{"paper_id":"10ae1f44-7826-4506-8c51-4fbd199903ff","body_text":"Endometriosis is a disease which pathogenesis is not fully known. Several factors\nare responsible for the onset of the disease. However, the etiology of all forms\nof endometriosis has not been fully explained to date ( Bulletti  et al ., 2010 ). It’s the most\ncommon disease in the female population in reproductive age ( Broi  et al ., 2019 ).\nEndometriosis is the leading cause of infertility and poorer outcomes of\nassisted reproduction ( Marí-Alexandre\n et al ., 2018 ). From the appearance of the first\nsymptoms to the surgical diagnosis, it takes an average of 4 to 11 years. A\npossible reason is the non-specificity of the symptoms of the disease as well as\nthe impossibility of non-invasive diagnosis of endometriosis ( Agarwal  et al ., 2019 ).\nEctopic endometrial tissue interacting with an aberrant immune system leads to\nthe chronic inflammation that characterizes endometriosis ( Wang  et al ., 2020 ). Such an inflamed area\ncauses pain that is not the same in all patients, regardless of the size of the\nlesions. In addition to pain, endometriosis often causes irregular menstrual\nbleeding, dyspareunia, dysmenorrhea and, as is known, infertility. It is a\nchronic debilitating disease that significantly reduces the quality of life of a\nwoman with endometriosis ( Parasar  et\nal ., 2017 ).\nThe American Society for Reproductive Medicine has created a classification of\nendometriosis called the ASRM classification ( Metzemaekers  et al ., 2020 ). The ASRM classification\nwas then supplemented with the ENZIAN classification and is used as the rASRM\nclassification ( Lee  et al .,\n2021 ). The pathological processes that occur with the development of\nendometriosis are invasion, adhesion, proliferation, angiogenesis and impaired\nimmunity. The above-mentioned pathophysiological processes are mutually\nmaintained and lead to disorders of the immune and endocrinological systems and\nchanges in the anatomical relations of the reproductive organs ( Burney & Giudice, 2012 ).\nThe aim of this research is to compare the concentrations of total proteins in\nthe urine of subjects with endometriosis with those of the control group and\npossibly identify a biomarker for the diagnosis of endometriosis.\n\nFor this study, an ethical permit was obtained from the committee of the Rijeka\nClinical Hospital Center (Approval number: 003-05/21-1/124). All patients who were\nincluded in the research gave their consent by signing an Informed Consent Form.\nUrine samples were collected from 141 subjects who were treated at the Clinic for\nGynecology and Obstetrics of KBC Rijeka for suspected endometriosis and other cystic\novarian diseases. The day before the surgical treatment, the first morning urine was\nobtained from the subjects. All test subjects were between the ages of 25 and 35, in\nthe proliferative phase of their menstrual cycles. Subjects with kidney disease were\nexcluded from the study, which was determined by urea and creatinine levels.\nEndometriosis was diagnosed by histopathological diagnosis after surgical treatment.\nAccording to the histopathological diagnosis, the urine samples were divided into a\ngroup of subjects with endometriosis (n=84) and a control group of subjects without\nendometriosis (n=57). Subjects with endometriosis were divided into rASRM I-II\n(n=42) and rASRM III-IV (n=42) subgroups. The subgroups, that is, the stage of the\ndisease, was determined by surgery.\nThe urine is centrifuged at 3500 rpm for 30 min (Hettich centrifuge, Universal\n320/320 R Andreas Hettich GmbH & Co., Tuttlingen, Germany). 10% TCA and 3%\nDTT were added to the total volume of acetone. The urine supernatant (7.5 ml) is\nmixed with acetone solution (20 ml). The mixture of urine and acetone (ratio\n1:3) was kept at a temperature of -20°C for a minimum of 20 hours to a maximum\nof 24 hours. After repeated centrifugation and decantation, a protein sediment\nof urine was obtained, to which a 5 times larger amount of lysis buffer was\nadded, which was a mixture of 2.1g urea, 0.2g CHAPS, 0.7612g thiourea and ddH₂O\nup to 5 ml. The resulting volume was weighed and 1% protease inhibitors are\nadded to it.\nQualitative determination of protein in urine by one-dimensional electrophoresis\nwas performed to check the quality of the pre-analytical preparation of urine\nprotein precipitates. The method of one-dimensional protein gel electrophoresis\ngives us information about the purity of the samples, the amount and the\nmolecular weight of the protein. In this study, one-dimensional gel\nelectrophoresis was performed in the classical way. An electrophoresis system\n(Bio-Rad Hercules, CA, USA) was used.\nFor proteomic analyzes of proteins in urine, protein concentration was determined\nusing the Bradford reagent, which was prepared in the classic way. The resulting\nCoomassie Brilliant Blue G-250 solution has a very characteristic blue color and\nis known as Bradford’s reagent. The standard is bovine serum albumin BSA (from\nEng. bovine serum albumin) protein concentration (γ=1 mg/mL). We used a\ndevice for colorometric protein determination (Spectrometer Varian Cary 100 and\nsoftware Cary WinUV, USA).\n\nQualitative analysis of protein in urine-protein sediment samples was analyzed with\none-dimensional electrophoresis. All urine samples of the endometriosis group and\nthe control group were analyzed using this method. In all analyzed samples, proteins\nare clearly present and separated. In all samples of urine protein deposits, there\nis a sufficient amount of proteins of different molecular masses required for\nfurther analytical analyses.\nQuantitative determination of protein in urine-protein sediment samples was\nperformed using the Bradford method. A statistically significant difference in\nthe concentration of total proteins was recorded between the samples of the\ngroup with endometriosis and the samples of the control group. The Wilcoxon test\nwith a confidence level of 95% showed that the concentrations of secreted\nproteins (µg/µl) in the urine analyzed by the Bradford method in\nsamples with endometriosis (n=84) were statistically significantly higher than\nin samples of the control group (n=57),  p <0.05. After the\nconcentration of total proteins in the urine is greater than 5,000\n(µg/µl), there is a significant grouping of samples with\nendometriosis, while at concentrations lower than this value, samples of both\ngroups were grouped.\nThe analysis of samples of endometriosis subgroups rASRM I-II and rASRM III-IV\ndid not show a difference in the concentration of excreted total proteins in the\nurine. The Wilcoxon test with a confidence level of 95% showed that there was no\nstatistically significant difference in the values of the concentration of\nsecreted proteins (µg/µl) in the urine, analyzed by the Bradford\nmethod in subgroups of EME samples - rASRM I-II (n=42) and rASRM III-IV IV\n(N=42) ( Figure 1 ,  Table 1 ).\nParameters of the comparison of total protein concentration\n(µg/µl) in the urine of the group of test subjects with\nendometriosis and the control group.\nFigure 1 Presentation of the proportion of urine samples in each group\ndepending on the concentration range of total proteins\n(µg/µl) in the group of subjects with endometriosis\nand subjects of the control group (Bradford method).\nPresentation of the proportion of urine samples in each group\ndepending on the concentration range of total proteins\n(µg/µl) in the group of subjects with endometriosis\nand subjects of the control group (Bradford method).\n\nIn addition to the fact that endometriosis is a disease that affects a number of\nphysiological reactions in the body, it also plays an unquestionable role in the\ninfertility of patients. There are different levels of causes of infertility\nassociated with endometriosis. We often see the disruption of anatomical\nrelationships through the formation of cystic formations, adhesions and fibrosis,\nwhich disrupts the tubo-ovarian relationship, reduced ovarian reserves and impaired\nreceptivity of the endometrium ( Bulletti  et\nal ., 2010 ). The results of this study show a statistically\nsignificant association of endometriosis with different types of proteins in the\nurine. The concentration of total excreted proteins in the first morning urine is\nstatistically significantly higher in the group of samples with endometriosis\ncompared with the control group. Higher concentrations of total excreted proteins in\nurine were shown in all stages of disease extension according to the rASRM criteria;\nhowever, no statistically significant difference was shown in protein concentration\nin urine depending on the degree of endometriosis extension.\nLessey  et al . (2015) did not show a difference in the concentration\nof total proteins in the urine in samples from the group with endometriosis compared\nto the samples from the control group. Contrary to the results obtained in the\naforementioned study, in our investigation we noticed a statistically significantly\nhigher concentration of total proteins in the urine samples with endometriosis. The\ndifference in recorded results was probably due to the different pre-analytical\npreparation of urine in this study, in contrast to  Lessey  et al . (2015 ). With the development of\nendometriosis, a number of proteins and their precursors are secreted with the aim\nof achieving an equilibrium state in the areas of ectopic invasion and proliferation\nof endometrial cells ( Xiao  et al .,\n2017 ). It is believed that one of the most important roles in the\netiopathogenesis of endometriosis is played by the pro-inflammatory activated immune\nsystem. In the peritoneal space, immune factors are secreted in an increased amount,\nthey modulate the immune response and open space for extrauterine implantation and\nproliferation of endometrial cells.\nDespite numerous studies, it cannot be asserted whether the pro-inflammatory\nactivated immune system is the cause of endometriosis, or a factor in its\nprogression ( Ahn  et al .,\n2015 ). Peritoneal fluid in endometriosis is rich in pro-inflammatory cells.\nThe resulting anaphylatoxins stimulate peritoneal macrophages and mast cells to\nincrease secretion of cytokines and histamine ( Agostinis  et al ., 2021 ). Proteases hydrolyze peptide\nbonds and break down protein chains into smaller fragments. With such reactions,\nproteins lose their controlled primary activity and cause the breakdown of the\nextracellular space, which opens up space for the development of endometriosis\n( Bałkowiec  et al.,  2018 ).\nProtease inhibitors have the opposite effect and suppress their activity by binding\nto the receptors of enzymatically active proteins ( Porter  et al ., 2016 ).\nIn ectopic endometrial cells, there is an inadequate relationship between apoptosis\nfactors and those that promote cell growth ( Li\n et al ., 2020 ). In addition to the uneven\nrelationship between growth factors and apoptosis, ectopic endometrial cells are\nvery resistant to apoptosis. Altered cells of the peritoneum enable better\nefficiency of proteins that promote proliferation and reduced function of apoptotic\nproteins ( Renner  et al .,\n2015 ). These are just some of the processes that occur during the\ndevelopment of endometriosis. The body continuously tries to achieve homeostasis.\nSince homeostasis in endometriosis is impossible to achieve, the increased\nproduction of all proteins occurs. Both those that serve the proliferation of\nendometriotic cells, as well as those proteins that try to suppress such processes\n( Zhang  et al ., 2006 ).\nThe result is an increased production of proteins that are eventually excreted in\nthe urine and it is possible to detect them and determine their concentration.\nProliferation, invasion and apoptosis of ectopic endometrial cells with the\nformation of adhesions in a chronically inflammatory changed medium is the basis of\nendometriosis ( Agostinis  et al .,\n2021 ;  Ferrero, 2019 ). All the\npathological processes that occur during this have as a result the increased\nproduction of proteins, and it is not surprising that we can detect them in the\nurine ( Grande  et al .,\n2020 ).\nHowever, although we notice an increased concentration of protein in the urine in\nsubjects with endometriosis, the progression of the disease does not lead to even\ngreater protein production. It is possible that an increase in the degree of\nprogression of endometriosis leads to a kind of exhaustion of the organism, which is\nmanifested by a reduced possibility of further expression of proteins aimed at\nestablishing homeostasis. Such a condition opens the way for the development of\nendometriosis with a severe clinical picture.\n\nEndometriosis is a disease for which it is necessary to find a specific non-invasive\nbiomarker so that treatment can be started at the very beginning of the disease\ndevelopment. By postponing diagnosis, and thus treatment, the processes that take\nplace in the development of endometriosis, which among other things has infertility\nas a reason for its progress. The concentration of total proteins in the urine could\nbe a possible biomarker for endometriosis diagnosis.","source_license":"CC0","license_restricted":false}