卵巢组织冷冻移植小鼠模型的子代安全性研究

Diseases and gonad toxic therapy may lead to female premature ovarian failure. Ovarian tissue cryopreservation and transplantation are the most important methods to reconstruct ovarian function. The two most frequently used technics including slow-freezing and vitrification. The carrier of ovarian tissue had been improved in our previous study, and a novel vitrification method with higher effecacy and better outcome named NIV was raised by current study team. It is considered that the cryopreserved transplanted ovarian tissue influenced by some physi-chemical factors could not be avoided through the whole procedure. Thus, potential safety problems may raise in offspings according to the above effects. None study is focusing in offspring's safety problems derived from ovarian tissue cryopreservation. Current study is designed to evaluate the safety issue of cryopreservation. The animal model of allogeneil graft would be constructed used the ovarian tissue cryopreserved by slow-freezing and NIV vitrification. Eligible filial generation mice would be selected to be compared with the normal gestation group and the fresh ovarian tissue transplantation group. Birth defect would be investigated. Abnormal methylation of the key genomic imprinting as IGF2/H19 will be detected by the epigenetic approach. Variability analysis in protein expression in the important organs as brain, heart and liver will be detected by proteomics methods among groups. This study is to evaluate the safety issue of offspring derived from ovarian tissue cryopreservation primaryly. Further improvement of the technology of fertility preservation will be warranted by the prospective results of current study.

疾病和性腺毒性治疗可以导致女性卵巢早衰。卵巢组织冷冻移植技术是保存和重建卵巢功能的重要手段。我们前期改良了卵巢组织冷冻载体，提出了效率更高、冻存效果更好的玻璃化冷冻新方法（NIV法）。由于卵巢组织冷冻过程中的各种体外因素会对生殖细胞造成影响，可能导致后代潜在的健康风险。目前对于卵巢组织冷冻移植技术的子代安全性问题尚缺乏研究。本项目拟采用NIV玻璃化冷冻法和传统程序化冷冻法建立小鼠卵巢组织同种异体移植模型，筛选出可供研究的子代小鼠，观察出生缺陷，运用表观遗传学的研究方法对关键印记基因甲基化水平进行检测；同时运用蛋白组学的研究方法对子代小鼠的重要脏器（脑、心脏、肝脏）蛋白表达进行差异性分析，通过与正常妊娠和新鲜卵巢组织移植组子代小鼠对比，初步探讨两种主要的卵巢组织冷冻技术的子代安全性问题。本项目首次从后代安全性角度对卵巢组织冷冻技术进行评估，为改善和优化女性生育力保存技术提供研究基础。

疾病和性腺毒性治疗可以导致女性卵巢早衰。卵巢组织冷冻移植技术是保存和重建卵巢功能的重要手段，常用冷冻方法主要为程序化冷冻和玻璃化冷冻。我们前期改良了卵巢组织冷冻载体，提出了效率更高、冻存效果更好的玻璃化冷冻新方法（NIV法）。由于卵巢组织冷冻过程中的各种体外因素会对生殖细胞造成影响，可能导致后代潜在的健康风险。目前对于卵巢组织冷冻移植技术的安全性问题尚缺乏研究。本项目采用了NIV玻璃化冷冻法和传统程序化冷冻法建立小鼠卵巢组织同种异体移植模型，筛选出可供研究的子代小鼠，观察子代生长发育、重要脏器解剖及形态分析，并对子代小鼠脑和肝脏关键印记基因甲基化H19/igf2r, Snrpn、Plagl1水平和基因表达量进行检测，通过与正常妊娠和新鲜卵巢组织移植组子代小鼠对比，探讨两种主要的卵巢组织冷冻技术的子代安全性问题。得出结论：小鼠卵巢组织玻璃化冷冻与程序化慢速冷冻相比，移植后卵巢功能恢复更快；诞生子代小鼠发育情况、行为和组织脏器形态分析，冷冻组与对照组相比无明显差异，新生鼠死亡率冷冻组高于新鲜组，两个冷冻组均有发现存活至成年发育畸形小鼠。通过对生长发育关键印记基因的甲基化水平检测和差异表达检测发现：冷冻组移植后代脑和肝脏组织存在H19基因印记区甲基化丢失，igf2r、Snrpn基因甲基化水平增高，Plagl1基因印记区甲基化水平四组无明显差别。冷冻组H19基因mRNA水平降低，两个冷冻组之间无差别。本项目首次从后代安全性角度对卵巢组织冷冻技术进行评估，为改善和优化女性生育力保存技术提供研究基础。