人卵巢组织3种超低温冷冻方法比较

Comparison of three kinds of cryopreservation methods of human ovarian tissue

目的:探讨3种玻璃化冷冻降温方案对成人卵巢中各级卵泡和卵巢间质的形态学及分离卵泡活性的影响。方法:采集14例人卵巢组织,随机分成3组:自制冷冻环组、筛网组、微滴组,均采用乙二醇(EG)、二甲基亚砜(DMSO)、蔗糖作为冷冻保护剂,上述3组采用不同载体降温实现玻璃化,冷冻前后卵巢组织通过苏木素-伊红(H-E)染色检测人卵巢中各级卵泡和卵巢间质的形态学改变并计数卵泡数目;用台盼兰染色进一步观察冷冻前后卵巢组织中分离卵泡活性。结果:冷冻前3组新鲜卵巢组织中形态正常卵泡百分比差异无统计学意义(P>0.05)。冷冻环组复苏后形态正常卵泡百分率与冷冻前比较差异无统计学意义(P>0.05);筛网组和微滴组复苏后均较冷冻前明显下降(P<0.01,P<0.05),冷冻环组复苏后形态正常卵泡百分率高于筛网组和微滴组;光镜下形态学分析提示冷冻环较筛网可以更好地保存人卵巢间质形态(P<0.01);分离卵泡台盼兰染色提示冷冻后卵泡活性明显下降(P=0.016)。结论:用自制冷冻环可以较好地保存人卵巢组织中的始基卵泡和初级卵泡及卵巢间质形态。玻璃化冷冻卵巢组织后分离获得的总卵泡中有活性卵泡比例较新鲜分离卵泡下降。

Objective： To explore the effects of three kinds of vitrification cryopreservation methods on morphology of follicles at all levels and ovarian interstitial tissues in adult ovary and activity of dissociative follicles. Methods： 14 samples of human ovarian tissue were collected and divided into selfmade cryoloop group, metal mesh group and microdrop group, and ethylene glycol, dimethyl suphoxide and sucrose were adopted as cryoprotectants, then different carriers were used in the three groups, HE staining was used before cryopreservation to detect the morphological changes of follicles at all levels and ovarian interstitial tissues, and the amount of follicles was calculated; trypan blue staining was used to observe the activities of dissociative follicles in ovarian tissue before and after cryopreservation. Results： Before cryopreservation, there was no significant difference in percentage of follicles with normal morphology in fresh ovarian tissue among the three groups （ P 〉 0. 05 ） . In cryoloop group, there was no significant difference in percentage of follicles with normal morphology before and after cryopreservation （P 〉 0. 05） ; the percentages of follicles with normal morphology after thawing were significantly lower than those before cryopreservation in metal mesh group and microdrop group （ P 〈 0. 01, P 〈 0. 05 ） ; after thawing, the percentage of follicles with normal morphology in cryoloop group was significantly higher than those in metal mesh group and microdrop group; compared with metal mesh, cryoloop preserved the morphology of ovarian interstitial tissues better （P 〈0. 01 ）; after cryopreservation, the activity of follicles decreased （P = 0. 016） . Conclusion： Selfmade cryoloop can preserve pri mordial follicles and primary follicles and morphology of ovarian interstitial tissues commendably, the percentage of active follicles in total follicles after cryopreservation is lower than that of fresh follicles.