Transcript
Page 1: Expression Pattern of G-Protein-Coupled Estrogen Receptor in Myometrium of Uteri …downloads.hindawi.com/journals/bmri/2017/5974693.pdf · 2018-11-19 · ResearchArticle Expression

Research ArticleExpression Pattern of G-Protein-Coupled Estrogen Receptor inMyometrium of Uteri with and without Adenomyosis

Jin-Jiao Li1 Hua Duan1 ShaWang1 Fu-Qing Sun1 Lu Gan1 Yi-Qun Tang1

Qian Xu1 and Tin-Chiu Li2

1Department of Gynecology Minimally Invasive Center Beijing Obstetrics and Gynecology HospitalCapital Medical University Beijing 100006 China2Department of Obstetrics and Gynecology The Chinese University of Hong Kong Shatin Hong Kong

Correspondence should be addressed to Hua Duan duanhuasci163com

Received 7 May 2017 Accepted 27 August 2017 Published 4 October 2017

Academic Editor Sven Becker

Copyright copy 2017 Jin-Jiao Li et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

ObjectiveTo compare the expression ofG-protein-coupled estrogen receptor (GPER) in the junctional zone and outermyometriumof the proliferative and secretory phases of women with and without adenomyosisMethods A total of 76 women were included inthis study 42 with adenomyosis (proliferative phase 119899 = 23 secretory phases 119899 = 19) and 34 controls (proliferative phase 119899 = 16secretory phases 119899 = 18) Protein and total RNA were extracted from the junctional zone (JZ) and outer myometrium (OM)GPER protein and mRNA expression levels were evaluated by the use of western blotting and real-time quantitative polymerasechain reaction (RT-qPCR) Results The expression of GPER protein and mRNA in women with adenomyosis was significantlyhigher than that of control subjects both in the junctional zone and in the outer myometrium and both in the proliferative andin the secretory phases Conclusion The significant and consistent increase in GPER expression in adenomyosis compared withcontrol subjects regardless of whether it was in the proliferative or secretory phases and regardless of whether it was in the JZ orOM suggests that GPER plays an important role in the pathogenesis of the adenomyosis

1 Introduction

Adenomyosis is characterized by the extension of endo-metrium into the myometrium along with myometrialsmooth muscle cells hyperplasia and hypertrophy It iscommon in women of reproductive age and often regressesafter menopause [1 2] Estrogen plays a crucial role in thepathogenesis of adenomyosis The effects of estrogen aremediated by two types of estrogen receptors nuclear estrogenreceptors (ER-120572 andER-120573) which aremembers of the nuclearreceptor family of intracellular receptors and membraneestrogen receptor (GPER or G-protein-coupled estrogenreceptor) [3] Adenomyosis is known to be associated withchanges in the expression of ER-120572 and ER-120573 A recentstudy showed that ER-120572 expression was reduced in the mid-secretory phase endometrium of women with adenomyosiswhereas ER-120573 was increased not only in the endometriumbut also in the inner myometrium and outer myometriumof women with adenomyosis compared with control subjects[4] However the expression of GPER in the uterus of women

with adenomyosis has not been previously investigated Inthis study we studied the expression of GPER in the outerand inner myometrium (junctional zone) of women withadenomyosis and compared the results to a group of controlsubjects

2 Materials and Methods

21 Subjects All subjects recruited in this study were pre-menopausal women who underwent hysterectomy at theBeijing Obstetrics and Gynecology Hospital Capital MedicalUniversity Beijing China

The inclusion criteria included the following(1) Regular 25ndash35 daysrsquo cycles(2) No hormonal treatment or having not used an

intrauterine device for 3 months prior to hysterec-tomy

(3) No evidence of leiomyoma

HindawiBioMed Research InternationalVolume 2017 Article ID 5974693 6 pageshttpsdoiorg10115520175974693

2 BioMed Research International

There were 2 groups of women recruited Group I con-sisted of 42 women with adenomyosis histologically con-firmed In this study the diagnosis of adenomyosis was basedon histological confirmation of the presence of endometrialtissue more than 25mm below the endomyometrial junctionor a JZ thickness of more than 12mm [5] The additionalinclusion criterion for women in this group was the presenceof dysmenorrhea with or without menorrhagia prior to thesurgery In this respect we have included only subjects withsymptomatic adenomyosis which was the underlying reasonfor their request for hysterectomy Group II consisted of 34subjects with no evidence of adenomyosis who underwenthysterectomy due to early cervical cancer or ovarian cancerThe exclusion criteria for this group of women were pre-operative radiotherapy or chemotherapy and involvement ofthe endometrium or myometrium by neoplasm As the studywas based on histological confirmation of adenomyosis onhysterectomy specimens and the collection of biopsy fromdifferent parts of the excised uterine specimen it was nec-essary to recruit control subjects who required hysterectomyfor a different clinical reason However it was also considerednecessary to exclude uterus with significant myometrialpathology such as myoma which could have altered histolog-ical findings in the junctional zone or myometrium Giventhat it was unusual for women of reproductive age to have anormal uterus removed other than those who suffered fromearly cervical or ovarian cancer we have chosen to includethis group of women as control subjects As it happenedall subjects included in the study belonged to early cervicalcancer

Excisional biopsy of the junctional zone and outer 13 ofthe myometrium was performed from the anterior fundalwall of each hysterectomy specimen The biopsy of theouter 13 myometrium was undertaken from 2mm beneaththe serosal surface of the uterus whereas the biopsy fromthe junctional zone was undertaken 2-3mm beneath theendometrium taking special care not to include any normalendometrial tissue in the biopsy

22 Histological Dating Histological dating of theendometrium from the uterine specimens was performed byan experienced pathologist with the use of standard criteria[6] The specimens were then classified into proliferative(PP) or secretory phases (SP)

23 Western Blotting Homogenized tissues were lysed forprotein extractions Protein extracts (30ndash50mg) were sep-arated by electrophoresis on 12 sodium dodecyl sulfate-polyacrylamide gels Proteins were transferred to polyvinyli-dene fluoride (PVDF) membranes incubated with rab-bit anti-GPER (ab39742 from Abcam Cambridge Mas-sachusetts) as a primary antibody and then developedwith a secondary anti-rabbit antibody (obtained from CellSignaling Technology Danvers Massachusetts) The signalwas detected using a LI-COR Biosciences Odyssey InfraredImaging System (LI-COR Lincoln Nebraska) Equivalentprotein loading and transfer efficiency were verified by stain-ing for GAPDH (D16H11 from Cell Signaling TechnologyDanvers Massachusetts)

Table 1 Primer sequences for GPER

Primer Sequence

GPER Forward 51015840-TGACCATCCCCGACCTGTAC-31015840

Reverse 51015840-CAGGTGAGGAAGAAGACGCT-31015840

GAPDH Forward 51015840-CTCCTCCACCTTTGACGCTG- 31015840

Reverse 51015840-TCCTCTTGTGCTCTTGCTGG-31015840

24 Quantitative Real-Time Polymerase Chain ReactionTotal RNA was extracted and purified from the tissuespecimens 10 120583g RNA was reverse transcribed with randomprimers using Superscript II reverse transcriptase (Life Tech-nologies Inc Melbourne Australia) Quantitative PCR wasperformed in the presence of SYBR Green (Tiangen BiotechChina) and amplicon yield was monitored during cyclingin an ABI 7500 Real-Time Polymerase Chain ReactionSystem (Applied Biosystems Grand Island New York) thatcontinually measured fluorescence caused by the binding ofthe dye to double-strandedDNAThe cycling conditionswere95∘C for 15minutes 40 cycles at 95∘C for 10 seconds and 60∘Cfor 32 seconds The cycle at which the fluorescence reacheda set threshold (cycle threshold) was used for quantitativeanalyses The cycle threshold in each assay was set at a levelat which the exponential increase in amplicon abundancewas approximately parallel between all samples Relative geneexpressionwas calculated in relation to an internal control fornormalization (glyceraldehyde 3-phosphate dehydrogenaseGAPDH) using the comparative cycle threshold methodPrimer sequences for GPER and GAPDH are presented inTable 1

25 Ethics This study was approved by Ethical Committeeof Clinical Research of Beijing Obstetrics and GynecologyHospital Capital Medical University Beijing China Writteninformed consent was obtained from all patients who partic-ipated in this study This study was conducted in accordancewith the Declaration of Helsinki (1964)

26 Statistical Analysis Normally distributed data were pre-sented as mean plusmn standard deviation Student t-test was usedto test for differences between groups Statistical analyseswere performed with the use of SPSS 190 for WindowsFor all tests a 119901 value of lt005 was considered statisticallysignificant

3 Results

31 Demographics A total of 42 women with adenomyosisand 34 women without adenomyosis (control subjects) wereincluded in the study In group I 23 specimens were classifiedas proliferative and 19 as secretory In group II 16 specimenswere classified as proliferative and the remaining 18 assecretory The demographic details of these two groups ofwomen are compared in Table 2 There was no significantdifference in any of the features between the two groups

Using western blotting GPER protein expression signalsappeared at approximately 42 kd (Figure 1) The position of

BioMed Research International 3

Table 2 Demographic details of patients with adenomyosis andcontrol subjects

Adenomyosis Control 119901 valueAge (years) 441 plusmn 29 430 plusmn 44 007Parity 13 plusmn 01 14 plusmn 01 045Miscarriage 21 plusmn 14 18 plusmn 11 006Body mass index kgm2 232 plusmn 27 241 plusmn 33 009Values are given in mean plusmn standard deviation

GPER

GPER

GAPDH

GAPDH

Adenomyosis Control

JZ

OM

PPPP SP SP

Figure 1 Representative western blotting of G-protein-coupledestrogen receptor (GPER) and glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) in biopsy specimens from the junctional zone(JZ) and outer myometrium (OM) in the proliferative phase (PP)and secretory phase (SP)

the GPER signal was the same as that observed in MCF-7breast cancer cells (data not shown)

32 Comparison of GPER Expression between ProliferativePhase and Secretory Phase In Table 3 the GPER proteinand mRNA expression in the proliferative and secretoryphase were compared separately in women with and withoutadenomyosis and in the JZ and OM

In the control subjects the expressions of both proteinand mRNA in the JZ in the proliferative phase were bothsignificantly (119901 lt 005) higher than those of the secretoryphase whereas the expressions of both protein and mRNAin the OM in the proliferative phase and the secretory phasewere similar

In women with adenomyosis in contrast there was nosignificant difference in the expression of GPER protein andmRNA level between the proliferative phase and secretoryphase in both the JZ and OM

33 Comparison of GPER Expression between JunctionalZone and Outer Myometrium In Table 3 the GPER proteinand mRNA expression in the junctional zone and outermyometrium were compared separately in women with andwithout adenomyosis and in the proliferative and secretoryphases

In the control group in the proliferative phase theexpression of both protein and mRNA in the JZ was higherthan that of the OM In the secretory phase however theexpression of both protein and mRNA in the JZ was similarto that of the OM

In women with adenomyosis on the other hand theexpression of both protein and mRNA in the JZ were higherthan that of the OM in both the proliferative and secretoryphases

34 Comparison of GPER Expression between Adenomyosisand Control Groups In Table 3 the GPER protein andmRNA expression in women with and without adenomyosiswere compared separately in the junctional zone and outermyometrium and in the proliferative and secretory phase

In the JZ the GPER protein expression and the mRNAlevel in women with adenomyosis were significantly higherthan that of control subjects in both the proliferative phaseand secretory phase

In the OM the GPER protein expression and the mRNAlevel in women with adenomyosis were also significantlyhigher than that of control subjects in both the proliferativephase and secretory phase

4 Discussion

In this study we found that the expression of GPER proteinand mRNA in women with adenomyosis was significantlyhigher than that of women without adenomyosis both in thejunctional zone and in the outermyometrium and both in theproliferative and in the secretory phases

Adenomyosis is often considered to be an estrogen-dependent disease with changes in structure and functionof the JZ [1] observed in the majority of cases The JZis responsible for uterine peristalsis which regulates spermtransport and implantation [7 8] Structural changes in theJZ in adenomyosis commonly include JZ hyperplasia andthickening [9] Ultrastructurally the myocytes in the JZof uterus affected by adenomyosis appeared smaller [10]Abnormal expression of oxytocin receptor in the JZ ofwomenwith adenomyosis has been observed [11] which may explainthe occurrence of hyperperistalsis and dysperistalsis in turnleading to dysmenorrhea and disturbance of reproductivefunction However the outer myometrium whose primaryfunction is involvedwith parturition [12] is often not affectedin adenomyosis until a later stage

There are two different types of estrogen receptors Thenuclear estrogen receptors (ER-120572 and ER-120573) mediate geneexpression through binding to estrogen receptor elementsin the promotor and regulatory regions of the target genesin contrast GPER the other type of estrogen receptormediates rapid cellular effects GPER is a membrane estrogenreceptor a 7-transmembrane spanning G-protein-coupledreceptor also called G-protein-coupled receptor 30 (GPR30)[13] GPER is structurally and genetically unrelated to ER-120572 and ER-120573 and expressed independently [3] It binds toestrogen with high affinity whereas binding affinities ofGPER for other steroid hormones are very low [14] In earlierstudies estrogen effects can be mimicked by selective GPERagonist or antagonist whereas inGPERknockoutmice theseeffects are absent or reduced suggesting that GPER plays anessential role [15] Expression of GPER has been describedin multiple physiological systems and tissues including thebreast heart endothelium brain bone adrenal kidney

4 BioMed Research International

Table 3 Relative GPER protein and mRNA expression levels (GPERGAPDH)

Adenomyosis group (119899 = 42) Control group (119899 = 34)JZ OM JZ OM

Relative GPER protein expression levels

PP 112 plusmn 0072bce(119899 = 23)

096 plusmn 0043bce(119899 = 23)

095 plusmn 0027ace(119899 = 16)

083 plusmn 0051bce(119899 = 16)

SP 109 plusmn 0076bce(119899 = 19)

094 plusmn 0052bce(119899 = 19)

082 plusmn 0097ade(119899 = 18)

084 plusmn 0086bde(119899 = 18)

Relative GPER mRNA expression levels

PP 152 plusmn 012bce(119899 = 23)

131 plusmn 009bce(119899 = 23)

128 plusmn 007ace(119899 = 16)

104 plusmn 01bce(119899 = 16)

SP 151 plusmn 014bce(119899 = 19)

129 plusmn 018bce(119899 = 19)

109 plusmn 013ade(119899 = 18)

1bde(119899 = 18)

Values expressed as mean plusmn standard deviation aComparison between proliferative phase and secretary phase (same group and zone) 119901 lt 005 Student 119905-test bcomparison between proliferative phase and secretary phase (same group and zone) 119901 ge 005 Student 119905-test ccomparison between outer myometrialzone and junctional zone (same group and phase) 119901 lt 005 Student 119905-test dcomparison between outer myometrial zone and junctional zone (same group andphase) 119901 ge 005 Student 119905-test ecomparison between adenomyosis and control group (corresponding phase and zone) 119901 lt 005 Student 119905-test

endometrium and ovary [15ndash20] The changes in GPERexpression in a number of gynecological conditions such asthe endometrium of women with endometriosis [21] andsmooth muscle of myoma [22] have been examined Onthe other hand whilst the expressions of nuclear estrogenreceptors (ER-120572 and ER-120573) have been examined in the innerand outer myometrium of adenomyosis [12] the expressionof GPER in adenomyosis has not previously been studiedEvidence has shown that GPER agonist G1 can lead toapoptosis in endometriosis and suppress proliferation ofendometriotic stromal cells [23] GPER is closely relatedto the outcome of estrogen therapy on various estrogen-dependent diseases [24ndash26] and selective GPER ligands(such as GPER agonist G1 and antagonist G15) have beenshown to exert control of these diseases [27ndash29] A betterunderstanding of the role played byGPER in the pathogenesisof adenomyosis may open up opportunity for GPER-targetedtherapy for the condition [15]

The special design of this study enabled us to examine thecyclical change and anatomical variation of GPER expressionin control subjects in addition to comparing GPER expres-sion between women with and without adenomyosis Onepossible limitation of our study was that biopsy specimensfrom the junctional zone of women with adenomyosis oftencontained foci of endometrial tissue which could have con-tributed to the observed difference in results between the twogroups of subjects

Cyclical Changes In our study in control subjects weobserved that the expression of GPER in the proliferativephase in the JZ was higher than that of secretory phase ofthe JZ however the cyclical change was not observed inthe OM In women with adenomyosis the cyclical changeappeared to have disappeared Similar cyclical changes in theultrastructure of myocytes in the JZ and OM were observedin our previous study [10]

Anatomical Variation As for the anatomical variation incontrol subjects the expression of GPER in the JZ was higher

than that of the OM in the proliferative phase but not thesecretory phases In contrast in women with adenomyosisthe expression of GPER in the JZ was higher than that of theOM in both the proliferative phase and the secretory phasesIn other words the anatomical variation between the JZ andOM in women with adenomyosis was consistently observedindependent of the phases of the cycle

Adenomyosis versus Control In contrast to the observationsrelating to the cyclical and anatomical variation of GPER inwhich significant difference in control subjects was foundonly in the proliferative phase and JZ there was consistentand significant difference in GPER expression regardless ofthe stage of the cycle (proliferative or secretory) or anatomy(JZ or OM)

Taken together the cyclical and anatomical variation ofGPER observed in control subjects in this study is consistentwith our current understanding that the effects of estrogen aremore dominant in the proliferative phase than the secretoryphase and more pronounced in the JZ than in the OM Inaddition the special design of our study enabled us to controlfor two important confounding variables (cyclical changesand anatomical variation) by doing so the variance in resultsdue to the effect of these confounding variables was reducedWhilst it is already known that the expressions of estrogennuclear receptors ER-120573 (but not ER-120572) and progesteronereceptor are different between women with and withoutadenomyosis [4] the additional finding in this study thatGPER expression is also altered in women with adenomyosisconfirms the notion that adenomyosis is associated withalteration in several different steroid receptors

Overall the finding observed in this study appears tohave provided a molecular basis for the smooth musclehyperplasia and hypertrophy observed in adenomyosis [1 2]Specifically it seems plausible that the abnormally elevatedGPER expression in the JZ is one mechanism by which thesmooth muscle cells continue to proliferate in adenomyosis

To conclude we have found significant and consis-tent increase in GPER expression in adenomyosis in both

BioMed Research International 5

proliferative and secretory phases and in both the JZ andOM suggesting that GPER plays an important role in thepathogenesis of the condition It remains to be seen iftreatment targeting the expression of GPER by the use ofselective GPER ligands may help to treat or prevent thecondition

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (no 81270680 no 81571412)and the Beijing Municipal Administration of Hospital Clin-ical Medicine Development of Special Funding Support(ZYLX201406)

References

[1] G BenagianoMHabiba and I Brosens ldquoThe pathophysiologyof uterine adenomyosis An updaterdquo Fertility and Sterility vol98 no 3 pp 572ndash579 2012

[2] G Benagiano I Brosens and S Carrara ldquoAdenomyosis Newknowledge is generating new treatment strategiesrdquo WomenrsquosHealth vol 5 no 3 pp 297ndash311 2009

[3] P Vrtacnik B Ostanek S Mencej-Bedrac and J Marc ldquoThemany faces of estrogen signalingrdquo Biochemia Medica vol 24no 3 pp 329ndash342 2014

[4] M K Mehasseb R Panchal A H Taylor L Brown S C Belland M Habiba ldquoEstrogen and progesterone receptor isoformdistribution through themenstrual cycle in uteri with andwith-out adenomyosisrdquo Fertility and Sterility vol 95 no 7 pp 2228ndash2235 2011

[5] J Struble S Reid andM A Bedaiwy ldquoAdenomyosis A ClinicalReview of a Challenging Gynecologic Conditionrdquo Journal ofMinimally Invasive Gynecology vol 23 no 2 pp 164ndash185 2016

[6] R W Noyes and J O Haman ldquoAccuracy of endometrial datingcorrelation of endometrial dating with basal body temperatureand mensesrdquo Fertility and Sterility vol 4 no 6 pp 504ndash5171953

[7] M K Mehasseb S C Bell J H Pringle and M A HabibaldquoUterine adenomyosis is associatedwith ultrastructural featuresof altered contractility in the inner myometriumrdquo Fertility andSterility vol 93 no 7 pp 2130ndash2136 2010

[8] I Brosens I Derwig J Brosens L Fusi G Benagiano and RPijnenborg ldquoThe enigmatic uterine junctional zone The miss-ing link between reproductive disorders and major obstetricaldisordersrdquo Human Reproduction vol 25 no 3 pp 569ndash5742010

[9] G Levy A Dehaene N Laurent et al ldquoAn update on adeno-myosisrdquo Diagnostic and Interventional Imaging vol 94 no 1pp 3ndash25 2013

[10] Y Zhang L Zhou T C Li H Duan P Yu and H Y WangldquoUltrastructural features of endometrial-myometrial interfaceand its alteration in adenomyosisrdquo International Journal ofClinical and Experimental Pathology vol 7 no 4 pp 1469ndash14772014

[11] Y Zhang P Yu F Sun T C Li J M Cheng and H DuanldquoExpression of oxytocin receptors in the uterine junctional zonein women with adenomyosisrdquo Acta Obstetricia et GynecologicaScandinavica vol 94 no 4 pp 412ndash418 2015

[12] M K Mehasseb S C Bell L Brown J H Pringle and MHabiba ldquoPhenotypic characterisation of the inner and outermyometrium in normal and adenomyotic uterirdquo Gynecologicand Obstetric Investigation vol 71 no 4 pp 217ndash224 2011

[13] CM Revankar D F Cimino L A Sklar J B Arterburn and ER Prossnitz ldquoA transmembrane intracellular estrogen receptormediates rapid cell signalingrdquo Science vol 307 no 5715 pp1625ndash1630 2005

[14] E J Filardo and P Thomas ldquoMinireview G protein-coupledestrogen receptor-1 GPER-1 Its mechanism of action and rolein female reproductive cancer renal and vascular physiologyrdquoEndocrinology vol 153 no 7 pp 2953ndash2962 2012

[15] E R Prossnitz and M Barton ldquoEstrogen biology new insightsinto GPER function and clinical opportunitiesrdquo Molecular andCellular Endocrinology vol 389 no 1-2 pp 71ndash83 2014

[16] A Madeo and M Maggiolini ldquoNuclear alternate estrogenreceptor GPR30 mediates 17120573-estradiol - Induced gene expres-sion and migration in breast cancer - Associated fibroblastsrdquoCancer Research vol 70 no 14 pp 6036ndash6046 2010

[17] T J Heino A S Chagin and L Savendahl ldquoThe novel estrogenreceptor G-protein-coupled receptor 30 is expressed in humanbonerdquo Journal of Endocrinology vol 197 no 2 pp R1ndashR6 2008

[18] P F Oliveira M G Alves A D Martins et al ldquoExpression pat-tern of G protein-coupled receptor 30 in human seminiferoustubular cellsrdquoGeneral and Comparative Endocrinology vol 201pp 16ndash20 2014

[19] S Heublein M Lenhard T Vrekoussis et al ldquoThe G-protein-coupled estrogen receptor (GPER) is expressed in normalhuman ovaries and is upregulated in ovarian endometriosis andpelvic inflammatory disease involving the ovaryrdquo ReproductiveSciences vol 19 no 11 pp 1197ndash1204 2012

[20] W Huang Y Chen Y Liu et al ldquoRoles of ER120573 and GPR30 inproliferative response of human bladder cancer cell to estrogenrdquoBioMed Research International vol 2015 Article ID 251780 10pages 2015

[21] B J Plante B A Lessey R N Taylor et al ldquoG protein-coupledestrogen receptor (GPER) expression in normal and abnormalendometriumrdquoReproductive Sciences vol 19 no 7 pp 684ndash6932012

[22] R Tian Z Wang Z Shi et al ldquoDifferential expression of G-protein-coupled estrogen receptor-30 in human myometrialand uterine leiomyoma smooth musclerdquo Fertility and Sterilityvol 99 no 1 pp 256ndash263 2013

[23] T Mori F Ito H Matsushima et al ldquoG protein-coupledestrogen receptor 1 agonist G-1 induces cell cycle arrest in themitotic phase leading to apoptosis in endometriosisrdquo Fertilityand Sterility vol 103 no 5 pp 1228ndash1235 2015

[24] L S G Ulrich ldquoEndometrial cancer types prognosis femalehormones and antihormonesrdquo Climacteric vol 14 no 4 pp418ndash425 2011

[25] C Krakstad J Trovik E Wik et al ldquoLoss of GPER identifiesnew targets for therapy among a subgroup of ER120572-positiveendometrial cancer patients with poor outcomerdquoBritish Journalof Cancer vol 106 no 10 pp 1682ndash1688 2012

[26] T Ignatov S Modl M Thulig et al ldquoGPER-1 acts as a tumorsuppressor in ovarian cancerrdquo Journal of Ovarian Research vol6 no 1 article 51 pp 51ndash61 2013

6 BioMed Research International

[27] EAAriazi E Brailoiu S Yerrumet al ldquoTheGprotein-coupledreceptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cellsrdquo Cancer Research vol 70 no 3 pp1184ndash1194 2010

[28] N Nass and T Kalinski ldquoTamoxifen resistance From cellculture experiments towards novel biomarkersrdquo PathologyResearch and Practice vol 211 no 3 pp 189ndash197 2015

[29] R Sirianni A Chimento C Ruggiero et al ldquoThe novel estrogenreceptor G protein-coupled receptor 30 mediates the prolifer-ative effects induced by 17120573-estradiol onmouse spermatogonialGC-1 cell linerdquo Endocrinology vol 149 no 10 pp 5043ndash50512008

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Page 2: Expression Pattern of G-Protein-Coupled Estrogen Receptor in Myometrium of Uteri …downloads.hindawi.com/journals/bmri/2017/5974693.pdf · 2018-11-19 · ResearchArticle Expression

2 BioMed Research International

There were 2 groups of women recruited Group I con-sisted of 42 women with adenomyosis histologically con-firmed In this study the diagnosis of adenomyosis was basedon histological confirmation of the presence of endometrialtissue more than 25mm below the endomyometrial junctionor a JZ thickness of more than 12mm [5] The additionalinclusion criterion for women in this group was the presenceof dysmenorrhea with or without menorrhagia prior to thesurgery In this respect we have included only subjects withsymptomatic adenomyosis which was the underlying reasonfor their request for hysterectomy Group II consisted of 34subjects with no evidence of adenomyosis who underwenthysterectomy due to early cervical cancer or ovarian cancerThe exclusion criteria for this group of women were pre-operative radiotherapy or chemotherapy and involvement ofthe endometrium or myometrium by neoplasm As the studywas based on histological confirmation of adenomyosis onhysterectomy specimens and the collection of biopsy fromdifferent parts of the excised uterine specimen it was nec-essary to recruit control subjects who required hysterectomyfor a different clinical reason However it was also considerednecessary to exclude uterus with significant myometrialpathology such as myoma which could have altered histolog-ical findings in the junctional zone or myometrium Giventhat it was unusual for women of reproductive age to have anormal uterus removed other than those who suffered fromearly cervical or ovarian cancer we have chosen to includethis group of women as control subjects As it happenedall subjects included in the study belonged to early cervicalcancer

Excisional biopsy of the junctional zone and outer 13 ofthe myometrium was performed from the anterior fundalwall of each hysterectomy specimen The biopsy of theouter 13 myometrium was undertaken from 2mm beneaththe serosal surface of the uterus whereas the biopsy fromthe junctional zone was undertaken 2-3mm beneath theendometrium taking special care not to include any normalendometrial tissue in the biopsy

22 Histological Dating Histological dating of theendometrium from the uterine specimens was performed byan experienced pathologist with the use of standard criteria[6] The specimens were then classified into proliferative(PP) or secretory phases (SP)

23 Western Blotting Homogenized tissues were lysed forprotein extractions Protein extracts (30ndash50mg) were sep-arated by electrophoresis on 12 sodium dodecyl sulfate-polyacrylamide gels Proteins were transferred to polyvinyli-dene fluoride (PVDF) membranes incubated with rab-bit anti-GPER (ab39742 from Abcam Cambridge Mas-sachusetts) as a primary antibody and then developedwith a secondary anti-rabbit antibody (obtained from CellSignaling Technology Danvers Massachusetts) The signalwas detected using a LI-COR Biosciences Odyssey InfraredImaging System (LI-COR Lincoln Nebraska) Equivalentprotein loading and transfer efficiency were verified by stain-ing for GAPDH (D16H11 from Cell Signaling TechnologyDanvers Massachusetts)

Table 1 Primer sequences for GPER

Primer Sequence

GPER Forward 51015840-TGACCATCCCCGACCTGTAC-31015840

Reverse 51015840-CAGGTGAGGAAGAAGACGCT-31015840

GAPDH Forward 51015840-CTCCTCCACCTTTGACGCTG- 31015840

Reverse 51015840-TCCTCTTGTGCTCTTGCTGG-31015840

24 Quantitative Real-Time Polymerase Chain ReactionTotal RNA was extracted and purified from the tissuespecimens 10 120583g RNA was reverse transcribed with randomprimers using Superscript II reverse transcriptase (Life Tech-nologies Inc Melbourne Australia) Quantitative PCR wasperformed in the presence of SYBR Green (Tiangen BiotechChina) and amplicon yield was monitored during cyclingin an ABI 7500 Real-Time Polymerase Chain ReactionSystem (Applied Biosystems Grand Island New York) thatcontinually measured fluorescence caused by the binding ofthe dye to double-strandedDNAThe cycling conditionswere95∘C for 15minutes 40 cycles at 95∘C for 10 seconds and 60∘Cfor 32 seconds The cycle at which the fluorescence reacheda set threshold (cycle threshold) was used for quantitativeanalyses The cycle threshold in each assay was set at a levelat which the exponential increase in amplicon abundancewas approximately parallel between all samples Relative geneexpressionwas calculated in relation to an internal control fornormalization (glyceraldehyde 3-phosphate dehydrogenaseGAPDH) using the comparative cycle threshold methodPrimer sequences for GPER and GAPDH are presented inTable 1

25 Ethics This study was approved by Ethical Committeeof Clinical Research of Beijing Obstetrics and GynecologyHospital Capital Medical University Beijing China Writteninformed consent was obtained from all patients who partic-ipated in this study This study was conducted in accordancewith the Declaration of Helsinki (1964)

26 Statistical Analysis Normally distributed data were pre-sented as mean plusmn standard deviation Student t-test was usedto test for differences between groups Statistical analyseswere performed with the use of SPSS 190 for WindowsFor all tests a 119901 value of lt005 was considered statisticallysignificant

3 Results

31 Demographics A total of 42 women with adenomyosisand 34 women without adenomyosis (control subjects) wereincluded in the study In group I 23 specimens were classifiedas proliferative and 19 as secretory In group II 16 specimenswere classified as proliferative and the remaining 18 assecretory The demographic details of these two groups ofwomen are compared in Table 2 There was no significantdifference in any of the features between the two groups

Using western blotting GPER protein expression signalsappeared at approximately 42 kd (Figure 1) The position of

BioMed Research International 3

Table 2 Demographic details of patients with adenomyosis andcontrol subjects

Adenomyosis Control 119901 valueAge (years) 441 plusmn 29 430 plusmn 44 007Parity 13 plusmn 01 14 plusmn 01 045Miscarriage 21 plusmn 14 18 plusmn 11 006Body mass index kgm2 232 plusmn 27 241 plusmn 33 009Values are given in mean plusmn standard deviation

GPER

GPER

GAPDH

GAPDH

Adenomyosis Control

JZ

OM

PPPP SP SP

Figure 1 Representative western blotting of G-protein-coupledestrogen receptor (GPER) and glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) in biopsy specimens from the junctional zone(JZ) and outer myometrium (OM) in the proliferative phase (PP)and secretory phase (SP)

the GPER signal was the same as that observed in MCF-7breast cancer cells (data not shown)

32 Comparison of GPER Expression between ProliferativePhase and Secretory Phase In Table 3 the GPER proteinand mRNA expression in the proliferative and secretoryphase were compared separately in women with and withoutadenomyosis and in the JZ and OM

In the control subjects the expressions of both proteinand mRNA in the JZ in the proliferative phase were bothsignificantly (119901 lt 005) higher than those of the secretoryphase whereas the expressions of both protein and mRNAin the OM in the proliferative phase and the secretory phasewere similar

In women with adenomyosis in contrast there was nosignificant difference in the expression of GPER protein andmRNA level between the proliferative phase and secretoryphase in both the JZ and OM

33 Comparison of GPER Expression between JunctionalZone and Outer Myometrium In Table 3 the GPER proteinand mRNA expression in the junctional zone and outermyometrium were compared separately in women with andwithout adenomyosis and in the proliferative and secretoryphases

In the control group in the proliferative phase theexpression of both protein and mRNA in the JZ was higherthan that of the OM In the secretory phase however theexpression of both protein and mRNA in the JZ was similarto that of the OM

In women with adenomyosis on the other hand theexpression of both protein and mRNA in the JZ were higherthan that of the OM in both the proliferative and secretoryphases

34 Comparison of GPER Expression between Adenomyosisand Control Groups In Table 3 the GPER protein andmRNA expression in women with and without adenomyosiswere compared separately in the junctional zone and outermyometrium and in the proliferative and secretory phase

In the JZ the GPER protein expression and the mRNAlevel in women with adenomyosis were significantly higherthan that of control subjects in both the proliferative phaseand secretory phase

In the OM the GPER protein expression and the mRNAlevel in women with adenomyosis were also significantlyhigher than that of control subjects in both the proliferativephase and secretory phase

4 Discussion

In this study we found that the expression of GPER proteinand mRNA in women with adenomyosis was significantlyhigher than that of women without adenomyosis both in thejunctional zone and in the outermyometrium and both in theproliferative and in the secretory phases

Adenomyosis is often considered to be an estrogen-dependent disease with changes in structure and functionof the JZ [1] observed in the majority of cases The JZis responsible for uterine peristalsis which regulates spermtransport and implantation [7 8] Structural changes in theJZ in adenomyosis commonly include JZ hyperplasia andthickening [9] Ultrastructurally the myocytes in the JZof uterus affected by adenomyosis appeared smaller [10]Abnormal expression of oxytocin receptor in the JZ ofwomenwith adenomyosis has been observed [11] which may explainthe occurrence of hyperperistalsis and dysperistalsis in turnleading to dysmenorrhea and disturbance of reproductivefunction However the outer myometrium whose primaryfunction is involvedwith parturition [12] is often not affectedin adenomyosis until a later stage

There are two different types of estrogen receptors Thenuclear estrogen receptors (ER-120572 and ER-120573) mediate geneexpression through binding to estrogen receptor elementsin the promotor and regulatory regions of the target genesin contrast GPER the other type of estrogen receptormediates rapid cellular effects GPER is a membrane estrogenreceptor a 7-transmembrane spanning G-protein-coupledreceptor also called G-protein-coupled receptor 30 (GPR30)[13] GPER is structurally and genetically unrelated to ER-120572 and ER-120573 and expressed independently [3] It binds toestrogen with high affinity whereas binding affinities ofGPER for other steroid hormones are very low [14] In earlierstudies estrogen effects can be mimicked by selective GPERagonist or antagonist whereas inGPERknockoutmice theseeffects are absent or reduced suggesting that GPER plays anessential role [15] Expression of GPER has been describedin multiple physiological systems and tissues including thebreast heart endothelium brain bone adrenal kidney

4 BioMed Research International

Table 3 Relative GPER protein and mRNA expression levels (GPERGAPDH)

Adenomyosis group (119899 = 42) Control group (119899 = 34)JZ OM JZ OM

Relative GPER protein expression levels

PP 112 plusmn 0072bce(119899 = 23)

096 plusmn 0043bce(119899 = 23)

095 plusmn 0027ace(119899 = 16)

083 plusmn 0051bce(119899 = 16)

SP 109 plusmn 0076bce(119899 = 19)

094 plusmn 0052bce(119899 = 19)

082 plusmn 0097ade(119899 = 18)

084 plusmn 0086bde(119899 = 18)

Relative GPER mRNA expression levels

PP 152 plusmn 012bce(119899 = 23)

131 plusmn 009bce(119899 = 23)

128 plusmn 007ace(119899 = 16)

104 plusmn 01bce(119899 = 16)

SP 151 plusmn 014bce(119899 = 19)

129 plusmn 018bce(119899 = 19)

109 plusmn 013ade(119899 = 18)

1bde(119899 = 18)

Values expressed as mean plusmn standard deviation aComparison between proliferative phase and secretary phase (same group and zone) 119901 lt 005 Student 119905-test bcomparison between proliferative phase and secretary phase (same group and zone) 119901 ge 005 Student 119905-test ccomparison between outer myometrialzone and junctional zone (same group and phase) 119901 lt 005 Student 119905-test dcomparison between outer myometrial zone and junctional zone (same group andphase) 119901 ge 005 Student 119905-test ecomparison between adenomyosis and control group (corresponding phase and zone) 119901 lt 005 Student 119905-test

endometrium and ovary [15ndash20] The changes in GPERexpression in a number of gynecological conditions such asthe endometrium of women with endometriosis [21] andsmooth muscle of myoma [22] have been examined Onthe other hand whilst the expressions of nuclear estrogenreceptors (ER-120572 and ER-120573) have been examined in the innerand outer myometrium of adenomyosis [12] the expressionof GPER in adenomyosis has not previously been studiedEvidence has shown that GPER agonist G1 can lead toapoptosis in endometriosis and suppress proliferation ofendometriotic stromal cells [23] GPER is closely relatedto the outcome of estrogen therapy on various estrogen-dependent diseases [24ndash26] and selective GPER ligands(such as GPER agonist G1 and antagonist G15) have beenshown to exert control of these diseases [27ndash29] A betterunderstanding of the role played byGPER in the pathogenesisof adenomyosis may open up opportunity for GPER-targetedtherapy for the condition [15]

The special design of this study enabled us to examine thecyclical change and anatomical variation of GPER expressionin control subjects in addition to comparing GPER expres-sion between women with and without adenomyosis Onepossible limitation of our study was that biopsy specimensfrom the junctional zone of women with adenomyosis oftencontained foci of endometrial tissue which could have con-tributed to the observed difference in results between the twogroups of subjects

Cyclical Changes In our study in control subjects weobserved that the expression of GPER in the proliferativephase in the JZ was higher than that of secretory phase ofthe JZ however the cyclical change was not observed inthe OM In women with adenomyosis the cyclical changeappeared to have disappeared Similar cyclical changes in theultrastructure of myocytes in the JZ and OM were observedin our previous study [10]

Anatomical Variation As for the anatomical variation incontrol subjects the expression of GPER in the JZ was higher

than that of the OM in the proliferative phase but not thesecretory phases In contrast in women with adenomyosisthe expression of GPER in the JZ was higher than that of theOM in both the proliferative phase and the secretory phasesIn other words the anatomical variation between the JZ andOM in women with adenomyosis was consistently observedindependent of the phases of the cycle

Adenomyosis versus Control In contrast to the observationsrelating to the cyclical and anatomical variation of GPER inwhich significant difference in control subjects was foundonly in the proliferative phase and JZ there was consistentand significant difference in GPER expression regardless ofthe stage of the cycle (proliferative or secretory) or anatomy(JZ or OM)

Taken together the cyclical and anatomical variation ofGPER observed in control subjects in this study is consistentwith our current understanding that the effects of estrogen aremore dominant in the proliferative phase than the secretoryphase and more pronounced in the JZ than in the OM Inaddition the special design of our study enabled us to controlfor two important confounding variables (cyclical changesand anatomical variation) by doing so the variance in resultsdue to the effect of these confounding variables was reducedWhilst it is already known that the expressions of estrogennuclear receptors ER-120573 (but not ER-120572) and progesteronereceptor are different between women with and withoutadenomyosis [4] the additional finding in this study thatGPER expression is also altered in women with adenomyosisconfirms the notion that adenomyosis is associated withalteration in several different steroid receptors

Overall the finding observed in this study appears tohave provided a molecular basis for the smooth musclehyperplasia and hypertrophy observed in adenomyosis [1 2]Specifically it seems plausible that the abnormally elevatedGPER expression in the JZ is one mechanism by which thesmooth muscle cells continue to proliferate in adenomyosis

To conclude we have found significant and consis-tent increase in GPER expression in adenomyosis in both

BioMed Research International 5

proliferative and secretory phases and in both the JZ andOM suggesting that GPER plays an important role in thepathogenesis of the condition It remains to be seen iftreatment targeting the expression of GPER by the use ofselective GPER ligands may help to treat or prevent thecondition

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (no 81270680 no 81571412)and the Beijing Municipal Administration of Hospital Clin-ical Medicine Development of Special Funding Support(ZYLX201406)

References

[1] G BenagianoMHabiba and I Brosens ldquoThe pathophysiologyof uterine adenomyosis An updaterdquo Fertility and Sterility vol98 no 3 pp 572ndash579 2012

[2] G Benagiano I Brosens and S Carrara ldquoAdenomyosis Newknowledge is generating new treatment strategiesrdquo WomenrsquosHealth vol 5 no 3 pp 297ndash311 2009

[3] P Vrtacnik B Ostanek S Mencej-Bedrac and J Marc ldquoThemany faces of estrogen signalingrdquo Biochemia Medica vol 24no 3 pp 329ndash342 2014

[4] M K Mehasseb R Panchal A H Taylor L Brown S C Belland M Habiba ldquoEstrogen and progesterone receptor isoformdistribution through themenstrual cycle in uteri with andwith-out adenomyosisrdquo Fertility and Sterility vol 95 no 7 pp 2228ndash2235 2011

[5] J Struble S Reid andM A Bedaiwy ldquoAdenomyosis A ClinicalReview of a Challenging Gynecologic Conditionrdquo Journal ofMinimally Invasive Gynecology vol 23 no 2 pp 164ndash185 2016

[6] R W Noyes and J O Haman ldquoAccuracy of endometrial datingcorrelation of endometrial dating with basal body temperatureand mensesrdquo Fertility and Sterility vol 4 no 6 pp 504ndash5171953

[7] M K Mehasseb S C Bell J H Pringle and M A HabibaldquoUterine adenomyosis is associatedwith ultrastructural featuresof altered contractility in the inner myometriumrdquo Fertility andSterility vol 93 no 7 pp 2130ndash2136 2010

[8] I Brosens I Derwig J Brosens L Fusi G Benagiano and RPijnenborg ldquoThe enigmatic uterine junctional zone The miss-ing link between reproductive disorders and major obstetricaldisordersrdquo Human Reproduction vol 25 no 3 pp 569ndash5742010

[9] G Levy A Dehaene N Laurent et al ldquoAn update on adeno-myosisrdquo Diagnostic and Interventional Imaging vol 94 no 1pp 3ndash25 2013

[10] Y Zhang L Zhou T C Li H Duan P Yu and H Y WangldquoUltrastructural features of endometrial-myometrial interfaceand its alteration in adenomyosisrdquo International Journal ofClinical and Experimental Pathology vol 7 no 4 pp 1469ndash14772014

[11] Y Zhang P Yu F Sun T C Li J M Cheng and H DuanldquoExpression of oxytocin receptors in the uterine junctional zonein women with adenomyosisrdquo Acta Obstetricia et GynecologicaScandinavica vol 94 no 4 pp 412ndash418 2015

[12] M K Mehasseb S C Bell L Brown J H Pringle and MHabiba ldquoPhenotypic characterisation of the inner and outermyometrium in normal and adenomyotic uterirdquo Gynecologicand Obstetric Investigation vol 71 no 4 pp 217ndash224 2011

[13] CM Revankar D F Cimino L A Sklar J B Arterburn and ER Prossnitz ldquoA transmembrane intracellular estrogen receptormediates rapid cell signalingrdquo Science vol 307 no 5715 pp1625ndash1630 2005

[14] E J Filardo and P Thomas ldquoMinireview G protein-coupledestrogen receptor-1 GPER-1 Its mechanism of action and rolein female reproductive cancer renal and vascular physiologyrdquoEndocrinology vol 153 no 7 pp 2953ndash2962 2012

[15] E R Prossnitz and M Barton ldquoEstrogen biology new insightsinto GPER function and clinical opportunitiesrdquo Molecular andCellular Endocrinology vol 389 no 1-2 pp 71ndash83 2014

[16] A Madeo and M Maggiolini ldquoNuclear alternate estrogenreceptor GPR30 mediates 17120573-estradiol - Induced gene expres-sion and migration in breast cancer - Associated fibroblastsrdquoCancer Research vol 70 no 14 pp 6036ndash6046 2010

[17] T J Heino A S Chagin and L Savendahl ldquoThe novel estrogenreceptor G-protein-coupled receptor 30 is expressed in humanbonerdquo Journal of Endocrinology vol 197 no 2 pp R1ndashR6 2008

[18] P F Oliveira M G Alves A D Martins et al ldquoExpression pat-tern of G protein-coupled receptor 30 in human seminiferoustubular cellsrdquoGeneral and Comparative Endocrinology vol 201pp 16ndash20 2014

[19] S Heublein M Lenhard T Vrekoussis et al ldquoThe G-protein-coupled estrogen receptor (GPER) is expressed in normalhuman ovaries and is upregulated in ovarian endometriosis andpelvic inflammatory disease involving the ovaryrdquo ReproductiveSciences vol 19 no 11 pp 1197ndash1204 2012

[20] W Huang Y Chen Y Liu et al ldquoRoles of ER120573 and GPR30 inproliferative response of human bladder cancer cell to estrogenrdquoBioMed Research International vol 2015 Article ID 251780 10pages 2015

[21] B J Plante B A Lessey R N Taylor et al ldquoG protein-coupledestrogen receptor (GPER) expression in normal and abnormalendometriumrdquoReproductive Sciences vol 19 no 7 pp 684ndash6932012

[22] R Tian Z Wang Z Shi et al ldquoDifferential expression of G-protein-coupled estrogen receptor-30 in human myometrialand uterine leiomyoma smooth musclerdquo Fertility and Sterilityvol 99 no 1 pp 256ndash263 2013

[23] T Mori F Ito H Matsushima et al ldquoG protein-coupledestrogen receptor 1 agonist G-1 induces cell cycle arrest in themitotic phase leading to apoptosis in endometriosisrdquo Fertilityand Sterility vol 103 no 5 pp 1228ndash1235 2015

[24] L S G Ulrich ldquoEndometrial cancer types prognosis femalehormones and antihormonesrdquo Climacteric vol 14 no 4 pp418ndash425 2011

[25] C Krakstad J Trovik E Wik et al ldquoLoss of GPER identifiesnew targets for therapy among a subgroup of ER120572-positiveendometrial cancer patients with poor outcomerdquoBritish Journalof Cancer vol 106 no 10 pp 1682ndash1688 2012

[26] T Ignatov S Modl M Thulig et al ldquoGPER-1 acts as a tumorsuppressor in ovarian cancerrdquo Journal of Ovarian Research vol6 no 1 article 51 pp 51ndash61 2013

6 BioMed Research International

[27] EAAriazi E Brailoiu S Yerrumet al ldquoTheGprotein-coupledreceptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cellsrdquo Cancer Research vol 70 no 3 pp1184ndash1194 2010

[28] N Nass and T Kalinski ldquoTamoxifen resistance From cellculture experiments towards novel biomarkersrdquo PathologyResearch and Practice vol 211 no 3 pp 189ndash197 2015

[29] R Sirianni A Chimento C Ruggiero et al ldquoThe novel estrogenreceptor G protein-coupled receptor 30 mediates the prolifer-ative effects induced by 17120573-estradiol onmouse spermatogonialGC-1 cell linerdquo Endocrinology vol 149 no 10 pp 5043ndash50512008

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: Expression Pattern of G-Protein-Coupled Estrogen Receptor in Myometrium of Uteri …downloads.hindawi.com/journals/bmri/2017/5974693.pdf · 2018-11-19 · ResearchArticle Expression

BioMed Research International 3

Table 2 Demographic details of patients with adenomyosis andcontrol subjects

Adenomyosis Control 119901 valueAge (years) 441 plusmn 29 430 plusmn 44 007Parity 13 plusmn 01 14 plusmn 01 045Miscarriage 21 plusmn 14 18 plusmn 11 006Body mass index kgm2 232 plusmn 27 241 plusmn 33 009Values are given in mean plusmn standard deviation

GPER

GPER

GAPDH

GAPDH

Adenomyosis Control

JZ

OM

PPPP SP SP

Figure 1 Representative western blotting of G-protein-coupledestrogen receptor (GPER) and glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) in biopsy specimens from the junctional zone(JZ) and outer myometrium (OM) in the proliferative phase (PP)and secretory phase (SP)

the GPER signal was the same as that observed in MCF-7breast cancer cells (data not shown)

32 Comparison of GPER Expression between ProliferativePhase and Secretory Phase In Table 3 the GPER proteinand mRNA expression in the proliferative and secretoryphase were compared separately in women with and withoutadenomyosis and in the JZ and OM

In the control subjects the expressions of both proteinand mRNA in the JZ in the proliferative phase were bothsignificantly (119901 lt 005) higher than those of the secretoryphase whereas the expressions of both protein and mRNAin the OM in the proliferative phase and the secretory phasewere similar

In women with adenomyosis in contrast there was nosignificant difference in the expression of GPER protein andmRNA level between the proliferative phase and secretoryphase in both the JZ and OM

33 Comparison of GPER Expression between JunctionalZone and Outer Myometrium In Table 3 the GPER proteinand mRNA expression in the junctional zone and outermyometrium were compared separately in women with andwithout adenomyosis and in the proliferative and secretoryphases

In the control group in the proliferative phase theexpression of both protein and mRNA in the JZ was higherthan that of the OM In the secretory phase however theexpression of both protein and mRNA in the JZ was similarto that of the OM

In women with adenomyosis on the other hand theexpression of both protein and mRNA in the JZ were higherthan that of the OM in both the proliferative and secretoryphases

34 Comparison of GPER Expression between Adenomyosisand Control Groups In Table 3 the GPER protein andmRNA expression in women with and without adenomyosiswere compared separately in the junctional zone and outermyometrium and in the proliferative and secretory phase

In the JZ the GPER protein expression and the mRNAlevel in women with adenomyosis were significantly higherthan that of control subjects in both the proliferative phaseand secretory phase

In the OM the GPER protein expression and the mRNAlevel in women with adenomyosis were also significantlyhigher than that of control subjects in both the proliferativephase and secretory phase

4 Discussion

In this study we found that the expression of GPER proteinand mRNA in women with adenomyosis was significantlyhigher than that of women without adenomyosis both in thejunctional zone and in the outermyometrium and both in theproliferative and in the secretory phases

Adenomyosis is often considered to be an estrogen-dependent disease with changes in structure and functionof the JZ [1] observed in the majority of cases The JZis responsible for uterine peristalsis which regulates spermtransport and implantation [7 8] Structural changes in theJZ in adenomyosis commonly include JZ hyperplasia andthickening [9] Ultrastructurally the myocytes in the JZof uterus affected by adenomyosis appeared smaller [10]Abnormal expression of oxytocin receptor in the JZ ofwomenwith adenomyosis has been observed [11] which may explainthe occurrence of hyperperistalsis and dysperistalsis in turnleading to dysmenorrhea and disturbance of reproductivefunction However the outer myometrium whose primaryfunction is involvedwith parturition [12] is often not affectedin adenomyosis until a later stage

There are two different types of estrogen receptors Thenuclear estrogen receptors (ER-120572 and ER-120573) mediate geneexpression through binding to estrogen receptor elementsin the promotor and regulatory regions of the target genesin contrast GPER the other type of estrogen receptormediates rapid cellular effects GPER is a membrane estrogenreceptor a 7-transmembrane spanning G-protein-coupledreceptor also called G-protein-coupled receptor 30 (GPR30)[13] GPER is structurally and genetically unrelated to ER-120572 and ER-120573 and expressed independently [3] It binds toestrogen with high affinity whereas binding affinities ofGPER for other steroid hormones are very low [14] In earlierstudies estrogen effects can be mimicked by selective GPERagonist or antagonist whereas inGPERknockoutmice theseeffects are absent or reduced suggesting that GPER plays anessential role [15] Expression of GPER has been describedin multiple physiological systems and tissues including thebreast heart endothelium brain bone adrenal kidney

4 BioMed Research International

Table 3 Relative GPER protein and mRNA expression levels (GPERGAPDH)

Adenomyosis group (119899 = 42) Control group (119899 = 34)JZ OM JZ OM

Relative GPER protein expression levels

PP 112 plusmn 0072bce(119899 = 23)

096 plusmn 0043bce(119899 = 23)

095 plusmn 0027ace(119899 = 16)

083 plusmn 0051bce(119899 = 16)

SP 109 plusmn 0076bce(119899 = 19)

094 plusmn 0052bce(119899 = 19)

082 plusmn 0097ade(119899 = 18)

084 plusmn 0086bde(119899 = 18)

Relative GPER mRNA expression levels

PP 152 plusmn 012bce(119899 = 23)

131 plusmn 009bce(119899 = 23)

128 plusmn 007ace(119899 = 16)

104 plusmn 01bce(119899 = 16)

SP 151 plusmn 014bce(119899 = 19)

129 plusmn 018bce(119899 = 19)

109 plusmn 013ade(119899 = 18)

1bde(119899 = 18)

Values expressed as mean plusmn standard deviation aComparison between proliferative phase and secretary phase (same group and zone) 119901 lt 005 Student 119905-test bcomparison between proliferative phase and secretary phase (same group and zone) 119901 ge 005 Student 119905-test ccomparison between outer myometrialzone and junctional zone (same group and phase) 119901 lt 005 Student 119905-test dcomparison between outer myometrial zone and junctional zone (same group andphase) 119901 ge 005 Student 119905-test ecomparison between adenomyosis and control group (corresponding phase and zone) 119901 lt 005 Student 119905-test

endometrium and ovary [15ndash20] The changes in GPERexpression in a number of gynecological conditions such asthe endometrium of women with endometriosis [21] andsmooth muscle of myoma [22] have been examined Onthe other hand whilst the expressions of nuclear estrogenreceptors (ER-120572 and ER-120573) have been examined in the innerand outer myometrium of adenomyosis [12] the expressionof GPER in adenomyosis has not previously been studiedEvidence has shown that GPER agonist G1 can lead toapoptosis in endometriosis and suppress proliferation ofendometriotic stromal cells [23] GPER is closely relatedto the outcome of estrogen therapy on various estrogen-dependent diseases [24ndash26] and selective GPER ligands(such as GPER agonist G1 and antagonist G15) have beenshown to exert control of these diseases [27ndash29] A betterunderstanding of the role played byGPER in the pathogenesisof adenomyosis may open up opportunity for GPER-targetedtherapy for the condition [15]

The special design of this study enabled us to examine thecyclical change and anatomical variation of GPER expressionin control subjects in addition to comparing GPER expres-sion between women with and without adenomyosis Onepossible limitation of our study was that biopsy specimensfrom the junctional zone of women with adenomyosis oftencontained foci of endometrial tissue which could have con-tributed to the observed difference in results between the twogroups of subjects

Cyclical Changes In our study in control subjects weobserved that the expression of GPER in the proliferativephase in the JZ was higher than that of secretory phase ofthe JZ however the cyclical change was not observed inthe OM In women with adenomyosis the cyclical changeappeared to have disappeared Similar cyclical changes in theultrastructure of myocytes in the JZ and OM were observedin our previous study [10]

Anatomical Variation As for the anatomical variation incontrol subjects the expression of GPER in the JZ was higher

than that of the OM in the proliferative phase but not thesecretory phases In contrast in women with adenomyosisthe expression of GPER in the JZ was higher than that of theOM in both the proliferative phase and the secretory phasesIn other words the anatomical variation between the JZ andOM in women with adenomyosis was consistently observedindependent of the phases of the cycle

Adenomyosis versus Control In contrast to the observationsrelating to the cyclical and anatomical variation of GPER inwhich significant difference in control subjects was foundonly in the proliferative phase and JZ there was consistentand significant difference in GPER expression regardless ofthe stage of the cycle (proliferative or secretory) or anatomy(JZ or OM)

Taken together the cyclical and anatomical variation ofGPER observed in control subjects in this study is consistentwith our current understanding that the effects of estrogen aremore dominant in the proliferative phase than the secretoryphase and more pronounced in the JZ than in the OM Inaddition the special design of our study enabled us to controlfor two important confounding variables (cyclical changesand anatomical variation) by doing so the variance in resultsdue to the effect of these confounding variables was reducedWhilst it is already known that the expressions of estrogennuclear receptors ER-120573 (but not ER-120572) and progesteronereceptor are different between women with and withoutadenomyosis [4] the additional finding in this study thatGPER expression is also altered in women with adenomyosisconfirms the notion that adenomyosis is associated withalteration in several different steroid receptors

Overall the finding observed in this study appears tohave provided a molecular basis for the smooth musclehyperplasia and hypertrophy observed in adenomyosis [1 2]Specifically it seems plausible that the abnormally elevatedGPER expression in the JZ is one mechanism by which thesmooth muscle cells continue to proliferate in adenomyosis

To conclude we have found significant and consis-tent increase in GPER expression in adenomyosis in both

BioMed Research International 5

proliferative and secretory phases and in both the JZ andOM suggesting that GPER plays an important role in thepathogenesis of the condition It remains to be seen iftreatment targeting the expression of GPER by the use ofselective GPER ligands may help to treat or prevent thecondition

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (no 81270680 no 81571412)and the Beijing Municipal Administration of Hospital Clin-ical Medicine Development of Special Funding Support(ZYLX201406)

References

[1] G BenagianoMHabiba and I Brosens ldquoThe pathophysiologyof uterine adenomyosis An updaterdquo Fertility and Sterility vol98 no 3 pp 572ndash579 2012

[2] G Benagiano I Brosens and S Carrara ldquoAdenomyosis Newknowledge is generating new treatment strategiesrdquo WomenrsquosHealth vol 5 no 3 pp 297ndash311 2009

[3] P Vrtacnik B Ostanek S Mencej-Bedrac and J Marc ldquoThemany faces of estrogen signalingrdquo Biochemia Medica vol 24no 3 pp 329ndash342 2014

[4] M K Mehasseb R Panchal A H Taylor L Brown S C Belland M Habiba ldquoEstrogen and progesterone receptor isoformdistribution through themenstrual cycle in uteri with andwith-out adenomyosisrdquo Fertility and Sterility vol 95 no 7 pp 2228ndash2235 2011

[5] J Struble S Reid andM A Bedaiwy ldquoAdenomyosis A ClinicalReview of a Challenging Gynecologic Conditionrdquo Journal ofMinimally Invasive Gynecology vol 23 no 2 pp 164ndash185 2016

[6] R W Noyes and J O Haman ldquoAccuracy of endometrial datingcorrelation of endometrial dating with basal body temperatureand mensesrdquo Fertility and Sterility vol 4 no 6 pp 504ndash5171953

[7] M K Mehasseb S C Bell J H Pringle and M A HabibaldquoUterine adenomyosis is associatedwith ultrastructural featuresof altered contractility in the inner myometriumrdquo Fertility andSterility vol 93 no 7 pp 2130ndash2136 2010

[8] I Brosens I Derwig J Brosens L Fusi G Benagiano and RPijnenborg ldquoThe enigmatic uterine junctional zone The miss-ing link between reproductive disorders and major obstetricaldisordersrdquo Human Reproduction vol 25 no 3 pp 569ndash5742010

[9] G Levy A Dehaene N Laurent et al ldquoAn update on adeno-myosisrdquo Diagnostic and Interventional Imaging vol 94 no 1pp 3ndash25 2013

[10] Y Zhang L Zhou T C Li H Duan P Yu and H Y WangldquoUltrastructural features of endometrial-myometrial interfaceand its alteration in adenomyosisrdquo International Journal ofClinical and Experimental Pathology vol 7 no 4 pp 1469ndash14772014

[11] Y Zhang P Yu F Sun T C Li J M Cheng and H DuanldquoExpression of oxytocin receptors in the uterine junctional zonein women with adenomyosisrdquo Acta Obstetricia et GynecologicaScandinavica vol 94 no 4 pp 412ndash418 2015

[12] M K Mehasseb S C Bell L Brown J H Pringle and MHabiba ldquoPhenotypic characterisation of the inner and outermyometrium in normal and adenomyotic uterirdquo Gynecologicand Obstetric Investigation vol 71 no 4 pp 217ndash224 2011

[13] CM Revankar D F Cimino L A Sklar J B Arterburn and ER Prossnitz ldquoA transmembrane intracellular estrogen receptormediates rapid cell signalingrdquo Science vol 307 no 5715 pp1625ndash1630 2005

[14] E J Filardo and P Thomas ldquoMinireview G protein-coupledestrogen receptor-1 GPER-1 Its mechanism of action and rolein female reproductive cancer renal and vascular physiologyrdquoEndocrinology vol 153 no 7 pp 2953ndash2962 2012

[15] E R Prossnitz and M Barton ldquoEstrogen biology new insightsinto GPER function and clinical opportunitiesrdquo Molecular andCellular Endocrinology vol 389 no 1-2 pp 71ndash83 2014

[16] A Madeo and M Maggiolini ldquoNuclear alternate estrogenreceptor GPR30 mediates 17120573-estradiol - Induced gene expres-sion and migration in breast cancer - Associated fibroblastsrdquoCancer Research vol 70 no 14 pp 6036ndash6046 2010

[17] T J Heino A S Chagin and L Savendahl ldquoThe novel estrogenreceptor G-protein-coupled receptor 30 is expressed in humanbonerdquo Journal of Endocrinology vol 197 no 2 pp R1ndashR6 2008

[18] P F Oliveira M G Alves A D Martins et al ldquoExpression pat-tern of G protein-coupled receptor 30 in human seminiferoustubular cellsrdquoGeneral and Comparative Endocrinology vol 201pp 16ndash20 2014

[19] S Heublein M Lenhard T Vrekoussis et al ldquoThe G-protein-coupled estrogen receptor (GPER) is expressed in normalhuman ovaries and is upregulated in ovarian endometriosis andpelvic inflammatory disease involving the ovaryrdquo ReproductiveSciences vol 19 no 11 pp 1197ndash1204 2012

[20] W Huang Y Chen Y Liu et al ldquoRoles of ER120573 and GPR30 inproliferative response of human bladder cancer cell to estrogenrdquoBioMed Research International vol 2015 Article ID 251780 10pages 2015

[21] B J Plante B A Lessey R N Taylor et al ldquoG protein-coupledestrogen receptor (GPER) expression in normal and abnormalendometriumrdquoReproductive Sciences vol 19 no 7 pp 684ndash6932012

[22] R Tian Z Wang Z Shi et al ldquoDifferential expression of G-protein-coupled estrogen receptor-30 in human myometrialand uterine leiomyoma smooth musclerdquo Fertility and Sterilityvol 99 no 1 pp 256ndash263 2013

[23] T Mori F Ito H Matsushima et al ldquoG protein-coupledestrogen receptor 1 agonist G-1 induces cell cycle arrest in themitotic phase leading to apoptosis in endometriosisrdquo Fertilityand Sterility vol 103 no 5 pp 1228ndash1235 2015

[24] L S G Ulrich ldquoEndometrial cancer types prognosis femalehormones and antihormonesrdquo Climacteric vol 14 no 4 pp418ndash425 2011

[25] C Krakstad J Trovik E Wik et al ldquoLoss of GPER identifiesnew targets for therapy among a subgroup of ER120572-positiveendometrial cancer patients with poor outcomerdquoBritish Journalof Cancer vol 106 no 10 pp 1682ndash1688 2012

[26] T Ignatov S Modl M Thulig et al ldquoGPER-1 acts as a tumorsuppressor in ovarian cancerrdquo Journal of Ovarian Research vol6 no 1 article 51 pp 51ndash61 2013

6 BioMed Research International

[27] EAAriazi E Brailoiu S Yerrumet al ldquoTheGprotein-coupledreceptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cellsrdquo Cancer Research vol 70 no 3 pp1184ndash1194 2010

[28] N Nass and T Kalinski ldquoTamoxifen resistance From cellculture experiments towards novel biomarkersrdquo PathologyResearch and Practice vol 211 no 3 pp 189ndash197 2015

[29] R Sirianni A Chimento C Ruggiero et al ldquoThe novel estrogenreceptor G protein-coupled receptor 30 mediates the prolifer-ative effects induced by 17120573-estradiol onmouse spermatogonialGC-1 cell linerdquo Endocrinology vol 149 no 10 pp 5043ndash50512008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Expression Pattern of G-Protein-Coupled Estrogen Receptor in Myometrium of Uteri …downloads.hindawi.com/journals/bmri/2017/5974693.pdf · 2018-11-19 · ResearchArticle Expression

4 BioMed Research International

Table 3 Relative GPER protein and mRNA expression levels (GPERGAPDH)

Adenomyosis group (119899 = 42) Control group (119899 = 34)JZ OM JZ OM

Relative GPER protein expression levels

PP 112 plusmn 0072bce(119899 = 23)

096 plusmn 0043bce(119899 = 23)

095 plusmn 0027ace(119899 = 16)

083 plusmn 0051bce(119899 = 16)

SP 109 plusmn 0076bce(119899 = 19)

094 plusmn 0052bce(119899 = 19)

082 plusmn 0097ade(119899 = 18)

084 plusmn 0086bde(119899 = 18)

Relative GPER mRNA expression levels

PP 152 plusmn 012bce(119899 = 23)

131 plusmn 009bce(119899 = 23)

128 plusmn 007ace(119899 = 16)

104 plusmn 01bce(119899 = 16)

SP 151 plusmn 014bce(119899 = 19)

129 plusmn 018bce(119899 = 19)

109 plusmn 013ade(119899 = 18)

1bde(119899 = 18)

Values expressed as mean plusmn standard deviation aComparison between proliferative phase and secretary phase (same group and zone) 119901 lt 005 Student 119905-test bcomparison between proliferative phase and secretary phase (same group and zone) 119901 ge 005 Student 119905-test ccomparison between outer myometrialzone and junctional zone (same group and phase) 119901 lt 005 Student 119905-test dcomparison between outer myometrial zone and junctional zone (same group andphase) 119901 ge 005 Student 119905-test ecomparison between adenomyosis and control group (corresponding phase and zone) 119901 lt 005 Student 119905-test

endometrium and ovary [15ndash20] The changes in GPERexpression in a number of gynecological conditions such asthe endometrium of women with endometriosis [21] andsmooth muscle of myoma [22] have been examined Onthe other hand whilst the expressions of nuclear estrogenreceptors (ER-120572 and ER-120573) have been examined in the innerand outer myometrium of adenomyosis [12] the expressionof GPER in adenomyosis has not previously been studiedEvidence has shown that GPER agonist G1 can lead toapoptosis in endometriosis and suppress proliferation ofendometriotic stromal cells [23] GPER is closely relatedto the outcome of estrogen therapy on various estrogen-dependent diseases [24ndash26] and selective GPER ligands(such as GPER agonist G1 and antagonist G15) have beenshown to exert control of these diseases [27ndash29] A betterunderstanding of the role played byGPER in the pathogenesisof adenomyosis may open up opportunity for GPER-targetedtherapy for the condition [15]

The special design of this study enabled us to examine thecyclical change and anatomical variation of GPER expressionin control subjects in addition to comparing GPER expres-sion between women with and without adenomyosis Onepossible limitation of our study was that biopsy specimensfrom the junctional zone of women with adenomyosis oftencontained foci of endometrial tissue which could have con-tributed to the observed difference in results between the twogroups of subjects

Cyclical Changes In our study in control subjects weobserved that the expression of GPER in the proliferativephase in the JZ was higher than that of secretory phase ofthe JZ however the cyclical change was not observed inthe OM In women with adenomyosis the cyclical changeappeared to have disappeared Similar cyclical changes in theultrastructure of myocytes in the JZ and OM were observedin our previous study [10]

Anatomical Variation As for the anatomical variation incontrol subjects the expression of GPER in the JZ was higher

than that of the OM in the proliferative phase but not thesecretory phases In contrast in women with adenomyosisthe expression of GPER in the JZ was higher than that of theOM in both the proliferative phase and the secretory phasesIn other words the anatomical variation between the JZ andOM in women with adenomyosis was consistently observedindependent of the phases of the cycle

Adenomyosis versus Control In contrast to the observationsrelating to the cyclical and anatomical variation of GPER inwhich significant difference in control subjects was foundonly in the proliferative phase and JZ there was consistentand significant difference in GPER expression regardless ofthe stage of the cycle (proliferative or secretory) or anatomy(JZ or OM)

Taken together the cyclical and anatomical variation ofGPER observed in control subjects in this study is consistentwith our current understanding that the effects of estrogen aremore dominant in the proliferative phase than the secretoryphase and more pronounced in the JZ than in the OM Inaddition the special design of our study enabled us to controlfor two important confounding variables (cyclical changesand anatomical variation) by doing so the variance in resultsdue to the effect of these confounding variables was reducedWhilst it is already known that the expressions of estrogennuclear receptors ER-120573 (but not ER-120572) and progesteronereceptor are different between women with and withoutadenomyosis [4] the additional finding in this study thatGPER expression is also altered in women with adenomyosisconfirms the notion that adenomyosis is associated withalteration in several different steroid receptors

Overall the finding observed in this study appears tohave provided a molecular basis for the smooth musclehyperplasia and hypertrophy observed in adenomyosis [1 2]Specifically it seems plausible that the abnormally elevatedGPER expression in the JZ is one mechanism by which thesmooth muscle cells continue to proliferate in adenomyosis

To conclude we have found significant and consis-tent increase in GPER expression in adenomyosis in both

BioMed Research International 5

proliferative and secretory phases and in both the JZ andOM suggesting that GPER plays an important role in thepathogenesis of the condition It remains to be seen iftreatment targeting the expression of GPER by the use ofselective GPER ligands may help to treat or prevent thecondition

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (no 81270680 no 81571412)and the Beijing Municipal Administration of Hospital Clin-ical Medicine Development of Special Funding Support(ZYLX201406)

References

[1] G BenagianoMHabiba and I Brosens ldquoThe pathophysiologyof uterine adenomyosis An updaterdquo Fertility and Sterility vol98 no 3 pp 572ndash579 2012

[2] G Benagiano I Brosens and S Carrara ldquoAdenomyosis Newknowledge is generating new treatment strategiesrdquo WomenrsquosHealth vol 5 no 3 pp 297ndash311 2009

[3] P Vrtacnik B Ostanek S Mencej-Bedrac and J Marc ldquoThemany faces of estrogen signalingrdquo Biochemia Medica vol 24no 3 pp 329ndash342 2014

[4] M K Mehasseb R Panchal A H Taylor L Brown S C Belland M Habiba ldquoEstrogen and progesterone receptor isoformdistribution through themenstrual cycle in uteri with andwith-out adenomyosisrdquo Fertility and Sterility vol 95 no 7 pp 2228ndash2235 2011

[5] J Struble S Reid andM A Bedaiwy ldquoAdenomyosis A ClinicalReview of a Challenging Gynecologic Conditionrdquo Journal ofMinimally Invasive Gynecology vol 23 no 2 pp 164ndash185 2016

[6] R W Noyes and J O Haman ldquoAccuracy of endometrial datingcorrelation of endometrial dating with basal body temperatureand mensesrdquo Fertility and Sterility vol 4 no 6 pp 504ndash5171953

[7] M K Mehasseb S C Bell J H Pringle and M A HabibaldquoUterine adenomyosis is associatedwith ultrastructural featuresof altered contractility in the inner myometriumrdquo Fertility andSterility vol 93 no 7 pp 2130ndash2136 2010

[8] I Brosens I Derwig J Brosens L Fusi G Benagiano and RPijnenborg ldquoThe enigmatic uterine junctional zone The miss-ing link between reproductive disorders and major obstetricaldisordersrdquo Human Reproduction vol 25 no 3 pp 569ndash5742010

[9] G Levy A Dehaene N Laurent et al ldquoAn update on adeno-myosisrdquo Diagnostic and Interventional Imaging vol 94 no 1pp 3ndash25 2013

[10] Y Zhang L Zhou T C Li H Duan P Yu and H Y WangldquoUltrastructural features of endometrial-myometrial interfaceand its alteration in adenomyosisrdquo International Journal ofClinical and Experimental Pathology vol 7 no 4 pp 1469ndash14772014

[11] Y Zhang P Yu F Sun T C Li J M Cheng and H DuanldquoExpression of oxytocin receptors in the uterine junctional zonein women with adenomyosisrdquo Acta Obstetricia et GynecologicaScandinavica vol 94 no 4 pp 412ndash418 2015

[12] M K Mehasseb S C Bell L Brown J H Pringle and MHabiba ldquoPhenotypic characterisation of the inner and outermyometrium in normal and adenomyotic uterirdquo Gynecologicand Obstetric Investigation vol 71 no 4 pp 217ndash224 2011

[13] CM Revankar D F Cimino L A Sklar J B Arterburn and ER Prossnitz ldquoA transmembrane intracellular estrogen receptormediates rapid cell signalingrdquo Science vol 307 no 5715 pp1625ndash1630 2005

[14] E J Filardo and P Thomas ldquoMinireview G protein-coupledestrogen receptor-1 GPER-1 Its mechanism of action and rolein female reproductive cancer renal and vascular physiologyrdquoEndocrinology vol 153 no 7 pp 2953ndash2962 2012

[15] E R Prossnitz and M Barton ldquoEstrogen biology new insightsinto GPER function and clinical opportunitiesrdquo Molecular andCellular Endocrinology vol 389 no 1-2 pp 71ndash83 2014

[16] A Madeo and M Maggiolini ldquoNuclear alternate estrogenreceptor GPR30 mediates 17120573-estradiol - Induced gene expres-sion and migration in breast cancer - Associated fibroblastsrdquoCancer Research vol 70 no 14 pp 6036ndash6046 2010

[17] T J Heino A S Chagin and L Savendahl ldquoThe novel estrogenreceptor G-protein-coupled receptor 30 is expressed in humanbonerdquo Journal of Endocrinology vol 197 no 2 pp R1ndashR6 2008

[18] P F Oliveira M G Alves A D Martins et al ldquoExpression pat-tern of G protein-coupled receptor 30 in human seminiferoustubular cellsrdquoGeneral and Comparative Endocrinology vol 201pp 16ndash20 2014

[19] S Heublein M Lenhard T Vrekoussis et al ldquoThe G-protein-coupled estrogen receptor (GPER) is expressed in normalhuman ovaries and is upregulated in ovarian endometriosis andpelvic inflammatory disease involving the ovaryrdquo ReproductiveSciences vol 19 no 11 pp 1197ndash1204 2012

[20] W Huang Y Chen Y Liu et al ldquoRoles of ER120573 and GPR30 inproliferative response of human bladder cancer cell to estrogenrdquoBioMed Research International vol 2015 Article ID 251780 10pages 2015

[21] B J Plante B A Lessey R N Taylor et al ldquoG protein-coupledestrogen receptor (GPER) expression in normal and abnormalendometriumrdquoReproductive Sciences vol 19 no 7 pp 684ndash6932012

[22] R Tian Z Wang Z Shi et al ldquoDifferential expression of G-protein-coupled estrogen receptor-30 in human myometrialand uterine leiomyoma smooth musclerdquo Fertility and Sterilityvol 99 no 1 pp 256ndash263 2013

[23] T Mori F Ito H Matsushima et al ldquoG protein-coupledestrogen receptor 1 agonist G-1 induces cell cycle arrest in themitotic phase leading to apoptosis in endometriosisrdquo Fertilityand Sterility vol 103 no 5 pp 1228ndash1235 2015

[24] L S G Ulrich ldquoEndometrial cancer types prognosis femalehormones and antihormonesrdquo Climacteric vol 14 no 4 pp418ndash425 2011

[25] C Krakstad J Trovik E Wik et al ldquoLoss of GPER identifiesnew targets for therapy among a subgroup of ER120572-positiveendometrial cancer patients with poor outcomerdquoBritish Journalof Cancer vol 106 no 10 pp 1682ndash1688 2012

[26] T Ignatov S Modl M Thulig et al ldquoGPER-1 acts as a tumorsuppressor in ovarian cancerrdquo Journal of Ovarian Research vol6 no 1 article 51 pp 51ndash61 2013

6 BioMed Research International

[27] EAAriazi E Brailoiu S Yerrumet al ldquoTheGprotein-coupledreceptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cellsrdquo Cancer Research vol 70 no 3 pp1184ndash1194 2010

[28] N Nass and T Kalinski ldquoTamoxifen resistance From cellculture experiments towards novel biomarkersrdquo PathologyResearch and Practice vol 211 no 3 pp 189ndash197 2015

[29] R Sirianni A Chimento C Ruggiero et al ldquoThe novel estrogenreceptor G protein-coupled receptor 30 mediates the prolifer-ative effects induced by 17120573-estradiol onmouse spermatogonialGC-1 cell linerdquo Endocrinology vol 149 no 10 pp 5043ndash50512008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: Expression Pattern of G-Protein-Coupled Estrogen Receptor in Myometrium of Uteri …downloads.hindawi.com/journals/bmri/2017/5974693.pdf · 2018-11-19 · ResearchArticle Expression

BioMed Research International 5

proliferative and secretory phases and in both the JZ andOM suggesting that GPER plays an important role in thepathogenesis of the condition It remains to be seen iftreatment targeting the expression of GPER by the use ofselective GPER ligands may help to treat or prevent thecondition

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (no 81270680 no 81571412)and the Beijing Municipal Administration of Hospital Clin-ical Medicine Development of Special Funding Support(ZYLX201406)

References

[1] G BenagianoMHabiba and I Brosens ldquoThe pathophysiologyof uterine adenomyosis An updaterdquo Fertility and Sterility vol98 no 3 pp 572ndash579 2012

[2] G Benagiano I Brosens and S Carrara ldquoAdenomyosis Newknowledge is generating new treatment strategiesrdquo WomenrsquosHealth vol 5 no 3 pp 297ndash311 2009

[3] P Vrtacnik B Ostanek S Mencej-Bedrac and J Marc ldquoThemany faces of estrogen signalingrdquo Biochemia Medica vol 24no 3 pp 329ndash342 2014

[4] M K Mehasseb R Panchal A H Taylor L Brown S C Belland M Habiba ldquoEstrogen and progesterone receptor isoformdistribution through themenstrual cycle in uteri with andwith-out adenomyosisrdquo Fertility and Sterility vol 95 no 7 pp 2228ndash2235 2011

[5] J Struble S Reid andM A Bedaiwy ldquoAdenomyosis A ClinicalReview of a Challenging Gynecologic Conditionrdquo Journal ofMinimally Invasive Gynecology vol 23 no 2 pp 164ndash185 2016

[6] R W Noyes and J O Haman ldquoAccuracy of endometrial datingcorrelation of endometrial dating with basal body temperatureand mensesrdquo Fertility and Sterility vol 4 no 6 pp 504ndash5171953

[7] M K Mehasseb S C Bell J H Pringle and M A HabibaldquoUterine adenomyosis is associatedwith ultrastructural featuresof altered contractility in the inner myometriumrdquo Fertility andSterility vol 93 no 7 pp 2130ndash2136 2010

[8] I Brosens I Derwig J Brosens L Fusi G Benagiano and RPijnenborg ldquoThe enigmatic uterine junctional zone The miss-ing link between reproductive disorders and major obstetricaldisordersrdquo Human Reproduction vol 25 no 3 pp 569ndash5742010

[9] G Levy A Dehaene N Laurent et al ldquoAn update on adeno-myosisrdquo Diagnostic and Interventional Imaging vol 94 no 1pp 3ndash25 2013

[10] Y Zhang L Zhou T C Li H Duan P Yu and H Y WangldquoUltrastructural features of endometrial-myometrial interfaceand its alteration in adenomyosisrdquo International Journal ofClinical and Experimental Pathology vol 7 no 4 pp 1469ndash14772014

[11] Y Zhang P Yu F Sun T C Li J M Cheng and H DuanldquoExpression of oxytocin receptors in the uterine junctional zonein women with adenomyosisrdquo Acta Obstetricia et GynecologicaScandinavica vol 94 no 4 pp 412ndash418 2015

[12] M K Mehasseb S C Bell L Brown J H Pringle and MHabiba ldquoPhenotypic characterisation of the inner and outermyometrium in normal and adenomyotic uterirdquo Gynecologicand Obstetric Investigation vol 71 no 4 pp 217ndash224 2011

[13] CM Revankar D F Cimino L A Sklar J B Arterburn and ER Prossnitz ldquoA transmembrane intracellular estrogen receptormediates rapid cell signalingrdquo Science vol 307 no 5715 pp1625ndash1630 2005

[14] E J Filardo and P Thomas ldquoMinireview G protein-coupledestrogen receptor-1 GPER-1 Its mechanism of action and rolein female reproductive cancer renal and vascular physiologyrdquoEndocrinology vol 153 no 7 pp 2953ndash2962 2012

[15] E R Prossnitz and M Barton ldquoEstrogen biology new insightsinto GPER function and clinical opportunitiesrdquo Molecular andCellular Endocrinology vol 389 no 1-2 pp 71ndash83 2014

[16] A Madeo and M Maggiolini ldquoNuclear alternate estrogenreceptor GPR30 mediates 17120573-estradiol - Induced gene expres-sion and migration in breast cancer - Associated fibroblastsrdquoCancer Research vol 70 no 14 pp 6036ndash6046 2010

[17] T J Heino A S Chagin and L Savendahl ldquoThe novel estrogenreceptor G-protein-coupled receptor 30 is expressed in humanbonerdquo Journal of Endocrinology vol 197 no 2 pp R1ndashR6 2008

[18] P F Oliveira M G Alves A D Martins et al ldquoExpression pat-tern of G protein-coupled receptor 30 in human seminiferoustubular cellsrdquoGeneral and Comparative Endocrinology vol 201pp 16ndash20 2014

[19] S Heublein M Lenhard T Vrekoussis et al ldquoThe G-protein-coupled estrogen receptor (GPER) is expressed in normalhuman ovaries and is upregulated in ovarian endometriosis andpelvic inflammatory disease involving the ovaryrdquo ReproductiveSciences vol 19 no 11 pp 1197ndash1204 2012

[20] W Huang Y Chen Y Liu et al ldquoRoles of ER120573 and GPR30 inproliferative response of human bladder cancer cell to estrogenrdquoBioMed Research International vol 2015 Article ID 251780 10pages 2015

[21] B J Plante B A Lessey R N Taylor et al ldquoG protein-coupledestrogen receptor (GPER) expression in normal and abnormalendometriumrdquoReproductive Sciences vol 19 no 7 pp 684ndash6932012

[22] R Tian Z Wang Z Shi et al ldquoDifferential expression of G-protein-coupled estrogen receptor-30 in human myometrialand uterine leiomyoma smooth musclerdquo Fertility and Sterilityvol 99 no 1 pp 256ndash263 2013

[23] T Mori F Ito H Matsushima et al ldquoG protein-coupledestrogen receptor 1 agonist G-1 induces cell cycle arrest in themitotic phase leading to apoptosis in endometriosisrdquo Fertilityand Sterility vol 103 no 5 pp 1228ndash1235 2015

[24] L S G Ulrich ldquoEndometrial cancer types prognosis femalehormones and antihormonesrdquo Climacteric vol 14 no 4 pp418ndash425 2011

[25] C Krakstad J Trovik E Wik et al ldquoLoss of GPER identifiesnew targets for therapy among a subgroup of ER120572-positiveendometrial cancer patients with poor outcomerdquoBritish Journalof Cancer vol 106 no 10 pp 1682ndash1688 2012

[26] T Ignatov S Modl M Thulig et al ldquoGPER-1 acts as a tumorsuppressor in ovarian cancerrdquo Journal of Ovarian Research vol6 no 1 article 51 pp 51ndash61 2013

6 BioMed Research International

[27] EAAriazi E Brailoiu S Yerrumet al ldquoTheGprotein-coupledreceptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cellsrdquo Cancer Research vol 70 no 3 pp1184ndash1194 2010

[28] N Nass and T Kalinski ldquoTamoxifen resistance From cellculture experiments towards novel biomarkersrdquo PathologyResearch and Practice vol 211 no 3 pp 189ndash197 2015

[29] R Sirianni A Chimento C Ruggiero et al ldquoThe novel estrogenreceptor G protein-coupled receptor 30 mediates the prolifer-ative effects induced by 17120573-estradiol onmouse spermatogonialGC-1 cell linerdquo Endocrinology vol 149 no 10 pp 5043ndash50512008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: Expression Pattern of G-Protein-Coupled Estrogen Receptor in Myometrium of Uteri …downloads.hindawi.com/journals/bmri/2017/5974693.pdf · 2018-11-19 · ResearchArticle Expression

6 BioMed Research International

[27] EAAriazi E Brailoiu S Yerrumet al ldquoTheGprotein-coupledreceptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cellsrdquo Cancer Research vol 70 no 3 pp1184ndash1194 2010

[28] N Nass and T Kalinski ldquoTamoxifen resistance From cellculture experiments towards novel biomarkersrdquo PathologyResearch and Practice vol 211 no 3 pp 189ndash197 2015

[29] R Sirianni A Chimento C Ruggiero et al ldquoThe novel estrogenreceptor G protein-coupled receptor 30 mediates the prolifer-ative effects induced by 17120573-estradiol onmouse spermatogonialGC-1 cell linerdquo Endocrinology vol 149 no 10 pp 5043ndash50512008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: Expression Pattern of G-Protein-Coupled Estrogen Receptor in Myometrium of Uteri …downloads.hindawi.com/journals/bmri/2017/5974693.pdf · 2018-11-19 · ResearchArticle Expression

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Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


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