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

胚胎和卵母细胞的低温保存在生殖科学领域广泛应用于安全性、冷冻保护剂、冷冻方法等方面的研究。本文就小鼠和人类胚胎及卵母细胞的冷冻保存结果进行综述，讨论冷冻和解冻的安全方法、保存期限、冷冻后卵母细胞的超微结构变化，并对人类胚胎冷冻保存在体外受精和胚胎移植中的应用前景进行展望。缓慢冷却（以每分钟0.3摄氏度的速度至零下80摄氏度）和缓慢解冻的组合比缓慢冷却和快速融化和快速冷却和缓慢融化的组合产生明显更高的存活率。快速冷却（以每分钟0.3度的速度冷却至零下40度，然后立即直接浸入液氮中）与快速解冻或直接浸入37度的预热水浴中结合也有效。在进一步解冻后，用光镜和电子显微镜对小鼠卵母细胞进行形态学观察，发现线粒体等重要细胞器发生了明显的超微结构变化，肿胀和退化。卵母细胞在光镜下基本正常。将5个不同发育阶段的胚胎和1个未受精的卵母细胞冷冻在1.5 M二甲基亚砜（DMSO）中，分三步添加，冷却速度为0.3℃。在人工播种程序后，每分钟加热至零下40摄氏度。然后直接放入液氮中储存。在解冻之后，将其浸入37摄氏度的温水中。，分五步去除冷冻保护剂并培养过夜。光镜下形态学检查显示3个胚胎（2个8细胞胚胎和1个3细胞胚胎）和1个未受精的卵母细胞，即约67%的细胞形态完整。随后的胚胎移植并没有按照日本妇产科协会发布的官方规定进行。少

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