{"paper_id":"39566313-dfd1-499c-84f8-dc4bcd27e839","body_text":"Dienogest reduces HSD17 b1\nexpression and activity in\nendometriosis\nTaisuke Mori, Fumitake Ito, Hiroshi Matsushima, Osamu Takaoka, Akemi Koshiba,\nYukiko Tanaka, Izumi Kusuki and Jo Kitawaki\nDepartment of Obstetrics and Gynecology, Graduate School of Medical Science, Kyoto Prefectural University of\nMedicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan\nCorrespondence\nshould be addressed\nto T Mori\nEmail\nmoriman@koto.kpu-m.ac.jp\nAbstract\nEndometriosis is an estrogen-dependent disease. Abnormally biosynthesized estrogens in\nendometriotic tissues induce the growth of the lesion and worsen endometriosis-associated\npelvic pain. Dienogest (DNG), a selective progesterone receptor agonist, is widely used to treat\nendometriosis and efficiently relieves the symptoms. However, its pharmacological action\nr e m a i n su n k n o w n .I nt h i ss t u d y ,w ee l u c i d a t e dthe effect of DNG on enzymes involved in local\nestrogen metabolism in endometriosis. Surgically obtained specimens of 23 ovarian endome-\ntriomas (OE) and their homologous endometrium (EE), ten OE treated with DNG (OE w/D), and\n19 normal endometria without endometriosis(NE) were analyzed. Spheroid cultures of\nstromal cells (SCs) were treated with DNG and progesterone. The expression of aromatase,\n17b-hydroxysteroid dehydrogenase 1 (HSD17b1), HSD17b2, HSD17b7, HSD17b12, steroid\nsulfatase (STS), and estrogen sulfotransferase (EST) was evaluated by real-time quantitative PCR.\nThe activity and protein level of HSD17b1 were measured with an enzyme assay using\nradiolabeled estrogens and immunohistochemistry respectively. OESCs showed increased\nexpression of aromatase, HSD17b1, STS, and EST, along with decreased HSD17b2 expression, when\ncompared with stromal cells from normal endometria without endometriosis (NESCs) (P!0.01)\nor stromal cells from homologous endometrium (EESCs) (P!0.01). In OESCs, DNG inhibited\nHSD17b1e x p r e s s i o na n de n z y m ea c t i v i t ya t1 0\nK7 M( P!0.01). Results of immunohistochemical\nanalysis displayed reduced HSD17b1 staining intensity in OE w/D (P!0.05). In conclusion, DNG\nexerts comprehensive inhibition of abnormal estrogen production through inhibition of\naromatase and HSD17b1, contributing to a therapeutic effect of DNG on endometriosis.\nKey Words\n\" 17b-hydroxysteroid\ndehydrogenase 1\n\" dienogest\n\" endometriosis\n\" ovarian endometrioma\n\" spheroid culture\nJournal of Endocrinology\n(2015) 225, 69–76\nIntroduction\nEndometriosis is defined as the presence of endometrium-\nlike tissues at extra-uterine sites. Clinical symptoms\nassociated with endometriosis include pelvic pain, dys-\nmenorrhea, dyspareunia, and infertility ( Giudice 2010 ).\nThere is marked relief of symptoms after menopause,\nclearly demonstrating the dependency of endometriosis\non estrogens. Besides syste mic circulating estrogens\nsecreted from the ovaries, abnormally biosynthesized\nestrogens in endometriotic tissues also contribute to\nthe growth of the lesion and worsening symptoms\n(Bulun 2009). In the eutopic and ectopic endometria of\nwomen with endometriosis, overexpressed aromatase\n(also known as estrogen synthase) biosynthesizes estro-\ngens, namely estrone and estradiol, from the androgens,\nJournal of Endocrinology\nResearch\nT MORI and others Dienogest inhibits HSD17 b1i n\nendometriosis\n225:2 69–76\nhttp://joe.endocrinology-journals.org /C2092015 Society for Endocrinology\nDOI: 10.1530/JOE-15-0052 Printed in Great Britain\nPublished by Bioscientifica Ltd.\nDownloaded from Bioscientifica.com at 06/23/2026 12:54:26PM\nvia free access\n\n\nandrostenedione, and testosterone respectively ( Noble\net al . 1996 , Kitawaki et al . 1997 , Matsuzaki et al . 2006 ,\nDassen et al . 2007 , Smuc et al . 2007 ). In these tissues,\nestradiol, the most potent estrogen, is predominantly\nsynthesized from less potent estrone by 17 b-hydroxy-\nsteroid dehydrogenase 1 (HSD17 b1), and the reverse\nreaction is catalyzed mainly by HSD17b2. In endometrio-\ntic tissues, the expression of HSD17b1 is higher than that\nof HSD17 b2; thus, the reaction is tilted in favor of\nproducing estradiol ( Zeitoun et al . 1998 , Dassen et al .\n2007). The other major source of estrogens is estrone\nsulfate, an inactive conjugated form abundant in the\ncirculation. Estrone sulfate is desulfated to estrone by\nsteroid sulfatase (STS) and estrone is inactivated by\nestrogen sulfotransferase (EST) ( Utsunomiya et al . 2004 ,\nColette et al . 2013 ). Understanding how the aberrant\nexpression of these enzymes in endometriosis contributes\nto local estrogen production and metabolism will allow\nthe development of improved therapeutic agents.\nDienogest (DNG), a selective progesterone (P\n4)\nreceptor (PR) agonist, is widely used to treat endometriosis\n(McCormack 2010) and efficiently relieves endometriosis-\nassociated pelvic pain (Harada et al. 2009, Momoeda et al.\n2009, Strowitzki et al . 2010 , Petraglia et al . 2012 ). DNG\ndirectly inhibits PR-mediated cell proliferation ( Okada\net al . 2001 , Fu et al . 2008 , Shimizu et al . 2009 ) and\nproduction of the inflammatory factors involved in the\npathology of endometriosis, such as prostaglandin estra-\ndiol (E\n2)( Shimizu et al . 2011 , Yamanaka et al . 2012 ),\ninflammatory cytokines ( Horie et al .2 0 0 5), Toll-like\nreceptor 4 ( Mita et al . 2011 ), and nerve growth factor\n(Mita et al . 2014 ). Supprerssion of these inflammatory\nfactors is considered to contribute, in part, to the\nimprovement of pain symptoms. DNG restores the\nantigen-presenting ability of peritoneal fluid macrophages\nby increasing human leukocyte antigen-DR expression\n(Maeda et al . 2014 ). DNG also suppresses aromatase\nexpression in human immortalized endometrial epithelial\ncells (Shimizu et al . 2011) and primary cultured stromal\ncells (SCs) derived from ovarian endometrioma (OE)\n(Yamanaka et al . 2012 ). However, the effect of DNG on\nother estrogen-metabolizing enzymes in endometriotic\ncells remains unknown, and a more detailed analysis is\nneeded to understand its cli nical effectiveness and\npharmacological function.\nThe purpose of this study was to investigate the effect\nof DNG on enzymes involved in estrogen metabolism\nusing spheroid cultures of primary cultured SCs derived\nfrom OE, endometrium with endometriosis (EE), and\nnormal endometrium without endometriosis (NE).\nPatients and methods\nPatients and samples\nPatient characteristics are given inTable 1. OE tissues from\npatients ( nZ23) who did not receive any hormonal\ntreatment and their homologous EE specimens ( nZ10),\nin addition to OE specimens from patients treated with\nDNG at a dose of 1 mg twice daily for 3–5 months (OE\ntreated with DNG (OE w/D)) (nZ11), were obtained from\nwomen undergoing surgery for OE. NE specimens were\nobtained from women undergoing surgery for uterine\nfibroids (nZ19). All women were of reproductive age, and\nall specimens, with the exception of OE w/D, were\ncollected at the proliferative phase of the regular\nmenstrual cycle. Women who had undergone hormonal\ntreatments within 6 months before surgery were excluded.\nOE w/D specimens were not used for in vitro experiments\nto avoid the effect of previous DNG exposure on the\nresults. The endometriosis stages were evaluated according\nto the American Society for Reproductive Medicine\nclassification of endometriosis. This study was conducted\nin accordance with the guidelines of the Declaration of\nTable 1 Clinical characteristics of study patients. Values are presented as means GS.E.M.\nNE ( nZ19) EE ( nZ10) OE ( nZ23) OE w/D ( nZ11)\nAge (years) 41.8 G4.3 41.4 G3.9 32.5 G7.0*,† 37.5G4.2\nCA-125 (U/ml) NA 68.9 G29.1 82.5 G83.4 68.0 G55.7\nr-ASRM stage (%)\nIII NA 6 (60) 14 (61) 6 (55)\nIV 4 (40) 9 (39) 5 (45)\nDuration of drug\nadministration (weeks)\nNA NA NA 13.4 G6.0\nP values were obtained by Kruskal–Wallis ANOVA followed by multiple comparisons using Scheffe’s procedure or\nc2 test. NE, normal endometrium; EE, endometrium with endometriosis; OE, ovarian endometrioma; OE w/D,\nOE treated with dienogest; r-ASRM, revised American Society for Reproductive Medicine; DNG, dienogest. * P!0.01\nversus NE and †P!0.05 versus EE.\nJournal of Endocrinology\nResearch T MORI and others Dienogest inhibits HSD17 b1i n\nendometriosis\n225:2 70\nhttp://joe.endocrinology-journals.org /C2092015 Society for Endocrinology\nDOI: 10.1530/JOE-15-0052 Printed in Great Britain\nPublished by Bioscientifica Ltd.\nDownloaded from Bioscientifica.com at 06/23/2026 12:54:26PM\nvia free access\n\n\nHelsinki and was approved by the institutional review\nboard of the Kyoto Prefectural University of Medicine.\nInformed consent was obtained from all patients.\nIsolation and culture of SCs\nThe isolation and culture of SCs was conducted as\ndescribed previously (Yamanaka et al. 2012). Briefly, tissue\ndigestion was performed with 2.5% collagenase (Nacalai\nTesque, Kyoto, Japan) and 15 IU/ml of DNase I (Takara\nShuzo, Tokyo, Japan). After filtering through a nylon cell\nstrainer, the digested cells were centrifuged in lymphocyte\nseparation solution (Nacalai Tesque) to remove the red\nblood cells. The O95% purity of SC preparations was\nconfirmed by positive staining for CD10 and vimentin and\nnegative staining for cytokeratin, CD31, and CD45. The\ncells were cultured in DMEM/Ham’s F-12 (Nacalai Tesque)\nsupplemented with 10% fetal bovine serum (FBS; Invitro-\ngen) and 1% penicillin and streptomycin (100 mg/ml),\nunder a humidified atmosphere at 37 8Ci n5 %C O 2. The\ncells that reached subconfluence were dispersed using\n0.1% trypsin (Nacalai Tesque) and resuspended in\nphenol-red-free DMEM/Ham’s F-12 (Nacalai Tesque) sup-\nplemented with 10% dextran-coated charcoal-treated\nFBS and 1% penicillin and streptomycin (100 mg/ml). For\nmRNA analysis, SCs were subcultured in U-bottom 96-well\nculture plates (Sumilon) at a density of 4 !104 cells/well\nto form spheroids. For HSD17 b1 activity assays, SCs\nwere plated into six-well culture plates at a density of\n4!105 cells/well to form monolayers. The OESC spheroid\nexpression of estrogen receptora (ERa (ESR1)), ERb (ESR2),\nPR, aromatase, cyclooxygenase-2 (COX2), and nuclear\nfactor-k B (NF kB) p50 subunit nuclear localization was\nvalidated by immunocytochemistry as described pre-\nviously (Yamanaka et al. 2012).\nTreatment of cultured SCs with DNG or P 4\nThe culture medium was replaced either after 72 h\n(spheroid culture for RNA extraction) or when cells\nreached subconfluence (HSD17 b1 activity assay) by\nmedium with or without DNG (10 K8,1 0 K7,a n d\n10K6 M; Bayer Schering Pharma, Berlin, Germany) or P 4\n(10K8,1 0 K7, and 10 K6 M; Sigma–Aldrich), and the cells\nwere incubated for a further 48 h.\nRNA extraction, cDNA preparation, and real-time PCR\nTotal RNA was extracted from cultured SCs using the\nRNeasy Mini Kit (Qiagen). After quantification and\ndetermination of the quality of the RNA by u.v. absorption\n(OD 260 nm/280 nm) using a NanoDrop Spectropho-\ntometer (Thermo Scientific, Waltham, MA, USA), cDNA\nwas synthesized using the SuperScript III first-strand\nsynthesis system (Invitrogen) and a GeneAmp PCR 9700\nmachine (Applied Biosystems). Quantitative real-time PCR\nwas conducted using TaqMan Fast Universal PCR Master\nMix (Applied Biosystems) and a StepOne Real-Time PCR\nSystem (Applied Biosystems) with TaqMan assay primer/\nprobe sets (Applied Biosystems) for the target genes:\naromatase ( CYP19A1 ) (Hs00240671_m1), HSD17 b1\n(Hs00166219_g1), HSD17b2 (Hs00157993_m1), HSD17b7\n(Hs00367686_m1), HSD17 b12 (Hs00275054_m1), STS\n(Hs00996676_m1), EST (SULT1E1) (Hs00960941_m1),\nand endogenous control GAPDH (Hs03929097_g1). Real-\ntime quantitative PCR was performed under the following\nthermal cycling conditions: denaturing at 95 8C for 60 s;\n3 s at 95 8C; 40 cycles of 30 s at 608C. Threshold cycle (Ct)\nvalues were calculated using the DDCt method.\nHSD17b1 enzyme assays\nHSD17b1 activity was measured using thin layer chroma-\ntography, as described previously ( Kitawaki et al . 2000 ).\nBriefly, cells were washed twice with phenol-red-free\nDMEM/Ham’s F-12, and then incubated at 37 8C/5% CO\n2\nfor 6 h with 0.5 ml of serum-free medium containing\n[6,7- 3H]estrone (Perkin Elmer, Waltham, MA, USA)\n(1.8!106 dpm, 37 mM). The reaction was stopped by\ntransferring the medium to the test tubes containing\n2 ml chloroform and the corresponding carrier steroids:\n[4-\n14C]estradiol (Perkin Elmer) (1.3 !104 dpm) and non-\nradioactive estrone and est radiol (0.2 mg each). The\nsteroids were isolated by thin-layer chromatography\nusing Silicagel 60 F254 (0.25 mm; Merck) in a system of\nchloroform:ethyl acetate (4:1, v/v). The aliquot was mixed\nwith Clear-sol I (Nacalai Tesque), and radioactivity was\nmeasured using a scintillation counter (Beckman Coulter,\nFullerton, CA, USA). Enzyme activity was calculated and\nnormalized according to the ratios of the estradiol formed.\nProtein concentration (pmol/mg protein per h) was\nmeasured by the Bradford method.\nImmunohistochemistry\nSpecimens from OE, OE w/D, EE, and NE were stained\nimmunohistochemically as described previously (Yamanaka\net al. 2012) using an anti-HSD17 b1 antibody (200 mg/ml;\nAbcam, Cambridge, UK). Normal term placenta tissue was\nused as a positive control. Because the cell components\nJournal of Endocrinology\nResearch T MORI and others Dienogest inhibits HSD17 b1i n\nendometriosis\n225:2 71\nhttp://joe.endocrinology-journals.org /C2092015 Society for Endocrinology\nDOI: 10.1530/JOE-15-0052 Printed in Great Britain\nPublished by Bioscientifica Ltd.\nDownloaded from Bioscientifica.com at 06/23/2026 12:54:26PM\nvia free access\n\n\nof epithelial cells and SCs were considerably different\nbetween eutopic endometrium and OE, we compared\nthe immunostaining intensity in SCs using the H-score,\na semi-quantitative index involving an algorithm\ndescribed previously ( Yamanaka et al . 2012). Briefly, two\nindependent observers evaluated approximately 500 cells/\nslide and scored them as follows: 3 !percentage of\nstrongly staining cells C2!percentage of moderately\nstaining cells Cpercentage of weakly staining cells. The\nH-score was calculated as the mean of the two scores.\nStatistical analyses\nThe mRNA expression levels of enzymes in the three types\nof SCs were analyzed by Kruskal–Wallis ANOVA followed\nby multiple comparisons using Scheffe’s procedure\nbecause of the unequal variances in the results. Results\nof real-time PCR and the HSD17 b1 activity assay measur-\ning the drugs’ effects on mRNA expression and enzyme\nactivity levels in OESCs were assessed by repeated\nmeasures ANOVA followed by multiple comparisons\nusing Dunnett’s procedure. Statistical analysis of the\nimmunohistochemical results was performed using an\nunpaired t-test. Each assay for individual experiments was\nperformed in triplicate. Data are presented as means G\nS.E.M. P values of !0.05 were considered statistically\nsignificant.\nResults\nmRNA expression of enzymes in spheroid-cultured SCs\nIn OESCs, the mRNA expression levels of aromatase\n(P!0.01), HSD17b1 (P!0.01), STS (P!0.01), and EST\n(P!0.01) were greater compared with those in NESCs and\nEESCs. In NESCs or EESCs, we detected neither aromatase\nnor EST mRNA expression and an extremely low level of\nHSD17b1 mRNA expression. In contrast, HSD17b2 mRNA\nexpression was lower in OESCs compared with that in\nNESCs (P!0.01) and EESCs (P!0.01) (Fig. 1A).\nEffects of drugs on enzymes in OESCs\nIncubating the spheroids for 48 h with DNG (10 K7 M\n(P!0.01) and 10 K6 M( P!0.01)) and P 4 (10K7 M\n(P!0.05) and 10 K6 M( P!0.01)) significantly decreased\nControl\nControl\nHSD17β1\nHSD17β1 HSD17 β2 STS EST\nNE\nEE\nOE\nNDNDNDND\nAromatase\nHSD17β7 HSD17 β12 HSD17 β2 EST\nDNG P 4 DNG P 4DNG P 4 DNG P 4DNG\nDNG (M) P 4 (M)\nP4 DNG P 4\nSTS\n2.0\n1.8\n1.6\n1.4\n1.2\n1.0\n**\n**\n**\n*\n**\n**\n**\n**\n**\n**\n**\n**\n**\n**\n** **\n**\n**\n*\nGene ¥GAPDH (relative to control value)\n0.8\n0.6\n0.4\n0.2\n0.0\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\nHSD17β1 enzyme activity\n(relative to control value)\n1.2\nA\nC\nB\n1\n10\n100\nmRNA level/GAPDH\n1000\n×10–5\n10 000\n10\n–8\n10–8 10–7 10–6 10–8 10–7 10–6\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\n10\n–8\n10\n–7\n10\n–6\nFigure 1\n(A) The baseline mRNA expression levels of estrogen-metabolizing\nenzymes in spheroid-cultured stromal cells (SCs) derived from normal\nendometrium (NE), endometrium with endometriosis (EE), and OE.\n(B) Effects of dienogest (DNG) and progesterone (P\n4) on mRNA expression\nlevels of enzymes in OESCs (at least seven separate experiments\nrespectively) and on (C) 17 b-hydroxysteroid dehydrogenase 1 (HSD17 b1)\nactivity in OESCs ( nZ8). All assays were performed in triplicate, and data\nare presented as means GS.E.M. P values for the statistical analysis of the\nmRNA expression of enzymes in SCs in spheroid culture are based on\nKruskal–Wallis ANOVA followed by multiple comparisons using Scheffe’s\nprocedure, and effects of drugs on OESC enzyme mRNA expression\nand activity are based on repeated measures ANOVA followed by\nmultiple comparisons using Dunnett’s procedure. ND, not detectable;\n*P!0.05 and ** P!0.01 versus control.\nJournal of Endocrinology\nResearch T MORI and others Dienogest inhibits HSD17 b1i n\nendometriosis\n225:2 72\nhttp://joe.endocrinology-journals.org /C2092015 Society for Endocrinology\nDOI: 10.1530/JOE-15-0052 Printed in Great Britain\nPublished by Bioscientifica Ltd.\nDownloaded from Bioscientifica.com at 06/23/2026 12:54:26PM\nvia free access\n\n\nthe expression of HSD17b1 mRNA compared with the\ncontrols. However, DNG or P 4 did not significantly\nsuppress HSD17b2, HSD17b7, HSD17b12, STS,o r EST\nmRNA expression ( Fig. 1 B). In parallel with the mRNA\nresults, DNG (10 K7 M( P!0.01) and 10 K6 M( P!0.01))\nand P4 (10K8 M( P!0.05), 10K7 M( P!0.01), and 10K6 M\n(P!0.01)) significantly suppressed the catalytic activity\nof HSD17b1( Fig. 1C).\nImmunohistochemistry\nImmunohistochemical analysis showed HSD17 b1\nexpression in the cytoplasm of epithelial cells and SCs\nof NE ( Fig. 2 A), EE ( Fig. 2 B), and OE ( Fig. 2 C). The\nimmunostaining intensity of HSD17 b1 was greater in\nO E S C sw h e nc o m p a r e dw i t hN E(P!0.01) and EE\n(P!0.01). Moreover, there was a significant reduction\nin the immunostaining intensity in the OE w/D group\n(P!0.05) (Fig. 2E).\nDiscussion\nIn this study, we demonstrated DNG-mediated inhibition\nof mRNA expression, catalyt ic activity, and protein\nexpression of HSD17 b1 in endometriosis. Taken together\nwith the previous findings that DNG inhibits aromatase in\nendometriosis (Shimizu et al. 2011, Yamanaka et al. 2012),\nDNG exerts comprehensive inhibition of abnormal\nestrogen production by the inhibition of two key enzymes\nthat regulate estradiol production ( Fig. 3). These actions\nof DNG contribute, in part, to its therapeutic effect on\nendometriosis.\nIn order to comprehensively examine the expression\npatterns of estrogen-metabolizing enzymes in OESCs\ncompared with those in EESCs or NESCs, we employed a\nthree-dimensional spheroid culture system characterized\nby multicellular aggregates of cells and extracellular\nmatrices. This culture system produces sufficient baseline\nlevels of proinflammatory factors ( Enzerink et al . 2009 ,\nVaheri et al. 2009), and spheroids of human immortalized\nendometrial epithelial cells ( Shimizu et al . 2011 ) and\nprimary cultured OESCs ( Yamanaka et al . 2012 ) express\nhigher levels of aromatase, COX2, and prostaglandin E 2\ncompared with the corresponding monolayer cultures.\nThe results of this study indicated that HSD17b1 localizes\nin the cytoplasm of both epithelial cells and SCs. In OE,\nhowever, the majority of cells are SCs. Thus, the OESC\nAB\nCD\nE\n100\nHSD17β1 H-score\n50\n0\nNE EE\n** **\n*\nOE OWwD\nFigure 2\nRepresentative immunohistochemical staining of 17 b-hydroxysteroid\ndehydrogenase 1 (HSD17 b1) in (A) normal endometrium (NE), (B)\nendometrium with endometriosis (EE), (C) ovarian endometrioma (OE), and\n(D) normal term placenta as a positive control. (E) The immunostaining\nintensity of NE ( nZ19), EE ( nZ10), OE ( nZ23), and OE treated with\ndienogest (OE w/D) ( nZ11) was scored with semi-quantitative index\nH-scores. Areas shown at a higher magnification are indicated by\nrectangles. Data are presented as means G\nS.E.M. P values are based on\nan unpaired t-test. * P!0.05 and ** P!0.01 versus OE.\nAndrostenedione Testosterone\nAromatase\nEstradiol\nSTS\nEST\nEstroneEstrone\nsulfate\nHSD17β2\nHSD17β1\nDienogest\nFigure 3\nScheme summarizing dienogest inhibition of estrogen production in\nendometriosis. Dienogest inhibits both aromatase and 17 b-hydroxysteroid\ndehydrogenase 1 (HSD17 b1), the key enzymes in estradiol biosynthesis.\nSTS, steroid sulfatase; EST, estrogen sulfotransferase; HSD17 b2,\n17b-hydroxysteroid dehydrogenase 2.\nJournal of Endocrinology\nResearch T MORI and others Dienogest inhibits HSD17 b1i n\nendometriosis\n225:2 73\nhttp://joe.endocrinology-journals.org /C2092015 Society for Endocrinology\nDOI: 10.1530/JOE-15-0052 Printed in Great Britain\nPublished by Bioscientifica Ltd.\nDownloaded from Bioscientifica.com at 06/23/2026 12:54:26PM\nvia free access\n\n\nspheroids are considered to mimic the local environment\nof enzyme expression.\nIn the spheroid-cultured OESCs, we detected very low\nlevel of aromatase mRNA expression, whereas it was not\ndetectable in EESCs or NESCs. This is in agreement with\nresults from earlier studies, in which aromatase is only\ndetectable in studies using immunohistochemistry and\nthose using homogenized specimens just after sampling\nbut not in monolayer cultured cells ( Kitawaki et al. 1997,\nDassen et al. 2007, Smuc et al. 2007). In OESCs, we detected\noverexpression of HSD17 b1 mRNA and protein levels,\nresponsible for activating estrogenic potency, whereas very\nlow levels of mRNA and protein expression were observed\nin EESCs and NESCs. In contrast, the mRNA expression of\nHSD17b2, responsible for weakening estrogenic potency,\nwas significantly lower in OESCs compared with EESCs\nand NESCs. This balance between the expressions of the\ntwo enzymes indicates that estradiol is more likely to be\nproduced in OESCs compared with eutopic endometrium,\nwhich is concordant with results from previous studies\n(Zeitoun et al. 1998, Matsuzaki et al. 2006).\nFurthermore, the expression of STS, also responsible\nfor activating estrogenic potency, was significantly higher\nin OESCs compared with EESCs and NESCs, which is also\nconsistent with previous findings (Utsunomiya et al. 2004,\nColette et al. 2013). Dassen et al. (2007) reported high STS\nmRNA expression in both eutopic and ectopic endome-\ntrium, but no difference between the two tissues.\nIn contrast to the high levels of expression of STS, we\ndetected very low levels of mRNA expression of EST,\nresponsible for inactivating estrone in OESCs, whereas it\nwas not detectable in EESCs or NESCs.Colette et al. (2013)\nreported very low levels of EST mRNA expression in both\neutopic and ectopic endometrium, but found no differ-\nence between the two tissues. Utsunomiya et al . (2004)\nshowed that EST was expressed in the endometrium but\nonly during the secretory phase. In this study, we obtained\nspecimens during the proliferative phase to eliminate the\neffect of P\n4. This balance of the STS and EST expression\nindicates that estrone is more favorably produced in\nOESCs as well as EESCs and NESCs during the proliferative\nphase. The significance of the differences in expression of\nSTS and EST between these cells remains to be elucidated.\nUsing this experimental model, it was determined that\nDNG significantly inhibited HSD17b1 mRNA expression\nand its enzyme activity at 10\nK7 M in OESCs. These\nconcentrations are equivalent to the blood level of mice\nadministered 1 mg of DNG twice daily and patients\nadministered 2 mg of DNG daily ( Meriggiola et al . 2002,\nSasagawa et al. 2008). The in vitro inhibitory effects of DNG\nare supported by the in vivo data from this study,\ndemonstrating that DNG treatment for 3–5 months\nresulted in decreased HSD17b1 protein expression in OE.\nThe conversion of estrone to estradiol is also mediated\nthough HSD17b7 and HSDb12 as well as HSDb1( Moeller &\nAdamski 2006 ). DNG inhibited only HSD17b1 but not\n17HSDb7 or 12 mRNA expression, which indicates that\nDNG reduces local estrogen production by the suppression\nof HSD17b1 in human OESCs.\nWe and other researchers have shown that the PR\nis involved in the mechanism of DNG-inhibited cell\nproliferation (Okada et al. 2001, Shimizu et al. 2009) and\nthe expression of inflammatory factors ( Mita et al. 2011),\nnerve growth factor, ( Mita et al . 2014 ), and aromatase\n(Yamanaka et al. 2012). Although the HSD17b1 gene lacks\naP\n4-responsive element in its promoter region, progestins\nincluding DNG downregulated HSD17b1 and upregulated\nHSD17b2 expression in immortalized endometriotic epi-\nthelial cells ( Beranic & Rizner 2012 ). DNG inhibits the\nDNA-binding activity of NF kB, a key regulator of various\npathological and inflammatory responses in endometrio-\nsis such as interleukin-8 production in human OESCs\n(Horie et al . 2005 , Shimizu et al . 2011 , Yamanaka et al .\n2012). Bulun proposed a vicious cycle of an estrogen-\ndependent mechanism of endometriosis growth ( Bulun\n2009). Estradiol produced locally by aromatase stimulates\ntissue growth of endometriosis and upregulates COX2 via\nERb activation. COX2 overexpression results in an excess\nof prostaglandin E\n2, which further stimulates aromatase\nexpression via the orphan nuclear receptor steroidogenic\nfactor 1. Furthermore, ER b activation downregulates PR,\nwhich leads to reduced induction of HSD17b2 via retinoic\nacid (Zeitoun et al . 1998). The combination of upregula-\ntion of aromatase and downregulation of HSD17 b2\ncontributes to the abnormally high levels of estradiol in\nendometriotic tissue. In addition to inhibition of aroma-\ntase, DNG inhibits HSD17 b1, resulting in further\nreductions in local estradiol concentration. This interrupts\nthe vicious cycle of endometriosis growth and also relieves\nendometriosis-associated pelvic pain by inhibiting prosta-\nglandin E\n2 production. The effect of inhibiting HSD17 b1\non endometriosis has been demonstrated ( Delvoux et al .\n2014)a n ds e v e r a lH S D 1 7b1i n h i b i t o r sh a v eb e e n\ndeveloped and used in preclinical studies ( Day et al .\n2008, Poirier 2011). DNG is widely used in clinics to treat\nendometriosis with fewer side effects. We believe that the\nidentification of the molecular mechanisms behind the\ntherapeutic effect of DNG described here will lead to better\nunderstanding of the pathophysiology of endometriosis.\nJournal of Endocrinology\nResearch T MORI and others Dienogest inhibits HSD17 b1i n\nendometriosis\n225:2 74\nhttp://joe.endocrinology-journals.org /C2092015 Society for Endocrinology\nDOI: 10.1530/JOE-15-0052 Printed in Great Britain\nPublished by Bioscientifica Ltd.\nDownloaded from Bioscientifica.com at 06/23/2026 12:54:26PM\nvia free access\n\n\nDeclaration of interest\nThe authors declare that there is no conflict of interest that could be\nperceived as prejudicing the impartiality of the research reported.\nFunding\nThis work was supported in part by a Grant-in-Aid for Scientific Research\n24592480 and 23197849 from the Ministry of Education, Culture, Sports,\nScience and Technology (Japan).\nReferences\nBeranic N & Rizner TL 2012 Effects of progestins on local estradiol\nbiosynthesis and action in the Z-12 endometriotic epithelial cell line.\nJournal of Steroid Biochemistry and Molecular Biology 132 303–310.\n(doi:10.1016/j.jsbmb.2012.07.004)\nBulun SE 2009 Endometriosis. 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(doi:10.1210/jcem.83.12.5301)\nReceived in final form 11 March 2015\nAccepted 12 March 2015\nAccepted Preprint published online 12 March 2015\nJournal of Endocrinology\nResearch T MORI and others Dienogest inhibits HSD17 b1i n\nendometriosis\n225:2 76\nhttp://joe.endocrinology-journals.org /C2092015 Society for Endocrinology\nDOI: 10.1530/JOE-15-0052 Printed in Great Britain\nPublished by Bioscientifica Ltd.\nDownloaded from Bioscientifica.com at 06/23/2026 12:54:26PM\nvia free access","source_license":"CC0","license_restricted":false}