Identifying Molecular Signatures of Genomic Imprinting Errors

识别基因组印记错误的分子特征

• 批准号: 10595043
• 负责人: Mellissa Rae Wigle Mann
• 金额: $ 37.42万
• 依托单位: MAGEE-WOMEN'S RES INST AND FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10595043
• 关键词: Affect; Assisted Reproductive Technology; Biological; Biological Markers; Biopsy; Cells; Chemicals; Child; Clinic; Couples; DNA Methylation; Data; Defect; Development; Disease; Embryo; Embryonic Development; Embryonic Induction; Epigenetic Process; Fetal Growth Retardation; Frequencies; General Population; Genes; Genetic; Genomic Imprinting; Germ Cells; Human; Impairment; Importins; Incidence; Incubated; Infertility; Knowledge; Lead; Low Birth Weight Infant; Maintenance; Mammals; Mediating; Medical; Methods; Methylation; Modality; Molecular Profiling; Mus; Mutagenesis; North America; Nuclear Import; Oocytes; Oogenesis; Outcome; Pathway interactions; Population; Prader-Willi Syndrome; Pre-implantation Embryo Development; Predisposition; Premature Birth; Procedures; Proteins; Proxy; Publishing; Regulation; Reporter; Reporting; Research Proposals; Risk; Screening procedure; Silver; Silver-Russell syndrome; Superovulation; TRIM Gene; Testing; Transcript; Unsafe Sex; bisulfite; blastocyst; blastomere structure; clinical practice; embryo cell; embryo culture; imprint; improved; infertility treatment; nucleocytoplasmic transport; pharmacologic; predictive marker; preimplantation; protein degradation; single-cell RNA sequencing; transcriptome sequencing; trophoblast; trying to conceive; whole genome

Abstract Alarmingly, ~50 million couples worldwide are unable to conceive after 5 years of unprotected sex. Medically assisted reproductive technologies (ARTs) constitute important treatment modalities for infertile couples trying to conceive. While generally considered safe, ARTs are associated with higher incidences of preterm birth, intrauterine growth restriction, and low birth weight. Moreover, there are more Beckwith-Wiedemann, Silver- Russell, Angelman and Prader-Willi Syndrome children in the ART population harboring imprinted methylation errors compared to those in the general population. Since ART usage coincides with important stages of imprinted DNA methylation programming during gamete and preimplantation development, it is possible that ARTs lead to imprinting errors. Studies in fertile mice support this. Superovulation during oogenesis and/or embryo culture during preimplantation, two nearly universal ART procedures, lead to significant numbers of embryos with imprinted methylation errors. To date, little is known about the regulation of imprinted methylation maintenance during preimplantation development, and moreover, how ARTs lead to disruption in this maintenance. This proposal will address these knowledge gaps. In Aim 1, we will test the hypothesize that embryos are predisposed to imprinted methylation errors because ARTs disrupt crucial maternal-effect transcripts in oocytes that are required in embryos to maintain imprinted methylation. Candidate maternal- effect transcripts will be identified using single cell RNA-seq on control and superovulated oocyte, 1-cell and 2- cell embryos, representing the period of oocyte-to-embryo transition. Candidate function will then be determined though protein degradation methods. Next, we will determine whether altered maternal-effect transcripts in polar bodies, as a proxy for oocytes, correlates with imprinted methylation errors in resulting embryos, ultimately leading to identification of transcript biomarkers that are predictive of imprinted methylation errors. Aim 2 will test the importance of nuclear import in imprint maintenance using pharmacological and protein degradation methods, and whether it is disrupted by ARTs. In Aim 3, we hypothesize that the 4 to 8-cell stages represent a window of susceptibility when fast developing ART embryos acquire DNA methylation errors, particularly in outer, trophoblast cells. We will determine when embryo development is altered by ARTs using time-lapse analysis, and then compare DNA methylation perturbation in slow and fast developing embryos using whole genome bisulfite mutagenesis. This will distill a molecular signature of DNA methylation errors. Next, we will investigate this molecular signature during early development to determine when DNA methylation errors arise and in which lineages of ART embryos, as well as in cells induced to trophoblast fate. Finally, we will test the predictive power of developmental and morphokinetic parameters as a noninvasive procedure for embryos with a molecular signature of imprinted methylation errors. Results from this proposal will provide the biological basis for improvements to fertility treatments aimed at identifying at-risk embryos.

摘要 令人担忧的是，全世界约有5000万对夫妇在无保护措施的性行为5年后无法怀孕。医学上 辅助生殖技术（ART）是不孕夫妇尝试 怀孕虽然一般认为是安全的，但ART与早产的发生率较高有关， 宫内生长受限和低出生体重。此外，还有更多的贝克维-威德曼，银- ART人群中携带印迹甲基化的Russell、Angelman和Prader-Willi综合征儿童 与一般人群相比，这些错误。由于ART的使用与 在配子和植入前发育过程中，有可能 ART会导致印记错误。对可育小鼠的研究支持这一点。卵子发生期间的超数排卵和/或 植入前胚胎培养，两种几乎普遍的ART程序，导致大量的 有印迹甲基化错误的胚胎。到目前为止，对印迹甲基化的调控知之甚少 在胚胎植入前发育过程中的维持，此外，ART如何导致这一过程的中断， 上维护本提案将弥补这些知识差距。在目标1中，我们将测试假设， 胚胎易于发生印迹甲基化错误，因为ART破坏了关键的母体效应， 卵母细胞中的转录本，这些转录本是胚胎中维持印迹甲基化所必需的。候选人母亲- 将使用单细胞RNA-seq在对照和超排卵的卵母细胞、1-细胞和2-细胞上鉴定有效转录物。 细胞胚胎，代表卵母细胞向胚胎过渡的时期。候选函数将是 通过蛋白质降解方法测定。接下来，我们将确定是否改变母体效应 作为卵母细胞的替代物，极体中的转录物与印迹甲基化错误相关， 胚胎，最终导致识别预测印迹甲基化的转录生物标志物 错误.目的2将测试核输入的重要性，在印记维护使用药理学和 蛋白质降解方法，以及它是否被ART破坏。在目标3中，我们假设4到8个细胞 当快速发育的ART胚胎获得DNA甲基化时， 错误，特别是在外部，滋养层细胞。我们将确定胚胎发育何时被ART改变 利用延时分析，比较了DNA甲基化扰动在慢速和快速发展过程中的变化 使用全基因组亚硫酸氢盐诱变胚胎。这将提取DNA甲基化的分子特征 错误.接下来，我们将在早期发育过程中研究这种分子特征，以确定DNA何时 甲基化错误的出现和ART胚胎的谱系，以及在细胞中诱导滋养层的命运。 最后，我们将测试发育和形态动力学参数作为非侵入性指标的预测能力。 程序的胚胎与印迹甲基化错误的分子签名。本提案的结果 将为改进旨在识别风险胚胎的生育治疗提供生物学基础。