印记基因DLK1对冻融胚胎移植妊娠后子痫前期发病的影响及机制研究

Frozen embryo transfer may provide a more physiological environment for embryo implanting since it could avoid placing embryos into the hyper-estrogenic uterine cavity. Freeze-all and frozen embryo transfer policy has been increasingly applied in clinic. The result of our multicenter randomized controlled trial, which has recently been published in NEJM, showed that frozen embryo transfer resulted in a higher rate of live birth but also a higher risk of preeclampsia compared with fresh embryo transfer. The mechanism underlying the increased risk of preeclampsia is unknown. However, our preliminary study showed that the imprinting gene DLK1 was closely associated with preeclampsia. The expression level of DLK1 was significantly lower in placenta resulted from frozen embryo transfer than that in placenta from fresh embryo transfer. Thus, it is hypothesized that the altered expression of DLK1 may mediate the effect of embryo cryopreservation on preeclampsia. In this study, we will further investigate the effect of embryo cryopreservation on placental DLK1 expression and its mechanism by comparison among placentas that are yielded by fresh or frozen embryo transfer or natural conception and by animal experiments. We will also delineate the role of DLK1 on trophoblasts’ proliferation, migration and invasion by manipulating DLK1 expression and evaluating downstream gene expression with transcriptome sequencing, qRT-PCR, etc.

冻胚移植可避免将胚胎置于高雌激素的子宫环境中，为胚胎着床提供更接近生理的条件。全胚冷冻-冻胚移植策略在临床的应用日益广泛。我们课题组刚发表于NEJM的一项随机对照研究显示，与鲜胚移植比较，冻胚移植可增加活产率，却同时增加子痫前期的发生率，机制不明。我们前期研究显示印记基因DLK1与子痫前期的发病密切相关，冻胚移植妊娠的胎盘组织中DLK1的表达水平明显低于鲜胚移植妊娠的胎盘。我们推测DLK1的表达异常可能介导了胚胎冷冻-冻胚移植对妊娠后子痫前期发病的影响。在本研究中，我们将在前期研究的基础上扩大样本量，并收集自然妊娠胎盘作为对照，同时收集供卵鲜胚、冻胚移植妊娠后的胎盘组织，结合动物模型进一步明确胚胎冷冻对DLK1基因表达的影响及机制；我们还将通过过表达及敲降DLK1，利用转录组测序及qRT-PCR等技术检测相关信号通路及下游效应分子的变化，明确DLK1对滋养细胞增殖、迁移侵袭的影响及机制。

随着胚胎冷冻保存技术的发展以及移植胚胎数目的下降，冻胚移植在辅助生殖临床的应用日益广泛。大样本随机对照临床试验的结果显示冻胚移植后子痫前期的发生风险高于鲜胚移植，但机制尚不明确。在本项目的资助和支持下，我们继续收集冻胚移植后子痫前期胎盘组织、冻胚移植后正常妊娠胎盘组织、以及鲜胚移植后正常妊娠胎盘组织，扩大样本量，并通过转录组测序、甲基化芯片检测、以及RT-PCR与Western-Blot等方法检测，发现印记基因DLK1在不同来源胎盘中的表达水平及甲基化水平的差异不再有统计学意义，因此我们推测DLK1基因可能不是介导冻胚移植后子痫前期发病的主要机制。.同时，通过对转录组测序结果的进一步分析，我们发现，与鲜胚移植后正常妊娠胎盘比较，前列腺六次跨膜表皮抗原3（STEAP3）基因在冻胚移植后正常妊娠胎盘及冻胚移植后子痫前期胎盘中的表达水平呈进行性下降。STEAP3基因编码的蛋白为细胞内3价铁离子的还原酶，且为P53信号通路的下游蛋白。通过体外实验，在人绒毛外滋养细胞系HTR-8细胞中降调STEAP3表达后发现，细胞的增殖、迁移侵袭、以及血管形成均明显下降，蛋白质组学的结果提示STEAP3表达下调可能通过影响铁代谢以及Gas/AXL通路影响绒毛外滋养细胞的功能，进而影响了子痫前期的发生。