体外受精-胚胎移植通过影响DNMT3B表达水平影响小鼠妊娠中晚期胎盘生长动力学轨迹

In vitro fertilization embryo transfer affects the placenta growth dynamics of mice by affecting the expression level of DNMT3B

目的:从分子信号通路水平更多地揭示体外受精(IVF)致胎盘生长动力轨迹改变的机制,为促进和改善IVF的体外操作提供理论支持和预警靶点,降低子代远期代谢疾病的发病风险。方法:构建IVF小鼠模型,比较IVF组与自然妊娠组(NC组)小鼠胎盘在妊娠12.5、14.5、16.5天与18.5天的胎盘质量与胎盘效率,绘制IVF小鼠胎盘与NC组小鼠胎盘妊娠中晚期胎盘质量与胎盘效率曲线。Q-RT-PCR检测两组胎盘在妊娠中晚期不同时间H19及其衍生的miR-675表达水平,IGFs及其受体表达水平。Western blot法检测两组胎盘在妊娠中晚期不同时间甲基化酶类蛋白水平。Chip-q-PCR检测两组胎盘在妊娠12.5天及18.5天DNMT3B与H19ICR区的结合丰度。结果:IVF组小鼠胎盘质量在妊娠12.5天显著低于NC组,胎盘效率显著低于NC组,而在妊娠16.5天及18.5天胎盘质量显著高于NC组,胎盘效率显著低于NC组,差异有统计学意义(P<0.05)。IVF组小鼠胎盘H19及其衍生的miR-675在妊娠12.5天显著高于NC组,而在妊娠16.5天及18.5天显著低于NC组,差异有统计学意义(P<0.05)。IVF组小鼠胎盘IGFs及其受体mRNA相对表达量在妊娠12.5天显著低于NC组,而在妊娠16.5天及18.5天显著高于NC组,差异有统计学意义(P<0.05)。IVF组小鼠在妊娠12.5天胎盘甲基化酶DNMT3B及其与H19ICR区的结合丰度显著低于NC组,而在妊娠18.5天胎盘甲基化酶DNMT3B及其与H19ICR区的结合丰度显著高于NC组,差异有统计学意义(P<0.05)。结论:IVF通过妊娠末期下调H19及其衍生的miR-657激活了IGFs及其下游信号通路,致小鼠妊娠末期胎盘生长曲线偏离。IVF通过调节DNMT3B表达水平及其与H19ICR区的结合水平引起小鼠妊娠中晚期胎盘生长动力学轨迹改变。

Objective:From the level of molecular signaling pathways,trying to reveal more about the mechanism of the change of placental growth dynamic trajectory caused by IVF,so as to provide theoretical support and early warning targets for the promotion and improvement of IVF operation in vitro,and to reduce the risk of long-term metabolic diseases in offspring.Methods:IVF mouse model was established to compare the placental mass and placental efficiency of IVF mouse placenta and NC mouse placenta at 12.5,14.5,16.5 and 18.5 days of pregnancy,and to draw the curve of placental mass and placental efficiency of IVF mouse placenta and NC mouse placenta in the middle and late stages of pregnancy.Q-RT-PCR was used to detect the expression levels of H19 and its derived miR-675,and the expression levels of IGFs and their receptors in the placenta of the two groups at different times of the second and third trimester of pregnancy.Western blot were used to detect the protein levels of methylases in the placenta of the two groups at different times during the middle and late trimester of pregnancy.The binding abundance of DNMT3b and H19ICR region at 12.5 and 18.5 days of gestation in both groups was detected by Chip-q-PCR.Results:The placental mass of mice in IVF group was significantly lower than that in NC group at 12.5 days of gestation,and the placental efficiency was significantly higher than that in NC group,while the placental mass of mice in IVF group was significantly higher than that in NC group at 16.5 and 18.5 days of gestation,and the placental efficiency was significantly lower than that in NC group,the difference was statistically significant(P<0.05).The placental H19 and its derived miR-675 in IVF group were significantly higher than those in NC group at 12.5 days of gestation,and were significantly lower than those in NC group at 16.5 and 18.5 days of gestation,the difference was statistically significant(P<0.05).The relative expression levels of IGFs and their receptor mRNA in the IVF group were significantly lower than those in the NC group at 12.5 days of gestation,and were significantly higher than those in the NC group at 16.5 and 18.5 days of gestation,with statistically significant differences(P<0.05).The abundance of placental methylase DNMT3B and its binding to H19ICR region in IVF group was significantly lower than that in NC group at 12.5 days of gestation,while the abundance of placental methylase DNMT3B and its binding to H19ICR region at 18.5 days of gestation was significantly higher than that in NC group,the difference was statistically significant(P<0.05).Conclusion:IVF activated IGFs and its downstream signaling pathway by down-regulating H19 and its derived miR-657 at the end of pregnancy,and caused the deviated placenta growth curve in mice at the end of pregnancy.IVF induced the change of placental growth dynamics in mice in the middle and late pregnancy by regulating the expression level of DNMT3b and its binding level with H19ICR region.