4种冷冻-解冻方法对家兔卵巢组织形态学的影响

目的:探讨适宜的卵巢组织冻存方案。方法:采用PROH(A2组)及DMSO(B2组)慢速程序化冷冻和DMSO+PROH(C2组)及DMSO+EG(D2组)玻璃化冷冻方法,冻存家兔卵巢组织,解冻复苏后,以相应的4组新鲜组织为对照(A1-D1组),做HE染色,行组织形态学分析。结果:A1-D1组始基卵泡的形态正常率分别为90.1%、91.5%、91.8%、92.2%,其相对应的A2-D2组分别下降为67.6%、69.7%、70.5%、80.1%,差异均有统计学意义(P均<0.05)。冷冻组中A2组始基卵泡形态正常率最高,与B2、C2、D2组比,差异有显著性(P<0.05)。C2、D2组间比,差异无显著性(P>0.05)。各冷冻组合并后形态正常率始基卵泡为72.7%,初级卵泡为55.7%,两者比较有统计学差异(P<0.05)。4个冷冻组中均可见卵巢组织结构受损的表现。结论:4种冷冻解冻方法对卵巢皮质中各级卵泡及卵巢组织结构均造成一定程度的损害,使各级卵泡的形态正常率明显下降,卵巢间质细胞连接变得疏松;PROH慢速程序化冷冻法明显优于DMSO法及玻璃化法,较适合卵巢组织中始基卵泡的保存;冻存卵巢组织对初级卵泡的影响大于始基卵泡。

Effects of Four Kinds of Freezing Methods on the Morphology of Rabbit Ovarian Tissue

Objective: To investigate the better method to cryopreserve ovarian tissue. Methods: Rabbitovarian tissues were cryopreserved by procedure slow-freezing protocols including PROH(A2 group) and DMSO(B2 group) method, and vitrification protocols including DMSO PROH(C2 group) and DMSO EG(D2 group) method.After thawing, the frozen-thawed tissue was compared with the fresh tissue in order to analyze their morphologicalchanges. Results: The rates of the healthy primordial follicles were 90.1%, 91.5%, 91.8% and 92.2% in 4 freshcontrol groups (A1-D1 groups), respectively, while those in freezing groups (A2-D2 groups) were 80.1%, 70.5%,67.6%, 69.7%, respectively (P<0.05). The differences between the fresh control groups and the freezing groups weresignificant. In all freezing groups, the rate of the healthy primordial follicles in A2 group was the highest. Comparingthe rate of the healthy primordial follicles in A2 group with that of B2, C2 and D2 group, the difference was significant.There was no significant difference of the rate between two vitrification groups. After merging the freezing groups,the rate of the healthy primordial follicles was 72.7%. The rate of the healthy primary follicles was 55.7%(P<0.05).The difference was significant. Conclusions: Four kinds of frozen-thawed protocols have bad effects on differentstages of follicles and the structure of ovarian tissue. PROH slow procedure freezing method is an optimal protocolfor cryopreserving ovarian tissue. Primary follicles are relatively easier to be damaged than primordial follicles inovarian tissue cryopreservation.