The genome integrity and epigenome of sperm from men with recurrent pregnancy loss

项目总结/摘要：流产是最常见的妊娠并发症， 在美国，每年有75万到100万例流产。多达5%的夫妇 经历过两次或两次以上的流产，并有复发性流产（RPL）。RPL是一个重要的临床 即使在诊断检查后，50%的夫妇也找不到他们经常性损失的原因， 无法解释的RPL（URPL）因此，调查RPL的新原因至关重要， 与RPL结合，以确定原因，从而确定可能的治疗方案。uRPL的男性因素原因 在临床上，对uRPL男性进行的唯一诊断测试是染色体核型， 排除染色体易位/倒位。我们的建议非常重要，因为它解决了这一重大差距 关于RPL的父亲原因的知识。有几条证据表明异常精子 通过印迹基因中的高DNA片段化和异常DNA甲基化客观地测量质量 与uRPL相关。该项目将改变目前RPL夫妇的临床诊断模式， 主要是女性为中心的方法，以平衡的方法，包括精子的分子分析。我们的项目将 包括对来自大量uRPL夫妇精子进行全面分析，以研究DNA片段化 (Aim 1）、DNA甲基化（Aim 2）和组蛋白模式（Aim 3）。在目标1中，我们的实验将研究 人类精子DNA断裂增加可导致RPL的分子机制， 研究发生的DNA损伤类型和受影响的基因组位点。我们的一项重大创新 一种独特的方法是平行研究高DNA片段化导致的流产的基因组， 精子来阐明潜在的缺陷。虽然精子基因组的完整性是确保精子质量的一个重要方面， 健康的怀孕，另一个方面是精子表观基因组，这也在怀孕中起着至关重要的作用 生存能力先前的研究表明，在uRPL男性中，印记位点的精子DNA甲基化受到干扰， 已经观察到来自uRPL男性的精子中组蛋白的保留增加。在此基础上， 在目标2中，我们将同时追求候选基因（印记基因）和无偏见的全基因组 使用最新的全面DNA甲基化阵列技术研究精子甲基化组的方法。 此外，我们还将利用DNA甲基化作为父亲衰老的标志物的先验预测模型 以评估来自uRPL男性的精子中是否存在过早老化。这项工作将确定表位突变， 精子可能在RPL中起机制作用。在目标3中，我们将扩展我们的表观遗传分析， uRPL男性精子组蛋白模式的全基因组分析。最后，我们将联合收割机 数据集来比较和对比受影响的基因组位点。总之，该项目将有助于 重要的是确定RPL的新的男性因素原因，这将最终导致显着的 RPL夫妇的诊断和治疗进展。

PROJECT SUMMARY/ABSTRACT: Miscarriage is the most frequent pregnancy complication, and around 750,000 to 1,000,000 miscarriages occur annually in the United States. Up to five percent of couples will experience two or more miscarriages and have recurrent pregnancy loss (RPL). RPL is a significant clinical problem as even after a diagnostic workup, 50% of couples will not find a cause for their recurrent losses and will be diagnosed with unexplained RPL (uRPL). Therefore, investigating new causes for RPL is crucial to help couples with RPL to identify a cause and, therefore, possible therapeutic options. Male factor causes of uRPL are understudied, and clinically, the only diagnostic testing performed on men with uRPL is a karyotype to exclude chromosomal translocation/inversion. Our proposal is highly significant as it addresses this major gap in our knowledge regarding paternal causes of RPL. Several lines of evidence show that abnormal sperm quality as objectively measured by high DNA fragmentation and aberrant DNA methylation in imprinted genes is associated with uRPL. This project will shift the present clinical diagnostic paradigm for RPL couples from a mainly female-centric approach to a balanced approach, including molecular analysis of sperm. Our project will involve a comprehensive analysis of sperm from a large cohort of uRPL couples to study DNA fragmentation (Aim 1), DNA methylation (Aim 2), and histone patterns (Aim 3). In Aim 1, our experiments will investigate the molecular mechanisms by which increased sperm DNA fragmentation in humans can lead to RPL by investigating the type of DNA damage occurring and the genomic loci affected. One major innovation in our approach is to uniquely study in parallel the genome of the miscarriages resulting from high DNA fragmented sperm to elucidate the underlying defect. Although sperm genomic integrity is one crucial aspect of ensuring a healthy pregnancy, another aspect is the sperm epigenome, which also plays a crucial role in pregnancy viability. Prior studies show that sperm DNA methylation at imprinted loci is perturbed in men with uRPL, and increased retention of histones in sperm from uRPL men has been observed. Building upon these observations, in aim 2, we will pursue both a candidate (imprinted genes) and an unbiased genome-wide approach to studying the sperm methylome using the latest comprehensive DNA methylation array technology. In addition, we will also leverage a prior predictive model using DNA methylation as a marker of paternal aging to assess if there is premature aging in the sperm from uRPL men. This work will identify epimutations in sperm that could play a mechanistic role in RPL. In aim 3, we will expand our epigenetic analysis to perform a genome-wide analysis of sperm histone patterns in uRPL men. Finally, we will combine our multi-omics datasets to compare and contrast the genomic loci affected. In summary, this project will contribute significantly to identifying novel male factor causes of RPL, which will ultimately lead to significant advancements in the diagnosis and treatment of couples with RPL.