多胎妊娠减胎后胎儿游离DNA的清除机制及影响因素研究

Multifetal pregnancy reduction is frequently occurrence in multiple pregnancy. The incidence of vanishing twin is 16%-30%. Multiple pregnancy is always suggested to execute selective reduction for protecting pregnant and reducing complications. The new technology of noninvasive prenatal testing can’t be used in multifetal pregnancy reduction due to knowledge gaps still existing in clearance mechanism of cffDNA. Using target region capture chip, we established approach to detect the cffDNA of vanishing twin. We confirmed that cell-free DNA in multiple pregnancy with vanished twin contain maternal DNA, developing fetus DNA and vanishing fetus DNA. In this study, analysis model would be established to detect DNA concentration of vanishing twin in pregnant women who implant two embryos by IVF-ET and vanish twin. To study the clearance mechanism of cffDNA, we dynamically detect vanishing fetus DNA concentration in twin reduction and multifetal pregnancy reduction, respectively. We would investigate the correlation between clinical factors and cffDNA clearance rates to explore relevant influence factors. This study would dynamically detect vanishing fetus DNA concentration during pregnancy, which can explore the clearance mechanism of cffDNA and determine the potential correlation factors in multifetal pregnancy reduction. Meanwhile, our study will provide a significantly clinical guidance for noninvasive prenatal testing in multifetal pregnancy reduction.

多胎减胎是多胎妊娠中的常见现象。双胎之一自然减胎发生的概率为16%-30%。多胎妊娠为了保护孕妇和减少并发症，一般都建议选择性减胎。但由于“消失”胎儿cffDNA清除机制存在未知性，近年来新兴的无创产前检测不可用于多胎减胎孕妇。我们用自行设计的捕获芯片，前期检测到“消失”胎儿cffDNA残留。确定多胎减胎孕妇的血浆为母亲、发育中胎儿和“消失”胎儿DNA的混合样本。本课题将采用IVF-ET植入双胚胎后发生减胎的孕妇，建立“消失”胎儿cffDNA浓度分析模型。动态分析双胎之一自然减胎和多胎选择性减胎后“消失”胎儿cffDNA浓度变化，并进一步研究其清除机制。通过样本的变量关联分析cffDNA清除速率，研究其影响因素。本课题将首次在妊娠过程中动态地监测多胎减胎孕妇“消失”胎儿cffDNA浓度变化，探究cffDNA清除机制及影响因素，将对无创产前检测在多胎减胎孕妇中的临床应用具有指导意义。

本课题通过多时间点收集了两类前瞻性双胎之一减胎的样本队列，包括4例双胎之一染色体存在非整倍体异常，进行减胎手术的样本，每位孕妇动态采集了1-4次血；辅助生育植入异卵双胎，其中一个胎儿自然消失的家系样本，每位孕妇动态采集了3次血。动态收集的时间间隔均约4周。通过两次离心法分离到血浆后，提取游离DNA。采用高通量测序4例双胎之一染色体存在非整倍体异常，进行减胎手术后的样本，共计不同时间点10个样本，测序深度为1X，平均有效数据为6.28M。通过生物信息方法，监测非整倍体信号，本研究成功监测到了“消失”胎儿cffDNA在母体血浆中的浓度变化趋势。经手术减胎的异常胎儿，其非整倍体信号在母体血浆中呈现先上升后下降的趋势。从我们的样本队列数据结果推断胎儿DNA在母体内的残留的时间是比较长的。在我们的样本队列中，于手术减胎后的4周-12周依旧可监测到“消失”胎儿cffDNA残留。由于实施减胎手术的孕周不同，可能手术减掉的胎儿DNA在母体残留的时间也是不一样的。我们的研究结果也进一步提示NIPT技术在临床上对于胎儿之一消失孕妇的应用也需要更加谨慎。因为“消失”胎儿游离DNA在母体内残留的时间存在不确定性。