FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080505512> ?p ?o ?g. }

• W2080505512 endingPage "2346" @default.
• W2080505512 startingPage "2343" @default.
• W2080505512 abstract "In a prospective, blinded, longitudinal study MMP-2, MMP-9, and MMP-9/neutrophil gelatinase-associated lipocalin were significantly more likely to be detected in the urine of patients with endometriosis than in controls. In a prospective, blinded, longitudinal study MMP-2, MMP-9, and MMP-9/neutrophil gelatinase-associated lipocalin were significantly more likely to be detected in the urine of patients with endometriosis than in controls. Clinical symptoms of endometriosis, defined as the presence of endometrium-like tissue outside of the uterine cavity, are often nonspecific and mimic those from other diseases such as pelvic inflammatory disease, interstitial cystitis, or inflammatory bowel disease (1Bulun S.E. Endometriosis.N Engl J Med. 2009; 360: 268-279Crossref PubMed Scopus (1437) Google Scholar). Due to a lack of reliable biomarkers, most cases can only be diagnosed by an invasive procedure such as a laparoscopy (2Giudice L.C. Kao L.C. Endometriosis. Lancet. 2004; 364: 1789-1799Abstract Full Text Full Text PDF PubMed Scopus (2469) Google Scholar).Matrix metalloproteinases (MMPs) are extracellular endopeptidases that play a pivotal role in many physiological and pathological processes including development, growth, and wound healing (3Mott J.D. Werb Z. Regulation of matrix biology by matrix metalloproteinases.Curr Opin Cell Biol. 2004; 16: 558-564Crossref PubMed Scopus (863) Google Scholar, 4Ravanti L. Kahari V.M. Matrix metalloproteinases in wound repair (review).Int J Mol Med. 2000; 6: 391-407PubMed Google Scholar, 5Roy R. Louis G. Loughlin K.R. Wiederschain D. Kilroy S.M. Lamb C.C. et al.Tumor-specific urinary matrix metalloproteinase fingerprinting: identification of high molecular weight urinary matrix metalloproteinase species.Clin Cancer Res. 2008; 14: 6610-6617Crossref PubMed Scopus (134) Google Scholar). Degradation and remodeling of extracellular matrix by MMPs are crucial steps for cellular migration and invasion into tissue. MMP-2 (gelatinase A, 72 kDa) and MMP-9 (gelatinase B, 92 kDa) degrade collagen IV, V, VII, and X, and fibronectin, all of which are constituents of the interstitial stroma and the subendothelial basement membrane (6Mignatti P. Rifkin D.B. Biology and biochemistry of proteinases in tumor invasion.Physiol Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar). The MMP-9/NGAL (neutrophil gelatinase-associated lipocalin) complex has been shown to protect MMP-9 from autodegradation in vitro and to increase aggressiveness in breast tumors in vivo (7Fernandez C.A. Yan L. Louis G. Yang J. Kutok J.L. Moses M.A. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients.Clin Cancer Res. 2005; 11: 5390-5395Crossref PubMed Scopus (237) Google Scholar, 8Yan L. Borregaard N. Kjeldsen L. Moses M.A. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.J Biol Chem. 2001; 276: 37258-37265Crossref PubMed Scopus (563) Google Scholar). It has been suggested that the differential expression of MMPs in endometriotic cells is responsible for the invasive properties of endometriotic lesions (9Bruner K.L. Matrisian L.M. Rodgers W.H. Gorstein F. Osteen K.G. Suppression of matrix metalloproteinases inhibits establishment of ectopic lesions by human endometrium in nude mice.J Clin Invest. 1997; 99: 2851-2857Crossref PubMed Scopus (269) Google Scholar, 10Sharpe-Timms K.L. Zimmer R.L. Jolliff W.J. Wright J.A. Nothnick W.B. Curry T.E. Gonadotropin-releasing hormone agonist (GnRH-a) therapy alters activity of plasminogen activators, matrix metalloproteinases, and their inhibitors in rat models for adhesion formation and endometriosis: potential GnRH-a-regulated mechanisms reducing adhesion formation.Fertil Steril. 1998; 69: 916-923Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Various MMPs have been demonstrated to be expressed in endometriotic tissue (reviewed in Ref.11Osteen K.G. Yeaman G.R. Bruner-Tran K.L. Matrix metalloproteinases and endometriosis.Semin Reprod Med. 2003; 21: 155-164Crossref PubMed Scopus (124) Google Scholar). MMP-2 and MMP-9 expression is up-regulated in eutopic endometrium of women with endometriosis and in endometriotic lesions, and MMP-2 is elevated in serum and peritoneal fluid (PF) of these patients (12Chung H.W. Lee J.Y. Moon H.S. Hur S.E. Park M.H. Wen Y. et al.Matrix metalloproteinase-2, membranous type 1 matrix metalloproteinase, and tissue inhibitor of metalloproteinase-2 expression in ectopic and eutopic endometrium.Fertil Steril. 2002; 78: 787-795Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 13Chung H.W. Wen Y. Chun S.H. Nezhat C. Woo B.H. Lake Polan M. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 mRNA expression in ectopic and eutopic endometrium in women with endometriosis: a rationale for endometriotic invasiveness.Fertil Steril. 2001; 75: 152-159Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 14Collette T. Maheux R. Mailloux J. Akoum A. Increased expression of matrix metalloproteinase-9 in the eutopic endometrial tissue of women with endometriosis.Hum Reprod. 2006; 21: 3059-3067Crossref PubMed Scopus (93) Google Scholar, 15Huang H.F. Hong L.H. Tan Y. Sheng J.Z. Matrix metalloproteinase 2 is associated with changes in steroid hormones in the sera and peritoneal fluid of patients with endometriosis.Fertil Steril. 2004; 81: 1235-1239Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 16Ria R. Loverro G. Vacca A. Ribatti D. Cormio G. Roccaro A.M. et al.Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 agree with progression of ovarian endometriomas.Eur J Clin Invest. 2002; 32: 199-206Crossref PubMed Scopus (59) Google Scholar, 17Sillem M. Prifti S. Koch A. Neher M. Jauckus J. Runnebaum B. Regulation of matrix metalloproteinases and their inhibitors in uterine endometrial cells of patients with and without endometriosis.Eur J Obstet Gynecol reprod Biol. 2001; 95: 167-174Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Szamatowicz J. Laudanski P. Tomaszewska I. Matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1: a possible role in the pathogenesis of endometriosis.Hum Reprod. 2002; 17: 284-288Crossref PubMed Scopus (50) Google Scholar).We have previously shown that MMPs are detectable in the urine of cancer patients and children suffering from vascular anomalies, and that their expression pattern correlates with the extent and stage of the disease (5Roy R. Louis G. Loughlin K.R. Wiederschain D. Kilroy S.M. Lamb C.C. et al.Tumor-specific urinary matrix metalloproteinase fingerprinting: identification of high molecular weight urinary matrix metalloproteinase species.Clin Cancer Res. 2008; 14: 6610-6617Crossref PubMed Scopus (134) Google Scholar, 7Fernandez C.A. Yan L. Louis G. Yang J. Kutok J.L. Moses M.A. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients.Clin Cancer Res. 2005; 11: 5390-5395Crossref PubMed Scopus (237) Google Scholar, 8Yan L. Borregaard N. Kjeldsen L. Moses M.A. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.J Biol Chem. 2001; 276: 37258-37265Crossref PubMed Scopus (563) Google Scholar, 19Marler J.J. Fishman S.J. Kilroy S.M. Fang J. Upton J. Mulliken J.B. et al.Increased expression of urinary matrix metalloproteinases parallels the extent and activity of vascular anomalies.Pediatrics. 2005; 116: 38-45Crossref PubMed Scopus (87) Google Scholar, 20Moses M.A. Wiederschain D. Loughlin K.R. Zurakowski D. Lamb C.C. Freeman M.R. Increased incidence of matrix metalloproteinases in urine of cancer patients.Cancer Res. 1998; 58: 1395-1399PubMed Google Scholar). In the current study, we hypothesized that this noninvasive assay could also be applied to patients with endometriosis. We show that the urinary expression of a panel of gelatinases can be used as a surrogate marker of disease progression. Consequently, urinary MMP expression may be useful [1] in facilitating clinical decision-making as to whether a patient suffers from endometriosis or not and [2] in monitoring therapeutic efficacy in patients with endometriosis.Urine samples were collected from patients who underwent laparoscopy either for suspected endometriosis or for reasons such as investigation of infertility or pelvic pain. Patients with a history of malignancies, irritable bowel syndrome, autoimmune or infectious disease, or patients using medical treatment for causes other than endometriosis were excluded from the study. Urine samples were collected prospectively before surgery (catheter) and, if available, during visits in an outpatient setting (midstream). The samples were then immediately aliquoted, frozen, and stored at -80 °C until further use. Frozen samples were randomly chosen by using a publicly available random numbers generator (www.random.org) and then sent for further processing to Children's Hospital Boston on dry ice. The study was approved by the local ethics committee of the Charité, Campus Benjamin Franklin, Berlin, Germany. Patients were classified into four groups depending on whether laparoscopic surgery was performed and endometriosis could be confirmed: group A, surgically and histologically verified endometriosis; group B, surgically or histologically verified no evidence of disease; group C, clinical diagnosis of endometriosis (patients with either a history of previously surgically identified endometriosis who had been symptomless after surgical or medical treatment or women with similar symptoms without previous abdominal surgery; symptoms included cyclical and noncyclical lower abdominal pain, dysmenorrhea, dysuria, and dyschezia); group D, clinical diagnosis of no evidence of disease (no symptoms).If surgery was performed, patients were evaluated for the presence and the extent of endometriosis or other possible causes for potential abdominal symptoms. Endometriosis was classified according to the revised guidelines of the American Fertility Society (AFS) (21American Society for Reproductive MedicineRevised American Society for Reproductive Medicine classification of endometriosis: 1996.Fertil Steril. 1997; 67: 817-821Abstract Full Text PDF PubMed Scopus (2229) Google Scholar). The experienced surgeons performing the procedures were not aware of the MMP data. Suspect lesions were sent for routine histologic evaluation to the Department of Pathology at the Charité, Berlin. Criteria for the verified diagnosis of endometriosis were the presence of both endometrial stromal cells and glandular epithelial cells.For analysis, samples were thawed on wet ice, aliquoted, and tested for blood, leukocytes, and protein using a Multistix 9 reagent strip (Miles, Elkhart, IN). Zymograms were conducted using an established procedure as described previously (5Roy R. Louis G. Loughlin K.R. Wiederschain D. Kilroy S.M. Lamb C.C. et al.Tumor-specific urinary matrix metalloproteinase fingerprinting: identification of high molecular weight urinary matrix metalloproteinase species.Clin Cancer Res. 2008; 14: 6610-6617Crossref PubMed Scopus (134) Google Scholar, 20Moses M.A. Wiederschain D. Loughlin K.R. Zurakowski D. Lamb C.C. Freeman M.R. Increased incidence of matrix metalloproteinases in urine of cancer patients.Cancer Res. 1998; 58: 1395-1399PubMed Google Scholar, 22Braunhut S.J. Moses M.A. Retinoids modulate endothelial cell production of matrix-degrading proteases and tissue inhibitors of metalloproteinases (TIMP).J Biol Chem. 1994; 269: 13472-13479Abstract Full Text PDF PubMed Google Scholar). In this assay, the proenzyme and the activated form of the gelatinase appear as bands of clearance of the substrate (gelatin) in the gel. Different MMPs were distinguished from each other according to their molecular weight and confirmed by immunoblot analysis. Zymograms were processed in a double-blinded manner, by two investigators who had no knowledge of the clinical status of the patient. Correlating MMP expression to creatinine clearance did not have an impact on the results as previously demonstrated in earlier studies (20Moses M.A. Wiederschain D. Loughlin K.R. Zurakowski D. Lamb C.C. Freeman M.R. Increased incidence of matrix metalloproteinases in urine of cancer patients.Cancer Res. 1998; 58: 1395-1399PubMed Google Scholar, 23Smith E.R. Zurakowski D. Saad A. Scott R.M. Moses M.A. Urinary biomarkers predict brain tumor presence and response to therapy.Clin Cancer Res. 2008; 14: 2378-2386Crossref PubMed Scopus (135) Google Scholar, 24Pories S.E. Zurakowski D. Roy R. Lamb C.C. Raza S. Exarhopoulos A. et al.Urinary metalloproteinases: noninvasive biomarkers for breast cancer risk assessment.Cancer Epidemiol Biomarkers Prev. 2008; 17: 1034-1042Crossref PubMed Scopus (79) Google Scholar).Sensitivity and specificity for each MMP biomarker were calculated using standard formulas for classifying patients with endometriosis and for correctly classifying patients verified with no evidence of disease. Logistic regression was applied to determine the significance of each biomarker and to calculate the odds ratios and 95% confidence intervals (CI) for differentiating surgically verified endometriosis from normal controls with the likelihood ratio test used to assess significance (25Hosmer D.W. Lemeshow S. Applied logistic regression.2nd ed. John Wiley and Sons, New York2000Crossref Google Scholar). A generalized estimating equations approach was used for the logistic regression modeling to account for multiple samples within the same patient. A receiver operating characteristic curve analysis was applied to determine the accuracy of the individual MMPs and the combined number of MMP biomarkers (0, 1, 2, 3) with area under the curve as a measure of discrimination between endometriosis and control. For longitudinal data, differences were assessed using the McNemar test for binary matched pairs. Two-tailed P<.05 were considered statistically significant. Statistical analysis was performed using SPSS software (version 16.0, SPSS Inc., Chicago, IL).The samples (1,541) were collected over the course of 3 years. Of these, 266 samples from 107 individual patients obtained at different time points were randomly chosen for analysis. Eighteen patients had multiple laparoscopies over time, partially with different stages of disease. The mean age of the patients included into the study was 33 years (range 29–38 years). The distribution of samples in the four groups was as follows: group A: 80 samples from 33 patients; group B: 51 samples from 19 patients; group C: 75 samples from 40 patients; and group D: 60 samples from 39 patients.Women with surgically and histologically verified endometriosis (group A) were more likely to have MMPs in their urine than women in whom disease was surgically excluded (group B). Logistic regression analysis using all samples from patients with endometriosis and controls indicated a significantly higher percentage of patients with endometriosis and positive MMPs compared with controls for all three gelatinases. The odds ratio of having a specific MMP in the urine and having endometriosis for MMP-2 was 4.8 (95% CI 1.8–13.2; P<.001), for MMP-9 was 7.8 (95% CI 2.5–25.1; P<.001), and for MMP-9/NGAL was 6.3 (95% CI 1.7–22.8; P<.001) (Table 1). This suggests that MMP-9 is the best single MMP biomarker of endometriosis which is confirmed as well by the highest likelihood ratio χ2 test of (likelihood ratio test = 15.05) among the three MMPs (logistic regression). The stage of the menstrual cycle was evenly distributed within and between the groups. In healthy volunteers, we detected urinary MMPs at the time of menstruation, but none when no blood was found in the urine. When samples from all groups containing leukocytes, traces of blood, or protein were excluded the differences between the groups remained significant. Given the urinary presence of any of these three gelatinases, the odds of endometriosis compared with control were more than eight times greater with a lower bound of three times higher based on the 95% CI. Using receiver operating characteristic analysis, the area under the curve for each MMP biomarker indicates moderately good discrimination between surgically verified endometriosis and normal controls. Based on the number of positive urinary MMP biomarkers (0, 1, 2, 3), the area under the curve is 0.755, which indicates good discrimination for the combination of all three MMP biomarkers (95% CI 0.645–0.865).Table 1Predictors of verified endometriosis based on logistic regression analysis.MMP biomarkerOdds ratio95% CIP valueMMP-9/NGAL6.31.7–22.8<.001aStatistically significant.MMP-97.82.5–25.1<.001aStatistically significant.MMP-24.81.8–13.2<.001aStatistically significant.Any MMP above8.33.0–22.7<.001aStatistically significant.Note: Analysis accounts for multiple samples from the same patient and is based on surgically verified endometriosis versus normal controls. CI = confidence interval; MMP = matrix metalloproteinase.a Statistically significant. Open table in a new tab In the case of 60 patients, more than one sample was collected in the course of the study, providing longitudinal data. Seventy-six percent of women with endometriosis or clinical symptoms (groups A and C) who had at least one gelatinase detectable in the urine, no longer had MMPs in their urine during a follow-up visit when either patients were asymptomatic at the follow-up visit (group D) or a second laparoscopy revealed no evidence of disease (group B) (P<.01, McNemar's test). In our randomly chosen cohort of samples, we had data of two patients who had verified endometriosis (with positive MMPs) during the first visit and had then surgically verified no evidence of disease status during the follow-up consultation (surgery because of symptoms). However, these two patients had no MMPs in their urine at their follow-up surgery. Conversely, some women with a previous history of endometriosis, who had no evidence of disease at surgery (group B) or were asymptomatic in clinic (group D), had no gelatinase initially detectable in the urine. Of them, 91% expressed either MMP-2, MMP-9, or MMP-9/NGAL in the urine when they became symptomatic (group C) or had a laparoscopy that verified the presence of endometriosis (group A) at a follow-up visit.Surgical identification of intra-abdominal endometriotic lesions by laparoscopy is currently the diagnostic gold standard (26Kennedy S. Bergqvist A. Chapron C. D'Hooghe T. Dunselman G. Greb R. et al.ESHRE guideline for the diagnosis and treatment of endometriosis.Hum Reprod. 2005; 20: 2698-2704Crossref PubMed Scopus (1191) Google Scholar). As with any surgical procedure, laparoscopy, even in experienced hands, is associated with some morbidity and mortality. Clinical symptoms, such as abdominal pain and dysmenorrhea, are highly nonspecific and can be caused by many disorders. As a result, diagnosis and treatment are often delayed leading to extended suffering, and unnecessary surgical procedures have been performed putting the patients' lives unnecessarily at risk. A biomarker that can more easily identify endometriosis in symptomatic patients is urgently needed. Studies show that serum CA-125 levels are significantly elevated in patients with moderate and severe disease compared with controls (27Mol B.W. Bayram N. Lijmer J.G. Wiegerinck M.A. Bongers M.Y. van der Veen F. et al.The performance of CA-125 measurement in the detection of endometriosis: a meta-analysis.Fertil Steril. 1998; 70: 1101-1108Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Currently, however, compared with laparoscopy, measuring serum CA-125 levels has demonstrated little value as a diagnostic tool (26Kennedy S. Bergqvist A. Chapron C. D'Hooghe T. Dunselman G. Greb R. et al.ESHRE guideline for the diagnosis and treatment of endometriosis.Hum Reprod. 2005; 20: 2698-2704Crossref PubMed Scopus (1191) Google Scholar).Along with MMP-2 and MMP-9 we detected a band at approximately 140 kDa corresponding to MMP-9 bound to human NGAL (MMP-9/NGAL). This complex formation protects MMP-9 from autodegradation (8Yan L. Borregaard N. Kjeldsen L. Moses M.A. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.J Biol Chem. 2001; 276: 37258-37265Crossref PubMed Scopus (563) Google Scholar). We have previously demonstrated that expression of MMP-9/NGAL in breast tumors leads to increased tumor growth with enhanced MMP-9 activity, tumor angiogenesis, and tumor cell proliferation (7Fernandez C.A. Yan L. Louis G. Yang J. Kutok J.L. Moses M.A. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients.Clin Cancer Res. 2005; 11: 5390-5395Crossref PubMed Scopus (237) Google Scholar). Thus far, no reports exist that have investigated the role of MMP-9/NGAL in endometriotic tissue. If it is detectable in endometriotic lesions, it will be interesting to determine whether it is differentially expressed at different stages of disease progression, location, and types of cells. It could be hypothesized that presence of NGAL in endometriotic tissue increases the invasive properties of the cells enhancing lesion formation.A biomarker of endometriosis would be especially useful for patients with minimal or mild disease as it is usually impossible to identify endometriotic lesions with imaging techniques, such as ultrasound or magnetic resonance imaging (MRI), or by clinical examination (28D'Hooghe T.M. Mihalyi A.M. Simsa P. Kyama C.K. Peeraer K. De Loecker P. et al.Why we need a noninvasive diagnostic test for minimal to mild endometriosis with a high sensitivity.Gynecol Obstet Invest. 2006; 62: 136-138Crossref PubMed Scopus (54) Google Scholar). Unfortunately, this study, using randomly chosen samples from a larger cohort, was not powered to distinguish between AFS stages I–IV in all four groups. The diagnostic power of MMPs in distinguishing between different disease stages will be investigated as part of a larger study.Many women in our study were observed in follow-up sessions during multiple visits to the clinic. In these patients' samples, we found that MMP presence in urine changed in ways consistent with the alteration in symptoms or presence/absence of endometriosis. In addition, in our randomly chosen samples, we monitored two women with surgically proven endometriosis who were followed during 2 years (data not shown). Although the number was too small to perform a statistical analysis, we found that the urinary MMP expression correlated with the presence or lack of symptoms over time during each visit (i.e., the existence of symptoms was associated with the presence of urinary MMPs and vice versa). We believe that these results are encouraging and we are currently analyzing larger cohorts of women in a prospective study to determine potential statistical significance. Clinical symptoms of endometriosis, defined as the presence of endometrium-like tissue outside of the uterine cavity, are often nonspecific and mimic those from other diseases such as pelvic inflammatory disease, interstitial cystitis, or inflammatory bowel disease (1Bulun S.E. Endometriosis.N Engl J Med. 2009; 360: 268-279Crossref PubMed Scopus (1437) Google Scholar). Due to a lack of reliable biomarkers, most cases can only be diagnosed by an invasive procedure such as a laparoscopy (2Giudice L.C. Kao L.C. Endometriosis. Lancet. 2004; 364: 1789-1799Abstract Full Text Full Text PDF PubMed Scopus (2469) Google Scholar). Matrix metalloproteinases (MMPs) are extracellular endopeptidases that play a pivotal role in many physiological and pathological processes including development, growth, and wound healing (3Mott J.D. Werb Z. Regulation of matrix biology by matrix metalloproteinases.Curr Opin Cell Biol. 2004; 16: 558-564Crossref PubMed Scopus (863) Google Scholar, 4Ravanti L. Kahari V.M. Matrix metalloproteinases in wound repair (review).Int J Mol Med. 2000; 6: 391-407PubMed Google Scholar, 5Roy R. Louis G. Loughlin K.R. Wiederschain D. Kilroy S.M. Lamb C.C. et al.Tumor-specific urinary matrix metalloproteinase fingerprinting: identification of high molecular weight urinary matrix metalloproteinase species.Clin Cancer Res. 2008; 14: 6610-6617Crossref PubMed Scopus (134) Google Scholar). Degradation and remodeling of extracellular matrix by MMPs are crucial steps for cellular migration and invasion into tissue. MMP-2 (gelatinase A, 72 kDa) and MMP-9 (gelatinase B, 92 kDa) degrade collagen IV, V, VII, and X, and fibronectin, all of which are constituents of the interstitial stroma and the subendothelial basement membrane (6Mignatti P. Rifkin D.B. Biology and biochemistry of proteinases in tumor invasion.Physiol Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar). The MMP-9/NGAL (neutrophil gelatinase-associated lipocalin) complex has been shown to protect MMP-9 from autodegradation in vitro and to increase aggressiveness in breast tumors in vivo (7Fernandez C.A. Yan L. Louis G. Yang J. Kutok J.L. Moses M.A. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients.Clin Cancer Res. 2005; 11: 5390-5395Crossref PubMed Scopus (237) Google Scholar, 8Yan L. Borregaard N. Kjeldsen L. Moses M.A. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.J Biol Chem. 2001; 276: 37258-37265Crossref PubMed Scopus (563) Google Scholar). It has been suggested that the differential expression of MMPs in endometriotic cells is responsible for the invasive properties of endometriotic lesions (9Bruner K.L. Matrisian L.M. Rodgers W.H. Gorstein F. Osteen K.G. Suppression of matrix metalloproteinases inhibits establishment of ectopic lesions by human endometrium in nude mice.J Clin Invest. 1997; 99: 2851-2857Crossref PubMed Scopus (269) Google Scholar, 10Sharpe-Timms K.L. Zimmer R.L. Jolliff W.J. Wright J.A. Nothnick W.B. Curry T.E. Gonadotropin-releasing hormone agonist (GnRH-a) therapy alters activity of plasminogen activators, matrix metalloproteinases, and their inhibitors in rat models for adhesion formation and endometriosis: potential GnRH-a-regulated mechanisms reducing adhesion formation.Fertil Steril. 1998; 69: 916-923Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Various MMPs have been demonstrated to be expressed in endometriotic tissue (reviewed in Ref.11Osteen K.G. Yeaman G.R. Bruner-Tran K.L. Matrix metalloproteinases and endometriosis.Semin Reprod Med. 2003; 21: 155-164Crossref PubMed Scopus (124) Google Scholar). MMP-2 and MMP-9 expression is up-regulated in eutopic endometrium of women with endometriosis and in endometriotic lesions, and MMP-2 is elevated in serum and peritoneal fluid (PF) of these patients (12Chung H.W. Lee J.Y. Moon H.S. Hur S.E. Park M.H. Wen Y. et al.Matrix metalloproteinase-2, membranous type 1 matrix metalloproteinase, and tissue inhibitor of metalloproteinase-2 expression in ectopic and eutopic endometrium.Fertil Steril. 2002; 78: 787-795Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 13Chung H.W. Wen Y. Chun S.H. Nezhat C. Woo B.H. Lake Polan M. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 mRNA expression in ectopic and eutopic endometrium in women with endometriosis: a rationale for endometriotic invasiveness.Fertil Steril. 2001; 75: 152-159Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 14Collette T. Maheux R. Mailloux J. Akoum A. Increased expression of matrix metalloproteinase-9 in the eutopic endometrial tissue of women with endometriosis.Hum Reprod. 2006; 21: 3059-3067Crossref PubMed Scopus (93) Google Scholar, 15Huang H.F. Hong L.H. Tan Y. Sheng J.Z. Matrix metalloproteinase 2 is associated with changes in steroid hormones in the sera and peritoneal fluid of patients with endometriosis.Fertil Steril. 2004; 81: 1235-1239Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 16Ria R. Loverro G. Vacca A. Ribatti D. Cormio G. Roccaro A.M. et al.Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 agree with progression of ovarian endometriomas.Eur J Clin Invest. 2002; 32: 199-206Crossref PubMed Scopus (59) Google Scholar, 17Sillem M. Prifti S. Koch A. Neher M. Jauckus J. Runnebaum B. Regulation of matrix metalloproteinases and their inhibitors in uterine endometrial cells of patients with and without endometriosis.Eur J Obstet Gynecol reprod Biol. 2001; 95: 167-174Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Szamatowicz J. Laudanski P. Tomaszewska I. Matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1: a possible role in the pathogenesis of endometriosis.Hum Reprod. 2002; 17: 284-288Crossref PubMed Scopus (50) Google Scholar). We have previously shown that MMPs are detectable in the urine of cancer patients and children suffering from vascular anomalies, and that their expression pattern correlates with the extent and stage of the disease (5Roy R. Louis G. Loughlin K.R. Wiederschain D. Kilroy S.M. Lamb C.C. et al.Tumor-specific urinary matrix metalloproteinase fingerprinting: identification of high molecular weight urinary matrix metalloproteinase species.Clin Cancer Res. 2008; 14: 6610-6617Crossref PubMed Scopus (134) Google Scholar, 7Fernandez C.A. Yan L. Louis G. Yang J. Kutok J.L. Moses M.A. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients.Clin Cancer Res. 2005; 11: 5390-5395Crossref PubMed Scopus (237) Google Scholar, 8Yan L. Borregaard N. Kjeldsen L. Moses M.A. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.J Biol Chem. 2001; 276: 37258-37265Crossref PubMed Scopus (563) Google Scholar, 19Marler J.J. Fishman S.J. Kilroy S.M. Fang J. Upton J. Mulliken J.B. et al.Increased expression of urinary matrix metalloproteinases parallels the extent and activity of vascular anomalies.Pediatrics. 2005; 116: 38-45Crossref PubMed Scopus (87) Google Scholar, 20Moses M.A. Wiederschain D. Loughlin K.R. Zurakowski D. Lamb C.C. Freeman M.R. Increased incidence of matrix metalloproteinases in urine of cancer patients.Cancer Res. 1998; 58: 1395-1399PubMed Google Scholar). In the current study, we hypothesized that this noninvasive assay could also be applied to patients with endometriosis. We show that the urinary expression of a panel of gelatinases can be used as a surrogate marker of disease progression. Consequently, urinary MMP expression may be useful [1] in facilitating clinical decision-making as to whether a patient suffers from endometriosis or not and [2] in monitoring therapeutic efficacy in patients with endometriosis. Urine samples were collected from patients who underwent laparoscopy either for suspected endometriosis or for reasons such as investigation of infertility or pelvic pain. Patients with a history of malignancies, irritable bowel syndrome, autoimmune or infectious disease, or patients using medical treatment for causes other than endometriosis were excluded from the study. Urine samples were collected prospectively before surgery (catheter) and, if available, during visits in an outpatient setting (midstream). The samples were then immediately aliquoted, frozen, and stored at -80 °C until further use. Frozen samples were randomly chosen by using a publicly available random numbers generator (www.random.org) and then sent for further processing to Children's Hospital Boston on dry ice. The study was approved by the local ethics committee of the Charité, Campus Benjamin Franklin, Berlin, Germany. Patients were classified into four groups depending on whether laparoscopic surgery was performed and endometriosis could be confirmed: group A, surgically and histologically verified endometriosis; group B, surgically or histologically verified no evidence of disease; group C, clinical diagnosis of endometriosis (patients with either a history of previously surgically identified endometriosis who had been symptomless after surgical or medical treatment or women with similar symptoms without previous abdominal surgery; symptoms included cyclical and noncyclical lower abdominal pain, dysmenorrhea, dysuria, and dyschezia); group D, clinical diagnosis of no evidence of disease (no symptoms). If surgery was performed, patients were evaluated for the presence and the extent of endometriosis or other possible causes for potential abdominal symptoms. Endometriosis was classified according to the revised guidelines of the American Fertility Society (AFS) (21American Society for Reproductive MedicineRevised American Society for Reproductive Medicine classification of endometriosis: 1996.Fertil Steril. 1997; 67: 817-821Abstract Full Text PDF PubMed Scopus (2229) Google Scholar). The experienced surgeons performing the procedures were not aware of the MMP data. Suspect lesions were sent for routine histologic evaluation to the Department of Pathology at the Charité, Berlin. Criteria for the verified diagnosis of endometriosis were the presence of both endometrial stromal cells and glandular epithelial cells. For analysis, samples were thawed on wet ice, aliquoted, and tested for blood, leukocytes, and protein using a Multistix 9 reagent strip (Miles, Elkhart, IN). Zymograms were conducted using an established procedure as described previously (5Roy R. Louis G. Loughlin K.R. Wiederschain D. Kilroy S.M. Lamb C.C. et al.Tumor-specific urinary matrix metalloproteinase fingerprinting: identification of high molecular weight urinary matrix metalloproteinase species.Clin Cancer Res. 2008; 14: 6610-6617Crossref PubMed Scopus (134) Google Scholar, 20Moses M.A. Wiederschain D. Loughlin K.R. Zurakowski D. Lamb C.C. Freeman M.R. Increased incidence of matrix metalloproteinases in urine of cancer patients.Cancer Res. 1998; 58: 1395-1399PubMed Google Scholar, 22Braunhut S.J. Moses M.A. Retinoids modulate endothelial cell production of matrix-degrading proteases and tissue inhibitors of metalloproteinases (TIMP).J Biol Chem. 1994; 269: 13472-13479Abstract Full Text PDF PubMed Google Scholar). In this assay, the proenzyme and the activated form of the gelatinase appear as bands of clearance of the substrate (gelatin) in the gel. Different MMPs were distinguished from each other according to their molecular weight and confirmed by immunoblot analysis. Zymograms were processed in a double-blinded manner, by two investigators who had no knowledge of the clinical status of the patient. Correlating MMP expression to creatinine clearance did not have an impact on the results as previously demonstrated in earlier studies (20Moses M.A. Wiederschain D. Loughlin K.R. Zurakowski D. Lamb C.C. Freeman M.R. Increased incidence of matrix metalloproteinases in urine of cancer patients.Cancer Res. 1998; 58: 1395-1399PubMed Google Scholar, 23Smith E.R. Zurakowski D. Saad A. Scott R.M. Moses M.A. Urinary biomarkers predict brain tumor presence and response to therapy.Clin Cancer Res. 2008; 14: 2378-2386Crossref PubMed Scopus (135) Google Scholar, 24Pories S.E. Zurakowski D. Roy R. Lamb C.C. Raza S. Exarhopoulos A. et al.Urinary metalloproteinases: noninvasive biomarkers for breast cancer risk assessment.Cancer Epidemiol Biomarkers Prev. 2008; 17: 1034-1042Crossref PubMed Scopus (79) Google Scholar). Sensitivity and specificity for each MMP biomarker were calculated using standard formulas for classifying patients with endometriosis and for correctly classifying patients verified with no evidence of disease. Logistic regression was applied to determine the significance of each biomarker and to calculate the odds ratios and 95% confidence intervals (CI) for differentiating surgically verified endometriosis from normal controls with the likelihood ratio test used to assess significance (25Hosmer D.W. Lemeshow S. Applied logistic regression.2nd ed. John Wiley and Sons, New York2000Crossref Google Scholar). A generalized estimating equations approach was used for the logistic regression modeling to account for multiple samples within the same patient. A receiver operating characteristic curve analysis was applied to determine the accuracy of the individual MMPs and the combined number of MMP biomarkers (0, 1, 2, 3) with area under the curve as a measure of discrimination between endometriosis and control. For longitudinal data, differences were assessed using the McNemar test for binary matched pairs. Two-tailed P<.05 were considered statistically significant. Statistical analysis was performed using SPSS software (version 16.0, SPSS Inc., Chicago, IL). The samples (1,541) were collected over the course of 3 years. Of these, 266 samples from 107 individual patients obtained at different time points were randomly chosen for analysis. Eighteen patients had multiple laparoscopies over time, partially with different stages of disease. The mean age of the patients included into the study was 33 years (range 29–38 years). The distribution of samples in the four groups was as follows: group A: 80 samples from 33 patients; group B: 51 samples from 19 patients; group C: 75 samples from 40 patients; and group D: 60 samples from 39 patients. Women with surgically and histologically verified endometriosis (group A) were more likely to have MMPs in their urine than women in whom disease was surgically excluded (group B). Logistic regression analysis using all samples from patients with endometriosis and controls indicated a significantly higher percentage of patients with endometriosis and positive MMPs compared with controls for all three gelatinases. The odds ratio of having a specific MMP in the urine and having endometriosis for MMP-2 was 4.8 (95% CI 1.8–13.2; P<.001), for MMP-9 was 7.8 (95% CI 2.5–25.1; P<.001), and for MMP-9/NGAL was 6.3 (95% CI 1.7–22.8; P<.001) (Table 1). This suggests that MMP-9 is the best single MMP biomarker of endometriosis which is confirmed as well by the highest likelihood ratio χ2 test of (likelihood ratio test = 15.05) among the three MMPs (logistic regression). The stage of the menstrual cycle was evenly distributed within and between the groups. In healthy volunteers, we detected urinary MMPs at the time of menstruation, but none when no blood was found in the urine. When samples from all groups containing leukocytes, traces of blood, or protein were excluded the differences between the groups remained significant. Given the urinary presence of any of these three gelatinases, the odds of endometriosis compared with control were more than eight times greater with a lower bound of three times higher based on the 95% CI. Using receiver operating characteristic analysis, the area under the curve for each MMP biomarker indicates moderately good discrimination between surgically verified endometriosis and normal controls. Based on the number of positive urinary MMP biomarkers (0, 1, 2, 3), the area under the curve is 0.755, which indicates good discrimination for the combination of all three MMP biomarkers (95% CI 0.645–0.865). Note: Analysis accounts for multiple samples from the same patient and is based on surgically verified endometriosis versus normal controls. CI = confidence interval; MMP = matrix metalloproteinase. In the case of 60 patients, more than one sample was collected in the course of the study, providing longitudinal data. Seventy-six percent of women with endometriosis or clinical symptoms (groups A and C) who had at least one gelatinase detectable in the urine, no longer had MMPs in their urine during a follow-up visit when either patients were asymptomatic at the follow-up visit (group D) or a second laparoscopy revealed no evidence of disease (group B) (P<.01, McNemar's test). In our randomly chosen cohort of samples, we had data of two patients who had verified endometriosis (with positive MMPs) during the first visit and had then surgically verified no evidence of disease status during the follow-up consultation (surgery because of symptoms). However, these two patients had no MMPs in their urine at their follow-up surgery. Conversely, some women with a previous history of endometriosis, who had no evidence of disease at surgery (group B) or were asymptomatic in clinic (group D), had no gelatinase initially detectable in the urine. Of them, 91% expressed either MMP-2, MMP-9, or MMP-9/NGAL in the urine when they became symptomatic (group C) or had a laparoscopy that verified the presence of endometriosis (group A) at a follow-up visit. Surgical identification of intra-abdominal endometriotic lesions by laparoscopy is currently the diagnostic gold standard (26Kennedy S. Bergqvist A. Chapron C. D'Hooghe T. Dunselman G. Greb R. et al.ESHRE guideline for the diagnosis and treatment of endometriosis.Hum Reprod. 2005; 20: 2698-2704Crossref PubMed Scopus (1191) Google Scholar). As with any surgical procedure, laparoscopy, even in experienced hands, is associated with some morbidity and mortality. Clinical symptoms, such as abdominal pain and dysmenorrhea, are highly nonspecific and can be caused by many disorders. As a result, diagnosis and treatment are often delayed leading to extended suffering, and unnecessary surgical procedures have been performed putting the patients' lives unnecessarily at risk. A biomarker that can more easily identify endometriosis in symptomatic patients is urgently needed. Studies show that serum CA-125 levels are significantly elevated in patients with moderate and severe disease compared with controls (27Mol B.W. Bayram N. Lijmer J.G. Wiegerinck M.A. Bongers M.Y. van der Veen F. et al.The performance of CA-125 measurement in the detection of endometriosis: a meta-analysis.Fertil Steril. 1998; 70: 1101-1108Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Currently, however, compared with laparoscopy, measuring serum CA-125 levels has demonstrated little value as a diagnostic tool (26Kennedy S. Bergqvist A. Chapron C. D'Hooghe T. Dunselman G. Greb R. et al.ESHRE guideline for the diagnosis and treatment of endometriosis.Hum Reprod. 2005; 20: 2698-2704Crossref PubMed Scopus (1191) Google Scholar). Along with MMP-2 and MMP-9 we detected a band at approximately 140 kDa corresponding to MMP-9 bound to human NGAL (MMP-9/NGAL). This complex formation protects MMP-9 from autodegradation (8Yan L. Borregaard N. Kjeldsen L. Moses M.A. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.J Biol Chem. 2001; 276: 37258-37265Crossref PubMed Scopus (563) Google Scholar). We have previously demonstrated that expression of MMP-9/NGAL in breast tumors leads to increased tumor growth with enhanced MMP-9 activity, tumor angiogenesis, and tumor cell proliferation (7Fernandez C.A. Yan L. Louis G. Yang J. Kutok J.L. Moses M.A. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients.Clin Cancer Res. 2005; 11: 5390-5395Crossref PubMed Scopus (237) Google Scholar). Thus far, no reports exist that have investigated the role of MMP-9/NGAL in endometriotic tissue. If it is detectable in endometriotic lesions, it will be interesting to determine whether it is differentially expressed at different stages of disease progression, location, and types of cells. It could be hypothesized that presence of NGAL in endometriotic tissue increases the invasive properties of the cells enhancing lesion formation. A biomarker of endometriosis would be especially useful for patients with minimal or mild disease as it is usually impossible to identify endometriotic lesions with imaging techniques, such as ultrasound or magnetic resonance imaging (MRI), or by clinical examination (28D'Hooghe T.M. Mihalyi A.M. Simsa P. Kyama C.K. Peeraer K. De Loecker P. et al.Why we need a noninvasive diagnostic test for minimal to mild endometriosis with a high sensitivity.Gynecol Obstet Invest. 2006; 62: 136-138Crossref PubMed Scopus (54) Google Scholar). Unfortunately, this study, using randomly chosen samples from a larger cohort, was not powered to distinguish between AFS stages I–IV in all four groups. The diagnostic power of MMPs in distinguishing between different disease stages will be investigated as part of a larger study. Many women in our study were observed in follow-up sessions during multiple visits to the clinic. In these patients' samples, we found that MMP presence in urine changed in ways consistent with the alteration in symptoms or presence/absence of endometriosis. In addition, in our randomly chosen samples, we monitored two women with surgically proven endometriosis who were followed during 2 years (data not shown). Although the number was too small to perform a statistical analysis, we found that the urinary MMP expression correlated with the presence or lack of symptoms over time during each visit (i.e., the existence of symptoms was associated with the presence of urinary MMPs and vice versa). We believe that these results are encouraging and we are currently analyzing larger cohorts of women in a prospective study to determine potential statistical significance." @default.
• W2080505512 created "2016-06-24" @default.
• W2080505512 creator A5011167993 @default.
• W2080505512 creator A5017815284 @default.
• W2080505512 creator A5028523107 @default.
• W2080505512 creator A5029802394 @default.
• W2080505512 creator A5036627839 @default.
• W2080505512 creator A5038393409 @default.
• W2080505512 creator A5058903340 @default.
• W2080505512 date "2010-11-01" @default.
• W2080505512 modified "2023-10-14" @default.
• W2080505512 title "Matrix metalloproteinases are elevated in the urine of patients with endometriosis" @default.
• W2080505512 cites W1579897036 @default.
• W2080505512 cites W1837228878 @default.
• W2080505512 cites W1949259756 @default.
• W2080505512 cites W1970232912 @default.
• W2080505512 cites W1975706974 @default.
• W2080505512 cites W1988449462 @default.
• W2080505512 cites W2012071401 @default.
• W2080505512 cites W2030776545 @default.
• W2080505512 cites W2032038471 @default.
• W2080505512 cites W2033046719 @default.
• W2080505512 cites W2058602477 @default.
• W2080505512 cites W2085585495 @default.
• W2080505512 cites W2094112735 @default.
• W2080505512 cites W2105925637 @default.
• W2080505512 cites W2110530600 @default.
• W2080505512 cites W2117204437 @default.
• W2080505512 cites W2122365868 @default.
• W2080505512 cites W2143752038 @default.
• W2080505512 cites W2157602823 @default.
• W2080505512 cites W2162317615 @default.
• W2080505512 cites W2167143360 @default.
• W2080505512 cites W2607215535 @default.
• W2080505512 cites W4211081176 @default.
• W2080505512 cites W4231488642 @default.
• W2080505512 cites W58421210 @default.
• W2080505512 doi "https://doi.org/10.1016/j.fertnstert.2010.02.040" @default.
• W2080505512 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20378112" @default.
• W2080505512 hasPublicationYear "2010" @default.
• W2080505512 type Work @default.
• W2080505512 sameAs 2080505512 @default.
• W2080505512 citedByCount "33" @default.
• W2080505512 countsByYear W20805055122012 @default.
• W2080505512 countsByYear W20805055122013 @default.
• W2080505512 countsByYear W20805055122014 @default.
• W2080505512 countsByYear W20805055122015 @default.
• W2080505512 countsByYear W20805055122016 @default.
• W2080505512 countsByYear W20805055122017 @default.
• W2080505512 countsByYear W20805055122018 @default.
• W2080505512 countsByYear W20805055122019 @default.
• W2080505512 countsByYear W20805055122020 @default.
• W2080505512 countsByYear W20805055122021 @default.
• W2080505512 countsByYear W20805055122023 @default.
• W2080505512 crossrefType "journal-article" @default.
• W2080505512 hasAuthorship W2080505512A5011167993 @default.
• W2080505512 hasAuthorship W2080505512A5017815284 @default.
• W2080505512 hasAuthorship W2080505512A5028523107 @default.
• W2080505512 hasAuthorship W2080505512A5029802394 @default.
• W2080505512 hasAuthorship W2080505512A5036627839 @default.
• W2080505512 hasAuthorship W2080505512A5038393409 @default.
• W2080505512 hasAuthorship W2080505512A5058903340 @default.
• W2080505512 hasBestOaLocation W20805055121 @default.
• W2080505512 hasConcept C106487976 @default.
• W2080505512 hasConcept C109523444 @default.
• W2080505512 hasConcept C126322002 @default.
• W2080505512 hasConcept C126894567 @default.
• W2080505512 hasConcept C185592680 @default.
• W2080505512 hasConcept C2779522080 @default.
• W2080505512 hasConcept C2780026642 @default.
• W2080505512 hasConcept C43617362 @default.
• W2080505512 hasConcept C71924100 @default.
• W2080505512 hasConceptScore W2080505512C106487976 @default.
• W2080505512 hasConceptScore W2080505512C109523444 @default.
• W2080505512 hasConceptScore W2080505512C126322002 @default.
• W2080505512 hasConceptScore W2080505512C126894567 @default.
• W2080505512 hasConceptScore W2080505512C185592680 @default.
• W2080505512 hasConceptScore W2080505512C2779522080 @default.
• W2080505512 hasConceptScore W2080505512C2780026642 @default.
• W2080505512 hasConceptScore W2080505512C43617362 @default.
• W2080505512 hasConceptScore W2080505512C71924100 @default.
• W2080505512 hasIssue "6" @default.
• W2080505512 hasLocation W20805055121 @default.
• W2080505512 hasLocation W20805055122 @default.
• W2080505512 hasOpenAccess W2080505512 @default.
• W2080505512 hasPrimaryLocation W20805055121 @default.
• W2080505512 hasRelatedWork W1582580594 @default.
• W2080505512 hasRelatedWork W1954766992 @default.
• W2080505512 hasRelatedWork W1988645453 @default.
• W2080505512 hasRelatedWork W2003635697 @default.
• W2080505512 hasRelatedWork W2414729050 @default.
• W2080505512 hasRelatedWork W3123462835 @default.
• W2080505512 hasRelatedWork W3208449664 @default.
• W2080505512 hasRelatedWork W4210756955 @default.
• W2080505512 hasRelatedWork W4321088342 @default.
• W2080505512 hasRelatedWork W88698947 @default.
• W2080505512 hasVolume "94" @default.
• W2080505512 isParatext "false" @default.

• next