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

项目摘要/摘要：流产是最常见的妊娠并发症， 美国每年发生75万至100万例流产。高达5%的夫妇将 经历两次或两次以上流产，并有反复妊娠丢失(RPL)。RPL是一种重要的临床 问题是即使在诊断检查之后，50%的夫妇也找不到反复丢失的原因，并且 将被诊断为不明原因的RPL(URPL)。因此，调查RPL的新原因对于帮助 与RPL配对，以确定原因，从而确定可能的治疗方案。男性因素引起uRPL的原因 在临床上，对患有uRPL的男性进行的唯一诊断测试是核型 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 在我们对RPL的父性病因的了解中。多条证据表明，异常精子 通过印迹基因中高DNA片段化和异常DNA甲基化客观衡量质量 与uRPL相关。该项目将改变目前RPL夫妇的临床诊断范式 主要是以女性为中心的平衡方法，包括精子的分子分析。我们的项目将 对一大群uRPL夫妇的精子进行综合分析，以研究DNA片段 (目标1)、DNA甲基化(目标2)和组蛋白模式(目标3)。在目标1中，我们的实验将研究 人类精子DNA片段化增加通过以下途径导致RPL的分子机制 调查发生的DNA损伤的类型和受影响的基因组位点。我们的一项重大创新 方法是以独特的平行方式研究由高度DNA片段化引起的流产的基因组 精子以阐明潜在的缺陷。尽管精子基因组完整性是确保 健康怀孕的另一个方面是精子表观基因组，它在怀孕过程中也起着至关重要的作用 生存能力。先前的研究表明，患有uRPL的男性精子DNA印迹基因甲基化受到干扰，并且 已经观察到uRPL男性精子中组蛋白的保留率增加。建立在这些基础上 观察，在目标2中，我们将在全基因组范围内同时追求候选(印迹基因)和无偏倚 利用最新的DNA甲基化阵列技术研究精子甲基化组的方法。 此外，我们还将利用先前的预测模型，使用DNA甲基化作为父亲衰老的标志 以评估uRPL男性的精子是否存在过早老化。这项工作将确定表位突变在 可能在RPL中起机械作用的精子。在目标3中，我们将扩展我们的表观遗传学分析，以执行 URPL男性精子组蛋白模式的全基因组分析。最后，我们将结合我们的多组学 数据集，以比较和对比受影响的基因组座位。总而言之，这个项目将有助于 对于确定新的男性因素导致RPL具有重要意义，这最终将导致显著的 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.