Identifying Molecular Signatures of Genomic Imprinting Errors

摘要 令人震惊的是，全球约有5000万对夫妇在经过5年无保护措施的性行为后无法怀孕。医学上的 辅助生殖技术(ARTS)是不育夫妇的重要治疗方式。 怀孕。虽然通常被认为是安全的，但抗逆转录病毒治疗与早产的高发生率有关， 胎儿宫内发育受限，出生体重偏低。此外，还有更多的贝克维斯-威德曼，西尔弗- ART人群中携带印记甲基化的Russell、Angelman和Prader-Willi综合征儿童 与普通人群中的误差相比。因为ART的使用恰好与 在配子和植入前发育过程中的印迹DNA甲基化编程，有可能 艺术会导致印记错误。对可育小鼠的研究支持这一观点。卵子发生和/或超数排卵 植入前的胚胎培养是两种几乎普遍的ART程序，导致大量的 带有印记甲基化错误的胚胎。到目前为止，对印迹甲基化的调节知之甚少。 在植入前发育期间的维持，此外，ART如何导致这一过程的中断 维修。这项提议将解决这些知识差距。在目标1中，我们将检验假设 胚胎容易发生印记甲基化错误，因为ART破坏了至关重要的母体效应 胚胎维持印迹甲基化所需的卵母细胞中的转录本。候选人母亲- 将使用单细胞rna-seq对对照和超排卵母细胞、1-细胞和2-细胞进行鉴定。 细胞胚胎，代表卵母细胞向胚胎过渡的时期。候选函数随后将为 通过蛋白质降解法测定。接下来，我们将确定改变的母性效应是否 极体中的转录本，作为卵母细胞的替代物，与导致 胚胎，最终导致识别预测印记甲基化的转录生物标记物 错误。目标2将测试核进口在印记维护中的重要性，使用药理学和 蛋白质的降解方法，以及它是否被ARTS破坏。在目标3中，我们假设4到8个细胞 阶段代表了快速发育的ART胚胎获得DNA甲基化的易感性窗口 错误，特别是在外层滋养层细胞。我们将确定胚胎发育何时会被ARTS改变 使用时移分析，然后比较缓慢和快速发展中的DNA甲基化扰动 使用全基因组亚硫酸氢盐诱变的胚胎。这将提炼出DNA甲基化的分子特征 错误。接下来，我们将研究早期发育过程中的这种分子特征，以确定DNA何时 甲基化错误出现，并在其中ART胚胎的谱系，以及在细胞中诱导滋养层命运。 最后，我们将测试发育和形态动力学参数作为非侵入性的预测能力 具有印记甲基化错误的分子签名的胚胎的程序。这项提案的结果 将为改进旨在识别高危胚胎的生育治疗提供生物学基础。

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.