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

体外受精和胚胎移植中卵母细胞和胚胎的冷冻保存

• 批准号: 63870067
• 负责人: KOBAYASHI Toshifumi
• 金额: $ 4.16万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B).
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63870067/
• 关键词: cryopreservation; oocyte; embryo; human; pregnancy; in vitro fertilization; 妊娠; 出産; 着床; ヒト未受精卵; ヒト受精卵; マウス受精卵; 本邦初の妊娠例; コンピュータ画像解析

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

这项研究表明，在不超过15分钟的时间内向小鼠胚胎中加入二甲基亚砜，DMSO和蔗糖的3步添加，以及在室温下在10分钟内最终平衡，允许超过95%的解冻胚胎在体外发育成扩张的囊胚。与保温0 ~ 60分钟相比，接种后保温30分钟也显著提高了胚胎的解冻后活力。除了这些时间因素外，冷冻和解冻步骤中的蔗糖对高活力有显著贡献，而单独冷冻或解冻步骤中的蔗糖则没有。基于这些基本知识，根据日本妇产科学会发布的人类胚胎冷冻保存伦理声明，在体外受精程序中进行人类胚胎冷冻保存。用1.5M DMSO和0.1M蔗糖作为冷冻保护剂， ...更多信息 tants. 42个冷冻胚胎在胚胎整合和活力的形态学评价后解冻并在7个自发周期中移植。结果，4次妊娠成功，其中一次于1989年12月25日在妊娠第37周时成功分娩双胞胎女婴，这是日本首例成功分娩冷冻解冻人类胚胎的病例。本研究的后半部分包括使用小鼠超数排卵卵母细胞冷冻保存。解冻后卵母细胞的细胞内细胞器，包括线粒体的超微结构研究显示了不同程度的结构变化，强烈建议潜在的细胞活力下降。由于成熟卵母细胞在中期II处于减数分裂停滞阶段，因此也可能存在染色体损伤。作为结论，本研究揭示了进一步调查和发展的冷冻/解冻协议的最佳断点温度，冷冻保护剂的选择，和接种程序的必要性。少

项目成果

飯塚 理八: 病態生理. 6. 433-439 (1987)

饭冢里八：病理生理学。6. 433-439 (1987)

Rui AOKI: "Study on cryopreservation of human and murine embryos : Successful pregnancy from cryopreserved human embryos" Journal of Keio Medical Society. Vol. 66, No. 5. 1113-1124 (1989)

青木锐：“人类和小鼠胚胎冷冻保存的研究：冷冻人类胚胎成功怀孕”庆应义塾医学会杂志。

飯塚 理八,青木 類: "ヒト受精卵の凍結保存" 凍結及び乾燥研究会会誌. 36. 3-9 (1990)

Rihachi Iizuka，Rui Aoki：“人类受精卵的冷冻保存”冷冻干燥研究会杂志 36. 3-9 (1990)。

青木 類 他: "ヒト凍結保存胚による体外受精妊娠成功例の検討" 日本梅精着床学会雑誌. 7. 229-233 (1990)

Rui Aoki 等：“使用人类冷冻胚胎的成功体外受精妊娠案例的研究”日本梅子精子植入学会杂志 7. 229-233 (1990)。

青木類: "ヒトおよびマウス胚の凍結保存に関する研究" 慶応医学. 66. 1113-1124 (1989)

青木：“人类和小鼠胚胎冷冻保存的研究”庆应义塾医学科学 66. 1113-1124 (1989)。