Deciphering molecular mechanisms controlling age-associated uterine adaptabilityto pregnancy

项目摘要 高龄产妇（即，≥ 35岁）被认为是出生缺陷的主要危险因素。在40岁以上的女性中 岁时，自然流产的发生率可增加至> 30%。许多注意力都集中在 卵巢功能和卵母细胞的质量，但我们提供的证据表明，子宫蜕膜化缺陷可能是一个 年龄相关性生殖衰退的主要原因。这个问题可能是由于钝孕激素（P4） 衰老子宫的反应性，通过其同源核受体PGR。PGR是 妊娠的建立和维持;然而，PGR的显著降低导致 随着子宫年龄的增长，荷尔蒙的反应。减少PGR表达的潜在分子机制 和PGR靶基因的失调可能是子宫衰老的原因，这是一种仍然难以捉摸的机制。我们 最近发现子宫Sirtuin 1（SIRT 1）是年龄相关性PGR作用的关键驱动因素， 基质细胞蜕膜化影响我们对子宫生物学和生殖衰老的认识。删除子宫 Sirt1基因在小鼠体内的表达，我们建立了一个用于研究子宫早衰的遗传模型（PgrCre/+ Sirt1f/f，即Sirt1d/d 这是由于与生理老化相关的PGR反应迟钝。因此，本研究将 首次尝试使用生理（46 - 54周龄）和遗传（Sirt1缺陷）衰老小鼠模型 发现哺乳动物生殖衰老的新子宫视角。该提案将首次提供 在子宫过早老化的背景下着床和蜕膜化的分子特征（遗传学） 老化; Sirt1d/d）通过多合一多模式单细胞ATAC-seq/RNA-seq和与 生理老化，确定共同的基因签名，顺式调控元件和转录辅因子， 子宫内膜细胞是建立和维持妊娠所必需的，但易受生殖衰老的影响。 我们还将使用hTERT永生化的人子宫内膜将小鼠中的发现转化为人类生物学 间质细胞（T-HESC）作为间质细胞的蜕膜化是生理和遗传过程中的主要缺陷， 衰老结合人类和小鼠模型之间的多模式单细胞数据集，以及功能性 通过CRISPR缺失和激活验证候选顺式调控元件和转录辅因子 本提案将描述PGR行动所需的SIRT1响应监管网络 随着子宫年龄的增长而失调。我们坚信，我们的研究将为我们提供一个更深入、更全面的 深入了解生殖衰老过程中对孕酮耐药的子宫内膜。有了这些新知识，研究 可以采取策略来抵消衰老对怀孕结果的不利影响。

PROJECT SUMMARY Advanced maternal age (i.e., ≥35 years old) is considered a major risk factor for birth defects. In women over 40 years of age, the incidence of spontaneous abortion can increase to >30%. Much attention has been focused on ovarian function and oocyte quality, but we provide evidence that uterine decidualization defects could be a major cause of age-related reproductive decline. This problem is likely due to a blunted progesterone (P4) responsiveness of the aging uterus, via its cognate nuclear receptor PGR. PGR is the master regulator for the establishment and maintenance of pregnancy; however, a significant diminution in PGR results in a blunted hormonal response as the uterus ages. The underlying molecular mechanisms that diminish expression of PGR and deregulate PGR target genes likely account for uterine aging, a mechanism that has remained elusive. Our recent discovery of uterine Sirtuin 1 (SIRT1) as a critical driver of age-related PGR action by which endometrial stromal cells decidualize impacts our knowledge of uterine biology and reproductive aging. By deleting uterine SIRT1 in mice, we generated a genetic model (PgrCre/+Sirt1f/f, i.e. Sirt1d/d) for research on premature uterine aging due to blunted PGR response that are similar to those associated with physiologic aging. Thus, this study will be the first attempt to use physiological (46-54 weeks of age) and genetic (Sirt1-deficient) aging mouse models to discover a novel uterine perspective of mammalian reproductive aging. This proposal will provide the first molecular characterization of implantation and decidualization in the context of premature uterine aging (genetic aging; Sirt1d/d) by all-in-one multimodal single-cell ATAC-seq/RNA-seq and comparative analyses with physiologic aging, identifying common gene signatures, cis-regulatory elements and transcriptional co-factors in endometrial cells required for establishment and maintenance of pregnancy but susceptible to reproductive aging. We will also translate the findings in mice to human biology using hTERT-immortalized human endometrial stromal cells (T-HESCs) as decidualization of stromal cells are major defects during physiological and genetic aging. Combined with multimodal single cell datasets between human and mouse models, as well as functional validation of candidate cis-regulatory elements and transcriptional co-factors by CRISPR deletion and activation systems, respectively, this proposal will delineate SIRT1-responsive regulatory network required for PGR actions that deregulate as uterus ages. We strongly believe that our study will give a deeper and more comprehensive insight into progesterone-resistant endometrium during reproductive aging. With that new knowledge, research can pursue strategies to counteract adverse effects of aging on outcomes of pregnancy.