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

项目摘要 全世界有数百万婴儿通过辅助生殖技术（ART）出生。虽然 大多数ART妊娠会产生健康的婴儿，ART与许多不良反应有关， 包括低出生体重、胎盘发育异常和罕见的印记疾病的风险增加。 来自动物研究的证据支持ART程序可以诱导这些效应， 潜在的不孕症重要的是适当的发展和增长，印记基因是一个独特的子集， 对起源亲本特异性单等位基因表达的基因。值得注意的是，印记基因表达 由DNA甲基化在称为印记控制区（ICR）的离散元件调节。Term 来自体外受精（IVF）产生的小鼠的胎盘显示异常的双等位基因表达， 与ICR低甲基化的几个印记基因。此外，IVF诱导异常胎盘 过度生长该提案的主要目的是测试个体IVF程序如何影响胎盘 功能，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， 观察到的表观遗传和形态学变化可能不会导致生理后果。解决 胎盘的形成、细胞组成、病理学和营养转运功能将在 目的1中的自然、有或无超数排卵的胚胎移植、胚胎培养和IVF受孕。因为 不同的细胞类型具有不同的表观遗传和表达谱，目前存在一些限制， 复杂组织的分子分析，比如胎盘，很困难。为了解决这个问题，激光捕获 显微切割（LCM）和单分子定量荧光原位杂交（FISH）将是 利用。为了确定可能导致胎盘异常的基因表达变化， 表型、转录组分析将在Aim 2中对外胎盘锥和绒毛膜（E7.5）进行， 通过LCM从自然和IVF孕体分离的结合区和迷路（E14.5）。在目标3中，基因 表达和单分子FISH方法将被用来测试的假设，特别是印迹 基因在导致IVF诱导的胎盘过度生长的潜在机制中起作用。确定如何 表观遗传变化影响胎盘形成不仅在IVF的背景下很重要，因为胎盘 对后代和母亲的整体健康至关重要。此外，在本研究金期间开发的管道可能 证明是不可或缺的其他复杂组织的未来表观遗传研究。

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.