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

胚胎和卵母细胞的冷冻保存在生殖科学领域已得到广泛应用，对冷冻的安全性、冷冻保护剂、冷冻方法等进行了研究。本文报道了小鼠和人胚胎和卵母细胞的冷冻保存结果，讨论了更安全的冷冻和解冻方法、保存期的长短、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-胚胎移植中的应用前景进行了展望。结合缓慢冷却（以每分钟0.3摄氏度的速度冷却至零下80摄氏度）缓慢解冻的成活率明显高于缓冷快融和急冷慢融。快速冷却（以0.3摄氏度的速率）每分钟下降到零下40度随后立即直接投入液氮中）与快速解冻或直接投入37 ℃的预热水浴相结合时也是有效的。C.小鼠卵母细胞aft ...更多信息 光镜和电镜观察显示，解冻后的卵母细胞线粒体肿胀、变性等重要细胞器发生明显的超微结构改变，光镜下基本正常。对于人的情况，将处于不同发育阶段的5个胚胎和1个未受精的卵母细胞冷冻在1.5M二甲基亚砜（DMSO）中，以0.3 ℃的冷却速率分三步加入。每分钟在Nimus 40 ℃下进行。然后直接浸入液氮中储存。在通过浸入37 ℃的预热水中进行解冻后，冷冻保护剂分五步取出并培养过夜。光学显微镜下的形态学检查显示了三个胚胎（两个八细胞胚胎和一个三细胞胚胎）和一个未受精的卵母细胞，i.d.，约百分之六十七的细胞形态完整。随后的胚胎移植未根据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