Cryopreservation of Oocytes and Embryos in in Vitro Fertilization and Embryo Transfer

这项研究表明，在小鼠胚胎中添加不超过15分钟的二甲基亚砜、DMSO和蔗糖，再加上在室温下10分钟内的最后平衡，可以使95%以上的解冻胚胎在体外发育成扩张的囊胚。与0~60min相比，种子后保温30min显著提高了胚胎的解冻后活力。除了这些时间因素外，在冷冻和解冻步骤中的蔗糖对高存活率有显著的贡献，而单独在冷冻或解冻步骤中的蔗糖对高活力没有贡献。在这些基础知识的基础上，根据日本妇产科学会发布的《人类胚胎冷冻保存伦理声明》，在体外受精计划中进行了人类胚胎的冷冻保存。用1.5M二甲基亚砜和0.1M蔗糖作为冷冻蛋白…对52个人胚胎进行冷冻处理更多的蚂蚁。对42枚冷冻胚胎进行了7个自然周期的解冻和移植，对胚胎的完整性和存活率进行了形态评价。结果，已确定4例妊娠，其中1例在1989年12月25日妊娠第37周时顺利产下双胞胎女婴，这是日本首例成功分娩冻融人类胚胎的案例。本研究的后半部分包括用小鼠超排卵卵母细胞冷冻保存卵母细胞。对解冻后卵母细胞胞内细胞器包括线粒体的超微结构研究表明，卵母细胞有不同程度的结构变化，这强烈地表明细胞活力的潜在下降。由于成熟卵母细胞在中期II处于减数分裂停滞阶段，染色体也可能受到损害。因此，本研究揭示了进一步研究和发展冷冻/解冻方案的必要性，包括最佳的断点温度、冷冻保护剂的选择和播种程序。较少

This study revealed that 3-step addition of dimethylsulfoxide, DMSO, and sucrose to murine embryos over no more than 15 minutes, together with final equilibration at room temperature for within a 10-minute period, allowed more than 95 per cent of thawed embryos to develop into expanded blastocyts in vitro. Temperature holding for 30 minutes after seeding also improved post-thaw viability of embryos significantly as compared with holding for 0 to 60 minutes. In addition to those time factors, sucrose in both freezing and thawing steps contributed significantly to the high viability, whereas sucrose in either freezing or thawing steps alone did not. Being based upon these basic knowledges, cryopreservation of human embryos was performed in in vitro fertilization program according to the Ethical Statements on Human Embryo Cryopreservation issued by the Japan Society of Obstetrics and Gynecology. Fifty-two human embryos were tehn cryopreseved with 1.5 M DMSO and 0.1 M sucrose as cryoprotec … More tants. 42 frozen embryos were thawed and transferred in 7 spontaneous cycles after morphological evaluation of embryonal integration and viability. As a result, 4 pregnancies have been established, and one of them led to a successful delivery of twin female babies at 37th week of gestation on December the 25th in 1989, which was the first case of successful delivery of frozen-thawed human embryos in Japan. The latter part of this study consisted of oocyte cryopreservation using murine superovulated oocytes. Ultrastructural investigation of the intracellular organellas including mitochondira of post-thaw oocytes revealed a various degrees of structural changes which strongly suggested latent decrease in cellular viability. Chromosomal damages could also be suggested since mature oocytes was at the stage of meiosis arrest in the metaphase II. As a conclusion, this study revealed the necessity of further investigation and development of freeze/thaw protocols in terms of the optimal breakpoint temperature, choice of cryoprotectants, and seeding procedures. Less