MOLECULAR AND METABOLIC ASPECTS OF IMPLANTATION

描述（由申请人提供）：本研究将首次尝试将雌二醇和雄激素水平升高/黄体酮水平降低与多囊卵巢综合征妇女流产联系起来，并将其与异常葡萄糖利用机制联系起来。了解子宫间质在脱个体化过程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的发展，以提高这类妇女的成功怀孕率。这项建议分为三个具体目标。在Aim 1中，我们将研究高水平雌二醇/雄激素和低水平黄体酮对小鼠子宫内膜基质细胞中葡萄糖转运蛋白表达、葡萄糖利用和去细胞化的影响。我们还将在人类子宫内膜基质细胞中进行同样的研究。我们假设高E2和低P4会下调mESCs和hESCs的GLUT1和葡萄糖摄取。在Aim 2中，我们将分析改变GLUT1表达和改变葡萄糖代谢对这些细胞脱个体化过程的影响。我们将使用siRNA/shRNA慢病毒敲除GLUT1，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中的脱细胞化。我们假设葡萄糖在脱个体化过程中主要通过戊糖磷酸途径代谢；上游抑制（GLUT1表达）或下游抑制（PPP抑制）都会对去个体化产生不利影响。我们将在小鼠和人类ESCs中验证这一假设。最后，在Aim 3中，我们将通过DHEA升高的小鼠模型，重点分析过量雌二醇/雄激素对子宫内膜间质去个体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。我们还将使用来自多囊卵巢综合征患者或卵母细胞供体患者的剩余样本来检测人类ESCs。我们预测1)暴露于脱氢表雄酮的小鼠将出现异常的葡萄糖利用，从而导致异常脱糖化，幼崽数量减少；2)通过PPP增加葡萄糖利用的药物将逆转脱糖化异常；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。

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.