MOLECULAR AND METABOLIC ASPECTS OF IMPLANTATION

描述（由申请人提供）：本研究将是首次尝试将多囊卵巢综合症女性中雌二醇和雄激素水平升高/黄体酮水平降低与流产与关注葡萄糖利用异常的机制联系起来。了解蜕膜化期间子宫间质中葡萄糖摄取和代谢的激素调节将有可能促进新型药物干预措施的开发，以提高该女性群体的成功妊娠率。 该提案分为三个具体目标。在目标 1 中，我们将研究高水平雌二醇/雄激素和低水平孕酮对小鼠子宫内膜基质细胞中葡萄糖转运蛋白表达、葡萄糖利用和蜕膜化的影响。我们还将在人类子宫内膜基质细胞中进行相同的研究。我们假设高 E2 和低 P4 将下调 mESC 和 hESC 中的 GLUT1 和葡萄糖摄取。在目标 2 中，我们将分析改变 GLUT1 表达以及改变葡萄糖代谢对这些细胞蜕膜化过程的影响。我们将使用 siRNA/shRNA 慢病毒敲低 GLUT1，进行葡萄糖通量研究以评估这些细胞中的葡萄糖利用率，然后抑制这些途径并评估 ESC 的蜕膜化。我们假设在蜕膜化过程中葡萄糖主要通过戊糖磷酸途径代谢；并且上游（GLUT1 表达）或下游（PPP 抑制）的抑制会对蜕膜化产生不利影响。我们将在小鼠和人类胚胎干细胞中测试这一假设。最后，在目标 3 中，我们将通过使用增加 DHEA 的小鼠模型，重点分析过量雌二醇/雄激素对子宫内膜基质中蜕膜化和葡萄糖利用的体内影响。我们将尝试通过暴露于体内和体外使用的 PPP 激活剂来纠正这些影响。我们还将使用 PCOS 患者或卵母细胞捐赠者的剩余样本来检查人类 ESC。我们预测：1) 暴露于 DHEA 的小鼠将出现葡萄糖利用异常，从而导致蜕膜化异常，幼仔数量减少；2) 通过 PPP 增加葡萄糖利用的药物将逆转蜕膜化异常；3) 来自这些患者群体的人类 ESC 将出现类似的葡萄糖利用异常。 公共健康相关性：患有多囊卵巢综合征 (PCOS) 以及其他雌二醇或雄激素升高的内分泌疾病的女性都会经历胚胎着床失败和子宫内膜功能障碍。人们普遍认为，这是由于雌激素/雄激素过多或孕激素缺乏造成的，这都是该患者群体的特征。我们希望，通过研究高雌激素/雄激素水平对子宫葡萄糖利用的影响，可能会发现针对多囊卵巢综合症患者着床失败和这些内分泌疾病的新治疗干预措施。

DESCRIPTION (provided by applicant): This study will be one of the first attempts to link the increased levels of estradiol and androgens/decreased levels of progesterone with miscarriages seen in women with PCOS to a mechanism which focuses on aberrant glucose utilization. Understanding the hormonal regulation of glucose uptake and metabolism in the uterine stroma during decidualization will potentially progress into the development of novel pharmacologic interventions to increase the rate of successful pregnancies in this population of women. This proposal is organized into three specific aims. In Aim 1, we will investigate the effects of high levels of estradiol/androgens and low levels of progesterone on expression of glucose transporters, glucose utilization and decidualization in murine endometrial stromal cells. We will also perform the same studies in human endometrial stromal cells. We hypothesize that high E2 and low P4 will downregulate GLUT1 and glucose uptake in mESCs and hESCs. In Aim 2, we will analyze the effects of changing GLUT1 expression as well as altering glucose metabolism on the process of decidualization of these cells. We will use siRNA/shRNA lentivirus to knockdown GLUT1, glucose flux studies to assess glucose utilization in these cells and then inhibit these pathways and assess decidualization in ESCs. We hypothesize that glucose is primarily metabolized via the pentose phosphate pathway during decidualization; and that inhibition either upstream (GLUT1 expression) or downstream (PPP inhibition) adversely affects decidualization. We will test this hypothesis in mouse and human ESCs. Finally, in Aim 3 we will focus on analyzing the in vivo effects of excess estradiol/androgen on decidualization and glucose utilization in the endometrial stroma by using a mouse model of increased DHEA. We will attempt to correct these effects by exposure to PPP activators used both in vivo and in vitro. We will also use leftover samples from patients with PCOS or oocyte donor patients and examine human ESCs. We predict that 1) DHEA exposed mice will have abnormal glucose utilization and thus abnormal decidualization with decreased numbers of pups, 2) the agents used to increase glucose utilization via the PPP will reverse decidualization abnormalities, and 3) human ESCs from these populations of patients will have similar abnormalities in glucose utilization. PUBLIC HEALTH RELEVANCE: Women with polycystic ovary syndrome (PCOS), as well as other endocrine disorders with elevated estradiol or androgens, all experience embryo implantation failure and endometrial dysfunction. The general belief is that this is due to the effects of excess estrogen/androgen or progesterone deficiency, both characteristic of this patient population. Our hope is that as a result of our studies examining the effects of high estrogen/androgen levels on glucose utilization in the uterus, new therapeutic interventions for implantation failure in patients with PCOS, and these endocrine disorders, may be discovered.