Identifying Molecular Signatures of Genomic Imprinting Errors

识别基因组印记错误的分子特征

基本信息

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
项目类别：
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

项目摘要

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.

抽象的令人担忧的是，全球约有 5000 万对夫妇在五年无保护性行为后无法怀孕。医学上辅助生殖技术（ART）是不孕夫妇尝试的重要治疗方式怀孕。虽然普遍认为 ART 是安全的，但 ART 与早产发生率较高有关，宫内生长受限和低出生体重。而且，还有更多的Beckwith-Wiedemann、Silver- ART 人群中的 Russell、Angelman 和 Prader-Willi 综合征儿童携带印迹甲基化与一般人群相比的错误。由于 ART 的使用恰逢在配子和植入前发育过程中印记DNA甲基化编程，有可能 ART 会导致印记错误。对可育小鼠的研究支持了这一点。卵子发生过程中的超数排卵和/或植入前胚胎培养是两种几乎普遍的 ART 程序，可产生大量具有印迹甲基化错误的胚胎。迄今为止，人们对印记甲基化的调控知之甚少植入前发育期间的维护，此外，ART 如何导致这种破坏维护。该提案将解决这些知识差距。在目标 1 中，我们将检验以下假设：胚胎容易出现印记甲基化错误，因为 ART 会破坏重要的母体效应卵母细胞中的转录物是胚胎维持印记甲基化所需的。候选母亲- 将使用单细胞 RNA-seq 对对照和超排卵母细胞、1-细胞和 2- 细胞胚胎，代表卵母细胞到胚胎的转变时期。候选函数将是通过蛋白质降解方法测定。接下来，我们将确定母体效应是否改变极体中的转录本作为卵母细胞的代表，与印记甲基化错误相关，从而导致胚胎，最终导致鉴定出可预测印迹甲基化的转录生物标志物错误。目标 2 将使用药理学和方法来测试核导入在印记维护中的重要性。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 至 8 单元阶段代表快速发育的 ART 胚胎获得 DNA 甲基化时的易感性窗口错误，特别是在外部滋养层细胞中。我们将确定 ART 何时改变胚胎发育使用延时分析，然后比较慢速和快速发展中的 DNA 甲基化扰动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 何时甲基化错误会出现，其中 ART 胚胎谱系以及细胞会诱导滋养层命运。最后，我们将测试发育和形态动力学参数作为无创性参数的预测能力。具有印记甲基化错误分子特征的胚胎的程序。该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。