玻璃化冷冻和程序化冷冻人卵巢组织的效果

Effects of vitrification and programmed freezing on human ovarian tissue

目的探讨玻璃化冷冻和程序化冷冻两种方法冻存人卵巢皮质的效果。方法收集因卵巢疾病患者需手术切除的卵巢组织标本,并随机分成玻璃化冷冻组、程序化冷冻组和新鲜对照组,分别进行组织学分析、窦前卵泡的分类计数和卵巢皮质的培养,比较3组卵巢组织内卵泡形态学、卵巢组织混悬液中卵泡计数和卵巢皮质培养上清液的雌二醇(E_(2))水平。结果(1)组织学分析:两冷冻组卵巢皮质中始基卵泡完整率明显低于新鲜对照组(84.62%、80.21%vs.97.93%,P<0.001),但两冷冻组之间差异均无统计学意义(P>0.05);程序化冷冻组次级卵泡完整率明显低于新鲜对照组(37.5%vs.88.24%,P<0.05);(2)窦前卵泡的计数:玻璃化冷冻组和程序化冷冻组单位体积卵巢组织混悬液中始基卵泡计数均明显低于新鲜对照组[(76.50±8.91)&(79.67±10.40)vs.(91.50±8.70),P=0.001];程序化冷冻组每100μL卵巢组织混悬液中初级卵泡和次级卵泡计数均明显低于新鲜对照组[(22.83±3.74)vs.(27.33±3.02),(9.50±3.46)vs.(12.55±3.25),P<0.05];(3)卵巢组织培养液中的E_(2)水平:体外培养的初期阶段(D2⁃D6),冻融卵巢组织分泌的E_(2)水平低于新鲜组织(P<0.05),之后阶段各组间上清液E_(2)水平差异无统计学意义(P>0.05)。结论玻璃化冷冻和程序化冷冻均能有效地保存人卵巢组织的活性;玻璃化冷冻方法简单易行,对于次级卵泡的保存效果优于程序化冷冻组,更适合人卵巢组织的冷冻保存。

Objective To investigate the effects of vitrification and programmed cryopreservation on human ovarian cortex.Methods Ovarian tissue samples were collected from patients with ovarian disease and were randomly divided into vitrification freezing group,programmed freezing group and control group.We carried out histological analysis of ovarian tissue,isolation and counting of preantral follicles,and in vitro culture of ovarian cortex.The follicle morphology of ovarian tissue,the number of follicles in suspension of ovarian tissue and the estradiol level in the supernatant of ovarian cortical culture were compared among the three groups.Results(1)The intact rate of primordial follicles in the ovarian cortex of the two freezing groups was significantly lower than that of the control group(84.62%&80.21%vs.97.93%,P<0.001),but there was no statistical significance between the two freezing groups(P>0.05).Normal rate of secondary follicles in programmed freezing group was significantly lower than that in the control group(37.5%vs.88.24%,P<0.05).(2)The number of primary follicles in suspension of ovarian tissue per unit volume in two freezing groups was significantly lower than that in the control group[(76.50±8.91)&(79.67±10.40)vs.(91.50±8.70),P=0.001].There were significant differences in the number of primary follicles and secondary follicles in the 100 microliters in suspension of ovarian tissue between programmed freezing group and the control group[(22.83±3.74)&(9.50±3.46)vs.(27.33±3.02)&(12.55±3.25),P<0.05].(3)At the initial stage of in vitro culture(D2⁃D6),the level of E_(2) secreted by freeze⁃thawing tissue was lower than that of fresh tissue(P<0.05),and there was no statistical significance in the E_(2) level of supernatant among each group(P>0.05).Conclusion Both vitrification and programmed freezing can effectively preserve the activity of ovarian tissue.The vitrification method is more effective in preserving secondary follicles than the programmed method,and it is simple and easy to operate,which is more suitable for cryopreservation of ovarian tissue.