玻璃化冷冻和程序化冷冻方法保存猕猴卵巢组织的存活效果比较  

Comparison of the effects of vitrification and programmed freezing protocol on cryopreservation of macaque ovarian tissue

作　　者：赵华[1] 张宇晖[1] 李蕾[1] 王璐[1] 李净羽 梁琳琳[1] Zhao Hua;Zhang Yuhui;Li Lei;Wang Lu;Li Jingyu;Liang LinlinHenan(Provincial People’s Hospital Reproductive Medicine Center,Zhengzhou 450000,China)

机构地区：[1]河南省人民医院生殖中心,郑州450000

出　　处：《中华生殖与避孕杂志》2020年第6期469-475,共7页Chinese Journal of Reproduction and Contraception

摘　　要：目的探讨玻璃化冷冻和程序化冷冻对猕猴卵巢组织的冷冻保存效果。方法取新鲜的猕猴卵巢组织随机分成对照组(新鲜卵巢组织)、玻璃化冷冻组和程序化冷冻组,分别进行HE染色、窦前卵泡的分类计数及免疫染色,比较3组卵巢组织内卵泡形态学、各级卵泡正常率及磷酸化组蛋白H3(PHH3)的表达量的差异。结果①HE染色:3组中,始基卵泡、初级卵泡正常率差异均无统计学意义(P均>0.05);程序化冷冻组单层次级卵泡正常率[40.6%(28/69)]均低于玻璃化冷冻组[53.5%(38/71)]、对照组[60.7%(34/56)],3组间差异有统计学意义(P=0.044),但玻璃化冷冻组、对照组间差异无统计学意义(P>0.05);玻璃化冷冻组、程序化冷冻组多层次级卵泡正常率[23.4%(11/47),16.7%(7/42)]均明显低于对照组[57.3%(39/68)](P均<0.001),玻璃化冷冻组多层次级卵泡正常率高于程序化冷冻组,但差异无统计学意义(P>0.05)。②分离卵泡比较:玻璃化冷冻组、程序化冷冻组初级卵泡正常形态率均接近对照组,差异无统计学意义(P>0.05);玻璃化冷冻组和程序化冷冻组次级卵泡正常率[38.2%(34/89),23.7%(23/97)]明显较低,与对照组[56.4%(61/108)]比较差异均有统计学意义(P均<0.001);玻璃化冷冻组的次级卵泡正常形态率明显高于程序化冷冻组,差异有统计学意义(P<0.001);③ PHH3染色:玻璃化冷冻组颗粒细胞PPH3的阳性表达率接近对照组(P>0.05),程序化冷冻组卵泡颗粒细胞PHH3阳性表达率(58.72%±12.31%)明显低于对照组(67.58%±8.45%,P=0.04)和玻璃化冷冻组(62.87%±9.94%,P=0.03)。结论玻璃化冷冻和程序化冷冻均能有效地保存卵巢组织,但玻璃化冷冻对卵巢组织冷冻和复融过程中各级卵泡及颗粒细胞形态学的损伤明显低于程序化冷冻,更适合下一步人类卵巢组织的冷冻保存的研究。Objective To investigate the cryopreservation effects of vitrification and programmed freezing protocol on ovarian tissue of macaque.Methods Fresh macaque ovarian tissues were randomly divided into three groups:control group(fresh ovarian issue),vitrification freezing group and programmed freezing group.HE staining,preantral follicle classification and immunostaining were performed in the three groups.The morphology of follicles,the normal rate of follicles at different stages and the difference in the expression of phosphohistone H3(PHH3)in three groups of ovarian tissues were compared.Results 1)HE staining showed that there were no significant differences in the normal rate of primary follicles and primordial follicles among the three groups(P>0.05).The normal rate of single-layer secondary follicles in the programmed freezing group[40.6%(28/69)]was lower than that in vitrification freezing group[53.5%(38/71)]and control group[60.7%(34/56)],and the difference was statistically significant(P=0.044),but there was no significant difference between vitrification freezing group and control group(P>0.05).The normal rate of multi-layer secondary follicles in vitrification freezing group[23.4%(11/47)and the programmed freezing group[16.7%(7/42)]was significantly lower than that of control group[57.3%(39/68)](P<0.001).The normal rate of multi-layer secondary follicles in vitrification freezing group was higher than that in programmed freezing group,but the difference was not statistically significant(P>0.05).2)Comparison of isolated follicles:the normal morphology of primary follicles in the vitrification freezing group and the programmed freezing group was close to control group,the difference was not statistically significant(P>0.05).The normal rate of secondary follicles in vitrification freezing group[38.2%(34/89)]and programmed freezing group[23.7%(23/97)]was obvious lower than that in control group[56.4%(61/108)].The difference was statistically significant(P<0.001).The normal morphology of secondary follicles in

关 键 词：玻璃化冷冻 程序化冷冻 卵巢组织冷冻 磷酸化组蛋白H3 

分 类 号：R714.8[医药卫生—妇产科学]