Izumi Kusuki

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.

Dienogest inhibits aromatase and cyclooxygenase-2 expression and prostaglandin E2 production in human endometriotic stromal cells in spheroid culture

OBJECTIVE To determine the effect of dienogest (DNG) on the expression of aromatase and cyclooxygenase-2 (COX-2) and the production of prostaglandin E(2) (PGE(2)) in human endometriotic stromal cells (ESCs). DESIGN Experimental study in vitro. SETTING University hospital. PATIENT(S) Seventeen patients with ovarian endometrioma. INTERVENTION(S) ESCs from chocolate cyst linings of ovaries were treated with DNG. MAIN OUTCOME MEASURE(S) Expression of aromatase and COX-2 evaluated in spheroid cultures of human ESCs by real-time quantitative polymerase chain-reaction and immunocytochemistry, production of PGE(2) quantified by enzyme-linked immunosorbent assay (ELISA), and nuclear factor kappa B (NF-κB) DNA-binding examined by ELISA and immunocytochemistry. RESULT(S) The pharmaceutical actions of DNG on the expression of aromatase and COX-2 and the production of PGE(2) were examined using spheroid cultures of human ESCs. More aromatase, COX-2, and PGE(2) were expressed in spheroid cultures than in conventional ESCs monolayers. In the spheroid cultures, DNG (10(-7) M) and progesterone (10(-7) M) inhibited the expression of aromatase, COX-2, and PGE(2). DNG also inhibited NF-κB DNA-binding activity and reduced the immunocytochemical protein expression of aromatase, COX-2, and NF-κB p50 nuclear localization. CONCLUSION(S) Because DNG inhibits aromatase and COX-2 expression as well as PGE(2) production in ESCs, these pharmacologic features might contribute to a therapeutic effect of DNG on endometriosis.

Genetic Contribution of the Interleukin-10 Promoter Polymorphism in Endometriosis Susceptibility

PROBLEM: Interleukin‐10 (IL‐10) is an important immunomodulatory cytokine. The aim of this study was to investigate whether polymorphisms of the IL‐10 gene promoter polymorphisms may be responsible in part for genetic susceptibility to endometriosis.

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.

Maintenance therapy with dienogest following gonadotropin-releasing hormone agonist treatment for endometriosis-associated pelvic pain

OBJECTIVE To examine whether long-term administration of dienogest following gonadotropin-releasing hormone agonist (GnRH-a) therapy would prolong the relief of pelvic pain while reducing the amount of irregular uterine bleeding. STUDY DESIGN This was a prospective, non-randomized clinical trial. Among the patients suffering from chronic pelvic pain associated with recurrent endometriosis, Group G (n=38) received GnRH-a for 4-6 months and then dienogest (1 mg/day) for 12 months. The dose of dienogest was increased to 1.5 or 2 mg/day when a patient had uncontrollable uterine bleeding {n=15 (39%)}. Group D (n=33) received only dienogest (2 mg/day) for 12 months. Pelvic pain was assessed using a visual analog scale (VAS). Uterine bleeding was semi-quantified using a pictorial blood loss assessment chart (PBAC). RESULTS In Group G, GnRH-a significantly reduced the VAS score for pelvic pain, and alleviation was maintained during the 12-month therapy with dienogest. There was no significant difference in pain reduction between Group G and Group D. The PBAC score during the first 6 months on dienogest was significantly smaller in Group G than in Group D. CONCLUSION Treatment with a GnRH-a followed by long-term dienogest therapy maintains the relief of endometriosis-associated pelvic pain achieved with GnRH-a therapy for at least 12 months. This regimen reduces the amount of irregular uterine bleeding that often occurs during the early phase of dienogest therapy.

Possible involvement of loss of heterozygosity in malignant transformation of ovarian endometriosis

OBJECTIVE Although histological and epidemiological studies have suggested that endometriosis has malignant potential, the molecular mechanism underlying the malignant transformation of endometriosis is poorly understood. Ovarian cancer arising from endometriosis (OCEM) may provide an ideal model for genetic studies. To investigate the genetic alterations during transformation of ovarian endometriosis into cancer, we analysed loss of heterozygosity (LOH) and mutations of tumour-related genes in OCEM cases (n=12) that fulfilled the histological criteria and in solitary ovarian endometriosis (n=12). METHODS Each paraffin-embedded section was microdissected to isolate the endometriotic epithelium, transitional epithelium, and cancer cells. Extracted DNA was amplified by nested PCR. LOH was identified with fluorescence-labelled microsatellite markers. Mutations of tumour-related genes PTEN, TP53, and K-ras were examined by bidirectional DNA sequencing. RESULTS With 13 microsatellite markers on six chromosomes, we detected 31 and eight LOH events in OCEMs and in solitary ovarian endometriosis, respectively. In the OCEM group, 18 LOH events were found in cancer cells alone, while 13 LOH events were found in all cancer, transitional, and endometriotic tissues. High frequencies of LOH were detected on chromosomes 9p, 10q, and 13q. However, only two point mutations were detected in cancer cells in one case. CONCLUSION Our study suggested that accumulated LOH events on some chromosome loci may be involved in malignant transformation of endometriosis, while mutations in certain tumour-related genes are not.

Dienogest reduces HSD17β1 expression and activity in endometriosis

Endometriosis is an estrogen-dependent disease. Abnormally biosynthesized estrogens in endometriotic tissues induce the growth of the lesion and worsen endometriosis-associated pelvic pain. Dienogest (DNG), a selective progesterone receptor agonist, is widely used to treat endometriosis and efficiently relieves the symptoms. However, its pharmacological action remains unknown. In this study, we elucidated the effect of DNG on enzymes involved in local estrogen metabolism in endometriosis. Surgically obtained specimens of 23 ovarian endometriomas (OE) and their homologous endometrium (EE), ten OE treated with DNG (OE w/D), and 19 normal endometria without endometriosis (NE) were analyzed. Spheroid cultures of stromal cells (SCs) were treated with DNG and progesterone. The expression of aromatase, 17β-hydroxysteroid dehydrogenase 1 (HSD17β1), HSD17β2, HSD17β7, HSD17β12, steroid sulfatase (STS), and estrogen sulfotransferase (EST) was evaluated by real-time quantitative PCR. The activity and protein level of HSD17β1 were measured with an enzyme assay using radiolabeled estrogens and immunohistochemistry respectively. OESCs showed increased expression of aromatase, HSD17β1, STS, and EST, along with decreased HSD17β2 expression, when compared with stromal cells from normal endometria without endometriosis (NESCs) (P<0.01) or stromal cells from homologous endometrium (EESCs) (P<0.01). In OESCs, DNG inhibited HSD17β1 expression and enzyme activity at 10(-7) M (P<0.01). Results of immunohistochemical analysis displayed reduced HSD17β1 staining intensity in OE w/D (P<0.05). In conclusion, DNG exerts comprehensive inhibition of abnormal estrogen production through inhibition of aromatase and HSD17β1, contributing to a therapeutic effect of DNG on endometriosis.

Antiproliferative and apoptotic effects of norethisterone on endometriotic stromal cells in vitro

OBJECTIVES Low-dose estrogen-progestin (LEP) agents are often used for relieving endometriosis-associated chronic pelvic pain, but a direct effect of LEP on endometriotic lesions remains to be demonstrated. The objective of this study is to investigate the antiproliferative and apoptotic effects of the synthetic progestin norethisterone (NET) against human endometriotic stromal cells (ESCs). STUDY DESIGN Ovarian endometrioma specimens were obtained at laparoscopy from 19 patients with endometriosis, and ESCs were isolated. The antiproliferative effect of compounds against cultured ESCs was evaluated by measuring the inhibition of [(3)H]thymidine incorporation. The ability of compounds to induce apoptosis in the cultured cells was evaluated by the measurement of caspase 3/7 activity and by nuclear staining. The cytotoxicity of compounds was evaluated by measuring the leakage of lactate dehydrogenase (LDH) into the supernatant of the cell culture. RESULTS NET and progesterone (P4) at concentrations of greater than 10nM significantly inhibited [(3)H]thymidine incorporation in a dose-dependent manner. Co-treatment with 17β-estradiol at 0.1 ng/mL did not affect the inhibition of [(3)H]thymidine incorporation by NET. At concentrations greater than 100 nM, NET significantly increased caspase 3/7 activity and the numbers of apoptotic cells, whereas P4 did not. Treatment of ESCs for 24h with NET or P4 at concentrations of up to 1000 nM did not cause LDH leakage. CONCLUSIONS NET inhibits the proliferation of ESCs and induces their apoptosis. These results suggest a possible direct effect of NET on endometriotic lesions in patients with endometriosis.

Peroxisome proliferator-activated receptor gamma, coactivator 1α enhances local estrogen biosynthesis by stimulating aromatase activity in endometriosis.

CONTEXT Endometriosis is an estrogen-dependent disease, and estrogen is overproduced by abnormally elevated aromatase in endometriotic tissues. Peroxisome proliferator-activated receptor gamma, coactivator 1α (PGC-1α) is a transcriptional coactivator-modulating steroid hormone. OBJECTIVE To investigate the effect of PGC-1α on aromatase activity in endometriosis. DESIGN Specimens from ovarian endometrioma (OE), endometrium with endometriosis (EE), and normal endometrium (NE) were analyzed for PGC-1α and aromatase expression. PGC-1α-dependent changes in aromatase expression in primary cultured stromal cells (SCs) were identified using luciferase and enzymatic assays, exon I-specific RT-PCR, and real-time PCR. Environmental stimulus-induced changes in PGC-1α were also examined. RESULTS PGC-1α was more highly expressed in OE than in EE and NE (P < .01). In OE, PGC-1α was coexpressed with aromatase, and their mRNA expressions were also correlated (r = 0.56, P = .02). PGC-1α was recruited to the nuclear receptor half-site between PI.3 and PII in the aromatase promoter. PGC-1α overexpression enhanced aromatase promoter activity (P < .01), mRNA expression (P < .05), and enzymatic activity (P < .01) in SCs from OE, but not in SCs from EE or NE. The levels of PI.3, PII, and exon II mRNA increased and transcriptional enhancement was abolished by mutation of the PGC-1α-interacting site. PGC-1α expression was enhanced in SCs from OE by tumor necrosis factor (TNF)-α (P < .05) but not by hypoxia or 17β-estradiol. CONCLUSIONS PGC-1α stimulated by TNF-α regulates aromatase expression and activity to promote local estrogen biosynthesis in OE, suggesting that PGC-1α is a promising candidate for novel targeted therapies in endometriosis treatment.

Clear Cell Carcinoma Arising from Cesarean Section Scar Endometriosis: Case Report and Review of the Literature

Introduction. The incidence of endometriosis affecting skin tissue represents only 0.5–1.0% of all endometriosis cases. A malignancy in the abdominal wall arising from endometriosis following cesarean section is even rarer; only 21 cases have previously been reported. The therapeutic strategy has not been determined because of the limited cases. We report a case of clear cell adenocarcinoma arising in the abdominal wall from endometriosis tissues following cesarean section and review previous literature to achieve the optimal treatment and better prognosis. Case Presentation. A 60-year-old woman presented with a growing mass at the left side of a cesarean section scar. Radical resection of the abdominal wall mass was performed. Histopathological examination showed a clear cell adenocarcinoma. Benign endometrium-like tissues were found adjacent to the cancer lesion in the excised specimen, suggesting malignant transformation from endometriosis of the abdominal wall. Discussion. Local resection was performed in 10 cases (47.6%) and total abdominal hysterectomy or oophorectomy was conducted in 11 cases (52.4%). No malignant lesions were observed in either the uterus or adnexa that were resected. These cases may be expected to increase with increasing incidence of cesarean section. The significance of the extensional resection should be further elucidated.