Mdecular Mechanism of Oogenesis-Assisted Reproductive Technology and genomic imprinting-

为了了解辅助生殖技术如何影响人类早期发育过程中的表观遗传学变化，我们将母体血浆和脐带血中的游离DNA和RNA作为解决这一问题的关键工具。首先，利用CA重复序列标记对3例父系限制性胎盘嵌合体(三体来自父系)进行分析。在所有三个病例中，在母体血浆中都检测到了父亲传递的外源等位基因，但在胎儿中未检测到，这表明游离DNA来自胎盘(Masuzaki J Med Genetics 2004)。此外，我们在日本使用了8个高度多态的标记来检测脐带血中的母体DNA。在阴道分娩的病例中，脐带血中孕妇DNA的检出率显著高于剖宫产者，这表明分娩增加了脐带血中的孕妇DNA污染(Masuzaki等人。第二，选择hCG和hPL基因作为胎盘表达的基因。采用实时荧光定量RT-PCR方法，检测母体血浆中两种基因的浓度随胎龄的变化。然后，我们将这一技术应用于一例前置胎盘-预置性胎盘(Clin Chemi，新闻中)的胎盘状态的评估。在这个项目(15591761)中，我们增加了三个新的发现。下面是三个新的发现，它们表明胎盘表达的印记基因是评估表观遗传状态的目标。1)母体血浆中的游离DNA来自胎盘，2)存在双向输血，这至少受分娩的影响，3)母体血浆中的胎盘mRNA有可能成为了解胎盘状态的指标，从而为了解辅助生殖技术在人类早期发育过程中对表观遗传学变化的影响提供了线索。

To know how assisted reproductive technology affects on the epigenetic change during early human development, we aimed at the cell free DNA and RNA in maternal plasma and cord blood as the key tool to resolve this question.First, three cases with paternal confined placental mosaicism (trisomy is from paternal origin) were analyzed by using CA repeats markers. In all three cases, paternal transmitted extra-alleles were detected in maternal plasma, but not in fetus, suggesting that cell free DNA is from placenta (Masuzaki J Med Genetics 2004).Also, we detected maternal DNA in cord blood using 8 highly polymorphic markers in Japanese. Detection rate of maternal DNA in cord blood was significantly higher in cases with vaginal delivery than in cases with Cesarean Section, indicating that labor increases maternal DNA contamination in cord blood (Masuzaki et al. Clin Chemi 2004).Second, hCG and hPL genes were selected as genes expressed from placenta. By using real time quantitative RT-PCR, changing concentration of both mRNA in maternal plasma was measured as the progress of gestational age. And then, we applied this technique to the evaluation of placental status in a case with placenta previa-percreta (Clin Chemi, in press).In this project(15591761), we add three novel findings to our knowledge. Three novel findings are below and suggested that imprinting gene expressed from placenta is a target to evaluate the epigenetic status. 1)Cell free DNA in maternal plasma is from placenta, 2)Bidirectional blood transfusion exists and this is affected by labor at least, and 3)there is the possibility that placental mRNA in maternal plasma is a target to know the placental status.Therefore, we got a clue to know how assisted reproductive technology affects on the epigenetic change during early human development.