{"paper_id":"fe63270d-c218-4bbd-be01-6ec16912cee3","body_text":"1 3\nJ Endocrinol Invest (2016) 39:785–791\nDOI 10.1007/s40618-016-0435-7\nORIGINAL ARTICLE\nAberrant expression and hormonal regulation of Galectin‑3 \nin endometriosis women with infertility\nH. Yang1 · J. Yin2 · K. Ficarrotta3 · S. H. Hsu4 · W. Zhang1 · C. Cheng5 \nReceived: 2 September 2015 / Accepted: 21 January 2016 / Published online: 17 February 2016 \n© The Author(s) 2016. This article is published with open access at Springerlink.com\nwomen. The changes in expression of Gal-3 were more dra-\nmatic in EECs than ESCs. Moreover, estrogen (E2) and pro-\ngesterone (P4) induced Gal-3 expression in EECs of healthy \ngroups, and P4 was more significant than E2 and combined \nE2 and P4 (E2P4). However, in the endometriosis group, P4 \nfailed to induce a similar increase in Gal-3 expression.\nConclusions Our results suggest that aberrant expres -\nsion of Gal-3 might contribute to infertility in patients with \nendometriosis due to progesterone resistance.\nKeywords Endometriosis · Galectin-3 (Gal-3) · Uterine \nreceptivity · Sex hormone\nIntroduction\nEndometriosis is a benign gynecological disorder with \nmalignant biological characteristics and is defined by the \npresence of endometrial glands and stroma outside the uter-\nine cavity. Ectopic endometrial cells can spread to pelvic \norgans such as rectum, bladder, and ovaries. This spread -\ning contributes to various clinical manifestations includ -\ning irregular uterine bleeding, dyspareunia, chronic pelvic \npain, and infertility: a condition affecting 10  % of women \nof reproductive age [1–3]. Statistically, 30–50 % of females \nwith endometriosis are infertile and 25–50  % of females \nwith infertility have endometriosis [ 4]. Although the asso -\nciation between endometriosis and infertility has been well \nestablished, the underlying mechanisms remain unknown.\nIt has been proposed that abnormal endometrial devel -\nopment in patients with endometriosis contributes to \nendometriosis-related infertility [ 5]. It is well known that \nendometrial receptivity is vital in women’s fertility. Human \ntissue and mouse model studies have demonstrated that the \nmolecular markers of endometrial receptivity are altered \nAbstract \nObjective To investigate the role and potential molecular \nmechanism of Galectin-3 (Gal-3) in the etiology of endo -\nmetriosis-associated infertility.\nMethods We detected Gal-3 expression in eutopic endo -\nmetrium from women with endometriosis-associated infer -\ntility and healthy women without endometriosis or infer -\ntility. We then evaluated Gal-3 expression in endometrial \nglandular epithelial cells (EECs) and endometrial stromal \ncells (ESCs) and investigated its response to hormone stim-\nulation in EECs and ESCs from both groups of women.\nResults Results of real-time PCR and western blot analysis \nshowed Gal-3 expression in both proliferative and secretory \nstages of the menstrual cycle decreased significantly in women \nwith endometriosis-associated infertility compared to healthy \nH. Yang and J. Yin contributed equally to this work.\n * W. Zhang \n zhang.wei.fudan@gmail.com\n * C. Cheng \n Chao.Cheng@dartmouth.edu\n H. Yang \n leaftaney@hotmail.com\n1 Department of Reproductive Endocrinology, Obstetrics \nand Gynecology Hospital, Fudan University, 413 Zhaozhou \nRoad, Shanghai 200011, China\n2 Department of Gynecology, Chongqing Ninth People’s \nHospital, Chongqing, China\n3 Department of Chemical and Biomedical Engineering, \nUniversity of South Florida, Tampa, FL 33620, USA\n4 Department of Medicine, SUNY Downstate Medical Center, \nNew York, NY , USA\n5 Department of Genetics, Geisel School of Medicine \nat Dartmouth, Hanover, NH, USA\n\n786 J Endocrinol Invest (2016) 39:785–791\n1 3\nin both humans and mice with endometriosis. Integrins, \nmatrix metalloproteinases (MMPs), and homeobox genes \n(HoxA10) display aberrant expression patterns in the \neutopic endometrium of women with endometriosis [6, \n7]. Other alterations of biochemical or molecular markers \nhave also been reported, including changes in the levels of \nvascular endothelial growth factor (VEGF) [8] and inter -\nleukin 6 (IL-6) [9]. Galectin-3 (Gal-3), a β-galactoside-\nbinding protein, has been related to endometrial receptiv-\nity during embryonic implantation recently [10, 11]. Gal-3 \nwith a ~31 kDa lectin contains a collagen-α-like domain, \nan N-terminal domain, and the carbohydrate recognition \ndomain (CRD) [12]. These three structures allow Gal-3 to \npossess specific biological functions, including cell adhe-\nsion, migration, cell-extracellular matrix interactions [13], \nimmune response, [14] and signal transduction [15]. It has \nbeen shown that Gal-3 is expressed in many cell types, \nincluding endometrial cells and trophoblast cells [16–19]. \nPrevious studies have reported that Gal-3 is specifically \nexpressed in endometrial cells in the secretory phase [10, \n20], in placental tissue during early pregnancy, and in \ndecidua surrounding the site of implantation. Due to these \nfindings, we speculated that the expression of Gal-3 might \nbe aberrant in endometriosis patients with infertility.\nAdditionally, Gla-3 has been known as a component of \na nuclear and cytoplasmic complex, shuttling between the \nnucleus and cytoplasm [21]. Estrogen and progesterone \nbelong to the nuclear hormone superfamily transcription fac-\ntors, which play important role in the formation of receptive \nendometrium. Furthermore, it has been demonstrated that \nendometriosis-related infertility is associated with abnormal \nsex hormone regulation [22]. Thus, it would be interesting to \nexplore whether the expression of Gal-3 is regulated by sex \nhormones in secondary infertility of endometriosis patients.\nMaterials and methods\nTissue collection\nWe collected endometrial tissue samples from 34 women, \naged 28–35 years (mean age 31.51 ± 3.52), who con-\nsulted for infertility and were found to have surgically \nand histologically-confirmed endometriosis. As controls, \nwe also collected endometrial biopsy specimens from 34 \nhealthy women aged 30–35 years (mean age 32.55 ± 3.90). \nWomen in the control group underwent tubal sterilization \nand were laparoscopically confirmed to be free of endome-\ntriosis. These women displayed normal menstrual cycles \nand had not received anti-inflammatory or hormonal ther -\napy within 3 months prior to surgery. The phase of the sub-\njects’ menstrual cycle was determined according to the cri-\nteria of Noyes et al. [23].\nThe endometrial tissue samples from each group were \ndigested for cell culture, snap frozen and kept under \n−80 °C for subsequent real-time PCR and western blot \nanalysis, or fixed in 4 % paraformaldehyde for paraffin \nblocks. Informed consent was obtained in writing from all \nsubjects before surgery.\nCell culture\nPrimary proliferative phase endometrial stromal and glan-\ndular epithelial cells were obtained from six subjects in the \nendometriosis group and six subjects in the control group. \nFor each subject, secretory phase endometrial stromal and \nglandular epithelial cells were also obtained. Endometrial \ntissue was transported from the site of collection to the \nlaboratory in Hanks’ balanced salt solution. The tissue was \nthen minced and digested in Hanks’ balanced salt solution \ncontaining 1 % penicillin, 1 % streptomycin, 5 % colla-\ngenase, and 0.5 % deoxyribonuclease at 37 °C for 30 min \nwith agitation. The dispersed endometrial cells were sepa-\nrated by filtration through a wire sieve (73-mm diameter \npore, Sigma). The endometrial glands (largely undispersed) \nwere retained by the sieve, whereas the dispersed stromal \ncells passed through the sieve into the filtrate. Endometrial \nglandular epithelial cells (EECs) were plated on Madri-\ngal coated 12 well-plates, while endometrial stromal cells \n(ESCs) were plated on plastic 12-well plates. Both types \nof cells were plated in DMEM/F12 phenol red medium \n(Gibco Invitrogen,Carlsbad, CA, USA) containing 10 % \nfetal bovine serum (FBS) (Gibco). Cell cultures were main-\ntained at 37 °C in a humidified atmosphere (5 % CO\n2) and \nwere allowed to replicate to confluence. Thereafter, cells \nwere passaged by standard methods of trypsinization and \nallowed to replicate to confluence, which required approxi-\nmately 24–48 h. Cells after first passage were characterized \nas described previously [24].\nReal‑time PCR analysis\nTotal RNA was extracted from 34 samples from the endo-\nmetriosis group and 34 samples from the control group \nusing Trizol Reagent (Invitrogen). Total RNA (1 μg) was \nreverse transcribed using a PrimeScript RT reagent kit \n(Takara, Dalian, China). Reverse-transcription PCR was \nperformed prior to quantitative real-time PCR. The mRNA \nlevels were determined by real-time PCR using SYBR Pre-\nmix Ex Taq (TaKaRa) with the Applied Biosystems 7000 \nsystem SDS software as previous described [25]. Glyceral-\ndehyde 3-phosphate dehydrogenase (GAPDH) was used as \nan endogenous control to normalize for differences in the \namount of total RNA in each sample. The primer sequences \nand the sizes of the amplified fragments were as follows: \nGal-3 (93 bp) 5′-CTT CCA CTT TAA CCC ACG CTT \n\n787J Endocrinol Invest (2016) 39:785–791 \n1 3\nCAA-3′ (sense), 5′-TGT CTT TCT TCC CTT CCC CAG \nTTA TT-3′ (anti-sense); GAPDH (131 bp) 5′-ATG ACC \nCCT TCA TTG ACC-3′ (sense), 5′-GAA GAT GGT GAT \nGGG ATT TC-3′ (anti-sense).\nImmunohistochemistry\nThe paraffin blocks were cut into sections of 4 μm, \nmounted on polylysine-coated microslides, dewaxed, \nand rehydrated. Then, tissue sections were incubated in \n3 % hydrogen peroxide at room temperature. For antigen \nretrieval, tissue slides were immersed in citric acid and \nboiled in a microwave oven. The sections were washed \nin distilled water and phosphate buffered saline (PBS, \nGibco), and then subjected to bovine serum to block unspe-\ncific binding agents. This step was followed by overnight \nexposure (4 °C) to the mouse anti-human Gal-3 antibody \n(mab1154, R&D Systems Inc., Minneapolis, MN, USA) in \na humidified chamber. After being rinsed in PBS, the sec-\ntions were incubated with the bridging rabbit anti-mouse \nimmunoglobulins conjugated with horseradish peroxidase \n(HRP)-labelled dextran polymer for 1 h. After washing in \nPBS, diaminobenizidine tetrahydrochloride (DAB) solu-\ntion was applied, followed by running tap water as well as \nnuclear counterstaining with haematoxylin (Sigma, USA). \nThe stained slides were viewed under microscopy (OLYM-\nPUS OPTICAL CO., Ltd.) under 400× magnification. \nPositive cells were characterized by the brown staining of \nGal-3 antibody. The intensity and distribution of the stain-\ning reaction were evaluated by two blinded, independent \nobservers.\nWestern blot\nProtein samples were extracted from tissues or cells, and \nthe protein concentration was measured by bicinchoninic \nacid assay (BCA). Samples were run in SDS-PAGE gel, \ntransferred to nitrocellulose membranes, and immunob-\nlotted overnight with gentle shaking at 4 °C with primary \nantibodies. The primary antibodies included monoclonal \nmouse anti-human Gal-3 antibody (1:2500) and mono-\nclonal mouse anti-human GAPDH (Kangchen, Shanghai, \nChina) antibody (1:5000). This procedure was followed by \nincubation with horseradish peroxidase-conjugated second-\nary antibodies. The reactions were detected by enhanced \nchemiluminescence assay. GAPDH was used as an endog-\nenous control for normalization.\nHormonal stimulation protocol\nFor the hormone stimulation test, 5 × 10\n5 cells in 1 ml of \nmedia were plated on a 6-well plate and grown for 24 h in \nnormal medium containing 10 % FBS without antibiotics. \nThen, the media were replaced by serum-free and phenol \nred-free culture media for 24 h to prepare for the stimula-\ntion experiment. Stimulation was performed under vari-\nous concentrations of 17β-estradiol (E2, 0, 10\n−10–10−6 M, \nSigma) alone and under various concentrations of pro-\ngesterone (P4, 0, 10\n−9–10−5 M, Sigma) alone. The same \nexperiment was performed with E2, P4, or E2 combined \nwith P4 (E2P4, 10\n−8 M E2, 10−7 M P4, Sigma) for 24, 48, \nand 72 h. The control was plated in phenol red-free culture \nmedia containing charcoal-stripped 10 % FBS (Bioind, \nShanghai, China).\nStatistical analysis\nData were expressed in terms of mean ± SEM. One-way \nANOV A analyses were performed and least significant dif-\nference (LSD) tests were applied for post hoc testing using \nSPSS software version 15.0 with p < 0.05 considered statis-\ntically significant.\nResults\nGal‑3 expression in endometria of endometriosis \nand control groups\nTo verify whether expression of Gal-3 is abnormal in the \nendometria of the endometriosis group, we performed \nimmunochemistry analysis to display the expression pat-\ntern of Gal-3 proteins in endometria from both the healthy \nand endometriosis group (Fig. 1). Our results showed that \nGal-3 was presented in the endometrium from both the con-\ntrol group (Fig. 1b) and the endometriosis group (Fig. 1c).\nThen, Gal-3 mRNA expression levels in all endome-\ntrium samples were detected by real-time PCR analysis. As \nshown in Fig. 2a, Gal-3 showed significantly higher levels \nof expression in the secretory phase than the proliferative \nphase, regardless of group. Moreover, during both phases, \nGal-3 expression was significantly down-regulated (secre-\ntory stage 0.47 ± 0.02 vs 0.05 ± 0.01, p < 0.05; prolifera-\ntive phase 0.26 ± 0.02 vs 0.02 ± 0.01, p < 0.05) in eutopic \nendometrium from endometriosis patients compared to the \nnormal endometrium. This result was confirmed at the pro-\ntein level by western blot analysis (Fig. 2b).\nGal‑3 expression in epithelial cells and stromal cells \nof endometria\nThe endometrium becomes receptive to embryonic attach-\nment in the secretory phase of the menstrual cycle; thus, \nwe explored whether Gal-3 expression is different in \nEECs and ESCs during this phase. The results of real-\ntime PCR showed that Gal-3 expression was significantly \n\n788 J Endocrinol Invest (2016) 39:785–791\n1 3\ndown-regulated in EECs from the endometriosis group \ncompared to the healthy group (0.14 ± 0.06 vs 0.59 ± 0.07, \np < 0.05) (Fig. 3a). In contrast, there was no significant \nchange in Gal-3 expression in ESCs from the control group \ncompared to the endometriosis group; however, there was \na trend of down-regulation of Gal-3 in the endometriosis \ngroup. We further confirmed these results by western blot \nanalysis. As shown in Fig. 3b, a significant down-regulation \nof Gal-3 protein was observed in EECs but not ESCs of \neutopic endometrial samples from the endometriosis group \ncompared to the control group (0.22 ± 0.05 vs 0.58 ± 0.07, \np < 0.05).\nHormone‑stimulated Gal‑3 expression in endometrial \nepithelial cells\nTo further investigate the hormone regulation of Gal-3 \nexpression in EECs, various concentrations of E2 and P4 \nwere used to co-culture EECs from the control group for \n24 h before mRNA levels of Gal-3 were detected. Our \nresults showed that Gal-3 was up-regulated by E2 and \nP4 at all concentrations, reaching peak level at 10\n−8 and \n10−7 M, respectively (Fig. 4a, b) Thus, concentrations of \n10−8 M of E2 and 10 −7 M of P4 were used in the subse-\nquent experiments.\nThen, the expression of Gal-3 was detected in EECs pre-\ntreated with 10 −8 M E2, 10 −7 M P4, or 10 −8 M E2 com-\nbined with 10−7 M P4 for 24, 48, and 72 h, respectively. In \nthe control group, Gal-3 expression was induced by E2 or \nP4 treatment in a time dependent manner (Fig. 5a). Expres-\nsion of Gal-3 in the E2P4 group was lower than that in the \nP4 treatment group and higher than that in the E2 treatment \ngroup. These results suggest Gal-3 induction is more sensi-\ntive to P4 than E2 treatment; however, in EECs from the \neutopic endometria with endometriosis (Fig. 5b), Gal-3 \nexpression increased slightly after hormone pretreatment \nFig. 1  Immunohistochemistry analysis of Gal-3 expression. a Nega -\ntive control. b Secretory phase endometrium from healthy controls. \nc Secretory phase eutopic endometrium from endometriosis patients \nwith infertility. Mouse IgG was used in the negative control (a), while \nGal-3 antibody was used other analyses (b, c)\nFig. 2  Gal-3 mRNA and protein expression in endometria samples \nfrom patients and healthy women. a Relative expression levels of \nGal-3 mRNA in different samples measured by real-time PCR analy-\nsis. b Relative expression levels of Gal-3 protein in different samples \nmeasured by western blot analysis. Expression levels of Gal-3 were \nnormalized against those of GAPDH in matched samples. ES endo-\nmetria of patient with endometriosis in secretory phase, EP endo-\nmetria of patient with endometriosis in proliferative phase, CS endo-\nmetria of controls in secretory phase, CP endometria of controls in \nproliferative phase, Control control group, EMT endometriosis group; \n#p < 0.05\n\n789J Endocrinol Invest (2016) 39:785–791 \n1 3\nfor 24 h, and then decreased time-dependently. Further -\nmore, compared with the control group, the expression of \nGal-3 was lower in the endometriosis groups at all time \npoints.\nDiscussion\nEndometriosis shows a serious impact on female fertility, \nbut the etiology and pathogenesis of endometriosis-related \ninfertility are unknown. Thus, it is imperative to identify \nthe molecular mechanism of endometriosis to develop an \neffective therapy for endometriosis patients with infertility. \nSeveral studies have reported that endometriosis is a major \ncause of infertility due to its adverse effect on endometrial \nreceptivity to embryonic implantation [26].\nOur previous study verified that Gal-3 plays an impor -\ntant role in the process of embryonic implantation [25]. \nIntracellular Gal-3 promoted proliferation and adhe-\nsion in endometrial cells. Decreased expression of Gal-3 \nhindered embryonic adhesion to endometrial epithelial \ncells and delayed proliferation of endometrial stromal cells \nin achieving optimal status to accommodate the invad-\ning embryo, resulting in failed embryonic implantation. \nSecreted Gal-3 inhibited cell proliferation and induced \napoptosis of endometrial cells [27]. This study shows that \nGal-3 is expressed in the endometrium of both endometrio-\nsis and healthy women, but is reduced significantly in the \nformer. This suggests a defect in Gal-3 expression occurs \nin eutopic endometrium from endometriosis patients with \ninfertility. Decreased Gal-3 expression in eutopic endo-\nmetrium from patients with endometriosis may contribute \nto the defective formation of receptive endometrium, thus \nleading to infertility.\nHormonal regulation of cellular function impacts many \ndynamic biological changes occurring during the peri-\nimplantation stage of the menstrual cycle. Estrogen and pro-\ngesterone act coherently at certain time intervals to stimulate \nFig. 3  Expression of Gal-3 mRNA and protein in epithelial cells and \nstromal cells from secretory phase endometria. a Relative expres-\nsion levels of Gal-3 mRNA in EECs and ESCs. b Relative expression \nlevels of Gal-3 protein in EECs and ESCs; Ctrl control group, EMT \nendometriosis group, EECs endometrial glandular epithelial cells, \nESCs endometrial stromal cells; \n#p < 0.05\nFig. 4  Hormonal regulation of Gal-3 expression by E2 and P4 in \nEndometrial epithelial cells. EECs were treated with E2 (a) (Ctrl, \n10\n−10–10−6 M) and P4 (b) (Ctrl, 10 −9–10−5M) for 24 h, mRNA level \nof Gal-3 was determined by real-time PCR. Gal-3 expression was \nanalyzed by real-time PCR and GAPDH was used as an endogenous \ncontrol to normalize for differences. EECs endometrial glandular epi-\nthelial cells; \n#p < 0.05\n\n790 J Endocrinol Invest (2016) 39:785–791\n1 3\nthe expression of key molecules that regulate endometrial \nreceptivity. Our results showed that Gal-3 expression spe-\ncifically increased during the secretory phase of the men-\nstrual cycle in both groups, indicating that Gal-3 may be \nregulated by sex hormones. To confirm this relationship, \nwe investigated the effect of hormones on Gal-3 expression \nin both EECs and ESCs. We found that regulation of Gal-3 \nexpression by E2 and P4 could be detected in EECs but not \nESCs. This result indicates that Gal-3 may primarily con-\ntribute to the dynamic change of EECs during embryonic \nimplantation. Then, we explored the physiological dose of \nE2 (10\n−8 M) and P4 (10−7 M) that maximized Gal-3 expres-\nsion in EECs. The results suggested that Gal-3 is regulated \nby sex hormones, which is in agreement with our previous \nstudy [27]. In the control group, expression of Gal-3 was \nsignificantly increased when induced by P4 alone, compared \nto E2 alone or E2P4. From these results, we concluded that \nE2, to some degree, antagonized the increased expression of \nGal-3 by P4 in normal endometrium. In the endometriosis \ngroup, E2 alone, P4 alone, nor E2P4 could increase Gal-3 \nexpression to the level of the control group. This indicates \nthat there is no induction of Gal-3 expression in response \nto E2 or P4 treatment in the endometriosis group. Previ-\nous reports have shown that progesterone resistance is one \nimportant factor for endometriosis. Moreover, Gal-3 can \nbe detected in the peritoneal fluid of endometriosis patients \n[28–30]; thus, defective progesterone regulation in endome-\ntriosis women with infertility might account for decreased \nGal-3 expression in eutopic endometrium.\nIn summary, we found decreased expression of Gal-3 \nin eutopic endometrium from endometriosis, which may \naccount for the defective formation of receptive endome-\ntrium. We further showed that Gal-3 was regulated mainly \nby hormones in EECs. We also suggested that the failure \nof Gal-3 elevation by hormones in EECs from endometri-\nosis patients may contribute to progesterone resistance in \nendometriosis-related infertility. Although our study pre-\nliminarily indicates that the defective expression of Gal-3 \nmay contribute to infertility in patients with endometriosis, \nfurther research is needed to detail the pathways of Gal-3 in \neutopic endometrium from endometriosis.\nAcknowledgments This study was supported by the National Natu-\nral Science Foundation of China (Grant No. 81300467), the Program \nof Shanghai Subject Chief Scientist (Grant No. 12XD1401200), and \nthe Programs Foundation of Ministry of Education of China (Grant \nNo. 20120071110074).\nCompliance with ethical standards \nConflict of interest The authors declare no conflict of interest.\nEthical approval Consent forms and protocols were approved by the \nEthical Committee of Obstetrics and Gynecology Hospital at Fudan \nUniversity. All procedures performed in studies involving human par-\nticipants were in accordance with the ethical standards of the institu-\ntional and/or national research committee and with the 1964 Helsinki \ndeclaration and its later amendments or comparable ethical standards. \nThis article does not contain any studies with animals performed by \nany of the authors.\nInformed consent Informed consent was obtained from all individ-\nual participants included in the study.\nOpen Access This article is distributed under the terms of the \nCreative Commons Attribution 4.0 International License (http://crea-\ntivecommons.org/licenses/by/4.0/), which permits unrestricted use, \ndistribution, and reproduction in any medium, provided you give \nappropriate credit to the original author(s) and the source, provide a \nlink to the Creative Commons license, and indicate if changes were \nmade.\nFig. 5  Defective progesterone regulation in endometrial epithelial \ncells from endometriosis women with infertility. EECs from control \ngroup (a) and endometriosis group (b) were treated with 10\n−8 M E2 \nwith or without 10 −7 M P4 and Gal-3 expression was analyzed at 0, \n24, 48, 72 h. Gal-3 expression was analyzed by real-time PCR and \nGAPDH was used as an endogenous control to normalize for differ -\nences. EECs endometrial glandular epithelial cells; \n#p < 0.05\n\n791J Endocrinol Invest (2016) 39:785–791 \n1 3\nReferences\n 1. Giudice LC, Kao LC (2004) Endometriosis. Lancet \n364:1789–1799\n 2. Gao X, Outley J, Botteman M et al (2006) Economic burden of \nendometriosis. Fertil Steril 86:1561–1572\n 3. Cramer DW, Missmer SA (2002) The epidemiology of endo-\nmetriosis. Ann N Y Acad Sci 955:11–22 (discussion 34–16, \n396–406)\n 4. Meuleman C, Vandenabeele B, Fieuws S et al (2009) High prev-\nalence of endometriosis in infertile women with normal ovula-\ntion and normospermic partners. Fertil Steril 92:68–74\n 5. Browne H, Taylor H (2006) HOXA10 expression in ectopic \nendometrial tissue. 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