Cancer is the second major cause of death in the world. The problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic. Cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence is the potential key solution to this problem. The aim of this study was to test the viability of cryopreserved human ovarian cortex after long-term cooling in culture medium composed of permeable cryoprotectants. Ovarian fragments from sixteen patients were randomly divided into two groups. After the operation, tissue pieces assigned to both groups were cooled to 5 °C for 22-24 h, frozen and thawed. Group 1 pieces (n = 32) were cooled before cryopreservation in the standard culture medium, and Group 2 pieces (n = 32) were cooled in the freezing medium (culture medium+6% ethylene glycol+6% dimethyl sulfoxide+0.15 M sucrose). Freezing was performed in standard 5 ml cryo-vials with ice formation at -9 °C, cooling from -9 to -34 °C at a rate of -0.3 °C/min and plunging at -34 °C into liquid nitrogen. Thawing was performed in a 100 °C (boiling) water bath. The removal of cryoprotectants was performed in 0.5 M sucrose with 20 min exposure in sucrose and 30 min stepping rehydration. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development of follicles (histology). Six months after the autotransplantation, oocytes from the twenty-seven-year old, hormonally stimulated patient were retrieved and inseminated with her partner sperm through the intracytoplasmic spermatozoa injection (ICSI). For groups 1 and 2, 93.5 ± 1.9% and 96.4 ± 2.0% of the preantral follicles, respectively, were morphologically normal (P > 0.1) (with a tendency toward increasing in quality in Group 2). Six months after the auto-transplantation, two ICSI cycles resulted in the gathering and transplantation of high quality embryos, but no pregnancy had been established. Thirteen months after the auto-transplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term. Long-term (24 h) cooling of ovarian tissue to 5 °C before cryopreservation in the presence of permeable cryoprotectants simplifies the protocol of cryopreservation and has a tendency of increasing of the cells viability after thawing.

中文翻译：

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冷冻前在有渗透性冷冻保护剂的情况下对卵巢碎片进行长期（24 小时）冷却：自发妊娠和再移植后出生的婴儿

癌症是世界上第二大死亡原因。癌后不孕问题起着重要作用，因为化疗可能具有性腺毒性。在癌症治疗前冷冻保存卵巢组织并在康复后重新植入是解决这个问题的潜在关键。本研究的目的是测试冷冻保存的人卵巢皮质在由可渗透冷冻保护剂组成的培养基中长期冷却后的生存能力。来自 16 名患者的卵巢碎片被随机分为两组。手术后，分配给两组的组织切片冷却至5°C 22-24 h，冷冻和解冻。第 1 组（n = 32）在标准培养基中冷冻保存前冷却，第 2 组（n = 32）在冷冻培养基（培养基+6% 乙二醇+6% 二甲亚砜+0.15 M 蔗糖）中冷却。在标准的 5 ml 冷冻瓶中进行冷冻，在 -9 °C 下结冰，以 -0.3 °C/min 的速度从 -9 冷却到 -34 °C，并在 -34 °C 下浸入液氮中。在 100 °C（沸腾）水浴中进行解冻。冷冻保护剂的去除在 0.5 M 蔗糖中进行，在蔗糖中暴露 20 分钟，步进再水化 30 分钟。通过毛囊的发育（组织学）评估组织的预冷冻冷却的有效性。自体移植六个月后，通过卵胞浆内精子注射 (ICSI)，取回了 27 岁受激素刺激的患者的卵母细胞，并与她的伴侣精子一起受精。对于第 1 组和第 2 组，93。分别有 5 ± 1.9% 和 96.4 ± 2.0% 的窦前卵泡形态正常（P > 0.1）（在第 2 组中质量有提高的趋势）。自体移植六个月后，两个 ICSI 周期导致收集和移植高质量胚胎，但没有建立怀孕。自体移植后 13 个月，患者自然怀孕，足月产下一个健康的女婴。在存在渗透性冷冻保护剂的情况下，在冷冻保存前将卵巢组织长期 (24 小时) 冷却至 5°C，简化了冷冻保存的方案，并且在解冻后有增加细胞活力的趋势。1)（在第 2 组中质量有提高的趋势）。自体移植六个月后，两个 ICSI 周期导致收集和移植高质量胚胎，但没有建立怀孕。自体移植后 13 个月，患者自然怀孕，足月产下一个健康的女婴。在存在渗透性冷冻保护剂的情况下，在冷冻保存前将卵巢组织长期 (24 小时) 冷却至 5°C，简化了冷冻保存的方案，并且在解冻后有增加细胞活力的趋势。1)（在第 2 组中质量有提高的趋势）。自体移植六个月后，两个 ICSI 周期导致收集和移植高质量胚胎，但没有建立怀孕。自体移植后 13 个月，患者自然怀孕，足月产下一个健康的女婴。在存在渗透性冷冻保护剂的情况下，在冷冻保存前将卵巢组织长期 (24 小时) 冷却至 5°C，简化了冷冻保存的方案，并且在解冻后有增加细胞活力的趋势。病人自然怀孕，足月产下一个健康的女婴。在存在渗透性冷冻保护剂的情况下，在冷冻保存前将卵巢组织长期 (24 小时) 冷却至 5°C，简化了冷冻保存的方案，并且在解冻后有增加细胞活力的趋势。病人自然怀孕，足月产下一个健康的女婴。在存在渗透性冷冻保护剂的情况下，在冷冻保存前将卵巢组织长期 (24 小时) 冷却至 5°C，简化了冷冻保存的方案，并且在解冻后有增加细胞活力的趋势。