MOLECULAR AND METABOLIC ASPECTS OF IMPLANTATION

描述(由申请人提供)：这项研究将是首次尝试将多囊卵巢综合征患者流产与雌二醇和雄激素水平升高/孕酮水平下降联系起来，与一种专注于葡萄糖利用异常的机制有关。了解蜕膜化过程中子宫间质葡萄糖摄取和代谢的激素调节，可能会促进开发新的药物干预措施，以提高这一妇女群体的成功妊娠率。这项提议被组织成三个具体目标。目的1研究高水平雌雄激素和低水平孕酮对小鼠子宫内膜间质细胞葡萄糖转运蛋白表达、葡萄糖利用和蜕膜形成的影响。我们还将在人类子宫内膜间质细胞中进行同样的研究。我们假设高E2和低P4将下调mESCs和hESCs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形成过程的影响。我们将使用siRNA/shRNA慢病毒来敲除GLUT1，通过葡萄糖通量研究来评估这些细胞中的葡萄糖利用，然后抑制这些途径，并评估ESCs的蜕膜形成。我们假设，在蜕膜化过程中，葡萄糖主要通过磷酸戊糖途径代谢；抑制上游(GLUT1表达)或下游(PPP抑制)都会对蜕膜化产生不利影响。我们将在老鼠和人类的胚胎干细胞中测试这一假设。最后，在目标3中，我们将利用DHEA增加的小鼠模型，重点分析体内过量雌激素/雄激素对子宫内膜间质蜕膜形成和葡萄糖利用的影响。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。我们还将使用来自多囊卵巢综合征患者或卵子捐赠者患者的剩余样本，并检测人类胚胎干细胞。我们预测，1)DHEA暴露的小鼠将出现葡萄糖利用异常，从而导致蜕膜化异常，幼鼠数量减少；2)通过PPP增加葡萄糖利用的药物将逆转蜕膜化异常；以及3)来自这些患者群体的人类ESCs将出现类似的葡萄糖利用异常。 公共卫生相关性：患有多囊卵巢综合征(PCOS)的妇女，以及其他雌激素或雄激素升高的内分泌疾病，都会经历胚胎植入失败和子宫内膜功能障碍。普遍认为，这是由于雌激素/雄激素过多或黄体酮缺乏的影响，这两种情况都是这类患者的特征。我们希望，作为我们研究高雌激素/雄激素水平对子宫内葡萄糖利用的影响的结果，可能会发现针对多囊卵巢综合征患者植入失败和这些内分泌紊乱的新的治疗措施。

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.