryopreservation of embryos and oocytes has been widely performed in the field of reproductive science to study on safety, cryprotectants, freezing methods, etc. In this report, results of ceyopresercation of murine and human embryos and oocytes were pressnted to discuss safer mothods of freezing and thawing, length of storage period, ultrastructural changes in frozen-thawed oocytes, and a future prespective of human embryo cryopreservation in in vitro fertilization and embryo transfer. Combination of slow cooling ( at a rate of 0.3 dagress Centigrade per minute to minus 80 degrees C.) and slow thawing yielded apparently higher survival rate as compared with those of slow cooling and rapid thawing and rapid cooling and slow thawing. Rapid cooling (at a rate of 0.3 deg. per minute to minus 40 deg. followed by immediate firect plunging into liquid nitrogen ) was also effective when combined with rapid thawing, or direct plunging into pre-warmed water bath at 37 deg. C. Murine oocytes aft … More e thawing were eveluated morphologically both with light and electron microscopes to reveal marked ultrastructural changes in vital organellas such as mitochondria being bulged and degenrated, in the oocytes which appered to be almost normal under light mictoscopic examination. As for human cases, five embryos at vatrious developmental stages and one unfertilized oocyte were frozen in 1.5 M dimethyl sulfoxide (DMSO) added in three steps at a cooling rate of 0.3 deg.C. per minute after nanual seeding procedure to nimus 40 deg.C. and then plunged directly into liquid nitrogen for storage. Following thwaing by dipping then into pre-warmed water at 37 deg.C., the cryoprotectant was temoved in five steps and cultured overnight. Morphological examination with light microscope reveald three embryos (two eight-cell embryos and one three-cell embryo) and an unfertilized oocyte, i.d., about 67 per cent of the cells were morphologically intact. Subsequent embryonsl transfer was not carried out according to an official regulation issued by The Japan Association of Obstetricvs and Gynecology. Less