Daidzein-rich isoflavone aglycones inhibit cell growth and inflammation in endometriosis.

Endometriosis is an estrogen-dependent disease, and isoflavones interact with estrogen receptors. The purposes of this study are to investigate the in vitro and in vivo effects of daidzein-rich isoflavone aglycones (DRIAs), dietary supplements, on cellular proliferation in endometriosis. Stromal cells isolated from ovarian endometrioma (OESCs) and normal endometrium (NESCs) were cultured with DRIAs, i.e., each of the DRIA components (daidzein, genistein, or glycitein), or isoflavone glycosides (IG; DRIA precursors). A mouse model of endometriosis was established by transplanting donor-mouse uterine fragments into recipient mice. Our results showed that DRIAs (0.2-20 µM) inhibited the proliferation of OESCs (P < 0.05 for 0.2 µM; P < 0.01 for 2 and 20 µM) but not of NESCs. However, daidzein, genistein, glycitein, and IG did not inhibit their proliferation. DRIA-induced suppression was reversed by inhibition of the estrogen receptor (ER)ß by an antagonist, PHTPP, or by ERß siRNA (P < 0.05), but not by MPP, an ERα antagonist. In OESCs, DRIAs led to reduced expression of IL-6, IL-8, COX-2, and aromatase, as well as reduced aromatase activity, serum glucocorticoid-regulated kinase levels, and PGE2 levels (P < 0.05). Western blot and immunofluorescence assays revealed that DRIAs inhibited TNF-α-induced IκB phosphorylation and p65 uptake into the nuclei of OESCs. In the mouse model, a DRIA-containing feed significantly decreased the number, weight, and Ki-67 proliferative activity of endometriosis-like lesions compared to in mice fed with an IG-containing feed and the control feed (P < 0.01). In conclusion, DRIAs inhibit cellular proliferation in endometriosis, thus representing a potential therapeutic option for the management of endometriosis.

Immunohistochemical localization of aromatase and apoptosis-associated proteins in ovarian serous cystadenocarcinoma arising from ovarian endometriosis.

An immunohistochemical study was made of a case of serous cystadenocarcinoma that had been shown to have arisen from ovarian endometriosis. Aromatase cytochrome P450 (P450arom), an enzyme responsible for estrogen biosynthesis, was localized in the epithelial linings of the endometriosis and faintly in the transitional part, whereas it was not expressed in the carcinoma tissue. In contrast, estrogen receptors, progesterone receptors, and apoptosis-associated proteins, Fas, Fas ligand, and Bax were expressed in both endometriosis and carcinoma tissues of the tumor, whereas Bcl-2 was not expressed in either tissue of the tumor. It was suggested that the undifferentiated shift of the histologic grade might result in the loss of P450arom and that the malignant transformation was not caused by an altered balance of apoptosis-associated proteins. Accumulation of these studies may lead to a better understanding of the nature of malignant transformation of ovarian endometriosis.

Progesterone inhibition of functional leptin receptor mRNA expression in human endometrium.

Leptin is secreted by adipocytes and regulates appetite through interaction with hypothalamic leptin receptors (OB-R). Leptin is involved in the stimulation of reproductive functions, and local expression of leptin and OB-R in the ovary, oocyte, embryo, and placenta might play a role in early development. The mRNA and protein of the long form leptin receptor (OB-R(L)) but not of leptin are expressed in the human endometrium and the abundance of OB-R mRNA expression varies during the menstrual cycle with a peak in the early secretory phase. We examined the steroidal regulation of OB-R(L) mRNA expression. Northern blot analyses showed that in organ-cultured proliferative endometrial specimens, oestradiol (10(-9) and 10(-8) mol/l) had no acute effect on the OB-R(L) mRNA expression, whereas oestradiol plus progesterone (10(-8), 10(-7) and 10(-6) mol/l) or medroxyprogesterone acetate (10(-8) and 10(-7) mol/l) suppressed the expression by approximately 50%. This progestin-induced suppression was blocked by a concomitant addition of mifepristone. Additionally, incubation of endometrial specimens in the presence of leptin resulted in the phosphorylation of its intracellular target, STAT3 (signal transducer and activator of transcription 3). These results indicate that, in the human endometrium, progestins act via the progesterone receptors to suppress functional OB-R(L) mRNA expression, and may thereby alter the sensitivity of the endometrium to leptin.

Oestrogen receptor-alpha gene polymorphism is associated with endometriosis, adenomyosis and leiomyomata.

Endometriosis, adenomyosis and leiomyomata develop in women of reproductive age and regress after menopause or ovariectomy, suggesting that they grow in an oestrogen-dependent fashion. We investigated whether polymorphism in the oestrogen receptor-alpha (ERalpha) gene is related to oestrogen-dependent benign uterine disease. A total of 203 women with regular menstrual cycles underwent laparotomy or laparoscopy and were diagnosed histologically with endometriosis, adenomyosis and/or leiomyomata. Patients with cervical carcinoma in situ, tubal occlusion or adhesion but no other gynaecological disease were considered to be disease-free. A total of 179 women undergoing annual health examination were grouped as reference population. The distribution of PVUII genotypes (PP, Pp, and pp) of the ERalpha gene was different between each pair of the four groups of endometriosis, adenomyosis/leiomyomata, disease-free, and reference population (P = 0.022-0.0005), except between the former two groups. The PP genotype was less frequent in the groups of endometriosis (P = 0.0002) and adenomyosis/leiomyomata (P = 0.002) as compared to that in the disease-free group. In the endometriosis group, there was no difference in the distribution of PVUII genotypes due to complicating diseases (adenomyosis and/or leiomyomata) or severity of the clinical stages. These results suggest that the PVUII polymorphism of the ERalpha gene is associated with the risk for endometriosis, adenomyosis, and leiomyomata.

Progesterone induction of 17beta-hydroxysteroid dehydrogenase type 2 during the secretory phase occurs in the endometrium of estrogen-dependent benign diseases but not in normal endometrium.

In the human endometrium, inactivation of 17beta-estradiol to estrone is catalyzed by 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2). Previous studies have shown that the 17betaHSD2 activity in the endometrium is elevated during the secretory phase, as compared with the level during the proliferative phase, and that the elevation is in response to progesterone via the progesterone receptors. Recently, it has been demonstrated that aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis, is not present in the endometrium obtained from normal menstruating women with cervical cancer in situ showing no other gynecological disease (defined as "disease free"), but present in the endometrium obtained from patients with endometriosis, adenomyosis, and/or leiomyomas (defined as "diseased"). However, the previous 17betaHSD studies have been performed without distinguishing between disease-free and diseased endometria. We, therefore, analyzed 17betaHSD2 distinguishing between disease-free and diseased endometria. During the proliferative phase, the abundance of messenger RNA (mRNA) and activity of 17betaHSD2 were comparable in both disease-free and diseased endometrium. However, during the secretory phase, while the abundance of mRNA and activity of 17betaHSD2 increased 4- to 6-fold in diseased endometrium, the 17betaHSD2 remained unchanged in the disease-free endometrium. Kinetic studies showed that the Km was identical among the four groups of endometria, suggesting that the elevation of 17betaHSD2 simply resulted from increased mRNA transcription. Organ culture of proliferative endometria in the presence of progestins resulted in the stimulation of 17betaHSD2 in diseased endometria via the progesterone receptors, whereas disease-free endometrium was not stimulated by progestins. These results suggest that the previous paradigm that 17betaHSD2 activity in the endometrium is elevated during the secretory phase is confined to diseased endometrium but not to disease-free endometrium and that the estrogen metabolism is altered in the endometria of the patients with estrogen-dependent benign diseases.

Expression of leptin receptor in human endometrium and fluctuation during the menstrual cycle.

Leptin is secreted by adipocytes and regulates appetite through interaction with hypothalamic leptin receptors (OB-R). Accumulated evidence shows that leptin is involved in the stimulation of reproductive functions and that local expression of leptin and OB-R in the ovary, oocyte, embryo, and placenta plays a role in early development. To investigate the role of leptin in implantation, we examined the expression of OB-R and leptin in the human endometrium. Northern and Western blot analyses and RT-PCR showed that the long form of OB-R (OB-R(L)) messenger ribonucleic acid (mRNA) and protein were expressed. In contrast, leptin mRNA or protein was not detected. All of the splice variants of OB-R (OB-R(T)) and OB-R(L) transcripts were expressed in 90% and 84% of the cases, respectively. OB-R mRNA expression peaked in the early secretory phase. Decidual tissue of early gestation also expressed OB-R(T) and OB-R(L). Their incidence and abundance were comparable among endometria with benign uterine diseases and disease-free endometria and were not related to a body mass index within the normal range. The present results indicate that OB-R, but not leptin, is expressed in the human endometrium.

Detection of aromatase cytochrome P-450 in endometrial biopsy specimens as a diagnostic test for endometriosis.

OBJECTIVE: To evaluate the clinical usefulness of examining endometrial biopsy specimens for aromatase cytochrome P-450 as a diagnostic test for endometriosis. DESIGN: Retrospective, case-controlled study. SETTING: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto, Japan. PATIENT(S): One hundred five women of reproductive age with normal menstrual cycles underwent endometrial biopsy laparotomy or laparoscopy, and examination of their tissue revealed endometriosis, adenomyosis, and/or leiomyomas. Patients who had cervical carcinoma in situ but no other gynecologic disease were considered to be disease-free. INTERVENTION(S): Endometrial biopsy specimens were collected. MAIN OUTCOME MEASURE(S): The expression of aromatase cytochrome P-450 was examined by reverse transcription-polymerase chain reaction and immunohistochemical analysis. The distribution and intensity of the immunostaining was assessed using a semiquantitative index designed H-score. RESULT(S): Immunostaining for aromatase cytochrome P-450 was detected in biopsy specimens obtained from patients with endometriosis, adenomyosis, and/or leiomyomas but not in specimens obtained from disease-free patients (H-score <20), with a sensitivity and specificity of 91% and 100%, respectively. CONCLUSION(S): The expression of aromatase cytochrome P-450 in biopsy specimens of eutopic endometrium distinguishes between disease-free women and women with endometriosis, adenomyosis, and/or leiomyomas. This technique can be used at outpatient infertility clinics as an initial screening procedure to rule out the presence of estrogen-dependent disease.

Expression of aromatase cytochrome P450 in eutopic endometrium and its application as a diagnostic test for endometriosis.

Endometriotic implants, like other estrogen-dependent tumors, contain both estrogen receptors and aromatase cytochrome P450 (P450arom), suggesting that at a local level, endometriotic implants produce estrogens, which may be involved in tissue growth through interaction with the estrogen receptors. P450arom is also expressed in the eutopic endometria of patients with endometriosis, adenomyosis, and/or leiomyomas, whereas neither P450arom protein nor mRNA is expressed in the eutopic endometria of normal menstruating women with cervical carcinoma in situ yet showing no other gynecological disease (disease-free). Examination of P450arom expression in endometrial biopsy specimens enables the physician to discriminate between the presence and absence of endometriosis, and may be used as an initial screening at outpatient infertility clinics. Copyrightz1999S.KargerAG,Basel

Leptin directly stimulates aromatase activity in human luteinized granulosa cells.

Leptin, the obese (ob) gene product, is secreted by adipocytes and regulates appetite through interaction with hypothalamic leptin receptors. Leptin may also have a stimulatory effect on reproductive function. Furthermore, leptin receptor mRNA is expressed in the ovary, suggesting a direct effect on its function. The present study examines the direct role of leptin on the oestrogen-producing activity in human luteinized granulosa cells. The cells were obtained from in-vitro fertilization pre-ovulatory follicles, precultured for 24 h in the presence of 5% charcoal-treated serum, and incubated for 48-96 h in a serum-free medium containing recombinant human leptin, follicle stimulating hormone (FSH), and/or insulin-like growth factor-I (IGF-I). A single addition of leptin (0. 5-10 ng/ml) stimulated aromatase activity with the incubation time of up to 96 h. The addition of leptin (1 ng/ml) further augmented the stimulation by a single addition of FSH (100 ng/ml) or IGF-I (100 ng/ml), or a combination of both. A single addition of leptin (1 ng/ml) or a combination of leptin (1 ng/ml), FSH (100 ng/ml), and IGF-I (100 ng/ml) gave rise to an increase in each parameter of oestrogen-producing activity measured, i.e. P450arom mRNA level, P450arom protein level, aromatase specific activity, and the oestradiol concentration in the culture supernatant. However, the production of progesterone did not change. These results indicate that leptin stimulates oestrogen production by increasing P450arom mRNA and P450arom protein expression and, consequently, aromatase activity by its direct action on the human luteinized granulosa cells.