{"paper_id":"c6840520-da20-4b26-8f37-e66e2a5c1ba5","body_text":"21475\nCopyright@ Domenico Milardi | Biomed J Sci & Tech Res | BJSTR. MS.ID.004628.\nISSN: 2574 -1241\nIdentification of Novel Putative Urinary Markers \nof Endometriosis by High-Resolution Quantitative \nProteomics\n      DOI: 10.26717/BJSTR.2020.28.004628\nGrande Giuseppe1,2, Milardi Domenico1,2, Vincenzoni Federica3,4, Ricciardi Domenico1, Mancini \nFrancesca1, Di Nicuolo Fiorella 1, Castagnola Massimo 3,4, Pontecorvi Alfredo 1,2 and Marana \nRiccardo1,5\n1International Scientific Institute “Paul VI” , Italy \n2Division of Endocrinology, Fondazione Policlinico Universitario “ Agostino Gemelli” IRCCS, Italy\n3Institute of Biochemistry and Clinical Biochemistry, Catholic University, Italy \n4Department of Basic Biotechnological Sciences, Intensivological and Perioperative Clinics, Fondazione Policlinico Universitario Agosti-\nno Gemelli-IRCCS, Italy\n5Department of Women and Children Health, Fondazione Policlinico Universitario “ Agostino Gemelli” IRCCS, Italy\n*Corresponding author: Domenico Milardi, Division of Endocrinology, Fondazione Policlinico Universitario  “ Agostino Gemelli” \nIRCCS, Rome L.go F. Vito, 100168 Rome, Italy\nResearch Article\nIntroduction\nEndometriosis is a chronic gynecological inflammatory disease \ncharacterized by the presence of functional endometrial glands and \nstroma outside of the uterine cavity. Endometriosis affects 7-10% \nof women of reproductive age, 60% of women with pelvic pain and \nup to 50% of women with infertility [1]. The spectrum of symptoms \nincludes dysmenorrhea, dyspareunia, chronic pelvic pain, \ndyschezia, dysuria. However, recognition and conclusive diagnosis \nis only made on the average approximately 9 years following the  \n \nbeginning of the disease [2]. Early detection of the disease would \nallow patients earlier options for treatment, i.e. a sooner medical \nor surgical treatment and follow-up. The current gold standard \nfor the diagnosis of the disease requests laparoscopic evaluation \nand biopsy of the visualized lesions [3]. Although laparoscopy is \na minimally invasive procedure, it requires general anesthesia, \ndeveloped surgical skills and it has a high procedural cost. In \naddition, laparoscopy is associated with a, although rare, risk of \npotential intra-operative or post-operative complications [4,5]. The \nARTICLE INFO AbsTRACT\nReceived: \n   May 28, 2020\nPublished: \n   June 15, 2020\nCitation: Grande G, Milardi D, Vincenzoni \nD, Ricciardi D, Mancini F, et al., Identifica -\ntion of Novel Putative Urinary Markers of \nEndometriosis by High-Resolution Quanti -\ntative Proteomics. Biomed J Sci & Tech Res \n28(2)-2020. BJSTR. MS.ID.004628.\nKeywords: Endometriosis; Urine; Mark -\ners; Non-Invasive; Proteins; Proteomics\n \nEndometriosis is a chronic gynecological inflammatory disease characterized by \nthe presence of functional endometrial glands and stroma outside of the uterine cavity. \nIt is an estrogen-dependent disease affecting 7-10% of women of reproductive age. \nLaparoscopic diagnosis remains the gold standard approach, providing that the risks \nthese minimally invasive surgical procedures are understood and accepted by the \npatients. The aim of this study was to compare urine proteomic profiles in patients \nwith endometriosis versus controls, trying to identify a panel of specific proteins \nrepresenting novel non-invasive biomarkers of endometriosis. Eight 30-40 years old \nwomen with laparoscopically verified ovarian endometriotic cysts were enrolled. Five \nfertile women with non-endometriotic ovarian cystsand no visual signs of peritoneal \nendometriosis served as control group. Morning urine were collected in the day of the \nscheduled surgery and processed to be analyzed by proteomic. Eleven proteins were \nincreased in endometriosis, while prothrombin was reduced. Seven proteins were \nfound to fall into one large protein-protein interaction network, mainly involved with \ninflammation.  This is the first report on the application of quantitative proteomics \nin the search of an array of urinary proteins, which may guide further studies for the \nvalidation of novel non-invasive biomarkers of endometriosis.\n\nCopyright@ Domenico Milardi | Biomed J Sci & Tech Res | BJSTR. MS.ID.004628.\nVolume 28- Issue 2\nDOI: 10.26717/BJSTR.2020.28.004628\n21476\ndevelopment of high-throughput technologies, such as proteomics, \nhas led to an early diagnosis of medical diseases through the \nidentification of proteins linked to different pathological conditions \n[6]. In previous studies we have demonstrated that cervical mucus \nis a source of putative protein biomarkers [7]. We have also \ncharacterized a pattern of cervical mucus proteins involved in \nthe pathophysiology of endometriosis [8], which might represent \nputative non-invasive markers of endometriosis. Urine proteomic \nanalysis is a valuable source to identify specific proteins and \npeptides. Compared to cervical mucus collection, urine proteomic \nanalysis has the advantage to obtain proteins in a non-invasive, \nsimple method and in a large quantity. \nIt has been defined as a fluid biopsy of the urogenital tract, \nwhich provides considerable information about these organs [9]. \nConsequently, many changes in urogenital tract function may \nbe detected by urinary proteomic analysis. Currently, urinary \nproteomics is mainly used as diagnostic tool to identify biomarkers \nof kidney diseases, such as IgA nephropathy, renal cell carcinoma, \nand to detect biomarkers for chronic allograft dysfunction after \nkidney transplantation [10]. In addition, urinary proteomics \nhave been used to study a variety of diseases, such as obstructive \nsleep apnea and obesity [11] and brain [12] and cardiovascular \ndiseases [13]. Protein metabolites excreted in urine may reflect \nthe state of the body and serve as informative biomarkers for some \ndiseases. Through urinary proteomics, the molecule metabolites \nin urine can be comprehensively analyzed (metabolomics). \nUrinary metabolomics has been widely used in the study of \nvarious diseases, including diabetic nephropathy [14], chronic \nheart failure [15] and offers potential early diagnosis of ovarian \nand breast cancer [16]. Previous studies have been published \nattempted to identify the proteomic profiling of endometriosis in \nurine samples. Using two-dimension gel separation coupled with \nmass spectrometry, Tokushige et al. [17] demonstrated the unique \npresence of a highly up-regulated protein, the CK-19, in the urine of \nwomen with endometriosis compared to a control group of women \nwithout endometriosis. Recently, urine peptide biomarkers have \nbeen searched using magnetic beads-based matrix-assisted laser \ndesorption/ionization time-of-flight mass spectrometry (MALDI-\nTOF-MS). Wang et al. [18] identified in urine samples of patients \nwith endometriosis a specific cluster of peptides. However, an \nexhaustive identification of the list of putative urine markers of \nendometriosis has not been still provided. The aim of this study was \nto identify, in urine of patients affected by endometriosis, a panel \nof candidate proteins, which may be further validated as novel \nputative biomarkers of this pathological condition. \nMaterials and Methods\nEthical Approval\nThe Ethical board of “Fondazione Policlinico “ A. Gemelli” \napproved the protocol (registration number 0019148/16) on May, \n5th 2016.\nPatients\nEight 30 to 40 years old infertile women, who underwent \nlaparoscopy for ovarian endometriotic cysts, confirmed at \nhistological exam, were enrolled. They were compared with 5 \nfertile women, with non-endometriotic ovarian cysts, confirmed \nat histological exam. Patients and controls were all operated upon \nby a surgeon with a long previous experience in the laparoscopic \ndiagnostic and treatment of endometriosis (R.M.) [1,19,20]. The \ninclusion criterion for the study was: patients 30 to 40 years old \nfor both groups, with endometriotic or non-endometriotic ovarian \ncysts. Patients with non-endometriotic ovarian cysts had obtained \nprevious pregnancies. Final diagnosis was obtained by video \nlaparoscopy and was confirmed by histological exam of the cyst \n(endometriotic or non-endometriotic). The exclusion criteria for \nboth groups were the following: patients with other gynecologic, \nkidney and systemic diseases (known cancer, diabetes, thyroid \ndisease, cardiovascular disease). \nUrine Collection\nUrine culture was performed in the week preceding admission \nfor surgery, to exclude urinary tract infection.\nMorning midstream urine samples were collected by patients \nand controls after hospitalization in the morning of surgery. \nImmediately after urine collection, a tablet of cocktail of protease \ninhibitor (Roche-Basel, Switzerland) was added to 10 ml of samples. \nSample Preparation\nTen milliliters of each urine samples were centrifuged at \n1.500×g, 10 minutes at 4 °C. The supernatant has been collected \nand stored at -80 °C. One milliliter of each urine sample has been \nadded with acetonitrile (4 volumes), vortex mixed and incubated \nat -20 °C overnight. After centrifugation at 10.000×g, 20 minutes \nat 4°C, the supernatant was discharged and the precipitated pellet \nwere resuspended in 100µL of Urea 6M/Tris 100 mM pH 7.8 buffer.  \nTotal protein content was quantified by Bradford assay. 10 μg of \neach sample have been subjected to in-solution digestion protocol \nas described previously [21]. Briefly, each sample corresponding to \n10μg of total protein has been mixed with 100mM of ammonium \nbicarbonate at pH 8.0 and reduced with 200mM dithiothreitol \n(DTT , 10mM final; Sigma) for 5 minutes at 100 °C, 15 minutes at \n50°C, and alkylated with 200mM of iodoacetamide (55mM final; \nSigma) in the dark at room temperature for 60 minutes. The \nsamples were left to digest overnight at 37 °C by adding 100mM of \nammonium bicarbonate (pH 8) with sequencing grade-modified \nporcine trypsin (1:50, trypsin: protein concentration; Promega). To \nstop the digestion, the samples have been acidified with aqueous \ntrifluoroacetic acid (TFA/H2O 0.2%vol/vol), immediately frozen, \nand lyophilized.\nLC-MS/MS analysis\nFor proteomic analysis, the samples have been resuspended \nin 40 µL of aqueous formic acid solution (0.1% v/v) and equal \n\nVolume 28- Issue 2\nDOI: 10.26717/BJSTR.2020.28.004628\n21477\nCopyright@ Domenico Milardi | Biomed J Sci & Tech Res | BJSTR. MS.ID.004628.\nprotein quantity (3.5 µg) of each sample has been analyzed by \nUltimate RSLC nano apparatus coupled to Orbitrap Elite mass \nspectrometer (Thermo Fisher Scientific). Separation experiments \nhave been performed using a Zorbax C18 column (3.5 µm particle \ndiameter, 1mm i.d. x 15cm) (Agilent Technologies, Santa Clara, CA) \nusing following eluents: (A) 0.1% (v/v) aqueous formic acid and \n(B) acetonitrile: water (80:20) with 0.1% aqueous formic acid. The \ngradient applied, was linear from 0 to 70% of solvent B in 40 min, at \na flow rate of 50µl/min. The LTQ Orbitrap Elite has been operated \nin data dependent mode in which each full MS scan was followed by \nMS/MS scans where the five most intense multi-charged ions have \nbeen dynamically selected and fragmented by collision-induced \ndissociation (CID) at normalized collision energy of 35%. \nProtein Identification and Quantification\nTandem mass spectra have been elaborated by Proteome \nDiscoverer 1.4 software (version 1.4.1.14 licensed by Thermo \nFisher Scientific), based on SEQUEST HT cluster as search engine, \nagainst Homo Sapiens proteome (UniProtKB/SwissProt release \n2018-03). Data have been elaborated with following parameters: \ntrypsin enzyme with maximum of two missed cleavages, fixed \ncarbamidomethylation of cysteine and the oxidation of methionine \nas variable modification. In order to obtain a reliable identification \nof the proteins/peptides the following stringent criteria have been \nused: two unique peptide per protein, high peptide confidence \n(False discovery rate <1%). A 1% FDR and a minimum of two \npeptide matches per protein have been the criteria used for protein \nidentification. The dissociated or ‘ungrouping’ of proteins from \ntheir respective families has been used during the quantification \nprocess to avoid the possible ambiguity associated with different \nisoforms of the same protein. The label-free quantitative analysis \nhas been performed via Precursor Ions Area Detector Node during \nthe bioinformatic analysis using Proteome Discoverer software. \nThis quantification method has been used to define the relative \nquantities of all peptides in a sample. The Proteome Discoverer \napplication calculates peptide areas during processing, using them \nto automatically calculate protein areas for the proteins in the \nreport. It calculates the area of any given protein as the average of \nthe three most abundant distinct peptides identified in the protein. \nBioinformatics and Statistical Analysis\nThe relative protein level ratios between the group of controls \n(n=5) and the group of patients (n=8) have been determined from \nthe respective averages of protein abundances expressed for each \nprotein in the two groups. All the proteins detected with a ratio>1.5 \n(less abundant proteins in patients) or <0.67 (more abundant \nproteins in patients) have been considered for this study. Protein-\nprotein interaction network analysis has been performed by Search \nTool for the Retrieval of Interacting Genes/Proteins (STRING). \nSTRING is a database for predicted protein-protein interactions \nat EMBL clusters the extracted results from many protein-protein \ninteractions databases, like Mint, BioGrid, etc. It also uses the \ninformation from KEGG pathways and Reactome to provide the \nbest annotations for the interactions of one protein [22]. Analysis \nhas been performed considering a high confidence of interactions \n(0.7), evaluated for experiments, databases, co-occurrence and co-\nexpression. MCL clustering has been performed defining 3 as MCL \ninflation parameter.\nResults\nProtein identification led to the characterization in the \nendometriosis group of 65-92 different urinary proteins per  \nsample in the group of patients with endometriosis and of and \n32-77 proteins per sample in the control group. Eleven proteins \nwere found to be increased in patients with endometriosis (Table \n1), while one protein (prothrombin) was found to be decreased in \npatients with endometriosis versus the control group.\nTable 1: Differentially expressed proteins in endometriosis and controls.\nIncreased In Endometriosis\nAccession Description Gene ratio C/P # AAs MW [kDa] calc. pI\nP10451 Osteopontin OSTP 0,30 314 35,4 4,58\nP01619 Immunoglobulin kappa variable \n3-20 KV320 0,49 116 12,5 4,96\nP01042 Kininogen-1 KNG1 0,24 644 71,9 6,81\nP01833 Polymeric immunoglobulin receptor PIGR 0,45 764 83,2 5,74\nP10909 Clusterin CLUS 0,49 449 52,5 6,27\nP01133 Pro-epidermal growth factor EGF 0,57 1207 133,9 5,85\nP12109 Collagen alpha-1(VI) chain CO6A1 0,46 1028 108,5 5,43\nQ6EMK4 Vasorin VASN 0,43 673 71,7 7,39\nQ14624 Inter-alpha-trypsin inhibitor heavy \nchain H4 ITIH4 0,40 930 103,3 6,98\nP98160\nBasement membrane-specific \nheparan sulfate proteoglycan core \nprotein\nPGBM 0,57 4391 468,5 6,51\n\nCopyright@ Domenico Milardi | Biomed J Sci & Tech Res | BJSTR. MS.ID.004628.\nVolume 28- Issue 2\nDOI: 10.26717/BJSTR.2020.28.004628\n21478\nP98164 Low-density lipoprotein receptor-\nrelated protein 2 LRP2 0,54 4655 521,6 5,08\nReduced In Endometriosis\nAccession Description Gene ratio C/P # AAs MW [kDa] calc. pI\nP00734 Prothrombin THRB 2,47 622 70,0 5,90\nSeven out of the 12 differentially expressed proteins fall into a \nlarge protein-protein interaction network based on the prediction \nresults of STRING system (Figure 1). Two clusters have been built, \nthe major one composed by 5 proteins (kininogen 1, Epidermal \ngrowth factor (EGF), Inter-alpha-trypsin inhibitor heavy chain H4 \n(ITIH4), clusterin (CLU) and thrombin (F2), mainly involved with \ninflammation.\nFigure 1: Protein-protein network interaction analysis and clustering analysis.\nDiscussion\nIn the present study, the human urinary proteomic pattern \nwas investigated for the first time to search for altered molecules \nin patients with endometriosis. We identified a panel of 12 \ndifferentially expressed proteins, which might represent novel \nputative markers for non-invasive diagnosis of endometriosis. \nFurther high-scale specific studies are needed to confirm these \ndata.  Among these 12 proteins, we reported a network of 7 proteins \nstrictly related with inflammation. These proteins represent a \nsignature of the systemic chronic inflammatory status induced \nby endometriosis. Inflammatory processes have in fact a crucial \nrole in the pathophysiology of endometriosis [23], as suggested \nby the abnormal levels of immune system cells within the female \nreproductive tract reported in patients with endometriosis. The \nimmune system cells were moreover found to be dysfunctional \nin endometriosis [24]. As a consequence after the implantation \nof endometriotic cells within the abdominal cavity, there is an \nendometriosis-induced secretion of cytokines, chemokines, nitric \noxide, immunoglobulins. Supporting this physio-pathological \nhypothesis we previously demonstrated that IL-10, although at low \ndoses, exerts its action on endometrial cells in modulating anti-\ninflammatory and anti-apoptotic effects in vitro [25]. In the present \nstudy we identified a cluster of proteins linked with inflammatory \nreactions, constituted by 5 proteins (KNG1, ITIH4, CLU, EGF and \nF2).\nKininogen 1 is a protein that in humans is encoded by the \nKNG1 gene. The KNG1 gene uses alternative splicing to generate \ntwo different proteins: high-molecular-weight kininogen (HMWK) \nand low-molecular-weight kininogen (LMWK). HMWK in turn is \ncleared by the enzyme kallikrein to produce bradykinin. Kallikrein \nin the presence of HMWK stimulates mononuclear chemotaxis \n[26] and induces the release of neutrophil elastase. Bradykinin \nstimulates inflammation, releases prostaglandins and nitric oxide, \nand enhances microvascular flow and permeability [27]. KNG-\n\nVolume 28- Issue 2\nDOI: 10.26717/BJSTR.2020.28.004628\n21479\nCopyright@ Domenico Milardi | Biomed J Sci & Tech Res | BJSTR. MS.ID.004628.\n1 plays moreover an important role in regulating, at gene level, \nurokinase plasminogen activator receptor, which is involved in \ncell migration and proliferation. The increased urinary excretion \nin endometriosis of KNG1 might so reflect an increase of KNG1 \nproduction and release, as part of the inflammatory reaction \nwhich is observed in endometriosis. Inter-alpha-trypsin inhibitor \nheavy chain H4 (ITIH4) is an acute-phase inflammatory response \nprotein that belongs to a super family of protease inhibitors [28]. \nWinden et al. [29] reported ITIH4 detection in blood up to 3 years \nprior to the diagnosis of some diseases, such as breast cancer and \ncolorectal cancer, which supports its potential use as a diagnostic \nbiomarker. Since both KNG1 and ITIH4 have been reported in \nurines as reduced in ovarian cancer at early stage [30], while we \nreported their increase in ovarian endometriosis, their evaluation \nmight represent a non-invasive marker to be translated in clinical \npractice for a further evaluation of ovarian disease.\nClusterin (CLU) was previously reported as increased in \nperitoneal fluid and in cervical mucus in women with endometriosis \n[31] Clusterin is a ubiquitous protein that is arousing increasing \ninterest owing to its widespread diffusion and multifunctional \nrole. It is composed of two 40 kD subunits (NA1, NA2) encoded \nby a single gene, which are hold together by disulphide bonds. It \nis a component of the high-density lipoprotein (HDL) complex, \nwith putative functions in the transport of lipids, apoptosis \nand protection of cells from stress and, most important, in the \nregulation of complement activity. There is strong evidence that \nclusterin plays a role in renal diseases. In fact, clusterin co-localizes \nwith the C5b-9 complex within immune deposits in human \nmembranous glomerulonephritis [32] where it is supposed to \nact as a regulator of the C5b-9 cell injury. Depletion of clusterin \nenhances immune glomerular injury in the isolated perfused kidney \n[33] and clusterin -/- knockout mice are more prone to injury \ndue to immunocomplexes [34]. In 1995 Megalin was identified \nas the target of the C5b-9 injury in experimental membranous \nglomerulonephritis [35]. Megalin is the cell receptor for clusterin \nin many tissues [36], so that the presence of clusterin in glomeruli \nrepresents a host limitation to complement injury, as it prevents \nC5b-9 insertion into cell membrane, where the complement \ncomplex competes with clusterin for the access to the same \nreceptor, which is Megalin. Several previous studies reported that \ncomplement pathway is frequently altered in endometriosis, with \nan increase in the levels of C5b-9 component [37]. As a consequence \nwe may speculate that the increase in the levels of clusterin in the \nurines of patients with endometriosis might reflect the high levels \nof clusterin, as acute phase protein, present in endometriosis. On \nthe other hand, the higher excretion of both clusterin and megalin \nin urines of patients with endometriosis might reflect a protective \nmechanism for kidneys to face the possible injury due to increased \nlevel in complement complexes in endometriosis. Further studies \nare needed to clarify the role of clusterin-megalin system in patients \nwith endometriosis, particularly at the kidney level.\nUrinary EGF (epidermal growth factor) has been proposed as a \nmarker for different non-urological diseases [38]. Intriguingly, the \nEGF system is strictly involved in the pathogenesis of endometriosis \n[39]. It is known that the expression of EGF system in eutopic \nendometrium from women with endometriosis varies from the \nendometrium of healthy women, with significant quantitative \nand qualitative differences [40]. In addition, a dysregulation \nof EGF system has been demonstrated in the setting of severe \nversus mild endometriosis, suggesting functional and biochemical \ndissimilarities between these two types of endometriosis [41]. \nAs a consequence, the higher urinary EGF levels observed in \npatients with endometriosis might reflect the dysfunctional EGF \nsystem in this disease. Finally, in the present study we reported \nfor the first time the lower urinary prothrombin levels in patients \nwith endometriosis. It might reflect the changes in coagulation \nstatus that has been described in patients with endometriosis \n[42]. Recent evidences in fact have reported higher prevalence of \ntrombophilic disorders in endometriosis patients [43]. The lower \nlevels of prothrombin in urines of endometriosis patients might \nreflect this specific coagulation status. This is the first reported \napplication of quantitative high-resolution mass spectrometry – \nbased proteomics that detected a specific array of proteins inthe \nurines of patients with endometriosis. The findings of the present \nstudy confirm that endometriosis could be viewed as a disease with \nsystemic inflammatory involvement, as reflected by metabolites \nexcreted in urines. These data call for further confirming studies for \nthe validation of these non-invasive biomarkers of endometriosis.\nAcknowledgment\nThe authors thank Claudia Menaldino (New York, USA) for her \nkind and careful English editing.\nConflicts of Interest \nThe authors declare no conflict of interest.\nFunding Information \nNo specific funding was obtained for this project.\nReferences\n1. Marana R, Lecca A, Biscione A, Muzii L (2012) Endometriosis: The \ngynecologist’s opinion. 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MS.ID.004628.\nSubmission Link: https://biomedres.us/submit-manuscript.php\nAssets of Publishing with us\n• Global archiving of articles\n• Immediate, unrestricted online access\n• Rigorous Peer Review Process\n• Authors Retain Copyrights\n• Unique DOI for all articles\nhttps://biomedres.us/\nThis work is licensed under Creative\nCommons Attribution 4.0 License\nISSN: 2574-1241\nDOI: 10.26717/BJSTR.2020.28.004628\nDomenico Milardi. Biomed J Sci & Tech Res","source_license":"CC0","license_restricted":false}