{"paper_id":"1ff4fa6f-9d1e-4614-a463-4d1e7e8ef9c3","body_text":"R E S E A R C H Open Access\nDienogest increases the progesterone receptor\nisoform B/A ratio in patients with ovarian\nendometriosis\nAtsushi Hayashi, Akiko Tanabe *, Sachiko Kawabe, Mika Hayashi, Hiroko Yuguchi, Yoshiki Yamashita,\nKiyoji Okuda and Masahide Ohmichi\nAbstract\nBackground: The resistance of endometriotic tissue to progesterone can be explained by alterations in the\ndistribution of progesterone receptor (PR) and estrogen receptor (ER) isoforms. The aims of this study were to\nexamine the expressions of PR-A, PR-B, ER α and ER β in endometrioma and assess whether these expressions are\naffected by dienogest or leuprolide acetate (LA) treatment.\nMethods: We enrolled 60 females, including 43 patients with endometriosis (14 who received no medical\ntreatment, 13 who received dienogest and 16 who received LA before undergoing laparoscopic surgery) and 17\npatients with leiomyoma. The expression levels of PR and ER isoforms in eutopic and ectopic endometrium were\nassayed with quantitative real-time PCR, and confirmed with immunohistochemistry.\nResults: A decreased PR-B/PR-A ratio and an increased ER β/ERα ratio were demonstrated in ectopic endometrium\nderived from females with endometriosis compared with the ratios observed in eutopic endometrium obtained\nfrom females without endometriosis. Although LA treatment did not affect the PR-B/PR-A and ER β/ERα ratios,\ndienogest treatment increased the PR-B/PR-A ratio and decreased the ER β/ERα ratio in patients with\nendometriomas.\nConclusions: Dienogest may improve progesterone resistance in endometriotic tissue by increasing the relative\nexpressions of PR-B and PR-A, and decreasing the relative expressions of ER β and ER α.\nKeywords: Dienogest, Progesterone receptor isoforms, Estrogen receptor isoforms, Ovarian endometriosis,\nProgesterone resistance\nBackground\nEndometriosis is an estrogen-dependent inflammatory dis-\nease that affects 6-10% of females of reproductive age [1]. It\nis characterized by the presence of endometrium-like tissue\noutside the uterine cavity, primarily on the ovaries, and\nrepresents one of the most common causes of chronic pelvic\npain, dysmenorrhea and infer tility [2]. The main aims of\ntreatment are to alleviate pain and other symptoms, reduce\nthe size of the endometriotic lesions and improve the quality\nof life of affected individuals. Nonsteroidal anti-inflammatory\ndrugs are frequently used by patients with endometriosis in\nan attempt to relieve pelvic pain , although clinical trial evi-\nd e n c et os u p p o r tt h ee f f i c a c yo ft h e s ea g e n t si ne n d o m e t r i o -\ntic patients is lacking [3]. Gonadotropin-releasing hormone\n(GnRH) agonists are an established therapy for endometri-\nosis. Although GnRH agonists provide effective pain relief\nand reduce the progression of endometriotic implants [4],\nthe hypoestrogenic state they induce is associated with nega-\ntive effects such as accelerated bone mineral density loss, hot\nflashes and vaginal dryness [5]. Oral contraceptives (OCs)\nare widely used to treat the symptoms of endometriosis, al-\nthough they are not approved for this indication in the ma-\njority of countries due to the lack of supportive trial evidence\n[6]. Progestins have long been used for the treatment of\nendometriosis to relieve pain by suppressing ovarian estro-\ngen biosynthesis, in turn, suppressing growth and\n* Correspondence: gyn074@poh.osaka-med.ac.jp\nDepartment of Obstetrics and Gynecology, Osaka Medical College, 2-7\nDaigaku-machi, Takatsuki city, Osaka 569-8686, Japan\n© 2012 Hayashi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative\nCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and\nreproduction in any medium, provided the original work is properly cited.\nHayashi et al. Journal of Ovarian Research 2012, 5:31\nhttp://www.ovarianresearch.com/content/5/1/31\n\ninflammation [7]. Unfortunately, the relief of pain appears to\nbe relatively short-term [8], and approximately 9% of females\nwith endometriosis simply do not respond to progestin ther-\napy due to unknown reasons [9]. In fact, a general tendency\nfor relative progesterone resistance within the eutopic and\nectopic endometrium of females with endometriosis is well-\ndocumented [1,10].\nRecently, two major progesterone receptor (PR) iso-\nforms were identified, namely PR-A and PR-B (11). PR-A\nis a 94-kDa protein, whereas PR-B is a 114-kDa protein\nthat contains an additional NH2-terminal stretch of ap-\nproximately 165 amino acids containing a region encoding\na transactivation function. These isoforms may arise as a\nresult of either initiation of translation from alternative\nsites in the same messenger RNA (mRNA) [11] or by tran-\nscription from alternative promoters [12]. Although the\nexact functions of each of these isoforms remain unclear,\nthere is increasing evidence that they are functionally dif-\nferent [12,13]. PR-B tends to be a stronger activator of\nprogesterone target genes, whereas PR-A has been shown\nto act as a dominant repressor of PR-B [14,15].\nEndometriosis is associated with a reduced response to\nprogesterone in both eutopic and ectopic endometrium.\nAccording to recent reports, the resistance of endome-\ntriotic tissue to progesterone, evident in both laboratory\nand clinical observations, can be explained by alterations\nin the distribution of ER and PR isoforms and dysregula-\ntion of progesterone target genes [10,16-18]. Because the\neffects of progesterone on target genes are conferred pri-\nmarily by PR-B in the endometrium [19], the presence of\nthe inhibitor isoform-A and the absence of the stimula-\ntory isoform-B provide a possible explanation for pro-\ngesterone resistance in endometriotic implants. In fact, a\ndecreased PR-B/PR-A ratio has been demonstrated in\nectopic tissue [20,21], and recent reports suggest that\nthe tendency toward progesterone resistance in patients\nwith endometriosis is likely the result of the promotion\nof hypermethylation of PR-B, which renders PR-B either\nsilenced or downregulated [21]. Moreover, a number of\ninvestigators have reported markedly elevated levels of\nERβ and lower levels of ER α in human endometriotic\ntissues and primary stromal cells compared with that\nobserved in eutopic endometrial tissues and cells\n[16,22,23]. ER β, acting as an ER α suppressor, might con-\ntribute to the decreased PR levels and progesterone re-\nsistance observed in patients with endometriosis [24].\nDienogest (17a-cyanomethyl- 17b-hydroxyestra-4,9-dien-\n3-one) is an oral progestin t hat has been systematically\ninvestigated for the treatment of endometriosis in dose-\nranging [25], placebo-controlled [26,27], active comparator-\ncontrolled [28,29] and long-term trials [30] conducted in\nEurope and Japan. The main an ti-endometriotic effect of\ndienogest has been suggested to be attributable to central\ninhibition of ovulation. Furthermore, direct antiproliferative\neffects of dienogest have been demonstrated in human\neutopic endometrial stromal cells [31]. Recent studies dem-\nonstrate that dienogest inhibits the proliferation of endome-\ntriotic stromal cells [32], prostaglandin E2 production and\nthe aromatase mRNA expressionof the endometrial epithe-\nlial cell line [33]. However, there is no evidence regarding\nwhether dienogest improves progesterone resistance in\npatients with endometriosis. In the present study, therefore,\nwe examined the effects of dienogest on alterations in the\nratios of PR-B to PR-A and ER β to ER α in ectopic endo-\nmetrial tissue obtained from endometriotic females.\nMethods\nPatients and tissue collection\nSixty patients treated between January 2002 and July\n2010 were included in this study. All patients were\nunder treatment at the department of obstetrics and\ngynecology of Osaka Medical College. This was a retro-\nspective cross-sectional case-controlled study of human\ntissue samples approved by the Institutional Review\nBoard of Osaka Medical College. Written informed con-\nsent was obtained from all patients participating in the\nstudy.\nThe inclusion criteria were: older than 20 years age of\nand no more than 50 years of age at the time of the sur-\ngical procedure, the presence of regular menstrual cycles\n(24–35 days of interval) with the exception of those trea-\nted with leuprolide acetate (Leuplin; Takeda pharma-\nceutical, Osaka, Japan) for endometriosis, the absence of\nany evidence of past or recent pelvic inflammatory dis-\nease and no history of any hormonal treatment for at\nleast 12 months at baseline. Trans-vaginal ultrasonog-\nraphy was performed for all patients, and showed mainly\nhypoechoic cystic masses in the ovaries, and the pres-\nence of ovarian endometriomas were confirmed before\nsurgery by magnetic resonance imaging (MRI), which\nshowed high-intensity areas on both T1- and T2-\nweighted images. The serum level of CA125 was also\nmeasured before surgery using automated assays on a\nRoche Modular E170 instrument (Roche, Vilvoorde, Bel-\ngium) at the central laboratories of Osaka Medical Col-\nlege. Tissue specimens were obtained from females\n(n=43) treated with dienogest at a dose of 2 mg (dieno-\ngest group; n=13) for three to five months or leuprolide\nacetate (LA) at a dose of 3.75 mg (LA group; n=16)\nadministered before surgery. Simultaneous sampling of\novarian endometrioma capsules was performed during\nlaparoscopic surgery for indications of adnexal masses\nconsistent with ovarian endometrioma. The stage of\nendometriosis in each case was documented according\nto the revised American Society of Reproductive Medi-\ncine Criteria (r-AFS stage) [34]. The diagnosis of endo-\nmetriosis was confirmed histologically. Normal\nendometrial tissues (n=17) were obtained using biopsies\nHayashi et al. Journal of Ovarian Research 2012, 5:31 Page 2 of 8\nhttp://www.ovarianresearch.com/content/5/1/31\n\nduring the proliferative phase of the menstrual cycle in\npatients undergoing hysterectomy for uterine fibroids.\nThe samples obtained from the ectopic and eutopic\nendometrium were immediately frozen in liquid nitrogen\nfor further RT-PCR analyses, fixed in 10% formaldehyde\nand then routinely processed for paraffin embedding for\na histological analysis.\nOneStep real-time polymerase chain reaction\nTotal RNA was obtained using the RNeasy Mini kit\n(Qiagen, Germantown MD), and 2 μg were reverse tran-\nscribed with Superscript II RNase H-reverse transcript-\nase (Invitrogen) using random primers according to the\nmanufacturer’s instructions. Oligonucleotide primers for\nTaqMan probes were designed with the use of Primer\nExpress (version 1.0; Perkin-Elmer Applied Biosystems,\nTokyo, Japan) from the GeneBank database. Human\nGAPDH was purchased from Perkin-Elmer Applied Bio-\nsystems and used as an internal standard. The primers\nused in this study are shown in Table 1. The first primer\nset, termed PR-B, was designed to amplify sequences\nspecific for PR-B (upstream of the second AUG transla-\ntion initiation site), whereas the second primer set, total\nPR, was designed to amplify sequences downstream of\nthe second AUG translation initiation site. None of these\nprimer sets corresponded to sequences in any of the\nother steroid hormone receptors. The cDNA template\nwas amplified using quantitative real-time polymerase\nchain reaction (qRT-PCR), as previously described [35].\nBriefly, the cDNA template was amplified in a 20 μLr e a c -\ntion containing 1 x T aqMan Universal PCR Master Mix\n(Perkin-Elmer Applied Biosystems), 200 nM forward and\nreverse primers and 100 nM T aqMan probe. The T aqMan\nPCR conditions were: 95°C for 15 seconds followed by\n60°C for one minute for 45 cycles in each case on OneStep\nreal-time PCR (Perkin-Elmer Applied Biosystems). The\namplification of the target gene mRNA relative to\nGAPDH was compared using the ΔΔ Ct method. The\nabundance of PR-A was calculated by subtracting the\nrelative abundance of PR-B from that of total PR.\nHistology and immunohistochemistry\nAll specimens fixed in 10% paraformaldehyde solution\nwere embedded in paraffin blocks. The sections were\nstained with hematoxylin and eosin for histological evalu-\nation of the tissues. For deection of PR-A, a human PR-A-\nspecific mouse monoclonal antibody purchased from\nNovocastra (NCL-L-PGR-312; Vision BioSystems Inc.,\nNorwell, MA) was used [36,37]. For PR-B immunostain-\ning, mouse monoclonal antibody Ab-6 (NeoMarkers, Fre-\nmont, CA) was used [36,38]. For ER α and ER β\nimmunostaining, rabbit polyclonal antibodies (LS-B1470\nand LS-B945, respectively, LifeSpan Biosciences, Seattle,\nWA, USA) were used. The antigen-antibody complexes\nwere identified using the Universal DAKO LSAB2-labeled\nstreptavidin-biotin peroxidase kit (Lifespan Biosciences).\nStatistics\nAll experiments were performed in triplicate. The statis-\ntical calculations were performed using the StatView\nstatistical software package (SAS Institute, Cary, NC),\nand the statistical significance of each difference was\ndetermined using the Kruskal-Wallis and Mann-\nWhitney U test or paired t-test as appropriate. A P value\nof < 0.05 was considered to be statistically significant.\nResults\nPatient characteristics\nA total of 14 females who did not receive any medical\ntreatment, 13 females who received dienogest and 16\nfemales who received LA were evaluated in this study\n(Table 2). There were no relevant group differences in\nage or VAS at baseline. The use of concomitant medica-\ntions recorded in patient-maintained diaries, including\nanalgesic medications for endometriosis, did not differ\nrelevantly between the groups at baseline.\nAt baseline, the mean ± SD VAS score was 53.1 ± 29.9\nmm in the endometriosis-control group, 46.2 ± 22.6 mm\nin the endometriosis-dienogest group and 56.3 ± 41.0\nmm in the LA group. Following surgical treatment, the\nmean VAS score decreased to 20.6 ± 15.1 mm in the\ndienogest group and 21.1 ± 18.0 mm in the LA group,\ndemonstrating the non-inferiority of dienogest versus\nLA as measured by observed VAS score changes. At\nbaseline, the mean ± SD serum CA125 level was 60.3 ±\n34.1 U/ml in the endometriosis-control group, 60.6 ±\n35.8 U/ml in the endometiosis-dienogest group and\n52.9 ± 42.8 U/ml in the LA group. Following surgical\ntreatment, the mean serum CA125 level significantly\ndecreased to 34.8 ± 14.9 U/ml in the dienogest group\nand to 34.9 ± 20.8 U/ml in the LA group.\nPR isoform expression and PR-B/PR-a ratios in eutopic\nand ectopic endometrium\nPrevious studies have demonstrated that the expression\nof repressive PR-A and the apparent downregulation of\nTable 1 Primers used for TaqMan real-time polymerase\nchain reaction\nGene Primer\nPR-B 5 0-CTGGCCTATCCTGCCTGCCTCA-30\n50-TGTCCAAGACACTGTCCAGCAG-30\ntotal PR 5 0CGTGCCTATCCTGCCTCTA-3\n50CCGCCGTCGTAACTTTCGT-30\nERα 50-GGGAAGCTACTGTTTGCTCCTAAC-30\n50-CACCATGCCCTTACACATTC-30\nERβ 50-GATCGCTAGAACACACCTTACCTGTA-30\n50-GCGCAACGGTTCCCACTA-30\nHayashi et al. Journal of Ovarian Research 2012, 5:31 Page 3 of 8\nhttp://www.ovarianresearch.com/content/5/1/31\n\nstimulatory PR-B may explain the development of pro-\ngesterone resistance in patients with endometriosis\n[1,39]. This study investigated whether treatment with\ndienogest or leuprolide acetate attenuates the expression\nof PR isoforms in ectopic endometrium.\nThe expression of PR-B (Figure 1A) was significantly\ndysregulated in control ectopic endometrial samples\nobtained from females who did not receive any treatment\ncompared with that observed in the eutopic endometrium\nof patients with fibroids. In contrast, the endometrioma\nTable 2 Baseline patient characteristics\nPatients\nwith\nuterine\nfibroids\n(n=17)\nEndometriosis\nControl (n=14) Dienogest 2 mg (n=13) LA 3.75 mg (n=16)\nbaseline baseline baseline preoperation baseline preoperation\nAge (years, mean ± SD 44.2 ± 6.8 34.9 ± 8.1 34.3 ± 5.7 37.5 ± 7.2\nDuration of drug administration - - 21.6 ± 11.5 12.6 ± 2.8\nPelvic pain VAS (mm, mean ± SD) 23.8 ± 18.2 53.1 ± 29.9 46.2 ± 22.6\na 20.6 ± 15.1 b 56.3 ± 41.0 ab 21.1 ± 18.0 b\nCA-125 (U/ml, mean ± SD) 33.1 ± 20.4 60.3 ± 34.1 60.6 ± 35.8 34.8 ± 14.9 c 52.9 ± 42.8 34.9 ± 20.8\nr-AFS stage (n,%)\nI: minimal 10 (58%) 0 (0%) 0 (0%) 0 (0%)\nII: mild 3 (18%) 0 (0%) 0 (0%) 2 (13%)\nIII: moderate 4 (24%) 5 (36%) 8 (62%) 9 (56%)\nIV: severe 0 (0%) 9 (64%) 5 (38%) 5 (31%)\nLA, leuprolide acetate; VAS, visual analogue scale; r-AFS, revised American Fertility Society.\nSD, standard deviation.\nA: p <0.01 compared to patients with uterine fibroids, b: p <0.01 compared to endometriosis-control, c: p <0.05 compared to baseline.\n0\n5\n10\n15\n20\n25\n30\nEutopic Control Dienogest LA\nEndometriosis\n0\n5\n10\n15\n20\n25\nEutopic Control Dienogest LA\nEndometriosis\n0\n1\n2\n3\n4\n5\n6\n7\nEutopic Control Dienogest LA\nEndometriosis\nPR-B PR-A PR-B/PR-A CBA\nPR-B / GAPDH mRNA ratio\nPR-A / GAPDH mRNA ratio\nPR-B / PR-A mRNA ratio\nFigure 1 PRs expression and PR-B/PR-A ratios in eutopic/ectopic endometrium following treatment with dienogest or leuprolide\nacetate. Total RNA was isolated from eutopic endometrial samples (Eutopic, n=17) obtained with biopsies performed during the proliferative\nphase of the menstrual cycle in patients undergoing hysterectomy for uterine fibroids. Total RNA was also isolated from endometriotic samples\nobtained from females who did not receive medical treatment (Control, n=14) and endometriotic samples obtained from females treated with\ndienogest at a dose of 2 mg (Dienogest, n=13) or leuprolide acetate (LA, n=16). The total RNA was then reverse-transcribed. A: The relative\nexpression ratio of the PR-B gene was calculated in comparison to the GAPDH expression. B: The relative expression ratio of the PR-A gene was\ncalculated by subtracting the relative abundance of PR-B from that of total PR. C: For each experimental sample, the data are graphically\nillustrated as the ratio between the relative expressions of PR-B and PR-A. The center lines indicate the median ratio. The columns and vertical\nbars indicate the 25 –75 percentiles. Significant differences are indicated by an asterisk. *p<0.05, **p<0.01.\nHayashi et al. Journal of Ovarian Research 2012, 5:31 Page 4 of 8\nhttp://www.ovarianresearch.com/content/5/1/31\n\nsamples obtained from the patients who received dieno-\ngest or LA treatment showed a significantly greater ex-\npression of PR-B compared with that observed in the\ncontrol tissues. The expression of PR-A (Figure 1B) was\nalso significantly decreased in control ectopic endometrial\nsamples compared with that observed in eutopic endo-\nmetrium. Although the endometrioma samples obtained\nfrom the patients who received LA treatment showed a\nsignificantly greater expression of PR-A, dienogest treat-\nment did not alter the expression of PR-A. Typical mRNA\nexpression patterns of the PR isoforms are shown in the\nAdditional file 1: Figure A, and explained in the Add-\nitional file 2. Progesterone resistance may be explained by\nthe absence of PR-B and the dominant expression of PR-A\n[20,21]; therefore, we analyzed the PR-B/PR-A ratio at the\nmRNA level (Figure 1C). In the control ectopic endomet-\nrial samples, the PR-B/PR-A ratio was significantly\ndecreased compared with that observed in eutopic endo-\nmetrium. Interestingly, dienogest treatment improved the\nPR-B/PR-A ratio; however, there were no significant differ-\nences in endometrioma after LA treatment.\nER isoform expression and ER β/ERα ratios in eutopic and\nectopic endometrium\nThis study investigated whether treatment with dieno-\ngest or leuprolide acetate attenuates the expression of\nER isoforms in ectopic endometrium.\nThe expression of ERα (Figure 2A) was significantly dys-\nregulated in the control ectopic endometrioma samples\nobtained from females who did not receive any treatment\ncompared with that observed in the eutopic endometrium\nof patients with fibroids. In addition, the endometrioma\nsamples obtained from the patients who received dienogest\nor LA treatment showed a significantly lower expression of\nERα compared with that observed in eutopic endomet-\nrium. The expression of ER β (Figure 2B) was also signifi-\ncantly elevated in the control ectopic endometrial samples\ncompared with that observed in eutopic endometrium.\nDienogest, but not LA, treatment altered the expression of\nERβ. Typical mRNA expression pattern of the ER isoforms\nare shown in the Additional file 1: Figure B, and explained\nin Additional file 2. A number of investigators have\nreported markedly elevated levels of ER β and lower levels\nof ER α in human endometriotic tissues compared with\nthat observed in eutopic endometrial tissues and cells\n[16,22,23]. Therefore, we also analyzed the ER β/ERα ratio\nat the mRNA level (Figure 2C). In the control ectopic\nendometrial samples, the ER β/ERα ratio was significantly\nincreased compared with that observed in eutopic endo-\nmetrium. Interestingly, dienogest treatment significantly\nimproved the ER β/ERα ratio; however, there were no sig-\nnificant differences in endometrioma after LA treatment.\nExpression of PR and ER isoform proteins in the eutopic\nand ectopic endometriotic samples\nAn immunohistochemical analysis revealed that PR-B\nimmunoreactivity was strongly localized to the glandular\nepithelium and stromal cells in the eutopic endometrial\n0\n0.5\n1\n1.5\n2\n2.5\n3\nEutopic Control Dienogest LA\nPE Endometriosis\n0\n5\n10\n15\n20\n25\n30\n35\n40\nEutopic Control Dienogest LA\nPE Endometriosis\n0\n0.5\n1\n1.5\n2\n2.5\nEutopic Control Dienogest LA\nPE Endometriosis\nCBA\nERa / GAPDH mRNA ratio\nERβ/ GAPDH mRNA ratio\nERβ/ ERαmRNA ratio\nERα ERβ ERβ/ERα\nFigure 2 ERs expression and ER β/ERα ratios in eutopic/ectopic endometrium following treatment with dienogest or leuprolide acetate.\nTotal RNA was isolated from eutopic endometrial samples (Eutopic, n=17) obtained with biopsies performed during the proliferative phase of the\nmenstrual cycle in patients undergoing hysterectomy for uterine fibroids. Total RNA was also isolated from endometriotic samples obtained from\nfemales who did not receive medical treatment (Control, n=14) and endometriotic samples obtained from females treated with dienogest at a\ndose of 2 mg (Dienogest, n=13) or leuprolide acetate (LA, n=16). The total RNA was then reverse-transcribed. A: The relative expression ratio of\nthe ERα gene was calculated in comparison to the GAPDH expression. B: The relative expression ratio of the ER β gene was calculated in\ncomparison to the GAPDH expression. C: For each experimental sample, the data are graphically illustrated as the ratio between the relative\nexpressions of ER β and ERα. The center lines indicate the median ratio. The columns and vertical bars indicate the 25 –75 percentiles. Significant\ndifferences are indicated by an asterisk. *p<0.05, **p<0.01.\nHayashi et al. Journal of Ovarian Research 2012, 5:31 Page 5 of 8\nhttp://www.ovarianresearch.com/content/5/1/31\n\nsamples during the proliferative phase (Figure 3E) and\nfaintly localized to the glandular epithelium in the control\nectopic endometrium (Figure 3F). In contrast, PR-A\nimmunoreactivity appeared faintly in both the eutopic\nendometrial samples (Figure 3A) and the control ectopic\nendometrium (Figure 3B). After the patients were treated\nwith dienogest or LA, immunostaining of PR-B increased\nin the ectopic endometrial epithelium (Figure 3G and H).\nERα was strongly localized to the glandular epithelium\nand stromal cells in the eutopic endometrium (Figure 3I)\nand faintly localized in the control ectopic endometrium\n(Figure 3J). Dienogest and LA treatment had no effect on\nthe ER α expression (Figure 3K and L). ER β immunoreac-\ntivity appeared faintly in the eutopic endometrial samples\n(Figure 3M). In contrast, ER β was strongly localized to the\nepithelium and stromal cells in the ectopic endometrium\n(Figure 3N). Although dienogest treatment decreased the\nERβ expression in ectopic endometrioma (Figure 3O), LA\ntreatment seemed to have no effect on the ER β expression\n(Figure 3P). No immunostaining was found in eutopic or\nectopic endometrium when the primary antibodies were\nomitted (data not shown).\nDiscussion\nThe current study demonstrated statistically significant\ndecreases in both PR –B and PR-A messenger RNA and\nproteins in ectopic endometrium derived from females with\nendometrioma who did not receive any medical treatment\n(Figure 1A, 1B, 3B, and 3F). Furthermore, the relative\nexpressions of PR-B and PR-A were significantly lower in\ne c t o p i ce n d o m e t r i u m( F i g u r e1C). According to Attia et al.\n[20], the resistance of endometriotic tissue to progesterone,\nevident in both laboratory and clinical observations, can be\nexplained by the absence of PR-B transcripts and proteins\nand the presence of PR-A in ectopic lesions. Similar find-\nings have been reported in epithelial cells selected from a\nControl Dienogest LA\nPR-APR-BER-αER-β\nPE\nABCD\nE F G H\nIJ K L\nM N O P\nFigure 3 Typical example of PR and ER isoform expression in patients with ovarian endometriosis. PR-A and PR-B were detected using\nPR-A (A, B, S, D) and PR-B ( E, F, G, H) antibodies. ER α and ERβ were detected using ER α (I, J, K, L) and ER β (M, N, O, P) antibodies. The eutopic\nendometrium (A, E, I, M) in the proliferative phase was obtained from females undergoing hysterectomy for uterine fibroids. The ectopic\nendometrium was obtained from endometrioma of the control females ( B, F, J, N), dienogest-treated females ( C, G, K, O) and LA-treated females\n(D, H, L, P). Scale bar: 25 m.\nHayashi et al. Journal of Ovarian Research 2012, 5:31 Page 6 of 8\nhttp://www.ovarianresearch.com/content/5/1/31\n\nsmall number of ectopic samples [10]. Recently, independ-\nent investigators suggested tha t alterations in the relative\nexpressions of PR-A and PR-B in endometrial cells may also\nplay a pivotal role in the patho genesis of endometriosis. A\ndecreased PR-B/PR-A ratio has been demonstrated in ec-\ntopic tissue [5,10], and our findings are consistent with the\nresults of these studies.\nSeveral investigators have reported markedly higher\nlevels of ER β and lower levels of ER α in human endome-\ntriotic tissues and primary stromal cells compared with\nthat observed in eutopic endometrial tissues and cells\n[22,40]. Recently, Bulun [41] reported that the levels of the\nnuclear receptors ER α,E R β and PR are quite different in\nendometriotic tissue and endometrium. The high ER β/\nERα ratiosin endometriotic stromal cells in turn lead to\nincreased ER β binding to the PR promoter and mediate\ndownregulation of the expression of PR [21]. ER β acts as a\nsuppressor of ER α in both endometrial and endometriotic\nstromal cells by binding to regulatory elements of specific\npromoters of the ER α and PR genes [42]. Therefore, ER α\ndeficiency in endometriotic patients may be responsible\nfor the failure of E2 to induce the PR expression, thus\ncontributing to secondary PR deficiency and progesterone\nresistance in females with endometriosis. Although strik-\ningly high quantities of E2 produced via local aromatase\nactivity are observed in endometriotic tissue [43,44], the\nE2-dependent induction of PR is strongly inhibited [20].\nFindings consistent with these studies were observed in\nthe current study, which demonstrated statistically\nsignificant higher levels of ER β and lower levels of ER α in\nectopic endometrium derived from females with endome-\ntrioma (Figure 2 and Figure 3).\nThe endometrioma samples obtained from the patients\nwho received dienogest or LA treatment showed a\nsignificantly higher expression of PR-B compared with\nthat observed in the control endometrioma samples\n(Figure 1A and Figure 3). Although LA treatment\nincreased the PR-A expression in the endometrioma\nsamples, dienogest treatment did not alter the expres-\nsion of PR-A (Figure 1B). Consequently, dienogest treat-\nment improved the PR-B/PR-A ratio; however, there\nwere no significant differences in endometrioma after\nLA treatment (Figure 1C). Progesterone has recently\nbeen shown to reverse E2-stimulated increases in the\nERβ mRNA and protein expression in cultured hippo-\ncampal slices [45]. Our current study demonstrates that\ndienogest treatment alters the expression of ER β\n(Figure 2B and Figure 3) and significantly decreases the\nERβ/ERα ratio in endometrioma (Figure 2C). A recent\nreport demonstrated that the promoter region of PR-B,\nbut not PR-A, is hypermethylated in patients with endo-\nmetriosis [21]. Although further studies are needed to\nclarify whether medical treatments might be associated\nwith the methylation status of the PR-B promoter and to\nexplore the mechanisms underlying the ER β downregu-\nlation induced by dienogest, a decreased ER β/ERα ratio\nin the endometriotic tissues of patients treated with die-\nnogest may be responsible for the observed improve-\nments in the PR expressions.\nConclusions\nWe demonstrated that dienogest improves progesterone\nresistance in endometrial tissue. This finding enhances\nunderstanding of the anti-endometriotic effects of dieno-\ngest. It is possible that PR-B deficiency is only the tip of\nthe iceberg with regard to the pathogenesis of endomet-\nriosis and that numerous other molecular aberrations\nmay also contribute to the development of resistance to\nhormone treatments in females with endometriosis. Al-\nthough our findings may explain at least part of the\nmechanisms underlying the clinical improvements\nobserved in endometriotic patients using dienogest, the\nnormalization of the PR expression profile observed in\nthis study suggests that dienogest may be an effective\nand long-term treatment for endometriosis.\nAdditional files\nAdditional file 1: Typical mRNA expression patterns of the PR and\nER isoforms. A: A representative agarose gel showing amplicons of PR-B\n(upper panel), total-PR (middle panel), and β-actin (lower panel). B: A\nrepresentative agarose gel showing amplicons of ER α (upper panel), ER β\n(middle panel), and β-actin (lower panel).\nAdditional file 2: Supplemental Results.\nCompeting interests\nThe authors declare that they have no competing interests.\nAuthors’ contributions\nAll authors read and approved the final manuscript.\nAcknowledgment\nWe are grateful to Junko Hayashi and Kumiko Satoh for their secretarial\nassistance.\nReceived: 13 September 2012 Accepted: 29 October 2012\nPublished: 1 November 2012\nReferences\n1. Giudice LC, Kao LC: Endometriosis. Lancet 2004, 364:1789–1799.\n2. Eskenazi B, Warner ML: Epidemiology of endometriosis. Obstet Gynecol Clin\nNorth Am 1997, 24:235–258.\n3. Allen C, Hopewell S, Prentice A, Gregory D: Nonsteroidal anti-inflammatory\ndrugs for pain in women with endometriosis. Cochrane Database Syst Rev\n2009, 2:CD004753. doi:10.1002/14651858.\n4. Brown J, Pan A, Hart RJ: Gonadotrophin-releasing hormone analogues for\npain associated with endometriosis. Cochrane Database Syst Rev 2010,\n12:CD008475.\n5. Sagsveen M, Farmer JE, Prentice A, Breeze A: Gonadotrophin-releasing\nhormone analogues for endometriosis: bone mineral density. Cochrane\nDatabase Syst Rev 2003, 4:CD001297.\n6. Schindler AE: Non-contraceptive benefits of hormonal contraceptives.\nMinerva Ginecol 2010, 62:319–329.\n7. Olive DL, Pritts EA: Treatment of endometriosis. N Engl J Med 2001,\n345:266–275.\nHayashi et al. Journal of Ovarian Research 2012, 5:31 Page 7 of 8\nhttp://www.ovarianresearch.com/content/5/1/31\n\n8. Waller KG, Shaw RW: Gonadotropin-releasing hormone analogues for\nthe treatment of endometriosis: long-term follow-up. Fertil Steril 1993,\n59:511–515.\n9. Vercellini P, Cortesi I, Crosignani PG: Progestins for symptomatic\nendometriosis: a critical analysis of the evidence. Fertil Steril 1997,\n68:393–401.\n10. Bulun SE, Cheng YH, Yin P, Imir G, Utsunomiya H, Attar E, Innes J, Julie Kim\nJ: Progesterone resistance in endometriosis: link to failure to metabolize\nestradiol. Mol Cell Endocrinol 2006, 248:94–103.\n11. Conneely OM, Maxwell BL, Toft DO, Schrader WT, O ’Malley BW: The A and\nB forms of the chicken progesterone receptor arise by alternate\ninitiation of translation of a unique mRNA. Biochem Biophys Res Commun\n1987, 149:493–501.\n12. Kastner P, Bocquel MT, Turcotte B, Garnier JM, Horwitz KB, Chambon P,\nGronemeyer H: Transient expression of human and chicken progesterone\nreceptors does not support alternative translational initiation from a\nsingle mRNA as the mechanism generating two receptor isoforms. J Biol\nChem 1990, 265:12163–12167.\n13. Tora L, Gronemeyer H, Turcotte B, Gaub MP, Chambon P: The N-terminal\nregion of the chicken progesterone receptor specifies target gene\nactivation. Nature 1988, 333:185–188.\n14. Tung L, Mohamed MK, Hoeffler JP, Takimoto GS, Horwitz KB: Antagonist-\noccupied human progesterone B-receptors activate transcription\nwithout binding to progesterone response elements and are dominantly\ninhibited by A-receptors. Mol Endocrinol 1993, 7:1256–1265.\n15. Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O ’Malley BW, McDonnell DP:\nHuman progesterone receptor A form is a cell- and promoter-specific\nrepressor of human progesterone receptor B function. Mol Endocrinol\n1993, 7:1244–1255.\n16. Jackson KS, Brudney A, Hastings JM, Mavrogianis PA, Kim JJ, Fazleabas AT:\nThe altered distribution of the steroid hormone receptors and the\nchaperone immunophilin FKBP52 in a baboon model of endometriosis is\nassociated with progesterone resistance during the window of uterine\nreceptivity. Reprod Sci 2007, 14:137–150.\n17. Burney RO, Talbi S, Hamilton AE, Vo KC, Nyegaard M, Nezhat CR, Lessey BA,\nGiudice LC: Gene expression analysis of endometrium reveals\nprogesterone resistance and candidate susceptibility genes in women\nwith endometriosis. Endocrinology 2007, 148:3814–3826.\n18. Osteen KG, Bruner-Tran KL, Eisenberg E: Reduced progesterone action\nduring endometrial maturation: a potential risk factor for the\ndevelopment of endometriosis. Fertil Steril 2005, 83:529–537.\n19. Wardell SE, Edwards DP: Mechanisms controlling agonist and antagonist\npotential of selective progesterone receptor modulators (SPRMs). Semin\nReprod Med 2005, 23:9–21.\n20. Attia GR, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE: Progesterone\nreceptor isoform A but not B is expressed in endometriosis. J Clin\nEndocrinol Metab 2000, 85:2897–2902.\n21. Wu Y, Strawn E, Basir Z, Halverson G, Guo SW: Promoter hypermethylation\nof progesterone receptor isoform B (PR-B) in endometriosis. Epigenetics\n2006, 1:106–111.\n22. Brandenberger AW, Lebovic DI, Tee MK, Ryan IP, Tseng JF, Jaffe RB, Taylor\nRN: Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal\nendometrial and endometriosis-derived stromal cells. Mol Hum Reprod\n1999, 5:651–655.\n23. Xue Q, Lin Z, Cheng YH, Huang CC, Marsh E, Yin P, Milad MP, Confino E,\nReierstad S, Innes J, Bulun SE: Promoter methylation regulates estrogen\nreceptor 2 in human endometrium and endometriosis. Biol Reprod 2007,\n77:681–687.\n24. Trukhacheva E, Lin Z, Reierstad S, Cheng YH, Milad M, Bulun SE: Estrogen\nreceptor (ER) beta regulates ERalpha expression in stromal cells derived\nfrom ovarian endometriosis. J Clin Endocrinol Metab 2009, 94:615–622.\n25. Kohler G, Faustmann TA, Gerlinger C, Seitz C, Mueck AO: A dose-ranging\nstudy to determine the efficacy and safety of 1, 2, and 4mg of dienogest\ndaily for endometriosis. Int J Gynaecol Obstet 2010, 108:21–25.\n26. Strowitzki T, Faustmann T, Gerlinger C, Seitz C: Dienogest in the treatment\nof endometriosis-associated pelvic pain: a 12-week, randomized, double-\nblind, placebo-controlled study. Eur J Obstet Gynecol Reprod Biol 2010,\n151:193–198.\n27. Cosson M, Querleu D, Donnez J, Madelenat P, Konincks P, Audebert A,\nManhes H: Dienogest is as effective as triptorelin in the treatment of\nendometriosis after laparoscopic surgery: results of a prospective,\nmulticenter, randomized study. Fertil Steril 2002, 77:684–692.\n28. Strowitzki T, Marr J, Gerlinger C, Faustmann T, Seitz C: Dienogest is as\neffective as leuprolide acetate in treating the painful symptoms of\nendometriosis: a 24-week, randomized, multicentre, open-label trial.\nHum Reprod 2010, 25:633–641.\n29. Harada T, Momoeda M, Taketani Y, Aso T, Fukunaga M, Hagino H, Terakawa\nN: Dienogest is as effective as intranasal buserelin acetate for the relief\nof pain symptoms associated with endometriosis –a randomized, double-\nblind, multicenter, controlled trial. Fertil Steril 2009, 91:675–681.\n30. Petraglia F, Hornung D, Seitz C, Faustmann T, Gerlinger C, Luisi S, Lazzeri L,\nStrowitzki T: Reduced pelvic pain in women with endometriosis: efficacy\nof long-term dienogest treatment. Arch Gynecol Obstet 2012, 285:167–173.\n31. Okada H, Nakajima T, Yoshimura T, Yasuda K, Kanzaki H: The inhibitory\neffect of dienogest, a synthetic steroid, on the growth of human\nendometrial stromal cells in vitro. Mol Hum Reprod 2001, 7:341–347.\n32. Fu L, Osuga Y, Morimoto C, Hirata T, Hirota Y, Yano T, Taketani Y: Dienogest\ninhibits BrdU uptake with G0/G1 arrest in cultured endometriotic\nstromal cells. Fertil Steril 2008, 89:1344–1347.\n33. Shimizu Y, Mita S, Takeuchi T, Notsu T, Mizuguchi K, Kyo S: Dienogest, a\nsynthetic progestin, inhibits prostaglandin E2 production and aromatase\nexpression by human endometrial epithelial cells in a spheroid culture\nsystem. Steroids 2010, 76:60–67.\n34. : Revised American Society for Reproductive Medicine classification of\nendometriosis: 1996. Fertil Steril 1997, 67:817–821.\n35. Karita M, Yamashita Y, Hayashi A, Yoshida Y, Hayashi M, Yamamoto H,\nTanabe A, Terai Y, Ohmichi M: Does advanced-stage endometriosis affect\nthe gene expression of estrogen and progesterone receptors in\ngranulosa cells? Fertil Steril 2011, 95:889–894.\n36. Mote PA, Johnston JF, Manninen T, Tuohimaa P, Clarke CL: Detection of\nprogesterone receptor forms A and B by immunohistochemical analysis.\nJ Clin Pathol 2001, 54:624–630.\n37. Goldman S, Weiss A, Almalah I, Shalev E: Progesterone receptor expression\nin human decidua and fetal membranes before and after contractions:\npossible mechanism for functional progesterone withdrawal. Mol Hum\nReprod 2005, 11:269–277.\n38. Oh SY, Kim CJ, Park I, Romero R, Sohn YK, Moon KC, Yoon BH: Progesterone\nreceptor isoform (A/B) ratio of human fetal membranes increases during\nterm parturition. Am J Obstet Gynecol 2005, 193:1156–1160.\n39. Lessey BA, Metzger DA, Haney AF, McCarty KS Jr: Immunohistochemical\nanalysis of estrogen and progesterone receptors in endometriosis:\ncomparison with normal endometrium during the menstrual cycle and\nthe effect of medical therapy. Fertil Steril 1989, 51:409–415.\n40. Fujimoto J, Hirose R, Sakaguchi H, Tamaya T: Expression of oestrogen\nreceptor-alpha and -beta in ovarian endometriomata. Mol Hum Reprod\n1999, 5:742–747.\n41. Bulun SE: Endometriosis. N Engl J Med 2009, 360:268–279.\n42. Bulun SE, Cheng YH, Pavone ME, Xue Q, Attar E, Trukhacheva E, Tokunaga\nH, Utsunomiya H, Yin P, Luo X, et al: Estrogen receptor-beta, estrogen\nreceptor-alpha, and progesterone resistance in endometriosis. Semin\nReprod Med 2010, 28:36–43.\n43. Zeitoun KM, Bulun SE: Aromatase: a key molecule in the pathophysiology\nof endometriosis and a therapeutic target. Fertil Steril 1999, 72:961–969.\n44. Zeitoun K, Takayama K, Michael MD, Bulun SE: Stimulation of aromatase\nP450 promoter (II) activity in endometriosis and its inhibition in\nendometrium are regulated by competitive binding of steroidogenic\nfactor-1 and chicken ovalbumin upstream promoter transcription factor\nto the same cis-acting element. Mol Endocrinol 1999, 13:239–253.\n45. Aguirre C, Jayaraman A, Pike C, Baudry M: Progesterone inhibits estrogen-\nmediated neuroprotection against excitotoxicity by down-regulating\nestrogen receptor-beta. J Neurochem 2010, 115:1277–1287.\ndoi:10.1186/1757-2215-5-31\nCite this article as: Hayashi et al. : Dienogest increases the progesterone\nreceptor isoform B/A ratio in patients with ovarian endometriosis.\nJournal of Ovarian Research 2012 5:31.\nHayashi et al. Journal of Ovarian Research 2012, 5:31 Page 8 of 8\nhttp://www.ovarianresearch.com/content/5/1/31","source_license":"CC0","license_restricted":false}