Investigation of epigenetic and morphological placental abnormalities induced by in vitro fertilization

项目摘要 全世界已有数百万婴儿通过辅助生殖技术(ART)出生。尽管 大多数抗逆转录病毒治疗的妊娠会产生健康的婴儿，抗逆转录病毒治疗与一些不良反应有关， 包括低出生体重、胎盘发育异常和罕见的印迹障碍的风险增加。 来自动物研究的证据支持，抗逆转录病毒治疗程序可以独立于 潜在的不孕不育。印记基因对于正常的发育和生长很重要，它是 单等位基因专一表达于亲本的基因。值得注意的是，印记基因的表达是 在被称为印迹控制区(ICR)的离散元件上受DNA甲基化调控。术语 体外受精(IVF)小鼠胎盘显示双等位基因异常表达相关 对几个印记基因进行ICR低甲基化。此外，试管受精还会导致胎盘异常 过度生长。这项建议的主要目的是测试个别试管受精程序对胎盘的影响。 并阐明这种异常胎盘的潜在遗传和表观遗传机制。 由于胎盘已被证明能够补偿环境干扰，因此 观察到的表观遗传学和形态变化可能不会导致生理后果。致信地址 这一点，胎盘的形成，细胞成分，病理和营养物质运输功能将在 目标1中的自然胚胎移植、有无超数排卵、胚胎培养和试管受精。因为 不同的细胞类型有不同的表观遗传学和表达谱，目前存在一些限制，使 像胎盘这样的复杂组织的分子分析是很困难的。为了补救这一点，激光捕获 显微切割(LCM)和单分子定量荧光原位杂交(FISH)将 被利用了。以确定可能导致胎盘异常的基因表达变化 将在目标2中对外胎盘锥体和绒毛膜进行表型、转录组分析(e7.5)和 LCM分离自然受精和体外受精胚胎的交界区和迷路(E14.5)。在目标3中，基因 表达和单分子FISH方法将被用来检验特定印记的假设 基因在导致体外受精诱导的胎盘过度生长的潜在机制中发挥了作用。确定如何 表观遗传变化影响胎盘形成不仅在试管受精的背景下很重要，因为胎盘是 对子孙后代和产妇的整体健康至关重要。此外，在此期间开发的管道可能 事实证明，这对于未来对其他复杂组织的表观遗传学研究是不可或缺的。

Project Abstract Several million babies worldwide have been born via Assisted Reproductive Technologies (ART). Although the majority of ART pregnancies result in healthy babies, ART is associated with a number of adverse effects, including increased risk for low birth weight, abnormal placental development, and rare imprinting disorders. Evidence from animal studies support that ART procedures can induce these effects independent from underlying infertility. Important for proper development and growth, imprinted genes are a unique subset of genes that are monoallelically expressed specific to the parent-of-origin. Notably, imprinted gene expression is regulated by DNA methylation at a discrete element known as the imprinting control region (ICR). Term placentae from mice generated by in vitro fertilization (IVF) display abnormal biallelic expression correlated with ICR hypomethylation for several imprinted genes. Additionally, IVF induces abnormal placental overgrowth. The main objective of this proposal is to test how individual IVF procedures affect placental function and to elucidate the underlying genetic and epigenetic mechanisms of this abnormal placentation. Because the placenta has been shown to be capable of compensating for environmental perturbations, the epigenetic and morphological changes observed may not result in physiological consequences. To address this, formation, cell composition, pathology, and nutrient transport function of the placenta will be assessed in natural, embryo transfer with and without superovulation, embryo culture, and IVF concepti in Aim 1. Because different cell types have distinct epigenetic and expression profiles, several limitations currently exist making molecular analyses of complex tissues, like the placenta, difficult. To remedy this, laser capture microdissection (LCM) and single-molecule quantitative fluorescence in situ hybridization (FISH) will be utilized. To determine gene expression changes that may be responsible for the abnormal placental phenotype, transcriptome analyses will be performed in Aim 2 on ectoplacental cone and chorion (E7.5) and junctional zone and labyrinth (E14.5) isolated by LCM from natural and IVF concepti. In Aim 3, gene expression and single-molecule FISH methods will be employed to test the hypothesis that particular imprinted genes play a role in the underlying mechanism resulting in IVF-induced placental overgrowth. Determining how epigenetic changes influence placentation is important not only in the context of IVF, because the placenta is critical for overall offspring and maternal health. Furthermore, the pipeline developed during this fellowship may prove indispensible for future epigenetic investigations of other complex tissues.