Abstract
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.
Introduction
Endometriosis is defined as the presence of endometrium-like tissues at extra-uterine sites. Clinical symptoms associated with endometriosis include pelvic pain, dysmenorrhea, dyspareunia, and infertility (Giudice 2010). There is marked relief of symptoms after menopause, clearly demonstrating the dependency of endometriosis on estrogens. Besides systemic circulating estrogens secreted from the ovaries, abnormally biosynthesized estrogens in endometriotic tissues also contribute to the growth of the lesion and worsening symptoms (Bulun 2009). In the eutopic and ectopic endometria of women with endometriosis, overexpressed aromatase (also known as estrogen synthase) biosynthesizes estrogens, namely estrone and estradiol, from the androgens, androstenedione, and testosterone respectively (Noble et al. 1996, Kitawaki et al. 1997, Matsuzaki et al. 2006, Dassen et al. 2007, Smuc et al. 2007). In these tissues, estradiol, the most potent estrogen, is predominantly synthesized from less potent estrone by 17β-hydroxysteroid dehydrogenase 1 (HSD17β1), and the reverse reaction is catalyzed mainly by HSD17β2. In endometriotic tissues, the expression of HSD17β1 is higher than that of HSD17β2; thus, the reaction is tilted in favor of producing estradiol (Zeitoun et al. 1998, Dassen et al. 2007). The other major source of estrogens is estrone sulfate, an inactive conjugated form abundant in the circulation. Estrone sulfate is desulfated to estrone by steroid sulfatase (STS) and estrone is inactivated by estrogen sulfotransferase (EST) (Utsunomiya et al. 2004, Colette et al. 2013). Understanding how the aberrant expression of these enzymes in endometriosis contributes to local estrogen production and metabolism will allow the development of improved therapeutic agents.
Dienogest (DNG), a selective progesterone (P4) receptor (PR) agonist, is widely used to treat endometriosis (McCormack 2010) and efficiently relieves endometriosis-associated pelvic pain (Harada et al. 2009, Momoeda et al. 2009, Strowitzki et al. 2010, Petraglia et al. 2012). DNG directly inhibits PR-mediated cell proliferation (Okada et al. 2001, Fu et al. 2008, Shimizu et al. 2009) and production of the inflammatory factors involved in the pathology of endometriosis, such as prostaglandin estradiol (E2) (Shimizu et al. 2011, Yamanaka et al. 2012), inflammatory cytokines (Horie et al. 2005), Toll-like receptor 4 (Mita et al. 2011), and nerve growth factor (Mita et al. 2014). Supprerssion of these inflammatory factors is considered to contribute, in part, to the improvement of pain symptoms. DNG restores the antigen-presenting ability of peritoneal fluid macrophages by increasing human leukocyte antigen-DR expression (Maeda et al. 2014). DNG also suppresses aromatase expression in human immortalized endometrial epithelial cells (Shimizu et al. 2011) and primary cultured stromal cells (SCs) derived from ovarian endometrioma (OE) (Yamanaka et al. 2012). However, the effect of DNG on other estrogen-metabolizing enzymes in endometriotic cells remains unknown, and a more detailed analysis is needed to understand its clinical effectiveness and pharmacological function.
The purpose of this study was to investigate the effect of DNG on enzymes involved in estrogen metabolism using spheroid cultures of primary cultured SCs derived from OE, endometrium with endometriosis (EE), and normal endometrium without endometriosis (NE).
Patients and methods
Patients and samples
Patient characteristics are given in Table 1. OE tissues from patients (n=23) who did not receive any hormonal treatment and their homologous EE specimens (n=10), in addition to OE specimens from patients treated with DNG at a dose of 1 mg twice daily for 3–5 months (OE treated with DNG (OE w/D)) (n=11), were obtained from women undergoing surgery for OE. NE specimens were obtained from women undergoing surgery for uterine fibroids (n=19). All women were of reproductive age, and all specimens, with the exception of OE w/D, were collected at the proliferative phase of the regular menstrual cycle. Women who had undergone hormonal treatments within 6 months before surgery were excluded. OE w/D specimens were not used for in vitro experiments to avoid the effect of previous DNG exposure on the results. The endometriosis stages were evaluated according to the American Society for Reproductive Medicine classification of endometriosis. This study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the institutional review board of the Kyoto Prefectural University of Medicine. Informed consent was obtained from all patients.
Clinical characteristics of study patients. Values are presented as means±s.e.m.
| NE (n=19) | EE (n=10) | OE (n=23) | OE w/D (n=11) | |
|---|---|---|---|---|
| Age (years) | 41.8±4.3 | 41.4±3.9 | 32.5±7.0*,† | 37.5±4.2 |
| CA-125 (U/ml) | NA | 68.9±29.1 | 82.5±83.4 | 68.0±55.7 |
| r-ASRM stage (%) | ||||
| III | NA | 6 (60) | 14 (61) | 6 (55) |
| IV | 4 (40) | 9 (39) | 5 (45) | |
| Duration of drug administration (weeks) | NA | NA | NA | 13.4±6.0 |
P values were obtained by Kruskal–Wallis ANOVA followed by multiple comparisons using Scheffe's procedure or χ 2 test. NE, normal endometrium; EE, endometrium with endometriosis; OE, ovarian endometrioma; OE w/D, OE treated with dienogest; r-ASRM, revised American Society for Reproductive Medicine; DNG, dienogest. *P<0.01 versus NE and † P<0.05 versus EE.
Isolation and culture of SCs
The isolation and culture of SCs was conducted as described previously (Yamanaka et al. 2012). Briefly, tissue digestion was performed with 2.5% collagenase (Nacalai Tesque, Kyoto, Japan) and 15 IU/ml of DNase I (Takara Shuzo, Tokyo, Japan). After filtering through a nylon cell strainer, the digested cells were centrifuged in lymphocyte separation solution (Nacalai Tesque) to remove the red blood cells. The >95% purity of SC preparations was confirmed by positive staining for CD10 and vimentin and negative staining for cytokeratin, CD31, and CD45. The cells were cultured in DMEM/Ham's F-12 (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin and streptomycin (100 μg/ml), under a humidified atmosphere at 37 °C in 5% CO2. The cells that reached subconfluence were dispersed using 0.1% trypsin (Nacalai Tesque) and resuspended in phenol-red-free DMEM/Ham's F-12 (Nacalai Tesque) supplemented with 10% dextran-coated charcoal-treated FBS and 1% penicillin and streptomycin (100 μg/ml). For mRNA analysis, SCs were subcultured in U-bottom 96-well culture plates (Sumilon) at a density of 4×104 cells/well to form spheroids. For HSD17β1 activity assays, SCs were plated into six-well culture plates at a density of 4×105 cells/well to form monolayers. The OESC spheroid expression of estrogen receptor α (ERα (ESR1)), ERβ (ESR2), PR, aromatase, cyclooxygenase-2 (COX2), and nuclear factor-κ B (NFκB) p50 subunit nuclear localization was validated by immunocytochemistry as described previously (Yamanaka et al. 2012).
Treatment of cultured SCs with DNG or P4
The culture medium was replaced either after 72 h (spheroid culture for RNA extraction) or when cells reached subconfluence (HSD17β1 activity assay) by medium with or without DNG (10−8, 10−7, and 10−6 M; Bayer Schering Pharma, Berlin, Germany) or P4 (10−8, 10−7, and 10−6 M; Sigma–Aldrich), and the cells were incubated for a further 48 h.
RNA extraction, cDNA preparation, and real-time PCR
Total RNA was extracted from cultured SCs using the RNeasy Mini Kit (Qiagen). After quantification and determination of the quality of the RNA by u.v. absorption (OD 260 nm/280 nm) using a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA), cDNA was synthesized using the SuperScript III first-strand synthesis system (Invitrogen) and a GeneAmp PCR 9700 machine (Applied Biosystems). Quantitative real-time PCR was conducted using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and a StepOne Real-Time PCR System (Applied Biosystems) with TaqMan assay primer/probe sets (Applied Biosystems) for the target genes: aromatase (CYP19A1) (Hs00240671_m1), HSD17 β 1 (Hs00166219_g1), HSD17 β 2 (Hs00157993_m1), HSD17 β 7 (Hs00367686_m1), HSD17 β 12 (Hs00275054_m1), STS (Hs00996676_m1), EST (SULT1E1) (Hs00960941_m1), and endogenous control GAPDH (Hs03929097_g1). Real-time quantitative PCR was performed under the following thermal cycling conditions: denaturing at 95 °C for 60 s; 3 s at 95 °C; 40 cycles of 30 s at 60 °C. Threshold cycle (Ct) values were calculated using the ΔΔCt method.
HSD17β1 enzyme assays
HSD17β1 activity was measured using thin layer chromatography, as described previously (Kitawaki et al. 2000). Briefly, cells were washed twice with phenol-red-free DMEM/Ham's F-12, and then incubated at 37 °C/5% CO2 for 6 h with 0.5 ml of serum-free medium containing [6,7-3H]estrone (Perkin Elmer, Waltham, MA, USA) (1.8×106 dpm, 37 μM). The reaction was stopped by transferring the medium to the test tubes containing 2 ml chloroform and the corresponding carrier steroids: [4-14C]estradiol (Perkin Elmer) (1.3×104 dpm) and nonradioactive estrone and estradiol (0.2 mg each). The steroids were isolated by thin-layer chromatography using Silicagel 60 F254 (0.25 mm; Merck) in a system of chloroform:ethyl acetate (4:1, v/v). The aliquot was mixed with Clear-sol I (Nacalai Tesque), and radioactivity was measured using a scintillation counter (Beckman Coulter, Fullerton, CA, USA). Enzyme activity was calculated and normalized according to the ratios of the estradiol formed. Protein concentration (pmol/mg protein per h) was measured by the Bradford method.
Immunohistochemistry
Specimens from OE, OE w/D, EE, and NE were stained immunohistochemically as described previously (Yamanaka et al. 2012) using an anti-HSD17β1 antibody (200 μg/ml; Abcam, Cambridge, UK). Normal term placenta tissue was used as a positive control. Because the cell components of epithelial cells and SCs were considerably different between eutopic endometrium and OE, we compared the immunostaining intensity in SCs using the H-score, a semi-quantitative index involving an algorithm described previously (Yamanaka et al. 2012). Briefly, two independent observers evaluated approximately 500 cells/slide and scored them as follows: 3×percentage of strongly staining cells+2×percentage of moderately staining cells+percentage of weakly staining cells. The H-score was calculated as the mean of the two scores.
Statistical analyses
The mRNA expression levels of enzymes in the three types of SCs were analyzed by Kruskal–Wallis ANOVA followed by multiple comparisons using Scheffe's procedure because of the unequal variances in the results. Results of real-time PCR and the HSD17β1 activity assay measuring the drugs’ effects on mRNA expression and enzyme activity levels in OESCs were assessed by repeated measures ANOVA followed by multiple comparisons using Dunnett's procedure. Statistical analysis of the immunohistochemical results was performed using an unpaired t-test. Each assay for individual experiments was performed in triplicate. Data are presented as means±s.e.m. P values of <0.05 were considered statistically significant.
Results
mRNA expression of enzymes in spheroid-cultured SCs
In OESCs, the mRNA expression levels of aromatase (P<0.01), HSD17 β 1 (P<0.01), STS (P<0.01), and EST (P<0.01) were greater compared with those in NESCs and EESCs. In NESCs or EESCs, we detected neither aromatase nor EST mRNA expression and an extremely low level of HSD17 β 1 mRNA expression. In contrast, HSD17 β 2 mRNA expression was lower in OESCs compared with that in NESCs (P<0.01) and EESCs (P<0.01) (Fig. 1A).
(A) The baseline mRNA expression levels of estrogen-metabolizing enzymes in spheroid-cultured stromal cells (SCs) derived from normal endometrium (NE), endometrium with endometriosis (EE), and OE. (B) Effects of dienogest (DNG) and progesterone (P4) on mRNA expression levels of enzymes in OESCs (at least seven separate experiments respectively) and on (C) 17β-hydroxysteroid dehydrogenase 1 (HSD17β1) activity in OESCs (n=8). All assays were performed in triplicate, and data are presented as means±s.e.m. P values for the statistical analysis of the mRNA expression of enzymes in SCs in spheroid culture are based on Kruskal–Wallis ANOVA followed by multiple comparisons using Scheffe's procedure, and effects of drugs on OESC enzyme mRNA expression and activity are based on repeated measures ANOVA followed by multiple comparisons using Dunnett's procedure. ND, not detectable; *P<0.05 and **P<0.01 versus control.
Citation: Journal of Endocrinology 225, 2; 10.1530/JOE-15-0052
Effects of drugs on enzymes in OESCs
Incubating the spheroids for 48 h with DNG (10−7 M (P<0.01) and 10−6 M (P<0.01)) and P4 (10−7 M (P<0.05) and 10−6 M (P<0.01)) significantly decreased the expression of HSD17 β 1 mRNA compared with the controls. However, DNG or P4 did not significantly suppress HSD17 β 2, HSD17 β 7, HSD17 β 12, STS, or EST mRNA expression (Fig. 1B). In parallel with the mRNA results, DNG (10−7 M (P<0.01) and 10−6 M (P<0.01)) and P4 (10−8 M (P<0.05), 10−7 M (P<0.01), and 10−6 M (P<0.01)) significantly suppressed the catalytic activity of HSD17β1 (Fig. 1C).
Immunohistochemistry
Immunohistochemical analysis showed HSD17β1 expression in the cytoplasm of epithelial cells and SCs of NE (Fig. 2A), EE (Fig. 2B), and OE (Fig. 2C). The immunostaining intensity of HSD17β1 was greater in OESCs when compared with NE (P<0.01) and EE (P<0.01). Moreover, there was a significant reduction in the immunostaining intensity in the OE w/D group (P<0.05) (Fig. 2E).
Representative immunohistochemical staining of 17β-hydroxysteroid dehydrogenase 1 (HSD17β1) in (A) normal endometrium (NE), (B) endometrium with endometriosis (EE), (C) ovarian endometrioma (OE), and (D) normal term placenta as a positive control. (E) The immunostaining intensity of NE (n=19), EE (n=10), OE (n=23), and OE treated with dienogest (OE w/D) (n=11) was scored with semi-quantitative index H-scores. Areas shown at a higher magnification are indicated by rectangles. Data are presented as means±s.e.m. P values are based on an unpaired t-test. *P<0.05 and **P<0.01 versus OE.
Citation: Journal of Endocrinology 225, 2; 10.1530/JOE-15-0052
Discussion
In this study, we demonstrated DNG-mediated inhibition of mRNA expression, catalytic activity, and protein expression of HSD17β1 in endometriosis. Taken together with the previous findings that DNG inhibits aromatase in endometriosis (Shimizu et al. 2011, Yamanaka et al. 2012), DNG exerts comprehensive inhibition of abnormal estrogen production by the inhibition of two key enzymes that regulate estradiol production (Fig. 3). These actions of DNG contribute, in part, to its therapeutic effect on endometriosis.
Scheme summarizing dienogest inhibition of estrogen production in endometriosis. Dienogest inhibits both aromatase and 17β-hydroxysteroid dehydrogenase 1 (HSD17β1), the key enzymes in estradiol biosynthesis. STS, steroid sulfatase; EST, estrogen sulfotransferase; HSD17β2, 17β-hydroxysteroid dehydrogenase 2.
Citation: Journal of Endocrinology 225, 2; 10.1530/JOE-15-0052
In order to comprehensively examine the expression patterns of estrogen-metabolizing enzymes in OESCs compared with those in EESCs or NESCs, we employed a three-dimensional spheroid culture system characterized by multicellular aggregates of cells and extracellular matrices. This culture system produces sufficient baseline levels of proinflammatory factors (Enzerink et al. 2009, Vaheri et al. 2009), and spheroids of human immortalized endometrial epithelial cells (Shimizu et al. 2011) and primary cultured OESCs (Yamanaka et al. 2012) express higher levels of aromatase, COX2, and prostaglandin E2 compared with the corresponding monolayer cultures. The results of this study indicated that HSD17β1 localizes in the cytoplasm of both epithelial cells and SCs. In OE, however, the majority of cells are SCs. Thus, the OESC spheroids are considered to mimic the local environment of enzyme expression.
In the spheroid-cultured OESCs, we detected very low level of aromatase mRNA expression, whereas it was not detectable in EESCs or NESCs. This is in agreement with results from earlier studies, in which aromatase is only detectable in studies using immunohistochemistry and those using homogenized specimens just after sampling but not in monolayer cultured cells (Kitawaki et al. 1997, Dassen et al. 2007, Smuc et al. 2007). In OESCs, we detected overexpression of HSD17β1 mRNA and protein levels, responsible for activating estrogenic potency, whereas very low levels of mRNA and protein expression were observed in EESCs and NESCs. In contrast, the mRNA expression of HSD17β2, responsible for weakening estrogenic potency, was significantly lower in OESCs compared with EESCs and NESCs. This balance between the expressions of the two enzymes indicates that estradiol is more likely to be produced in OESCs compared with eutopic endometrium, which is concordant with results from previous studies (Zeitoun et al. 1998, Matsuzaki et al. 2006).
Furthermore, the expression of STS, also responsible for activating estrogenic potency, was significantly higher in OESCs compared with EESCs and NESCs, which is also consistent with previous findings (Utsunomiya et al. 2004, Colette et al. 2013). Dassen et al. (2007) reported high STS mRNA expression in both eutopic and ectopic endometrium, but no difference between the two tissues.
In contrast to the high levels of expression of STS, we detected very low levels of mRNA expression of EST, responsible for inactivating estrone in OESCs, whereas it was not detectable in EESCs or NESCs. Colette et al. (2013) reported very low levels of EST mRNA expression in both eutopic and ectopic endometrium, but found no difference between the two tissues. Utsunomiya et al. (2004) showed that EST was expressed in the endometrium but only during the secretory phase. In this study, we obtained specimens during the proliferative phase to eliminate the effect of P4. This balance of the STS and EST expression indicates that estrone is more favorably produced in OESCs as well as EESCs and NESCs during the proliferative phase. The significance of the differences in expression of STS and EST between these cells remains to be elucidated.
Using this experimental model, it was determined that DNG significantly inhibited HSD17 β 1 mRNA expression and its enzyme activity at 10−7 M in OESCs. These concentrations are equivalent to the blood level of mice administered 1 mg of DNG twice daily and patients administered 2 mg of DNG daily (Meriggiola et al. 2002, Sasagawa et al. 2008). The in vitro inhibitory effects of DNG are supported by the in vivo data from this study, demonstrating that DNG treatment for 3–5 months resulted in decreased HSD17β1 protein expression in OE.
The conversion of estrone to estradiol is also mediated though HSD17β7 and HSDβ12 as well as HSDβ1 (Moeller & Adamski 2006). DNG inhibited only HSD17 β 1 but not 17HSD β 7 or 12 mRNA expression, which indicates that DNG reduces local estrogen production by the suppression of HSD17β1 in human OESCs.
We and other researchers have shown that the PR is involved in the mechanism of DNG-inhibited cell proliferation (Okada et al. 2001, Shimizu et al. 2009) and the expression of inflammatory factors (Mita et al. 2011), nerve growth factor, (Mita et al. 2014), and aromatase (Yamanaka et al. 2012). Although the HSD17 β 1 gene lacks a P4-responsive element in its promoter region, progestins including DNG downregulated HSD17 β 1 and upregulated HSD17 β 2 expression in immortalized endometriotic epithelial cells (Beranic & Rizner 2012). DNG inhibits the DNA-binding activity of NFκB, a key regulator of various pathological and inflammatory responses in endometriosis such as interleukin-8 production in human OESCs (Horie et al. 2005, Shimizu et al. 2011, Yamanaka et al. 2012). Bulun proposed a vicious cycle of an estrogen-dependent mechanism of endometriosis growth (Bulun 2009). Estradiol produced locally by aromatase stimulates tissue growth of endometriosis and upregulates COX2 via ERβ activation. COX2 overexpression results in an excess of prostaglandin E2, which further stimulates aromatase expression via the orphan nuclear receptor steroidogenic factor 1. Furthermore, ERβ activation downregulates PR, which leads to reduced induction of HSD17β2 via retinoic acid (Zeitoun et al. 1998). The combination of upregulation of aromatase and downregulation of HSD17β2 contributes to the abnormally high levels of estradiol in endometriotic tissue. In addition to inhibition of aromatase, DNG inhibits HSD17β1, resulting in further reductions in local estradiol concentration. This interrupts the vicious cycle of endometriosis growth and also relieves endometriosis-associated pelvic pain by inhibiting prostaglandin E2 production. The effect of inhibiting HSD17β1 on endometriosis has been demonstrated (Delvoux et al. 2014) and several HSD17β1 inhibitors have been developed and used in preclinical studies (Day et al. 2008, Poirier 2011). DNG is widely used in clinics to treat endometriosis with fewer side effects. We believe that the identification of the molecular mechanisms behind the therapeutic effect of DNG described here will lead to better understanding of the pathophysiology of endometriosis.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported in part by a Grant-in-Aid for Scientific Research 24592480 and 23197849 from the Ministry of Education, Culture, Sports, Science and Technology (Japan).
References
Beranic N & Rizner TL 2012 Effects of progestins on local estradiol biosynthesis and action in the Z-12 endometriotic epithelial cell line. Journal of Steroid Biochemistry and Molecular Biology 132 303–310. (doi:10.1016/j.jsbmb.2012.07.004)
Bulun SE 2009 Endometriosis. New England Journal of Medicine 360 268–279. (doi:10.1056/NEJMra0804690)
Colette S , Defrère S , Van Kerk O , Van Langendonckt A , Dolmans MM & Donnez J 2013 Differential expression of steroidogenic enzymes according to endometriosis type. Fertility and Sterility 100 1642–1649. (doi:10.1016/j.fertnstert.2013.08.003)
Dassen H , Punyadeera C , Kamps R , Delvoux B , Van Langendonckt A , Donnez J , Husen B , Dunselman G & Groothuis P 2007 Estrogen metabolizing enzymes in endometrium and endometriosis. Human Reproduction 22 3148–3158. (doi:10.1093/humrep/dem310)
Day JM , Tutill HJ , Purohit A & Reed MJ 2008 Design and validation of specific inhibitors of 17β-hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer, and in endometriosis. Endocrine-Related Cancer 15 665–692. (doi:10.1677/ERC-08-0042)
Delvoux B , D'Hooghe T , Kyama C , Koskimies P , Hermans RJ , Dunselman GA & Romano A 2014 Inhibition of type 1 17β-hydroxysteroid dehydrogenase impairs the synthesis of 17β-estradiol in endometriosis lesions. Journal of Clinical Endocrinology and Metabolism 99 276–284. (doi:10.1210/jc.2013-2851)
Enzerink A , Salmenpera P , Kankuri E & Vaheri A 2009 Clustering of fibroblasts induces proinflammatory chemokine secretion promoting leukocyte migration. Molecular Immunology 46 1787–1795. (doi:10.1016/j.molimm.2009.01.018)
Fu L , Osuga Y , Morimoto C , Hirata T , Hirota Y , Yano T & Taketani Y 2008 Dienogest inhibits BrdU uptake with G0/G1 arrest in cultured endometriotic stromal cells. Fertility and Sterility 89 1344–1347. (doi:10.1016/j.fertnstert.2007.03.042)
Giudice LC 2010 Clinical practice. Endometriosis. New England Journal of Medicine 362 2389–2398. (doi:10.1056/NEJMcp1000274)
Harada T , Momoeda M , Taketani Y , Aso T , Fukunaga M , Hagino H & Terakawa N 2009 Dienogest is as effective as intranasal buserelin acetate for the relief of pain symptoms associated with endometriosis – a randomized, double-blind, multicenter, controlled trial. Fertility and Sterility 91 675–681. (doi:10.1016/j.fertnstert.2007.12.080)
Horie S , Harada T , Mitsunari M , Taniguchi F , Iwabe T & Terakawa N 2005 Progesterone and progestational compounds attenuate tumor necrosis factor alpha-induced interleukin-8 production via nuclear factor kappaB inactivation in endometriotic stromal cells. Fertility and Sterility 83 1530–1535. (doi:10.1016/j.fertnstert.2004.11.042)
Kitawaki J , Noguchi T , Amatsu T , Maeda K , Tsukamoto K , Yamamoto T , Fushiki S , Osawa Y & Honjo H 1997 Expression of aromatase cytochrome P450 protein and messenger ribonucleic acid in human endometriotic and adenomyotic tissues but not in normal endometrium. Biology of Reproduction 57 514–519. (doi:10.1095/biolreprod57.3.514)
Kitawaki J , Koshiba H , Ishihara H , Kusuki I , Tsukamoto K & Honjo H 2000 Progesterone induction of 17β-hydroxysteroid dehydrogenase type 2 during the secretory phase occurs in the endometrium of estrogen-dependent benign diseases but not in normal endometrium. Journal of Clinical Endocrinology and Metabolism 85 3292–3296. (doi:10.1210/jcem.85.9.6829)
Maeda N , Izumiya C , Taniguchi K , Matsushima S , Mita S , Shimizu Y & Fukaya T 2014 Dienogest improves human leucocyte antigen-DR underexpression and reduces tumour necrosis factor-α production in peritoneal fluid cells from women with endometriosis. European Journal of Obstetrics, Gynecology, and Reproductive Biology 177 48–51. (doi:10.1016/j.ejogrb.2014.03.019)
Matsuzaki S , Canis M , Pouly JL , Déchelotte PJ & Mage G 2006 Analysis of aromatase and 17β-hydroxysteroid dehydrogenase type 2 messenger ribonucleic acid expression in deep endometriosis and eutopic endometrium using laser capture microdissection. Fertility and Sterility 85 308–313. (doi:10.1016/j.fertnstert.2005.08.017)
McCormack PL 2010 Dienogest: a review of its use in the treatment of endometriosis. Drugs 70 2073–2088. (doi:10.2165/11206320-000000000-00000)
Meriggiola MC , Bremner WJ , Costantino A , Bertaccini A , Morselli-Labate AM , Huebler D , Kaufmann G , Oettel M & Flamigni C 2002 Twenty-one day administration of dienogest reversibly suppresses gonadotropins and testosterone in normal men. Journal of Clinical Endocrinology and Metabolism 87 2107–2113. (doi:10.1210/jcem.87.5.8514)
Mita S , Shimizu Y , Notsu T , Imada K & Kyo S 2011 Dienogest inhibits Toll-like receptor 4 expression induced by costimulation of lipopolysaccharide and high-mobility group box 1 in endometrial epithelial cells. Fertility and Sterility 96 1485–1489. (doi:10.1016/j.fertnstert.2011.09.040)
Mita S , Shimizu Y , Sato A , Notsu T , Imada K & Kyo S 2014 Dienogest inhibits nerve growth factor expression induced by tumor necrosis factor-α or interleukin-1β. Fertility and Sterility 101 595–601. (doi:10.1016/j.fertnstert.2013.10.038)
Moeller GM & Adamski J 2006 Multifunctionality of human 17β-hydroxysteroid dehydrogenases. Molecular and Cellular Endocrinology 248 47–55. (doi:10.1016/j.mce.2005.11.031)
Momoeda M , Harada T , Terakawa N , Aso T , Fukunaga M , Hagino H & Taketani Y 2009 Long-term use of dienogest for the treatment of endometriosis. Journal of Obstetrics and Gynaecology Research 35 1069–1076. (doi:10.1111/j.1447-0756.2009.01076.x)
Noble LS , Simpson ER , Johns A & Bulun SE 1996 Aromatase expression in endometriosis. Journal of Clinical Endocrinology and Metabolism 81 174–179. (doi:10.1210/jcem.81.1.8550748)
Okada H , Nakajima T , Yoshimura T , Yasuda K & Kanzaki H 2001 The inhibitory effect of dienogest, a synthetic steroid, on the growth of human endometrial stromal cells in vitro. Molecular Human Reproduction 7 341–347. (doi:10.1093/molehr/7.4.341)
Petraglia F , Hornung D , Seitz C , Faustmann T , Gerlinger C , Luisi S , Lazzeri L & Strowizki T 2012 Reduced pelvic pain in women with endometriosis: efficacy of long-term dienogest treatment. Archives of Gynecology and Obstetrics 285 167–173. (doi:10.1007/s00404-011-1941-7)
Poirier D 2011 Contribution to the development of inhibitors of 17β-hydroxysteroid dehydrogenase types 1 and 7: key tools for studying and treating estrogen-dependent diseases. Journal of Steroid Biochemistry and Molecular Biology 125 83–94. (doi:10.1016/j.jsbmb.2010.12.007)
Sasagawa S , Shimizu Y , Kami H , Takeuchi T , Mita S , Imada K , Kato S & Mizuguchi K 2008 Dienogest is a selective progesterone receptor agonist in transactivation analysis with potent oral endometrial activity due to its efficient pharmacokinetic profile. Steroids 73 222–231. (doi:10.1016/j.steroids.2007.10.003)
Shimizu Y , Takeuchi T , Mita S , Mizuguchi K , Kiyono T , Inoue M & Kyo S 2009 Dienogest, a synthetic progestin, inhibits the proliferation of immortalized human endometrial epithelial cells with suppression of cyclin D1 gene expression. Molecular Human Reproduction 15 693–701. (doi:10.1093/molehr/gap042)
Shimizu Y , Mita S , Takeuchi T , Notsu T , Mizuguchi K & Kyo S 2011 Dienogest, a synthetic progestin, inhibits prostaglandin E2 production and aromatase expression by human endometrial epithelial cells in a spheroid culture system. Steroids 76 60–67. (doi:10.1016/j.steroids.2010.08.010)
Smuc T , Pucelj MR , Sinkovec J , Husen B , Thole H & Lanisnik Rizner T 2007 Expression analysis of the genes involved in estradiol and progesterone action in human ovarian endometriosis. Gynecological Endocrinology 23 105–111. (doi:10.1080/09513590601152219)
Strowitzki T , Marr J , Gerlinger C , Faustmann T & Seitz C 2010 Dienogest is as effective as leuprolide acetate in treating the painful symptoms of endometriosis: a 24-week, randomized, multicentre, open-label trial. Human Reproduction 25 633–641. (doi:10.1093/humrep/dep469)
Utsunomiya H , Ito K , Suzuki T , Kitamura T , Kaneko C , Nakata T , Kiikura H , Okamura K , Yaegashi N & Sasano H 2004 Steroid sulfatase and estrogen sulfotransferase in human endometrial carcinoma. Clinical Cancer Research 10 5850–5856. (doi:10.1158/1078-0432.CCR-04-0040)
Vaheri A , Enzerink A , Rasanen K & Salmenpera P 2009 Nemosis, a novel way of fibroblast activation, in inflammation and cancer. Experimental Cell Research 315 1633–1638. (doi:10.1016/j.yexcr.2009.03.005)
Yamanaka K , Xu B , Suganuma I , Kusuki I , Mita S , Shimizu Y , Mizuguchi K & Kitawaki J 2012 Dienogest inhibits aromatase and cyclooxygenase-2 expression and prostaglandin E2 production in human endometriotic stromal cells in spheroid culture. Fertility and Sterility 97 477–482. (doi:10.1016/j.fertnstert.2011.11.005)
Zeitoun K , Takayama K , Sasano H , Suzuki T , Moghrabi N , Andersson S , Johns A , Meng L , Carr B & Bulun SE 1998 Deficient 17β-hydroxysteroid dehydrogenase type 2 expression in endometriosis: failure to metabolize 17β-estradiol. Journal of Clinical Endocrinology and Metabolism 83 4474–4480. (doi:10.1210/jcem.83.12.5301)
