{"paper_id":"677c8221-d49f-441b-a324-2e1d504f9155","body_text":"Research Article\nIntegrative Molecular Medicine\n Volume 6: 1-7\nIntegr Mol Med, 2019     doi: 10.15761/IMM.1000379\nISSN: 2056-6360\nExpression of the Wnt antagonist Dickkopf-1 in \nendometriosis\nMariz Kasoha1*, Laura Golbach 1, Panagiotis Sklavounos 1, Sebastian Findeklee 1, Ingolf Juhasz-Böss 1, Hermann Eichler 2 and Erich-Franz \nSolomayer1\n1Department of Gynecology, Obstetrics and Reproductive Medicine, University Medical School of Saarland, 66421 Homburg / Saar, Germany\n2Institute of Clinical Hemostaseology and Transfusion Medicine, University Medical School of Saarland, 66421 Homburg / Saar, Germany\nAbstract\nBackground: Dickkopf-1 protein (Dkk1), a major inhibitor of Wnt/β-catenin signaling, is a secretory glycoprotein that has been found to be involved in the \npathogenesis of several human diseases and to provide a promising diagnostic and therapeutic tool in diverse cancer entities. Endometriosis shares some characteristics \nwith invasive tumors. The present study aimed to investigate whether Dkk1 expression is dysregulated in endometriosis.\nMethods: 188 subjects including 109 women with histologically confirmed endometriosis and 79 healthy women were recruited. Serum Dkk1 levels were measured \nby ELISA in 97 and 75 women with- and without endometriosis respectively. Gene expression levels of Dkk1- and β-catenin were assessed by quantitative polymerase \nchain reaction in 28 different human tissue types, including 4 normal endometrium tissues, 8 normal peritoneal tissues, and 16 endometriosis tissues.\nResults: Endometriosis patient women showed significantly higher serum levels of Dkk1 compared with healthy women [2999 pg/ml (947-5104) vs 2216 pg/ml \n(1008-4109) respectively; p<0.0001]. No significant differences in Dkk1 gene expression levels were found between the different tissue types but it showed, within \nendometriosis tissue group, to be upregulated in cases with more severe disease. β-catenin gene expression was found to be down regulated in endometriosis tissues \ncompared with normal endometrium tissues.\nConclusions: To the best of our knowledge, this is the first study to investigate serum levels of Dkk1 in patients with endometriosis. Our results demonstrated \naberrant expression of two main components of Wnt/β-catenin signaling in endometriosis. Interventions in this signaling transduction pathway can contribute to \ndevelopment of new targets for disease control and therapy. Dkk1 could serve as diagnostic biomarker in endometriosis. However, further multicentre large-scaled \nstudies are required.\nIntroduction\nWnt/β‑catenin signaling pathway is mediated by the tight \nregulation of β‑catenin stability and widely known to be involved \nin a wide range of physiologic and pathologic processes throughout \nthe body. In the presence of Wnt, signaling is kept on by binding of \nWnt proteins to the N‑terminal extra‑cellular cysteine‑rich domain \nof a Frizzled (Fz) family receptor and other co‑receptors such as \nlipoprotein receptor‑related protein (LRP)‑5/6. This binding initiates \na signaling cascade that inhibits the so called “destruction complex” \nwhich targets β‑catenin for ubiquitin‑mediated proteasomal \ndegradation. Consequently, free cytoplasmic non phosphorylated \nβ‑catenin accumulates and relocates into the nucleus, where it binds \nto different transcription factors promoting transcription of a broad \nspectrum of Wnt downstream target genes including those that \npromote cell proliferation and survival. In the absence of Wnt, the \ndestruction complex induces phosphorylation of β‑catenin resulting \nin its degradation [1].\nIn the endometrium, Wnt/β‑catenin signaling pathway serves \na decisive role in endometrial gland formation and mesenchymal \ndevelopment and involves in the adhesion, invasion and angiogenesis \nof the ectopic endometrium [2]. Sanchez and colleagues showed that \nthe expression of specific members of Wnt pathway and its pivot \nmolecule β‑catenin were dysregulated in luteinized granulosa cells \nderived from endometriosis patients compared with those from control \n*Correspondence to: Mariz Kasoha, Department of Gynecology, Obstetrics \nand Reproductive Medicine, University Medical School of Saarland, 66421 \nHomburg / Saar, Germany, Tel: +49 (0) 6841/16‑28199; Fax: +49 (0) 6841/16‑\n28110; E‑mail: mariz.kasoha@uks.eu\nKey words: Wnt/β-catenin signaling, dickkopf-1 (Dkk1), endometriosis, biomarker, \npathogenicity\nReceived: July 30, 2019; Accepted: August 23, 2019; Published: August 27, 2019\nwomen. This aberrant expression was characterised by an increased \napoptosis suggesting that Wnt/β‑catenin signaling could be involved \nin causing granulosa cell atresia [3]. Furthermore, it has been showed \nthat Wnt/β‑catenin signaling trigger fibrogenesis associated with \nendometriosis via stimulating fibrotic markers expression of and these \neffects were prevented by treatment with small‑molecule antagonists of \ncertain components of this signaling pathway [4]. Zhang et al . showed \nthat stimulation of human endometrial stroma cells (HESCs) with \nE2 increased mRNA expression of vascular endothelial growth factor \n(VEGF) and this effect can be prevented when cells were transfected \nwith β‑catenin si‑RNA suggesting the plausible role of Wnt/β‑catenin \nsignaling in initiating endometriosis via angiogenesis stimulation [5].\nDickkopf (Dkk) proteins, exemplified by Dkk1, are LRP5/6 ligands \nantagonists and considered potent inhibitors for Wnt/β‑catenin \nsignaling [6]. Overexpression of Dkk1 is associated with several \n\nKasoha M (2019) Expression of the Wnt antagonist Dickkopf-1 in endometriosis\nVolume 6: 2-7\nIntegr Mol Med, 2019     doi: 10.15761/IMM.1000379\nconfirmed endometriosis and no recent pathological finding in uterus \ncavity. Serum samples were obtained from 97 patient women. Patients’ \ncharacteristics are outlined in Table 1. The median (Range) age for the \npatients at the time of the sample drawn was 34 years (16‑52). Disease \nstaging was determined according to the revised American Society \nfor Reproductive Medicine (rASRM) [15]. Serum of 75 age‑matched \nhealthy women [30 years (19‑52)] was also collected. These healthy \ncohorts were recruited from those who had undergone comprehensive \nmedical screening prior to blood donation at the Institute of Transfusion \nMedicine of our university hospital. All serum samples were frozen in \naliquots at ‑80°C until being analysed.\nTissue samples were collected from 22 women with endometriosis \nduring laparoscopic surgery. Specimens included 16 endometriosis \ntissues and 8 normal peritoneal tissues of which 2 matched normal \nperitoneal‑ and endometriosis tissues were collected from the same \npatient. Furthermore, normal endometrium tissue samples were \ncollected from 4 women who underwent laparoscopic surgery for \nbenign indications other than endometriosis. Endometrial pathology \nwas excluded in healthy controls. Tissues were directly flash‑frozen in \nliquid nitrogen and immediately transferred to ‑80°C until used.\nAll study participants were documented not to be pregnant and \nhave no history of malignancies. \nThis study was approved by the local Ethic Committee of the \nMedical Association of the Saarland (Reference number: 23/16). \nHuman blood samples and tissue samples were collected from all \nindividuals after signing a written and informed consent that is \napproved by the Ethic Committee .\nSerum analysis\nSerum protein levels of Dkk1 were measured by Enzyme‑Linked \nImmunosorbent Assay (ELISA) in 75 healthy women and in 97 patient \nwomen using Quantikine immunoassay control set 921 for human Dkk1 \n(QC241) and Human Dkk‑1 Quantikine ELISA Kit (DKK100B) from \ndiseases including various types of cancers [7] and anti‑Dkk1 antibodies \nare be ing tested experimentally and clinically as potential therapy \nagents in different bone diseases and cancers and preliminary results \ndemonstrated a favourable safety profile introducing promising therapy \noptions [8,9]. We have previously reported that protein levels of Dkk1 \nin peripheral blood are increased in breast cancer patients compared \nwith control women. In addition, increased protein expression of \nDkk1 in breast cancer cells and cancer surrounding tissues has been \ndocumented [10]. Moreover, elevated blood levels of Dkk1 were found \nto be positively correlated with poor prognosis in lung cancer and \ncervical cancer [11,12]. Therefore, blood levels of Dkk1 could be used \nas non‑invasive diagnostic‑ and prognostic biomarker in certain cancer \nentities. \nDiagnosis of endometriosis in many affected women is often \ndelayed resulting in reduced quality of life and decline in reproductive \npotential and fertility imposing a significant economic burden \n[13,14]. Although direct laparoscopic visualization and histologic \nconfirmation remains the gold standard for diagnosing endometriosis, \na non‑invasive tool could facilitate earlier diagnosis and intervention \nthat could ultimately improve disease consequences. Blood level of \nDkk1 is considered as an advantageous biomarker because it is non‑\ninvasive, has a simple detection protocol, and is inexpensive to quantify. \nOur present study aimed to investigate whether blood protein levels \nof Dkk1 and/or its tissue gene expression are dysregulated in women \nwith endometriosis compared with healthy women.\nMaterial and methods\nStudy participants and sampling\nOur study included 188 women consisting of 79 healthy women \nand 109 endometriosis patient women. Details on the study case \nseries are illustrated in Figure 1. Patient cohorts were recruited from \nwomen admitted to our department with signs of endometriosis from \nMay 2013 and December 2016. Inclusion criteria were histologically \nFigure 1. Details on the study case series\n\nKasoha M (2019) Expression of the Wnt antagonist Dickkopf-1 in endometriosis\nVolume 6: 3-7\nIntegr Mol Med, 2019     doi: 10.15761/IMM.1000379\nR&D Systems® (Germany) according to manufacturer´s instructions. \nOptical density was measured using 96‑well microplate reader (Sunrise‑\nTecan, Life Science). Dkk‑1 concentrations were obtained with a four‑\nparameter logistic curve fitted against a standard curve and multiplied \nby the dilution factor using Magellan 7.2 Ink Data Analysis Software \n(Life Science‑Tecan). All measurements were performed in duplicate.\nTissue analysis\nHistologic test: Cryosections were developed from obtained tissue \nsamples and consequently stained according to the Hematoxylin \nand eosins (H&E) protocol for frozen tissue. Then, stained sections \nwere analyzed under Zeiss microscope (Axioskop 40, Carl Zeiss, \nGermany) and selected pictures were captured with attached digital \ncamera (AxioCam MRC, Carl Zeiss, Germany) using Axiovision \nDocumentation Rel.4.8 program. Histological evaluation was done \nby an experienced pathologist from the department of Pathology at \nour university hospital. Histologic diagnosis of endometriosis was \nconfirmed by the presence of endometrial glands and stroma with or \nwithout haemosiderin‑laden macrophages. All tested peritoneal tissues \nshowed negative diagnosis for endometriosis (Figure 2). \nRNA extraction and cDNA synthesis: Levels of Dkk1‑ and β‑catenin \ngenes were analyzed in 4 normal human endometrium tissues, 8 \nnormal peritoneal tissues, and 16 endometriosis lesions. A maximum \namount of 15‑20 mg frozen tissues were distributed and homogenized \nin 300 µl of RNeasy lysis buffer (Qiagen, Valencia, CA, USA) using \nTissueLyser LT Adapter and stainless steel beads from Qiagen. RNA \nextraction was performed using RNeasy MiniKit (Qiagen, Valencia, \nCA, USA) following the manufacturer’s instructions. Thereafter, \nAmbion® TURBO DNA ‑free™ DNase kit (Life Technologies GmbH, \nDarmstadt, Germany) was used to remove contaminating DNA from \nRNA preparations, and to subsequently remove the DNase and divalent \ncations from the sample. Concentration and purity of extracted RNA \nwere measured using Thermo Scientific™ NanoDrop 2000. In addition, \nfurther detecting of RNA integrity was done using Agilent RNA 6000 \nNano Reagents Part I (Agilent  Technologies, Waldbronn, Germany) \nand Bioanalyser Agilent 2100 from Agilent Technologies. Afterward, \nreverse transcribed complementary DNA (cDNA) was synthesized \nwith the High Capacity cDNA Reverse Transcription Kit as described \nby the manufacturer (Applied Biosystem, Foster City, CA, USA).\nReal Time RT-PCR: The PCR efficiency was tested to confirm that \nthe results will not be influenced by amplification of contaminating \nDNA in the RNA samples and was found to be in a range between 90% \nand 100% for tested Dkk1‑ and β‑catenin genes. Glyceraldehyde‑3‑\nphosphate dehydrogenase gene (GAPDH) was used as a housekeeping \ngene. Quantitative real‑time PCR was performed in triplicates with \ndilutions of 10 ng cDNA using TaqMan ® Gene Expression Assays by \nLife Technologies (Table 2) on an Applied Biosystems 7500 Fast Real‑\nTime PCR System. All samples with a cycle threshold (Ct) coefficient of \nvariation value >5% were retested. In addition, a no template control \n(NTC) was included in each run, and the resulting C t values were \nnormalized to GAPDH mRNA expression. The RT ‑PCR data were \nanalyzed using the 7500 Software v.2.0.5 (Life Technologies GmbH, \nDarmstadt, Germany).\nStatistical analysis: All statistical tests were performed at a two‑\nsided comparison‑wise significance level of 5%. Continuous variables \nwere analysed descriptively using mean, standard deviation, median \nand range of the observations. Categorical variables were analysed \ndescriptively using absolute and relative frequencies. The null hypothesis \nwas tested using two tails Student's t‑test and Mann–Whitney U test/\nor Kruskal‑Wallis‑Test for parametric and non‑parametric continuous \ndata respectively. All data obtained from this study were analyzed using \nSPSS version 21 (IBM, Armonk, NY , USA). \nResults\nSerum levels of Dkk1 are increased in endometriosis patients\nSerum levels of Dkk1 were measured in 97 endometriosis patient \nwomen patients [Age: Mean (range): 34 (16‑52) years] and in 75 age‑\nmatched healthy women [30 years (19‑52)]. Our results revealed that \nendometriosis patients had significantly higher serum levels of Dkk1 \ncompared with healthy women [Mean (Range): 2999 pg/ml (947‑5104) \nvs 2216 pg/ml (1008‑4109) respectively; p<0.0001] (Figure 3). \nThen, we have compared serum levels of Dkk1 between different \nsubgroups according to disease occurrence and disease classification \nand no significant differences were reported (Table 3). \nGene expression levels of Dkk1 and β-catenin in normal \nendometrium tissues, in normal peritoneal tissues, and in \nendometriosis tissues\nFor this purpose, we have collected endometrium tissues from 4 \nwomen (aged 26, 44, 44, and 46 years) who underwent laparoscopic \nsurgery for benign indications other than endometriosis. In addition, \nwe collected normal peritoneal tissues and endometriosis tissues from \n22 patient women. The final tissues that were valid to be used for \nGene Taqman assay ID\ndkk1 Hs00183740_m1\nCTNNB1 (ß-catenin) Hs00355045_m1\nGAPDHa Hs03929097_m1\nTable 2. TaqMan® Gene expression assays used for quantitative RT-PCR\naEndogenous gene\nNumber of patients %\nAll patients\nAge: Median (Range)years\n97\n34 (16-52)\n100%\nDisease occurrence\nPatients with first time \ndiagnosis\nPatients with one recurrence\nPatients more than one \nrecurrence\n54\n30\n13\n55.7\n30.9\n13.4\nDisease classification†\nNot deep infiltrated\nDeep infiltrated\n83‡\n14\n85.6\n14.4\nTable 1. Characteristics of patients in whom Dkk1 levels were tested in serum\n†Cases were according to the revised American Society for Reproductive Medicine \n(rASRM). ‡ Included 47 cases with minimal-, 17 cases with mild-, and 19 cases with \nmoderate endometriosis.\nFigure 2. Hematoxylin and eosins (H&E) staining of cryosections of endometriosis tissues. \nHistologic diagnosis of endometriosis was confirmed by the presence of endometrial \nglands (Black arrows) and stroma (Green arrows) with or without haemosiderin-laden \nmacrophages\n\nKasoha M (2019) Expression of the Wnt antagonist Dickkopf-1 in endometriosis\nVolume 6: 4-7\nIntegr Mol Med, 2019     doi: 10.15761/IMM.1000379\nreal time PCR test included 16 endometriosis tissues and 8 normal \nperitoneal tissues, of which 2 matched normal peritoneal‑ and \nendometriosis tissues were available. The rest samples were excluded \nfrom the study because of the low purity and integrity of the extracted \nRNA. Characteristics of patients in whom gene expression tests were \nperformed are listed in Table 4.\nOur RT ‑PCR results showed no significant difference in dkk1 \nexpression levels within the three tested tissue groups (Figure 4A). \nHowever, in the endometriosis tissue group, we found that dkk1  \nexpression level in 5 deep infiltrated endometriosis tissues was \nsignificantly higher than it in 11 not deep infiltrated endometriosis \ntissues [Mean (Range) ∆C t: 3.4±2.8 vs 8.32±4.16 respectively; p=0.032] \n(Figure 4B). Expression levels of dkk1 did not differ according to disease \noccurrence status. \nOn the other hand, we found that β-catenin expression levels in \nendometriosis tissues was downregulated compared with normal \nCharacteristic Subgroup 1 Subgroup 2 Subgroup 3 Subgroup 4 p\nDisease occurrence Patients with first time diagnosis\n3031 (1408-4579)\nPatients with one recurrence\n2889 (1008-5104)\nPatients more than one recurrence\n3034 (974-4214) - NS\nDisease classification deep infiltrated\n2811 (1678-3820)\nMinimal\n3279 (1408-5104)\nMild\n2848 (1953-3647)\nModerate\n3145 (947-4597) NS\nTable 3. Serum levels of Dkk1 (pg/ml) in different patients subgroups\nResults are showed as [Median (Range)]. P value is tested with Kruskal-Wallis-Test. NS: not significant.\nNumber of patients %\nAll patients\nAge: Median (Range)years\n22\n35 (24-46)\n100%\nDisease occurrence\nPatients with first time diagnosis\nPatients with one recurrence\nPatients more than one recurrence\n9\n7\n6\n40.9\n31.8\n27.3\nDisease classification†\nNot deep infiltrated\nDeep infiltrated\n16‡\n6\n72.7\n27.3\nTable 4. Characteristics of patients in whom Dkk1- and ß-catenin gene expression tests were done\n†Cases were classified according to the revised American Society for Reproductive Medicine (rASRM). ‡ Included 7 cases with minimal-, 3 cases with mild-, and 6 cases with moderate \nendometriosis.\nendometrium tissues (Figure 5). No other significant differences in \nβ-catenin expression levels were reported between other tissue groups. \nIn addition, β-catenin expression levels in endometriosis tissues showed \nno significant alteration according to any of patient’s specifications \nincluding disease occurrence status and disease severity (data are not \nshown). \nDiscussion\nEndometriosis is considered as a multifactorial disease affected by \nimmunological, hormonal, and environmental factors. However, the \nSampson’s retrograde hypothesis remains the most widely accepted \ntheory, of which a main part implicates the epithelial to mesenchymal \ntransition (EMT) as the pathogenic mechanism [16,17]. Wnt/β‑catenin \nsignaling is considered essential in EMT activation and regulation [18]. \nAs aforementioned, aberrant Wnt/β‑catenin signaling has been linked \nto the pathogenesis of endometriosis via various mechanisms [3‑5]. \nFigure 3. Serum levels of Dkk1 (pg/ml) in healthy women and in endometriosis patients. Dkk1serum levels in patients [2999 pg/ml (947-5104)] were significantly higher than those in \ncontrols [2216 pg/ml (1008-4109)] (p<0.0001)]\n\nKasoha M (2019) Expression of the Wnt antagonist Dickkopf-1 in endometriosis\nVolume 6: 5-7\nIntegr Mol Med, 2019     doi: 10.15761/IMM.1000379\nIn this study, we showed that significant more abundances of Dkk1 \nprotein were determined in serum of endometriosis patients compared \nwith healthy cohorts. However, we found that Dkk1 serum levels in \nour study cohorts were not affected by disease severity or by disease \noccurrence. Serum Dkk1 is considered as advantageous biomarker as \nit has a simple non‑invasive detection protocol and is inexpensive to \nquantify. However, the great variability and non‑normal distribution \nof its levels affected the chances of identifying a clear cut‑off value or \nsignificant differences related to the clinical features of the groups. \nTherefore, further large‑scale and multicentre studies are absolutely \nrequired to confirm our findings introducing serum levels of Dkk1 as a \ndiagnostic biomarker in endometriosis.\nWe further tested weather Dkk1 expression is dysregulated \ntoo at mRNA level. We determined the expression level of dkk1  in \nFigure 4. Gene expression levels of Dkk1 in different tissue samples. A: Differences in gene expression levels of Dkk1 in normal endometrium-, in normal peritoneal- and in endometriosis \ntissue samples were not significant. B: Gene expression levels of Dkk1 in deep infiltrated endometriosis tissue samples was significantly lower than it in not deep infiltrated endometriosis \ntissue samples\nFigure 5. Gene expression levels of β-catenin in different tissue samples. Gene expression levels of Dkk1 in endometriosis tissue samples was significantly lower than it in normal \nendometrium tissue samples\n\nKasoha M (2019) Expression of the Wnt antagonist Dickkopf-1 in endometriosis\nVolume 6: 6-7\nIntegr Mol Med, 2019     doi: 10.15761/IMM.1000379\nendometriosis tissues and compared it to its levels either in normal \nendometrium tissues from women without endometriosis or in \nnormal peritoneal tissues from women with endometriosis. Moreover, \nwe tested β-catenin expression levels to investigate whether Dkk1is \nenrolled in endometriosis pathogenicity directly via its function as an \ninhibitor of the Wnt/β‑catenin signaling. Our results demonstrated \nno significant alteration in dkk1  expression levels within the tested \ntissue groups. However, unlike serum Dkk1 protein concentrations, \ndkk1 expression levels were increased in endometriosis tissues of \npatients with deep infiltrated endometriosis compared to them in \npatients with less disease severity. In addition, there was no significant \ncorrelation between serum levels of Dkk1 and dkk1  expression levels \nin endometriosis tissues from the same patient. These findings suggest \nthat transcript levels by themselves could not be sufficient to predict \nprotein levels in this scenario and to thus explain genotype‑phenotype \nrelationship. Moreover, as a secreted glycoprotein, a difference in \nsecondary modifications on it or its ligands could impact its biological \nproperties and functions.\nExpression profile of the Wnt/β-catenin signaling´s components \nin endometriosis, particularly Dkk1, has been investigated in a few, \nsmall, human studies and contradicted results were demonstrated. \nFindings of Pazhohan and co‑workers showed that while Dkk1 gene‑ \nand protein expression levels in both endometriotic lesions and eutopic \nendometrium of endometriosis patients were significantly lower than \nthem in the endometrium of healthy subjects, non‑phosphorylated \nβ‑catenin (active form of β‑catenin) was more expressed at protein \nlevel only [19]. Microarray analysis of eight endometrial biopsies from \npatients with endometriosis versus seven ones from women without \nendometriosis identified 91 genes that were significantly upregulated \nand 115 genes that were significantly downregulated including \nDkk1 gene [20]. Brueggmann and colleagues documented that gene \nexpression levels of dkk1  and E-cadherin  were downregulated in \novarian endometriosis tissues compared with matching endometrial \nsamples of patients with endometriosis [21]. Data from these studies \nprovided a proof of downregulated expression of dkk1in endometriosis \ntissues when compared to non‑endometriotic tissues from subjects \nwith‑ or without endometriosis. On the other hand, it has been showed \nthat β‑catenin mRNA expression levels in endometriosis tissues had no \nsignificant cyclical differences compared to eutopic endometrium from \nwomen with‑ or without endometriosis [19,22]. However, contradicted \nobservations regarding protein expression of β‑catenin were reported \nby the last two studies. In a rat model of peritoneal endometriosis, there \nwere no differences in β‑catenin mRNA levels and β‑catenin protein \nlevels in endometriosis samples and their matched endometrium \nsamples of twenty female rats [23]. \nIn our study, although dkk1 expression level in endometriosis \ntissues was almost the same as it in normal peritoneal tissues of \nendometriosis patients and lower than it in normal endometrium of \nsubjects without endometriosis, these observations were statistically \nnot significant. Nevertheless, β‑catenin mRNA levels in endometriosis \ntissues, but not in normal peritoneal samples, were downregulated \ncompared with normal endometrium tissues. These inconsistent \nobservations from our study with the others could be relevant to the \nvariances in cohorts’ characteristics such as number of tested cases, \ncycle phase, location of endometriosis lesions, and disease stages. \nIn conclusions, the present study demonstrated aberrant \nexpression of certain components of Wnt/β‑catenin signaling \nin endometriosis patients represented by decreased β‑catenin at \nmRNA expression levels and increased Dkk1 at protein expression \nlevels suggesting the enrollment of this transduction pathway in \nendometriosis pathogenicity. To the best of our knowledge, this is \nthe first study to investigate serum levels of Dkk1 in patients with \nendometriosis. Hence, Dkk1 could be a useful marker for diagnosing \nendometriosis. However, this hypothesis needs to be further explored \nby doing large‑scale, multicentre studies to prove the suggested role of \nDkk1 as a potential diagnostic protein biomarker for endometriosis.\nAcknowledgements\nThe authors would like to thank Mrs. Adriana Nistor from the \ndepartment of Pathology at the University Medical School of Saarland \nfor her assistant doing the histological evaluation of all H&E sections \nthat were included in this study.\nConflict of interest\nWe declare that we have no conflict of interest.\nFunding\nThis research did not receive any specific grant from any funding \nagency in the public, commercial or not‑for‑profit sector.\nReferences\n1. Clevers H (2006) Wnt/beta-catenin signaling in development and disease. Cell 127: \n469-480. [Crossref]\n2. van der Horst PH, Wang Y , van der Zee M, Burger CW, Blok LJ (2012) Interaction \nbetween sex hormones and WNT/b-catenin signal transduction in endome-trial \nphysiology and disease. Mol Cell Endocrinol 358: 176-184. \n3. Sanchez AM, Vigano P, Quattrone F, Pagliardini L, Papaleo E, et al. (2014) The Wnt/ \nß-catenin signalling pathway and expression of survival promoting genes in luteinized \ngranulosa cells: endometriosis as a paradigm for a dysregulated apoptosis pathway. \nFertil Steril 101: 1688-1696. \n4. Li J, Dai Y , Zhu H, Jiang Y , Zhang S (2016) Endometriotic mesenchymal stem cells \nsignificantly promote fibrogenesis in ovarian endometrioma through the Wnt/ß-catenin \npathway by paracrine production of TGF-ß1 and Wnt1. Hum Reprod 31: 1224-1235. \n5. Zhang L, Xiong W, Xiong Y , Liu H, Liu Y (2016) 17 ß-Estradiol promotes vascular \nendothelial growth factor expression via the Wnt/ß-catenin pathway during the \npathogenesis of endometriosis. Mol Hum Reprod 22: 526-535. \n6. Semënov MV , Tamai K, Brott BK, Kühl M, Sokol S, et al. (2001) Head inducer \nDickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr Biol 11: 951-961. [Crossref]\n7. Niehrs C (2006) Function and biological roles of the Dickkopf family of Wnt \nmodulators. Oncogene 25: 7469-7481. [Crossref]\n8. Jin H, Wang B, Li J, Xie W, Mao Q, et al. (2015) Anti-DKK1 antibody promotes bone \nfracture healing through activation of ß-catenin signaling. Bone 71: 63-75. \n9. Iyer SP, Beck JT, Stewart AK, Shah J, Kelly KR, et al. (2014) A Phase IB multicentre \ndose-determination study of BHQ880 in combination with anti-myeloma therapy and \nzoledronic acid in patients with relapsed or refractory multiple myeloma and prior \nskeletal-related events. Br J Haematol 167: 366-375. [Crossref]\n10. Kasoha M, Bohle RM, Seibold A, Gerlinger C, Juhasz-Böss I, et al. (2018) Dickkopf-1 \n(Dkk1) protein expression in breast cancer with special reference to bone metastases. \nClin Exp Metastasis 35: 763-775. [Crossref]\n11. Dong LL, Qu LY , Chu LY , Zhang XH, Liu YH (2014) Serum level of DKK-1 and its \nprognostic potential in non-small cell lung cancer. Diagn Pathol 9: 10-52. \n12. Jiang T, Huang L, Zhang S (2013) DKK-1 in serum as a clinical and prognostic factor \nin patients with cervical cancer. Int J Biol Markers 28: 221-225. \n13. Nnoaham KE, Hummelshoj L, Webster P, d´Hooghe T, de Cicco Nardone F, et al. \n(2011) World Endometriosis Research Foundation Global Study of Women's Health \nconsortium. Impact of endometriosis on quality of life and work productivity: a \nmulticenter study across ten countries. Fertil Steril 96: 366-373. \n14. Soliman AM, Yang H, Du EX, Kelley C, Winkel C (2016) The direct and indirect costs \nassociated with endometriosis: a systematic literature review. Hum Reprod 31: 712-\n722. \n\nKasoha M (2019) Expression of the Wnt antagonist Dickkopf-1 in endometriosis\nVolume 6: 7-7\nIntegr Mol Med, 2019     doi: 10.15761/IMM.1000379\n15. American Society for Reproductive Medicine (1997) Revised American Society for \nReproductive Medicine classification of endometriosis: 1996. Fertil Steril 67: 817-821. \n[Crossref]\n16. Albertsen HM, Ward K (2017) Genes linked to endometriosis by GWAS are integral to \ncytoskeleton regulation and suggests that mesothelial barrier homeostasis is a factor in \nthe pathogenesis of endometriosis. Reprod Sci 24: 803-811. [Crossref]\n17. Yang YM, Yang WX (2017) Epithelial-to-mesenchymal transition in the development \nof endometriosis. Oncotarget 8: 41679-41689. [Crossref]\n18. Zhang J, Tian XJ, Xing J (2016) Signal transduction pathways of EMT Induced by \nTGF-β, SHH, and WNT and their crosstalks. J Clin Med 5. [Crossref]\n19. Pazhohan A, Amidi F, Akbari-Asbagh F, Seyedrezazadeh E, Farzadi L, et al. (2018) \nThe Wnt/ß-catenin signaling in endometriosis, the expression of total and active forms \nof ß-catenin, total and inactive forms of glycogen synthase kinase-3ß, WNT7a and \nDICKKOPF-1. Eur J Obstet Gynecol Reprod Biol 220: 1-5. \nCopyright: ©2019 Kasoha M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted \nuse, distribution, and reproduction in any medium, provided the original author and source are credited.\n20. Kao LC, Germeyer A, Tulac S, Lobo S, Yang JP, et al. (2003) Expression profiling \nof endometrium from women with endometriosis reveals candidate genes for disease-\nbased implantation failure and infertility. Endocrinology 144: 2870-2881. \n21. Brueggmann D, Jaque JM, Lee AW, Pearce CL, Templeman C (2016) Expression of \nWnt signaling pathway genes in human endometriosis tissue - a pilot study. Eur J \nObstet Gynecol Reprod Biol 199: 214-215. \n22. Matsuzaki S, Darcha C, Maleysson E, Canis M, Mage G (2010) Impaired down-\nregulation of E-cadherin and beta-catenin protein expression in endometrial epithelial \ncells in the mid-secretory endometrium of infertile patients with endometriosis. J Clin \nEndocrinol Metab 95: 3437-3445. \n23. de Mattos RM, Pereira PR, Barros EG, da Silva JH, Palmero CY , et al. (2016) Aberrant \nlevels of Wnt/ß-catenin pathway components in a rat model of endometriosis. Histol \nHistopathol 31: 933-942.","source_license":"CC0","license_restricted":false}