Hypoxia-inducible factor-1alpha expression in human endometrium and its regulation by prostaglandin E-series prostanoid receptor 2 (EP2).

The menstrual cycle is a complex interaction of sex steroids, prostanoids, and cytokines that lead to coordinated tissue degradation, regeneration and repair. The transcription factor hypoxia-inducible factor (HIF-1) plays critical roles in cellular responses to hypoxia, the generation of an inflammatory response and vasculogenesis through transcriptional activation of angiogenic genes. We hypothesize that HIF-1 is expressed in human endometrium and that locally synthesized prostaglandins (PGE2 and PGF(2alpha)) regulate HIF-1 activity. Here we demonstrate that PGE2 up-regulates HIF-1alpha mRNA and protein via the E-series prostanoid receptor 2 (EP2), and this up-regulation is dependent on epidermal growth factor receptor kinase activity. We show the tight temporal-spatial confinement of HIF-1alpha protein expression in endometrium across the cycle. HIF-1alpha is expressed exclusively during the secretory and menstrual phases. Protein expression is maximal at progesterone withdrawal during the late secretory and menstrual phase. HIF-1alpha protein colocalizes with prostaglandin EP2 receptor in glandular cells. In contrast, HIF-1beta/aryl receptor nuclear translocator 1 expression occurs throughout the cycle but is maximal in glandular cells during the proliferative phase. This provides evidence for a role for HIF-1 in the menstrual cycle and demonstrates that HIF-1 activation in human endometrium may occur via a PGE2-regulated pathway and provides a coordinated pathway from progesterone withdrawal through to angiogenic gene expression via HIF-1.

11Beta-hydroxysteroid dehydrogenases in human endometrium.

Key reproductive events, such as menstruation and implantation, are considered to be inflammatory processes and glucocorticoids act as anti-inflammatory agents. The balance of expression of types 1 and 2 11beta-hydroxysteroid dehydrogenases (11betaHSD) controls the availability of cortisol to bind to the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Expression profiles of glucocorticoid-metabolising enzymes and their cognate receptors have been characterized in the reproductive tract. We propose that factors that peripherally promote glucocorticoid action are part of an anti-inflammatory response to tissue remodelling in human endometrium. Protein and mRNA expression in endometrium were investigated using immunohistochemistry and quantitative real-time PCR. There was up-regulated expression of 11betaHSD-1 at menstruation and in first trimester decidua. 11BetaHSD-2 and GR were expressed across the cycle. The MR expression pattern across the cycle and in decidua implies progesterone may also play a regulatory role. The precise roles and interactions of these proteins require further investigation.

Gene expression profiling of mid to late secretory phase endometrial biopsies from women with menstrual complaint.

OBJECTIVE: The purpose of this study was to test whether a quantitative high-throughput molecular screen can be used to probe human endometrium and initiate the development of molecular diagnostic tools with potential for identification of therapeutic targets in women with menstrual complaints. STUDY DESIGN: Endometrium was collected from 10 patients with complaint of heavy bleeding, classified into mid or late secretory phase of the menstrual cycle by histologic dating and serum progesterone concentration. Total RNA was extracted and gene activity assessed using high-density oligonucleotide arrays. RESULTS: Statistical testing identified 83 'signature' genes whose expression levels differentiated the mid and late secretory phases of the menstrual cycle. CONCLUSION: The results show that the endometrium, a complex heterogeneous tissue, is amenable to high-throughput molecular analyses and this work provides further support for the future application of molecular profiling to clinical diagnosis.

Leukocyte populations and steroid receptor expression in human first-trimester decidua; regulation by antiprogestin and prostaglandin E analog.

CONTEXT: Progesterone acting via its cognate receptor is critical to maintaining a viable endometrial environment for implantation and pregnancy. During medical termination of pregnancy, the biological effect of progesterone is pharmacologically withdrawn and prostaglandins administered exogenously. Leukocytes within the uterus are the effector cells of an inflammatory response and play important roles in both tissue breakdown and remodeling. OBJECTIVE: The aim of this study was to identify the separate and combined effects of the antiprogestin Mifepristone (single dose, 200 mg) and the prostaglandin E (PGE) analog (gemeprost) on leukocyte populations and steroid receptor expression in human first-trimester decidua. PATIENTS: Eighty women were recruited from the termination of pregnancy service with a gestational age of between 35 and 65 d at the time of surgical termination of pregnancy. MAIN OUTCOME MEASURES: Immunohistochemistry was used to measure macrophage (CD68 +ve), neutrophil (neutrophil elastase +ve), and uterine natural killer cell (CD56 +ve) populations and progesterone (PR(A) and PR(B)), estrogen (ERalpha and ERbeta), and androgen receptor (AR) expression. RESULTS: After administration of both antiprogestin and the PGE analog, macrophage and neutrophil numbers were significantly increased, whereas natural killer cell numbers were unchanged. Antiprogestin and PGE analog coadministration also significantly decreased PR and ERalpha immunoreactivity but had no effect on androgen receptor or ERbeta receptor expression. PGE analog alone was also capable of reducing PR expression. CONCLUSIONS: In this study, we demonstrate that the inflammatory response induced by antiprogestin in combination with PGE analog is accompanied by both increases in macrophages and neutrophils numbers and decreases in PR and ERalpha expression in human first-trimester decidua.

Mifepristone-induced vaginal bleeding is associated with increased immunostaining for cyclooxygenase-2 and decrease in prostaglandin dehydrogenase in luteal phase endometrium.

The mechanism of mifepristone-induced vaginal bleeding and endometrial shedding was investigated in 13 women who took 200 mg mifepristone in the midluteal phase on d 8 after the onset of the urinary LH surge (LH+8). Endometrial biopsies were collected, 6-24 h after mifepristone (group 1, n = 7) or 36-48 h after mifepristone (group 2, n = 6), and compared with those from a control group in the midluteal phase (n = 7). All women reported vaginal bleeding commencing 36-48 h after taking mifepristone. Treatment with mifepristone significantly reduced serum progesterone levels in all women, when compared with the controls (13.2 nM vs. 34.8 nM, P = 0.001). After mifepristone, a significant increase in cyclooxygenase-2 immunoreactivity was apparent at 36-48 h (P = 0.0018), whereas prostaglandin 15 dehydrogenase enzyme-positive immunostaining declined, to be virtually absent by 36-48 h in both glands and in stroma (P < 0.05). There was no change in intensity or distribution of staining for steroid receptors after mifepristone. The changes in immunostaining for cyclooxygenase-2 and prostaglandin 15 dehydrogenase strongly support the hypothesis that an increase in the local concentration of prostaglandins in the endometrium is involved in the mechanism of bleeding induced by mifepristone in the luteal phase.

Wild-type estrogen receptor (ERbeta1) and the splice variant (ERbetacx/beta2) are both expressed within the human endometrium throughout the normal menstrual cycle.

Estrogen action is mediated via two subtypes of the estrogen receptor (ER), usually referred to as ERalpha and ERbeta. We have previously compared the spatial and temporal expressions of ERalpha and ERbeta proteins in human endometrium and reported that endothelial cells exclusively express ERbeta. In the present study we have extended our investigations to compare the pattern of expression of wild-type (ERbeta1) and a newly identified ERbeta variant isoform (ERbetacx/beta2) that lacks the ability to bind steroids. mRNAs encoding both ERbeta1 and ERbetacx/beta2 receptors were identified in human endometrial extracts by RT-PCR. Quantitative TaqMan R-TPCR demonstrated that levels of total mRNAs were increased significantly premenstrually as circulating progesterone levels declined. ERbeta1 and ERbetacx/beta2 proteins were identified within multiple cell types within the endometrium using isotype-specific monoclonal antibodies; immunoexpression of ERbetacx/beta2 appeared less intense than that of ERbeta1 in endometrial glandular epithelium and endothelial cells. Immunoexpression of ERbeta1 appeared unchanged throughout the menstrual cycle. In contrast, levels of ERbetacx/beta2-specific immunoreactivity were specifically reduced in gland cells within the functional layer, but not in those of the basal layer, in the midsecretory phase. It is possible that coexpression of ERbetacx/beta2 in cells containing ERbeta1 and/or ERalpha may modulate the effects of estrogens on the endometrium.

Steroid receptor expression in uterine natural killer cells.

The endometrium contains a unique subset of uterine-specific natural killer (uNK) cells, the proposed functions of which include a role in decidualization, menstruation, and implantation. These cells increase in number during the mid-late secretory phase of the menstrual cycle and are also present in large numbers in early pregnancy. The cyclical nature of uNK cell appearance suggests hormonal regulation of these cells. To date, it has not been possible to localize either estrogen receptors (ERs) or progesterone receptors (PRs) to uNK cells. In the present study, we have investigated the steroid receptor expression of uNK cells, including not only ER alpha and PR but also wild-type ER beta 1, its variant form ER beta cx/beta 2, and glucocorticoid receptor (GR) using specific monoclonal antibodies and real-time quantitative RT-PCR. mRNA encoding ER alpha, PR, ER beta cx/beta 2, ER beta 1, and GR were identified in extracts of human endometrium across the menstrual cycle and in decidua. Quantitative real-time RT-PCR demonstrated an absence of ER alpha and PR mRNA in purified uNK cells. In contrast, mRNA for ER beta cx/beta 2, ER beta 1, and GR was present in uNK cells. ER alpha, PR, ER beta cx/beta 2, ER beta 1, and GR proteins were identified in endometrial and decidual biopsies. Colocalization using specific monoclonal antibodies confirmed that uNK cells were immunonegative for ER alpha and PR protein. These cells were also immunonegative for ER beta cx/beta 2 but did express ER beta 1 and GR proteins. These results raise the possibility that estrogens and glucocorticoids could be acting directly on uNK cells through ER beta and GR, respectively, to influence gene transcription in the endometrium and decidua.

Local levonorgestrel regulation of androgen receptor and 17beta-hydroxysteroid dehydrogenase type 2 expression in human endometrium.

BACKGROUND: The levonorgestrel-releasing intrauterine system (LNG-IUS) is a highly effective contraceptive. However, unscheduled breakthrough bleeding (BTB), leads to discontinuation in a proportion of users. The LNG-IUS down-regulates endometrial progesterone and estrogen receptors and this may play a role in the mechanism responsible for BTB. LNG is an androgenic progestogen and so we examined the regulation of the androgen receptor (AR) in endometrium exposed to intrauterine LNG. Furthermore, as the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2) regulates intracellular levels of estrogens, progestins and androgens, we evaluated the changes in expression of 17betaHSD2 in the same tissue endometrial samples. METHODS: Immunohistochemistry and real time quantitative RT-PCR were used to compare protein and mRNA expression of AR and 17betaHSD2 in endometrial biopsies from women with normal menstrual cycles and those using a LNG-IUS. RESULTS: Immunohistochemistry showed that AR and 17betaHSD2, which were immunolocalized to the stroma and glands of endometrium respectively, were both suppressed by LNG-IUS treatment, though moderate staining of 17betaHSD2 was evident 1 month after insertion of the LNG-IUS. AR mRNA expression was down-regulated in LNG-exposed endometrium when compared with the proliferative phase of the menstrual cycle. 17betaHSD2 mRNA was significantly increased 3 months (but not 6-12 months) after LNG-IUS insertion. CONCLUSIONS: Endometrial intracellular estradiol levels would have been suppressed by 17betaHSD2 during the first few, but not the later, months of LNG-IUS action, and the lowered endometrial estradiol level may contribute to the frequent BTB evident in the early months of LNG-IUS use. The subsequent decline in 17betaHSD2 would lead to elevated local intracellular estradiol in the later months, when the BTB tends to subside. The suppression of AR by the LNG-IUS may also play a role in BTB, as elevated AR has been associated with amenorrhoea.