不同冷冻方案保存人类卵巢组织的活性比较

背景：卵巢组织冷冻保存被认为是保存女性生殖内分泌功能安全、有效的方法，但目前尚无统一确定的方案。 目的：探讨3种冷冻方案对人类卵巢组织保存冷冻效果及卵泡活性的影响。方法：采用丙二醇慢速程序冷冻法、二甲基亚砜玻璃化冷冻法、液氮直投法对20例人类卵巢组织进行冷冻保存，复苏后采用细胞存活/死亡荧光分析法计数有活性的细胞，体外培养测定培养液雌二醇浓度及各级卵泡计数，判断3种不同冷冻方案对人类卵巢组织活性的影响。结果与结论：不同冷冻方案冻融后卵巢组织块的活性卵泡率均低于新鲜卵巢组（P 〈 0.05），二甲基亚砜组最低，液氮直投法组与慢速程序冷冻法组差异无显著性意义。体外培养各冷冻组分泌的雌二醇水平比较，第4天时二甲基亚砜组低于液氮直投法组和慢速程序冷冻法组（P 〈 0.05），至第8天时各冷冻组雌二醇水平与新鲜卵巢组一致。培养14 d组织学观察，各组卵巢组织内生长期卵泡比例增多，始基卵泡仍然占最主要的。新鲜卵巢组织正常卵泡的总数高于冷冻组（P 〈 0.05），二甲基亚砜组的正常卵泡数低于液氮直投法组和慢速程序冷冻法组（P 〈 0.05）。提示冷冻保存对卵巢组织卵泡有一定的损伤，但仍能保存大部分始基卵泡的活性，经体外培养后可进一步发育并具有分泌功能，在3种冷冻方案中，慢速程序冷冻法和液氮直投法的冷冻效果优于二甲基亚砜玻璃化冷冻法，液氮直投法操作简便，冷冻效果稳定。

Influence of different cryopreservation schemes on human ovarian tissue activity

BACKGROUND：Ovarian tissue cryopreservation is considered to be a safe and effective method to save female reproductive endocrine function, but there is no uniform scheme identified.
OBJECTIVE：To explore the effects of three freezing schemes on human ovarian tissue and the influence on follicle activity.METHODS： Twenty specimens of human ovarian tissue were frozen by propanediol slow freezing method, dimethyl sulfoxide vitrification method, and direct liquid nitrogen method, respectively. After cell resuscitation, the number of activated cells was counted using the live/dead fluorescence analysis, and estradiol level and follicles at different development stages were measured and counted respectively after cultured in vitro to evaluate the influence of three freezing schemes on human ovarian tissue activity .RESULTS AND CONCLUSION： The activated oocyte rates of three frozen-thawed ovarian tissue groups were lower than that of the fresh ovarian tissue group （P 〈 0.05）. Among the three frozen-thawed groups, the activated oocyte rate of the dimethyl sulfoxide group was the lowest, and there was no significant difference between the other two groups. After in vitro culture, the estradiol level of the dimethyl sulfoxide group was lower than that in the propanediol and liquid nitrogen groups at 4 days （P 〈 0.05）, and the estradiol levels showed no differences between the fresh ovarian tissue group and three frozen-thawed groups at 8 days. After 14 days of culture, the ratio of growing follicles was increased, but primordial follicles were still in the lead. The total number of normal occytes in the fresh ovarian tissue was higher than that in the three frozen-thawed groups （P 〈 0.05）, and the number of normal occytes in dimethyl sulfoxide group was lower than that in the other two frozen-thawed groups （P 〈 0.05）. These findings indicate that the cryopreservation can cause damage to ovarian tissue follicles but still can save most primordium follicle activity. The primordium follicle cultured in vitro can further develop to have secretion function. The dimethyl sulfoxide vitrification method is superior to the propanediol slow freezing method and direct liquid nitrogen method, but the direct liquid nitrogen method is simple and convenient with stable cryopreservation effects.