β2微球蛋白的表达降低影响人玻璃化冷冻卵母细胞发育潜能的机制研究

Oocyte cryopreservation is the most promising technology for female fertility preservation. In our previous research, the content of β2 microglobulin was decreased in subsequent embryo development medium originating from vitrified oocytes, and the early embryo developmental potential was impaired. In this study, the mouse knockout model and human immature oocytes were used as materials. We are firstly propose that β2- MG expression is associated with early embryonic development, and further observe the dynamics changes of HLA-G, DNA methylation, lncRNA, H3K4me3, H1foo, PADI6 and OCT4 in fertilized eggs and embryonic development stages with β2- MG decreased expression. The relationship between β2-MG and HLA-G, DNA methylation, lncRNA, H3K4me3, H1foo, PADI6 and OCT4expression in embryos was explored, and revealing the mechanism of β2-MG affecting the developmental potential of early embryos. We explore new ways to improve the developmental potential of oocytes after vitrification by up-regulating the expression of β2-MG for the first time. This is the initial study understanding the molecular mechanism of early embryo development from the perspective of immunity, and providing a new theoretical basis for rationally improving egg cryopreservation technology.

卵母细胞冷冻是女性生育力保存最具前景的技术。本课题以小鼠敲除模型以及人未成熟卵母细胞为材料，在前期冷冻卵母细胞后续胚胎发育培养基中β2微球蛋白含量降低，早期胚胎发育潜能受损的研究结果基础上，首次提出卵母细胞中β2-MG表达与早期胚胎发育的关联，进一步观察β2-MG含量降低和卵母细胞受精后受精卵和各胚胎发育时期直至囊胚期的HLA-G、DNA甲基化、lncRNA、H3K4me3、H1foo、PADI6和OCT4动态变化，揭示β2-MG影响早期胚胎发育潜能的机制,并首次通过上调卵母细胞中β2-MG表达来探索改善卵母细胞冷冻后发育潜能。本课题首次从免疫学角度理解早期胚胎发育的分子机制，为合理改进卵母细胞冷冻保存技术提供新的理论依据。

依据本项目研究结果，敲除β2-MG基因会影响小鼠生育性别比、HLA-G的表达、DNA甲基化、mRNA表达及生育能力。利用敲除β2-MG基因的小鼠模拟人卵子玻璃化冷冻复苏过程，结果显示敲除β2-MG基因的卵子在玻璃化冷冻复苏后的发育潜能显著降低。在人体外培养成熟的MII卵子中，上调玻璃化冷冻复苏后存活卵子中β2-MG水平，可以提高卵子的发育潜能，也能改善玻璃化冷冻复苏技术对卵子DNA甲基化、lncRNA表达影响。然而卵子本身质量、取卵-冷冻之间时间间隔也会显著影响卵子玻璃化冷冻复苏后的发育潜能。.A、经过繁殖维持种系，纯合型敲除β2-MG基因的C57BL/6N小鼠繁殖能力要显著降低，且纯合型敲除β2-MG基因的C57BL/6N小鼠更容易生雄鼠。.B、H3K4me3在纯合型敲除β2-MG基因和野生型C57BL/6N小鼠的卵子及各期胚胎中表达无显著性差异，而同源HLA-G表达存在差异，且2-细胞之前差异更明显。敲除β2-MG基因能够改变C57BL/6N小鼠mRNA 和DNA甲基化表达差异。因此敲除β2-MG基因能够显著影响小鼠HLA-G、mRNA及甲基化表达，从而影响小鼠的胚胎发育潜能及生育能力。.C、对来源于纯合型敲除β2-MG基因的C57BL/6N小鼠和野生型C57BL/6N小鼠的MII卵子模拟了卵子玻璃化冷冻复苏过程，统计分析发现敲除β2-MG基因会显著升高2-细胞胚胎发育阻滞，显著降低C57BL/6N小鼠的囊胚获得率，类似于人卵子玻璃化冷冻复苏后的临床表现。.D、收集本中心ICSI周期中未成熟卵母细胞（GV/MI），体外培养成熟和玻璃化冷冻复苏后，卵子中β2-MG（P=0.038）、HLA-G（P=0.033）基因表达显著降低。DNA甲基化、LncRNA表达都存在差异，主要表现在CPG岛位点增加，区域分布变化等。LncRNA表达差异主要表现在细胞分化、MARK通路等。.E、上调玻璃化冷冻复苏后存活卵子中β2-MG水平，卵子的发育潜能表现出上升趋势，会降低胚胎的DNA甲基化差异。.F、减数分裂纺锤体的特征可能预示着玻璃化后卵母细胞的临床结局。同时，取卵-冷冻之间的间隔时间也会明显影响卵母细胞冷冻复苏后的发育潜能，我们研究显示，取卵后2小时内是最优的卵子玻璃化冷冻时间，超过2小时将显著影响卵子的复苏成活率及发育潜能。