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Objective: To determine the effect of inhibiting aromatase activity on endometrial lesion growth and aromatase expression in a baboon model of induced endometriosis., Design: Prospective study., Setting: Primate research institute., Animal(s): Sixteen olive baboons., Intervention(s): Sixteen olive baboons with induced endometriosis were examined with laparoscopy 10 months after disease inoculation. Animals in group 1 (n = 10) were treated with 1.25 mg/d of the aromatase inhibitor (AI) letrozole, and animals in group 2 (n = 6) were given a placebo for a total of 6 months., Main Outcome Measure(s): Total number of endometriotic lesions, morphology, and volume of lesions, as well as semiquantitative reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction for levels of aromatase cytochrome messenger RNA were measured. Ovarian volumes were evaluated before treatment initiation and every 2 months during the study., Result(s): Treatment of group 1 animals with an AI significantly decreased lesion volume from baseline measurements, whereas the placebo-treated animals showed an increase in lesion volume. Aromatase messenger RNA levels in lesions in the AI-treated animals were significantly lower compared with the placebo-treated animals. Ovarian volumes were significantly increased at 6 months of AI treatment compared with pretreatment volumes., Conclusion(s): These findings suggest that suppression of aromatase cytochrome P450 may inhibit the in vivo growth of endometriotic lesions in baboons., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Endometriosis is a common and chronic disease characterized by persistent pelvic pain and infertility. Estradiol is essential for growth and inflammation in endometriotic tissue. The complete cascade of steroidogenic proteins/enzymes including aromatase is present in endometriosis leading to de novo estradiol synthesis. PGE(2) induces the expression of the genes that encode these enzymes. Upon PGE(2) treatment, coordinate recruitment of the nuclear receptor SF-1 to the promoters of these steroidogenic genes is the key event for estradiol synthesis. SF-1 is the key factor determining that an endometriotic cell will respond to PGE(2) by increased estradiol formation. The presence of SF-1 in endometriosis and its absence in endometrium is determined primarily by the methylation of its promoter. The key steroidogenic enzyme in endometriosis is aromatase encoded by a single gene because its inhibition blocks all estradiol biosynthesis. Aromatase inhibitors diminish endometriotic implants and associated pain refractory to existing treatments in affected women.

Context: Products of at least five specific steroidogenic genes, including steroidogenic acute regulatory protein (StAR), which facilitates the entry of cytosolic cholesterol into the mitochondrion, side chain cleavage P450 enzyme, 3beta-hydroxysteroid-dehydrogenase-2, 17-hydroxylase/17-20-lyase, and aromatase, which catalyzes the final step, are necessary for the conversion of cholesterol to estrogen. Expression and biological activity of StAR and aromatase were previously demonstrated in endometriosis but not in normal endometrium. Prostaglandin E2 (PGE2) induces aromatase expression via the transcriptional factor steroidogenic factor-1 (SF1) in endometriosis, which is opposed by chicken-ovalbumin upstream-transcription factor (COUP-TF) and Wilms' tumor-1 (WT1) in endometrium., Objective: The aim of the study was to demonstrate a complete steroidogenic pathway leading to estrogen biosynthesis in endometriotic cells and the transcriptional mechanisms that regulate basal and PGE2-stimulated estrogen production in endometriotic cells and endometrium., Results: Compared with normal endometrial tissues, mRNA levels of StAR, side chain cleavage P450, 3beta-hydroxysteroid-dehydrogenase-2, 17-hydroxylase/17-20-lyase, aromatase, and SF1 were significantly higher in endometriotic tissues. PGE2 induced the expression of all steroidogenic genes; production of progesterone, estrone, and estradiol; and StAR promoter activity in endometriotic cells. Overexpression of SF1 induced, whereas COUP-TFII or WT1 suppressed, StAR promoter activity. PGE2 induced coordinate binding of SF1 to StAR and aromatase promoters but decreased COUP-TFII binding in endometriotic cells. COUP-TFII or WT1 binding to both promoters was significantly higher in endometrial compared with endometriotic cells., Conclusion: Endometriotic cells contain the full complement of steroidogenic genes for de novo synthesis of estradiol from cholesterol, which is stimulated by PGE2 via enhanced binding of SF1 to promoters of StAR and aromatase genes in a synchronous fashion.

Background: The objective of this study is to investigate the effect of pyrrolidine dithiocarbamate [PDTC; a nuclear factor-kappaB (NF-kappaB) inhibitor] and bortezomib (Velcade; a proteasome inhibitor) on the development of experimental endometriotic implants in rats., Methods: Endometriosis was surgically induced in 30 rats using the method of Vernon and Wilson. Three weeks later the viability and volume of the implants were recorded and classified. Afterwards, rats were put into three groups with equal numbers. The groups were labelled as the control, the PDTC and the bortezomib groups. Seven days after treatment, a third laparotomy was done and the volume of implants was measured again. The animals were then sacrificed, and the implants were stained with Ki67, proliferating cell nuclear antigen (PCNA), CD34, CD31 and Masson's trichrome histochemical staining., Results: In 80% of the implanted rats, vesicles at the suture region were observed, and the rats graded according to average vesicle diameter (D) as: Grade 1 (no vesicle, 20% of rats), Grade 2 (D < 2 mm, 33.3% of rats), Grade 3 (2 mm 4.5 mm, 26.7% of rats) and Grade 4 (D > 4.5 mm, 20% of rats). After treatment with PDTC or bortezomib, these percentages were decreased for Grades 3 and 4, and increased in Grade 1. The post-treatment implant volumes were decreased in the PDTC and bortezomib groups (P < 0.002 and P < 0.001), and slightly increased in the control group (P = 0.279). In the PDTC and bortezomib groups, CD34, CD31, PCNA and Ki67 expression levels were similar but were significantly reduced compared with the control group., Conclusions: PDTC and bortezomib may represent a novel therapeutic strategy for treatment of endometriosis.

Endometrial tissue from uterine disease-free women does not exhibit aromatase activity. In contrast, aromatase enzyme activity and mRNA levels are readily detectable in endometriosis. PGE2 stimulates both aromatase expression and activity in endometriotic stromal cells via promoter II region of the aromatase gene. This results in local production of estradiol, which induces PGE2 formation and establishes a positive feedback cycle. This mechanism seems to contribute to continuous production of estradiol and PGE2. Aromatase mRNA levels and enzyme activity are also present in uterine leiomyomata that are estrogen-dependent benign tumors of the myometrium. Successful treatment of endometriosis and uterine leiomyomata using aromatase inhibitors by recent pilot trials underscores the clinical significance of these molecular studies.

Objective: To determine the role of P and its nuclear receptor PR in the growth of ectopic uterine tissue of mice with or without a disrupted PR gene., Design: Animal study., Setting: Academic medical center., Animal(s): Female wild-type (WT) and transgenic knockout mice for P receptor (PRKO)., Intervention(s): Endometriosis was induced in the following groups of ovariectomized adult mice: [1] untreated WT, [2] estradiol (E(2))-treated WT, [3] P-treated WT, [4] E(2) + P-treated WT, [5] untreated PRKO, [6] E(2)-treated PRKO, and [7] E(2) + P-treated PRKO (n = 5 in each group)., Main Outcome Measure(s): The size of ectopic uterine tissue in WT and PRKO mice were compared between the groups subjected to treatments with P or E(2). Tissue proliferating cell nuclear antigen (PCNA) levels were compared among these groups., Results: Treatment with P only significantly decreased the size of WT ectopic uterine tissue. The untreated PRKO ectopic uterine tissue was significantly larger than WT tissue. Estradiol increased the size of ectopic uterine tissues, and this E(2)-dependent growth could be suppressed by P in WT tissues but not in PRKO tissues. Finally, the hormone-dependent changes in ectopic uterine tissue size were accompanied by comparable alterations in PCNA levels., Conclusion(s): Intact PR in ectopic uterine tissue is essential to abolish E(2)-dependent or -independent proliferation. We also suggest that ectopic uterine tissue is associated with significantly increased resistance to P action and increased predisposition to E(2)-dependent proliferation in the absence of PR. Overall, these findings support the hypothesis that P resistance in human endometriosis may be due to the absence of sufficient levels of functional PR in this tissue.

Objective: To determine the role of an aromatase inhibitor, letrozole, in the treatment of reproductive-age women with endometriosis and associated chronic pelvic pain., Design: Phase 2, open-label, nonrandomized proof-of-concept study., Setting: Outpatient tertiary-care center., Patient(s): Ten patients with endometriosis, all previously treated both medically and surgically, with unsatisfactory results., Intervention(s): Endometriosis was diagnosed by biopsy and scored from an initial diagnostic laparoscopy performed within 1 month before treatment was begun. Oral administration of letrozole (2.5 mg), the progestin norethindrone acetate (2.5 mg), calcium citrate (1,250 mg), and vitamin D (800 IU) was done daily for 6 months. Within 1-2 months after completion of the treatment, a second-look laparoscopy was performed to score and biopsy endometriosis., Main Outcome Measure(s): Changes in American Society for Reproductive Medicine (ASRM) scores for endometriosis, pelvic pain assessed by visual analog scale, serum hormone levels (FSH, LH, E(2), and estrone [E(1)]), and bone density (DEXA scan)., Result(s): No histologically demonstrable endometriosis was present in any patient during the second-look laparoscopy. ASRM and pelvic pain scores decreased significantly in response to treatment. Overall, no significant change in bone density was detected. Gonadotropin levels were not significantly altered by treatment, and although circulating E(2) and E(1) levels were reduced, the decrease was not statistically significant., Conclusion(s): The combination of letrozole and norethindrone acetate achieved marked reduction of laparoscopically visible and histologically confirmed endometriosis in all 10 patients and significant pain relief in nine out of 10 patients who had not responded previously to currently available treatments. On this basis, letrozole should be a candidate for the medical management of endometriosis.

Aromatase p450 (p450arom) is the key enzyme for biosynthesis of estrogen, which is an essential hormone for the establishment and growth of endometriosis. There is no detectable aromatase enzyme activity in normal endometrium; therefore, estrogen is not locally produced in endometrium. Endometriosis tissue, however, contains very high levels of aromatase enzyme, which leads to production of significant quantities of estrogen. Moreover, one of the best-known mediators of inflammation and pain, prostaglandin E (2), strikingly induces aromatase enzyme activity and formation of local estrogen in this tissue. Additionally, estrogen itself stimulates cyclo-oxygenase-2 and therefore increases the formation of prostaglandin E (2) in endometriosis. We were able to target this positive feedback cycle in endometriosis using aromatase inhibitors. In fact, pilot trials showed that aromatase inhibitors could decrease pelvic pain associated with endometriosis.

Estrogen is an extremely potent mitogen for endometrium and endometriosis. Progesterone, on the other hand, inhibits the mitogenic action of estrogen on endometrium and enhances differentiation. These antiproliferative and differentiative effects of progesterone are less pronounced on endometriosis tissue compared with endometrium. Thus, endometriosis is, at least in part, resistant to progesterone action. The product of a single gene named aromatase synthesizes estrogen. The potent estrogen estradiol is metabolized and thus inactivated by an enzyme termed 17beta-hydroxysteroid dehydrogenase (HSD) type 2 that is normally induced by progesterone in endometrium. Progesterone action is mediated by its receptor subtypes progesterone receptor (PR)-A and PR-B. We found a number of abnormalities in the expression of aromatase, 17beta-HSD type 2, and the PR-B/PR-A ratio in endometriosis tissue. These abnormalities and their functional consequences are discussed in this review article.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) induces the expression of Müllerian inhibiting substance (MIS) and many steroidogenic genes, including aromatase P450 (P450arom). Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1) inhibits SF-1-mediated induction of MIS and other steroidogenic genes, whereas Wilms' tumor suppressor gene (WT1) augments SF-1-mediated MIS expression. The effects of WT1 on steroidogenesis or P450arom expression have not been explored to date. In human endometriotic stromal cells, extremely high levels of P450arom mRNA and enzyme activity are present. Prostaglandin E(2) stimulates cAMP formation, SF-1 binding activity, P450arom mRNA levels, and estrogen synthesis in endometriotic stromal cells. Stromal cells of eutopic endometrium from disease-free women, on other hand, do not contain readily detectable levels of P450arom mRNA. Thus, we evaluated herein the possible roles of WT1 and DAX-1 in cAMP/SF-1-mediated regulation of P450arom expression in endometriotic and endometrial stromal cells. We also determined the cellular distribution and levels of these transcription factors in pathological endometriotic vs. normal eutopic endometrial tissues by immunohistochemistry to understand their in vivo roles. In vitro transcriptional regulation studies showed that both WT1 and DAX-1 inhibited cAMP and/or SF-1-induced P450arom promoter activity in a dose-dependent fashion in cultured human endometriotic and endometrial stromal cells. Site-directed disruption of the SF-1 binding site (-136/-124 bp) in the P450arom promoter abolished basal or cAMP/SF-1-induced promoter activity in the presence or absence of WT1 or DAX-1. Immunohistochemistry and H-scoring showed that DAX-1 was ubiquitously present in epithelial and stromal cells of both tissues. WT1, on the other hand, was preferentially expressed in stromal (vs. epithelial) cells. Moreover, WT1 levels in endometriotic stromal cells are significantly down-regulated compared with normal endometrial stromal cells. In summary, WT1 or DAX-1 inhibits cAMP-SF-1 pathway-dependent P450arom expression in cultured human endometriotic and endometrial stromal cells. In vivo down-regulation of WT1 in endometriotic stromal cells (vs. normal endometrial stromal cells) may in part be responsible for aberrantly increased P450arom expression and estrogen formation in this pathological tissue.

Aromatase P450 (P450arom) is the key enzyme for the biosynthesis of estrogen that is essential for the growth of human endometriosis, a pathology characterized by endometrium-like tissue on the peritoneal surfaces of abdominal organs manifest by pelvic pain and infertility. Surgically transplanted autologous uterine tissue to ectopic sites on the peritoneum in mice has been used as an animal model to study endometriosis. Using this mouse model, we evaluated the roles of the P450arom gene and aromatase enzyme activity in the growth of endometriosis represented by ectopic uterine tissues in mice. Endometriosis was induced surgically in the following groups of mice: 1) untreated transgenic mice with disrupted P450arom gene (ArKO); 2) ArKO mice treated with systemic estrogen; 3) untreated wild-type (WT) mice; 4) WT mice treated with estrogen; 5) WT mice treated with the aromatase inhibitor, letrozole; and 6) WT mice treated with letrozole and estrogen. Each group contained eight mice; +/+ littermates of ArKO mice were used as WT controls. Treatment with estrogen increased the size of ectopic uterine tissues in ArKO and WT mice significantly. The ectopic uterine lesions in untreated and estrogen-treated ArKO mice were strikingly smaller than those in untreated and estrogen-treated WT controls, respectively. Systemic treatment of WT mice with letrozole significantly decreased the lesion size in a dose-dependent manner. The addition of estrogen to letrozole treatment increased the ectopic lesion size, although these lesions were significantly smaller than those in mice treated with estrogen only. As tissue controls, the effects of these conditions on normally located (eutopic) uterine tissue were evaluated. The effects of disruption of the P450arom gene and treatments with letrozole and estrogen seemed to be more profound on ectopic tissues, suggesting that ectopic tissues might be more sensitive to estrogen for growth. We conclude that both an intact P450arom gene and the presence of aromatase enzyme activity are essential for the growth of ectopic uterine tissue in a mouse model of endometriosis.

Estrogen is produced in a number of human tissues including the ovary, placenta and extraglandular sites such as adipose tissue, skin and the brain. Aromatase is the key enzyme that regulates estrogen formation in these tissues. Aromatase activity is not detectable in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis and is stimulated by PGE(2). This results in local production of estrogen, which induces PGE(2) formation and establishes a positive feedback cycle. Another abnormality in endometriosis, i.e. deficient 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE(2) in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis using an aromatase inhibitor.

In human endometriotic stromal cells, markedly high levels of aromatase P450 (P450arom) mRNA and promoter II activity are present and can be vigorously stimulated by PGE(2) via a cAMP-dependent pathway to give rise to physiologically significant estrogen biosynthesis. Stromal cells of eutopic endometrium, on the other hand, do not express sufficient levels of P450arom for detectable enzyme activity. Because P450arom is up-regulated in the ovaries of CCAAT/enhancer binding protein (C/EBP) beta knockout mice and activation of the ovarian-type P450arom promoter (II) is responsible for aberrant P450arom expression in endometriosis, we sought here to evaluate the possible roles of C/EBP isoforms in the regulation of P450arom expression in endometriotic vs. eutopic endometrial stromal cells. We previously found that the -517-bp flanking region of promoter II contained the critical cis-acting elements for baseline and cAMP (analog)-induced activity. In this study, we disrupted several potential sequences and found that mutations of a -211/-197-bp cAMP-response element (CRE) and a -317/-304-bp C/EBP binding site abolished both baseline and cAMP-induced promoter II activity. Ectopic expression of C/EBPalpha increased both baseline and cAMP-dependent promoter II activity significantly in endometriotic cells, whereas ectopic expression of C/EBPbeta or C/EBPdelta abolished promoter II activity in both untreated and cAMP-treated endometriotic stromal cells. Comparable changes in promoter II activity were observed using endometrial stromal cells, which showed, however, seemingly diminished levels of baseline and cAMP-induced promoter II activity in comparison with endometriotic cells. EMSA using a probe containing the critical -317/-304-bp C/EBP site upstream of promoter II demonstrated a distinct DNA-protein complex in endometriotic, but not in endometrial stromal cells. This specific complex, however, could not be altered using antibodies against C/EBPalpha, -beta, or -delta. Because CRE is another potential DNA motif that can bind C/EBP isoforms, we next used EMSA using a probe containing the -211/-197-bp CRE and demonstrated that specific DNA-protein complexes contained C/EBPalpha but not C/EBPbeta or C/EBPdelta in endometriotic stromal cells. In contrast, C/EBPbeta and C/EBPdelta but not C/EBPalpha were detected in DNA-protein complexes using nuclear extracts from endometrial stromal cells. Western blotting and immunohistochemistry demonstrated expression of C/EBPalpha, -beta, and -delta in human endometriotic and endometrial stroma and epithelium. Intriguingly, C/EBPbeta was expressed at increased levels in stromal cells of human eutopic endometrium compared with simultaneously biopsied endometriotic tissues. We conclude that both -317/-304 and -211/-197-bp elements in promoter II are critical for the robust cAMP-dependent induction in endometriosis. C/EBPalpha up-regulates, whereas C/EBPbeta and C/EBPdelta inhibit P450arom promoter activity via binding primarily to the -211/-197-bp CRE under in vitro conditions. In vivo down-regulation of C/EBPbeta in endometriotic stromal cells and its up-regulation in endometrial stromal cells may in part account for the induction of P450arom expression in endometriosis and its inhibition in endometrium.

Endometriosis is one of the most common causes of infertility and chronic pelvic pain and affects 1 in 10 women in the reproductive-age group. Although existence of this disease has been known for over 100 years, our current knowledge of its pathogenesis, the pathophysiology of related infertility, and its spontaneous evolution is limited. Several reasons contribute to our lack of knowledge, the most critical being the difficulty in carrying out objective long-term studies in women. Thus, we and others have developed the baboon as an appropriate nonhuman primate to study the etiology of this disease. We suggested that endometriosis develops in two distinct phases. Phase I is invasive and dependent on ovarian steroids. Phase II, which is the active phase of the disease, is characterized by endogenous estrogen biosynthesis. Following inoculation with menstrual endometrial tissues in two consecutive menstrual cycles, baboons develop lesions that are similar to those seen in humans. Laparoscopy at 1, 4, and 10 months revealed a preponderance of red raised nodules at the first month, while both red lesions and reddish-blue proliferative endometriotic lesions were evident at 4 and 10 months. The presence of glandular tissue and stromal fibroblasts in these lesions was confirmed by histology. Lesions obtained at 1 and 4 months expressed estrogen receptor beta (ERbeta), matrix metalloproteinase-7 (MMP-7), and vascular endothelial growth factor (VEGF) predominantly. However, aromatase expression was only readily evident at 10 months, although some lesions obtained at 4 months expressed low levels of aromatase. Therefore, our preliminary data suggest that endometriosis can be artificially induced in baboons, and the role of exogenous and endogenous estradiol in proliferation, angiogenesis, and immune modulations can now be evaluated in a potentially systemic manner.

Aromatase activity is absent in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis, which gives rise to strikingly high levels of aromatase activity in this tissue. Both aromatase expression and activity are stimulated by PGE2. This results in local production of estrogen, which induces PGE2 formation and establishes a positive feedback cycle. Another abnormality in endometriosis, that is, deficient 17beta-HSD type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE2 in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis using an aromatase inhibitor.

Objective. To determine the effects of Mullerian inhibiting substance (MIS) treatment on endometriosis cells through study of apoptosis and autophagy. Design. Experimental in vitro study. Setting. University research laboratory. Cell Line. CRL-7566 endometriosis cell line. This line was established from a benign ovarian cyst taken from a patient with endometriosis. Interventions. In vitro treatment with MIS. Main Outcome Measures. The main outcome measures were cellular viability, proliferation, cell-cycle arrest, and induction of apoptosis and autophagy in endometriotic cells. Results. MIS treatment inhibited proliferation of endometriosis cells and induced apoptosis, as indicated by Annexin V staining, and induced caspase-9 cleavage and cell-cycle arrest, as evidenced by increased expression of p27 CDK-inhibitor. MIS treatment also induced autophagy in endometriosis cells as demonstrated by a significant increase in LC3-II induction, a hallmark of autophagy. Conclusions. MIS inhibits cell growth and induces autophagy, as well as apoptosis, in ectopic endometrial cell lines. Our results suggest that MIS may have a potential as a novel approach for medical treatment of endometriosis. Further studies may be needed to test the efficacy of MIS treatment in animal models and to develop MIS treatment specifically targeted to the endometriosis. [ABSTRACT FROM AUTHOR]

Abstract: Pathogenesis and growth of three common women''s cancers (breast, endometrium and ovary) are linked to estrogen. A single gene encodes the key enzyme for estrogen biosynthesis named aromatase, inhibition of which effectively eliminates estrogen production in the entire body. Aromatase inhibitors successfully treat breast cancer, whereas their roles in endometrial and ovarian cancers are less clear. Ovary, testis, adipose tissue, skin, hypothalamus and placenta express aromatase normally, whereas breast, endometrial and ovarian cancers overexpress aromatase and produce local estrogen exerting paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kb regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. In cancers of breast, endometrium and ovary, aromatase expression is primarly regulated by increased activity of the proximally located promoter I.3/II region. Promoters I.3 and II lie 215bp from each other and are coordinately stimulated by PGE2 via a cAMP-PKA-dependent pathway. In breast adipose fibroblasts exposed to PGE2 secreted by malignant epithelial cells, PKC is also activated, and this potentiates cAMP-PKA-dependent induction of aromatase. Thus, inflammatory substances such as PGE2 may play important roles in inducing local production of estrogen that promotes tumor growth. [Copyright &y& Elsevier]