NEW BIOLOGICAL MARKERS IN THE DIAGNOSIS OF ENDOMETRIOSIS

Abstract

The invention relates to novel combinations of biological markers that can be used in the diagnosis of endometriosis. The aim of the invention is to detect biological markers that are expressed differently in eMCHs isolated from endometrial biopsy samples compared to healthy eMCHs, in the diagnosis of endometriosis.

Description

25418.120 TARIFF NEW BIOLOGICAL MARKERS IN THE DIAGNOSIS OF ENDOMETRIOSIS Technical Area This discovery relates to novel combinations of biological markers that can be used in the diagnosis of endometriosis. Previous Technique Endometriosis is a disease defined as the proliferation of endometrial tissue, which lines the inner lining of the uterus and thickens and sheds during menstruation each month, from puberty onwards, in women of reproductive age, and its implantation outside the uterus in other parts of the body [1], [2]. Looking at the prevalence of endometriosis, it is seen in approximately 10-15% of women of reproductive age and 9-50% in infertility groups. On the other hand, these values ​​can reach up to 50% in adolescent women with chronic pelvic pain and dysmenorrhea [3]. Headache, [4] arthralgia, myalgia, [5] allergies, hypothyroidism, fibromyalgia, chronic fatigue syndrome [6] and susceptibility to vaginal yeast infections [7] are frequently encountered clinical symptoms in endometriosis. These symptoms significantly reduce women's daily life activities and quality of life [8] and can even cause infertility, leading to feelings of inadequacy in women.Improving patients' daily quality of life and their psychological recovery requires the application of new diagnostic and treatment methods in endometriosis treatment. Previous studies in the literature and the report published by the American Society for Reproductive Medicine have shown that “laparoscopy”, in other words surgical intervention, has become the “gold standard” in the diagnosis of endometriosis [9] and brings with it many risks. For example, in addition to the risks carried by every surgical intervention, patients with endometriosis may also have risks related to the bladder, bowel, ureter, etc. In addition to the risks of injury to the abdominal organs and subsequent internal bleeding, damage to the ovaries leading to decreased ovarian reserve and reduced fertility,

[10] ,

[11] . Surgical intervention poses a high risk, especially in patients whose priority is to get rid of the infertility factor caused by endometriosis

[11] . On the other hand, although monoclonal antibody of CA125 glycoprotein, a serum marker, is used in the diagnosis of endometriosis, studies have shown that the results of this marker are questionable and its sensitivity is low, especially in endometriosis types that have spread to the peritoneal region

[12] . The information in the literature indicates a need for alternative diagnostic methods to the currently available techniques, which are expensive, cause postoperative complications, hinder the patient's ability to return to social life quickly, and have low sensitivity. In light of this information, the study for which a patent application is submitted aims to identify the biological markers necessary for developing a new diagnostic technique for endometriosis that is low-cost, painless, and does not burden the patient's psychology with the concept of "surgery." Endometriosis takes its name from the endometrium, and although endometriosis was defined in 1860 [1], its etiology and pathogenesis are still unclear today 1131,

[14] . Different definition tables and theories have been developed for the pathogenesis of endometriosis. These are; retrograde menstruation / transplantation [2],

[15] , coelomic metaplasia

[16] , altered cellular immunity

[17] ,

[18] ,

[19] ,

[20] , metastasis

[21] , genetic factors

[22] ,

[23] , environmental factors

[24] and the interaction of the environment with specific genes

[25] . The most emphasized theory is the one presented in the 1920s, which states that the disease develops from the spread of endometrial tissue into the peritoneal cavity through retrograde menstruation [2],

[18] . The presence of endometrial tissue in the form of subperitoneal implants means a pathological condition. This condition is called retrograde endometrial tissue. The attachment of 25418.120 fragments to the peritoneal surface and subsequent invasion leads to the development of the disease. According to Sampson, menstrual blood, which should be expelled with each menstrual cycle, contains living cells that continue to survive, and these living cells have the ability to migrate to different places instead of being expelled from the body, and to attach and grow to the places they go to and other neighboring organs [2]. The human endometrium structure, which Sampson mentioned in his studies and which was later studied in the literature, has a chimeric cell population consisting of many different cells

[26] . Within this chimeric structure, there are stromal cells, fibroblasts, endothelial cells, lymphoid cells and smooth muscle cells surrounding the endometrial wall, and mesenchymal stem cells.It has been hypothesized that in women who develop endometriosis, endometrial stem cells are shed into the peritoneal cavity along with their niche cells during retrograde menstruation

[27] ,

[28] ,

[29] ,

[30] and this supports Sampson's theory. In animal studies, it was shown that mesenchymal stem cells isolated from menstrual blood taken from baboons with ongoing menstrual cycles induced endometriosis in the experimental environment

[31] . The high adhesion and migration properties of endometrial mesenchymal cells (eMCCs) have led to the idea that tissue transglutaminase (dTG) enzyme loses its cross-linking property and is synthesized in high amounts in these cells as a specific cell adhesion molecule

[32] ,

[33] ,

[34] ,

[35] ,

[36] and thus the hypothesis has arisen that cells that should be located in the endometrium have the ability to migrate to another location and form endometriosis.Based on this information, differences in surface markers were examined and identified between eMCHs, which are abundant in the chimeric structure of the endometrium and have a high migration potential, in healthy individuals and those with endometriosis. Brief Description of the Invention The aim of the invention is to identify possible differential expression in eMSCs isolated from endometrial biopsy samples in the diagnosis of endometriosis, compared to healthy eMSCs. The aim is to list 25418.120 biological markers. Biological markers selected from the marker list, whose expression was observed to change in 5 patient eMKH samples, were pathologically evaluated in endometriosis foci in 17 patients diagnosed with endometriosis and identified as biomarkers. Another objective of the invention is to develop a diagnostic kit that utilizes the gene or protein expression of differentially expressed markers such as tissue transglutaminase (dTG), cluster of differentiation 146 (CD 146), platelet-derived growth factor receptor (PDGFR), Integrin Beta 1, and Sushi domain-containing protein 2 (SUSD2, or W5C5), either individually or in combination, in endometriosis eMKH. Thus, instead of surgery, which is necessary for a definitive diagnosis of endometriosis in patients, diagnosis can be made with a biopsy sample. Detailed Description of the Invention The findings of this discovery, titled "NEW BIOLOGICAL MARKERS IN THE DIAGNOSIS OF ENDOMETRIOSIS," are shown in the attached figures; these figures are as follows: Figure 1. Characterization of healthy control endometrial mesenchymal stem cells (cMSCs) from endometrial tissue samples taken from individuals without a diagnosis of endometriosis is shown in the black filled curve, and ± values ​​represent the standard deviation after flow cytometry for 5 different cMSCs. Cells incubated with isotype IgG antibody were used as negative controls (NC) and are presented in the hollow gray curve. Figure 2. Characterization of patient endometriosis mesenchymal stem cells (hMSCs) from endometrial tissues taken from five different patients diagnosed with endometriosis, shown with a curve filled in black ± The values ​​25418.120 represent the standard deviation after flow cytometry for 5 different hMCHs. Cells incubated with isotype TgG antibody were used as negative control (NC) and are presented with a hollow gray curve. Figure 3. dTG protein level in all isolated eMSCs, (a). dTG protein bands in five different groups were visualized by Western Blot technique, (b). Analysis of dTG protein isolated from different control (kMSC) and patient (hMSC) mesenchymal stem cells of five different groups, (c). Mean value and statistical analysis of dTG protein isolated from different kMSC and hMSCs of five different groups. All cells belong to passage number 3 and were isolated by non-enzymatic procedure. P <0.0001 value is symbolized by ****. Figure 4. Western immersion results of the level of silenced dTG protein applied by shRNA technique in all isolated eMKHs (a). Membrane images of SCR and shRNA results applied to control and patient samples in five different groups, (b). SCR and shRNA analysis applied to each control and patient sample in five different groups, (c). Mean analysis of SCR and shRNA applied to control and patient samples in five different groups. In the statistical analysis, a p-value that does not have statistical significance is indicated with “ns”, while a significant p-value <0.00001 is symbolized with *****. Figure 5. Mean (±) values ​​and standard deviations of flow cytometry of control (kMKH) and control-treated scrambled (kMKH + SCR) samples from all groups. Five different kMKH samples are represented by the black filled curve in the graph, while the hollow gray curve symbolizes the kMKH + SCR samples. Figure 6. All groups treated with scrambled hMKH+ SCR and dTG-targeting shRNA containing control lentiviral particles. Mean (±) values ​​and standard deviations of flow cytometry of 25418.120 treated hMCH + shRNA samples. Five different hMCH samples are represented by the black filled curve in the graph, while the hollow gray curve symbolizes hMCH + SCR samples. Figure 7. Effect of dTG, intentionally and controllably silenced with shRNA, on mean cell growth in five different groups of eMKH samples. Statistical analysis results were analyzed using ns for non-significant p-values ​​and * p <0.05 and **** p <0.00001 for significant p-values, respectively. Figure 8. Representative images of immunohistochemical staining of tissue sections taken from an endometriosis focus with endometrium-specific surface markers. Images are for CD 146 (A), integrin β-1 (B), PDGFR (C) and dTG (D) markers. Scale bar: 50 pm. The subject of the invention is the use of tissue transglutaminase (dTG) as a biomarker for the diagnosis of endometriosis in endometrial biopsy and / or isolated endometrial mensenchymal stem cells from menstrual blood in patients with endometriosis. This method is a non-invasive method compared to the "laparoscopy" method currently used in the diagnosis of endometriosis. In addition, tissue transglutaminase (dTG) along with at least one marker selected from a group consisting of "cluster of differentiation 146" (CD146), "Sushi domain containing protein 2" (SUSD2, or W5C5), "integrin beta 1 (ITGB1)" and "platelet derived growth factor receptor" (PDGFR) markers and combinations thereof, are used as biomarkers for the diagnosis of endometriosis in endometrial biopsy and / or isolated endometrial mensenchymal stem cells from menstrual blood in patients with endometriosis.Furthermore, the subject markers of the invention are unique, either being a combination of dTG or one or more of CD 146, SUSD2 (W5C5), ITGB1 and PDGFR. An endometriosis diagnostic kit containing markers 25418.120, either individually or in combination, and used for measuring gene and / or protein levels in endometrial mesenchymal cells (eMCCs) isolated from endometrial biopsy samples and / or menstrual blood, has also been developed within the scope of the invention. The markers in question, which are combinations of dTG or one or more of CD 146, SUSD2 (W5C5), ITGB1, and PDGFR, can also be used to determine the success and course of treatment with medications by measuring gene and protein expression levels in mesenchymal cells isolated from menstrual blood. Given that endometriosis is thought to be responsible for 9-50% of infertility cases, determining the risk of infertility due to endometriosis in young women is of great importance.Given that surgeries performed when endometriosis cysts reach large sizes significantly reduce ovarian reserve, the early and easy diagnosis that will be developed within the framework of this invention will make it possible to preserve the fertility of patients through egg freezing at a young age. Endometriosis takes its name from the endometrium, the inner lining of the uterus in women of reproductive age, which thickens and sheds during menstruation each month. In endometriosis, tissue resembling the endometrium, which should normally be found in the inner lining of the uterus, migrates outside the uterus and implants in other parts of the body

[37] . These ectopic tissues, located outside their proper location, function during each menstrual cycle thanks to the living cells they contain

[37] . The high adhesion and migration properties of these cells can be explained by the adhesion and migration-inducing surface proteins they express on their cell surfaces.In the literature, it has been shown that eMK cells express CD 146, PDGFR, W5C5 markers, unlike mesenchymal stem cells isolated from other organs

[38] , and it was presented as a conference paper by us that tissue transglutaminase (dTG) protein is also expressed in eMK cells, unlike these proteins

[39] . In our paper, dTG was expressed in eMK cells isolated from a healthy and a sick individual. The enzyme activity and dTG mRNA level were measured in 25418.120 cells, and the effect of dTG on matrix metaproteinase enzyme activity, as well as the levels of syndecan-4 and integrin beta-1, were determined. In laboratory studies, which are the subject of this patent application and which we conducted as a continuation of our conference presentation, it was shown that dTG, which increased in eMKH cells taken from five endometriosis patients, supported eMKH proliferation by controlling the expression of CD 146, SUSD2 (W5C5), 1TGB1 and PDGFR proteins, and that CD 146, ITGB1 and PDGFR increased in 17 biopsy samples obtained from endometriosis tissues along with dTG. Human tissue transglutaminase (dTG) enzyme, a member of the transglutaminase family, catalyzes Ca+2-dependent protein deamidation, transamidation and cross-linking like other members of the family

[40] ,

[41] .dTG's transamidase activity plays an extracellular role in matrix stabilization, which is important in wound healing, angiogenesis, and bone repair, and an intracellular role in cross-linking proteins, generally during apoptosis

[40] . Since the discovery of dTG in 1957, numerous enzymatic substrates have been identified in intracellular compartments including the cytosol, nucleus, and mitochondria, as well as in extracellular compartments within the intracellular and extracellular matrix (ECM)

[42] ,

[43] ,

[44] . dTG, also known as cytosolic type II or liver transglutaminase, is an enzyme that can bind and hydrolyze GTP in addition to Ca2+-dependent post-translational modification of proteins and may function as a G protein

[41] ,

[45] . Therefore, in terms of catalytic activity, dTG can be called a bifunctional enzyme due to its ability to catalyze Ca2+-dependent protein cross-linking activity and Ca2+-independent GTP hydrolysis

[41] ,

[46] ,

[47] ,

[45] ,

[48] . In the Ca+2-dependent transamidation reaction of dTG, an intermolecular isopeptide ε-(γ-glutamyl) fisin bond is formed, leading to internal cross-linking of monomeric protein units

[49] . These bonds are resistant to chemical and physical degradation, therefore they are known to have biological importance, especially in the stabilization of the extracellular matrix (ECM)

[50] ,

[51] . 25418.120 Studies have shown that dTG, which is dependent on the Ca+2 level in the transamidation reaction, loses its cross-linking activity at low Ca+2 levels and is converted into a G protein

[41] ,

[45] that plays an active role in cell adhesion and migration

[36] ,

[50] ,

[52] . The ability of dTG in adhesion function is mainly due to its cooperation with two transmembrane proteins, integrins (βΐ / β 3 / β5) and syndecans, and as a result of this cooperation, the proteins bind to the adhesion receptor in the ECM with non-covalent bonds

[50] ,

[53] . Both these receptors and dTG itself interact with fibronectin

[34] ,

[36] ,

[50] . Recent studies have suggested that dTG loses its “cross-linking” enzyme activity upon binding to the extracellular fibronectin (FN) matrix protein and acts as a co-receptor to integrin and syndecan-4 receptors as a specific cell adhesion molecule

[32] ,

[33] ,

[34] ,

[35] ,

[36] .In addition, it has been observed that dTG is responsible for preventing apoptosis in cells whose integrin receptors are blocked and which cannot bind to a surface in the complex it forms with the FN matrix protein

[54] , thus dTG is synthesized in increased amounts in melanoma, breast, lung and pancreatic cancers and it has been proven that dTG gives cancer cells drug resistance and metastatic properties

[55] ,

[56] ,

[57] ,

[58] ,

[59] ,

[60] ,

[61] ,

[62] . In patients with endometriosis, other molecules that may play a role in the migration of endometrial cells from the fallopian tubes to the peritoneum and abdominal organs during menstruation, creating foci of retroglandular menstruation, are the surface markers CD146, PDGFR, and W5C5. In our laboratory studies conducted as part of our patent application, eMKH cells isolated from endometriotic individuals were compared with eMKH cells isolated from healthy individuals, and it was determined that the CD146, PDGFR, and W5C5 surface markers were expressed at higher levels in eMKH cells with endometriosis compared to healthy individuals. The increased expression of these markers was shown to be under the control of dTG in eMKH cells where dTG expression was silenced using shRNA technology. 25418.120 In many studies in the literature, it has been observed that CD146, belonging to the immunoglobulin superfamily (TgSF), increases the expression of cells, acting as a cell adhesion molecule (CAM) and increasing the potential for cell migration and invasion

[63] ,

[64] but this marker has not been studied in patients with endometriosis except in healthy eMSCs. In addition to CD146, when comparing healthy endometrial mesenchymal stem cells (cMSCs) with endometriotic mesenchymal stem cells (hMSCs), PDFGR, another endometrial mesenchymal cell marker, is among the parameters included in our hypothesis that it may be expressed more in hMSCs. Studies have shown that PDFGR is expressed at high levels in ovarian

[65] ,

[66] ,

[67] ,

[68] and uterine

[69] cancers that develop in untreated late-phase endometriosis cases.In addition, studies in the literature have shown that PDGFR is important in various cellular processes such as cell proliferation, migration, transformation and survival during the development and pathogenesis of various endometrial diseases

[70] ,

[71] ,

[72] . However, although it has been clarified that PDFFR plays a role in the development of many different gynecological disorders, a comparative study of eMCHs of individuals with and without a prior diagnosis of endometriosis has not been conducted. This discovery encompasses another original study addressing this gap in the literature. Therefore, in light of this information, it is expected that cells with endometriosis contain higher PDGFR than healthy cells, and this finding is supported by the presented results. Apart from these markers, there is an antibody known as W5C5 that recognizes the SUSD2 protein isolated from healthy individuals and shown to be specific to endometrial mesenchymal cells.It is known that this marker is expressed at a basal level in endometrial mesenchymal stem cells, just like CD 146 and PDGFR

[38] . In their studies, Garget et al. showed that W5C5 is highly expressed in perivascular implantation foci in endometriosis patients

[73] ,

[74] . In another study in the literature, W5C5 levels were found to be higher in fibroblast cells isolated from individuals who were not diagnosed with endometriosis compared to fibroblast cells isolated from healthy individuals

[75] . Within the scope of the invention. Detection of W5C5 levels in eMK cells isolated from 25418.120 healthy and endometriosis patients has never been studied in the literature. Within the framework of our discovery, our studies on endometriotic and healthy eMCHs revealed the following: 1. CD146, PDGFR, and W5C5 expression was increased in eMCHs with endometriosis under dTG control, and 2. Studies have shown that dTG is the main actor driving the proliferation of these cells. In addition to our studies on CD146, Integrin β-1, PDGFR, dTG, and W5C5 markers in endometrial medulla obstetric tissues (EMTs), we also examined the expression levels of endometrial foci in 17 patients with different stages of endometriosis, using immunohistochemical staining. Staining in the glands and stroma of the tissues was evaluated separately, and it was determined that all endometrial foci showed more than 50% or very high (+++) expression of CD146, PDGFR, and dTG markers in the glands, similar to our findings in EMTs (Table 5). CD146, PDGFR, and dTG expression in the stroma was found to be lower compared to the glands in the endometrial foci. EXPERIMENTAL STUDY Determining the Cell Groups to be Used in Experiments All isolated cells used in our experiments were isolated from tissue samples of patients diagnosed with endometriosis and healthy volunteers. An ethics application for our study was submitted to the Yeditepe University Human Ethics Committee, and approval was received from the committee members under Decision No: 63 / 509. In the experiments conducted within the framework of our discovery, tissue samples were taken from five different healthy individuals (control groups) and five different patients diagnosed with endometriosis. 25,418,120 samples were studied. Healthy tissue samples were selected from women under 49 years of age who did not have reproductive problems, no endometrial polyps in the uterus, no endometrial hyperplasia, no endometrial cancer or submucosal fibroids, and no diagnosis of endometriosis. Endometrial biopsy samples were taken from healthy individuals who had not shown endometriosis in the pelvic peritoneum and organs during non-gynecological surgeries and were fertile. Volunteers for patient samples were women under 49 years of age and selected based on a diagnosis of moderate or severe endometriosis according to the American Society for Reproductive Medicine (ASRM) classification system. All patients underwent abdominal examination and laparoscopic surgery for total endometriotic tissue excision. Women with pathological conditions other than endometriosis that could affect the study results were excluded. Cell Isolation and Culture After German endometrial tissues were transferred to our laboratory in saline solution, the tissues were washed three times with 3% (volume / volume) penicillin-streptomycin phosphate buffer solution (FTS) to remove blood and sterilize them. Then, the samples were washed twice with serum-free growth culture medium (MEM), after which the tissue fragments were chopped into 1-2 mm³ pieces using a scalpel. Following this process, the chopped tissue fragments were shaken in 10 ml of serum-free MEM containing 0.05% trypsin enzyme at 37°C for 2 hours at 70 rpm. After shaking, the cells were centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the remaining pellet was transferred to 6-well cell plates, covered with coverslips, and grown for 5 days at 37 °C in a humidified environment with 5% CO2 in low glucose content (1g / L) DMEM culture medium containing 20% ​​(volume / volume) fetal bovine serum (FSS), 100 IU / ml penicillin, and 100 pg / ml streptomycin.At the end of the waiting period, the cells filling the surface of the 6-well plate are T-25 and... The 25418.120 cells were subsequently transferred to T-75 tissue culture dishes, grown, and then subjected to CD marker analysis to characterize stem cell properties. Characterization of eMKHs Flow cytometry analysis was performed to characterize control and patient eMHK cells. After fixing the cells with 4% (volume / volume) paraformaldehyde solution, they were washed three times with FTS and labeled with antibodies CD146, PDGFR, W5C5, CD44, CD29, CD73, Integrin-1, CD90, and CD105, which are surface markers for eMHK cells, for 16 hours at 4°C and analyzed using flow cytometry. Simultaneously, the isolated cells were also incubated with homeopathic stem cell surface markers CD31, CD34, and CD45 antibodies (to serve as a negative control). After sedimentation at 300xg, the cells were washed once with FTS and suspended in 1 ml of FTS, then analyzed using FL1 (green) and FL2 (red) channels in flow cytometry. Western Implantation: The dTG protein level in cells with reduced dTG levels due to kMKH, hMKH, and shRNA was determined using the Western Implantation method. Beta-actin was used as a control antibody to ensure that each sample was loaded with equal protein amounts. For this purpose, serum cells were plated in 6-well plates at a concentration of 300,000 cells per well after 4 hours of incubation. After 24 hours of incubation, the cells were incubated with 30μl RIPA buffer (1mM PMSF, 0.5% Non-idet, 0.1% SDS, 1mMNaF, 1mM Na3VO4, and a protease inhibitor cocktail) to lyse the cell membranes, and the protein amount was determined using the Lowry method. Following 24 hours of incubation, the cells... Proteins present in the cell homogenate (50 pg / well) were separated in gels with concentrations ranging from 8% to 12% using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Proteins were transferred to a nitrocellulose membrane using the wet-transfer method at a current of 200mA for 1 hour, and then the membranes were immersed for 1 hour in Tris-Tween (100mM) solution containing 5% milk powder. 25418.120 The membranes were immersed in Tris-HCl, 0.9% (weight / volume) NaCl, and 0.05% (weight / volume), pH 7.4 solution and incubated with appropriate antibodies at 4°C for 16 hours. The antibodies used in Western Implantation were prepared in Tris-Tween solution containing 5% milk powder at the concentrations specified by the purchasing company. Following the 16-hour incubation period, the membranes were washed three times for 5 minutes each with Tris-Tween solution and labeled with HRP-labeled anti-mouse antibody diluted 1:5000 or 1:10000 in Tris-Tween solution containing 5% milk powder for 2 hours at room temperature. After 2 hours, the membranes, washed three times for 5 minutes each with Tris-Tween solution, were immersed in ECL-HRP substrate solution and then photographed using a ChemiDoc XRS+ (BioRad) instrument. Reduction of dTG Expression by shRNA Technology: eMKHs were plated in 24-well plates (20,000 / well) and incubated for 24 hours in a culture medium containing 4% (volume / volume) FSS, 100 IU / ml penicillin, 100 pg / ml streptomycin (transduction culture medium), and 8 pg / ml polybrene. hMKHs were infected for 24 hours with lentiviral particles containing shdTG diluted in transduction culture medium at a treatment degree (MOI) of 4 and with control shRNA lentiviral particles, while kMKHs were transduced with control shRNA alone at an MOI of 2. Viral particles were manufactured by Santa Cruz (USA), and shdTG viral particles contained target-specific shRNA of 19-25 nucleotides (+ hairpin) that binds to dTG mRNA at at least 3 different sites.Control shRNA consisted of scrambled shRNA sequences within lentiviral particles that did not cause any specific disruption of cellular messaging and were used to determine whether the transduction process had any effect on eMCH. After treatment, cells were washed once with FTS and then incubated for 6 to 9 days in culture medium containing 12% (volume / volume) FSS, 100 IU / ml penicillin, 100 pg / ml streptomycin, and 2.5 pg / ml puromycin. The puromycin resistance gene was also present in the plasmids containing shRNAs. Since it is coded as 25418.120, cells transduced with the shRNA plasmid are also expected to develop puromycin resistance. After puromycin selection, the cells were cultured and banked in liquid nitrogen in FSS containing 10% (volume / volume) dimethyl sulfoxide (DMSO). Determination of Cell Proliferation Capacity by WST-1 Assay The 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt water solubel tetrazolium (WST-1) assay, a colorimetric measurement to test the proliferation capacity of dTG-guided eMK cells at 24, 48, 72 and 96 hours respectively, was performed on all isolated and shRNA-treated / untreated eMK cells

[76] . The working principle of the WST-1 cell proliferation assay is to measure the mitochondrial activity of the cells. First, the cells were incubated with WST-1 reagent, a colorimetric substrate

[77] . This technique is based on the reduction of tetrazolium salts catalyzed by mitochondrial enzyme systems. First, in the early morning hours, all isolated eMKHs were seeded into 96-well plates at 2000 cells / well, 5000 cells / well, 10000 cells / well, 15000 cells / well, and 20000 cells / well ratios to create a standard curve.Following this process, all isolated eMKHs were seeded in 96-well plates at a concentration of 2000 cells per well, and at predetermined time points, the cells were incubated for 1 hour with a mixture containing 5 µL WST-1 reagent and 45 µL growth medium. After the incubation period, the absorbance of the cells was measured at a wavelength of 420-480 nm (λ max 450 nm) to determine their proliferation capacity. Selection of Tissue Sections Obtained from Endometriosis Foci Reports of patient samples diagnosed with endometriosis by a pathologist were reviewed, and 17 patient samples at different stages were selected. The selection criterion was that the samples were from different stages. Tissue samples were preserved in paraffin after being collected from the patient. 25418.120 Immunohistochemical Staining of Tissue Sections Paraffin-embedded tissue samples were allowed to cool at -20°C to allow for the preparation of thin sections. Using a microtome, 5 µm thick sections were taken from the samples, floated over hot water, and placed on positively charged slides. The slides were dried to ensure the paraffin-embedded tissue section adhered to the slide surface. Before antibody staining, all slides were incubated at 70°C for one hour to remove paraffin from the tissues. Then, CD146, Integrin βΐ, PDGFR, W5C5, and dTG endometrial mesenchymal stem cell surface marker antibodies were prepared and treated according to the manufacturer's recommendations. All immunohistochemical staining procedures were performed using a Leica Bond Max Immunocytochemistry Stainer (Shanghai, China). After staining was complete, the samples were thoroughly dried, mounting medium was added, and coverslips were used.For each antibody staining of each sample, five images were taken from different regions using the 40x objective of a Zeiss fluorescence microscope's light module. Quantification and Evaluation of Immunohistochemical Staining Images The staining intensity of each marker was analyzed separately for glands and stroma using ImageJ software, and the staining intensities were expressed as percentages. Then, the least and most intense stainings were selected, and the median value was calculated using Excel. Staining values ​​between the least and the median were divided into two groups and evaluated as "0" and "+". Similarly, values ​​between the most and the median were divided into two groups and evaluated as "++" and "+++". The numerical evaluation table is as follows (Table 1). This procedure was performed for endometriosis foci obtained from 17 patients. Studies with W5C5 antibody were not included in the evaluation as they showed no staining. 25418.120 Table 1: Evaluation of immunohistobiochemical stainings. CD146 Integrin β-1 PDGFR dTG Minimum staining area percentage 1.597 1.063 1.653 5.420 Maximum staining area percentage 66.363 34.107 47.492 72.996 Median percentage staining area 22.550 8.655 16.499 35.986 0 1.597 -12.074 1.063 -4.859 1.653 -9.076 5.420 -20.703 + 12.074 -22.550 4.859 -8.655 9.076 -16.499 20.703 -35.986 ++ 22.550 -44.457 8,655 -21,381 16,499 -31,995 35,986 -54,491 +++ 44,457 -66,363 21,381 -34,107 31,995 -47,492 54,491 -72,996 Statistical analysis Graphs prepared for all data analyses were created using the GraphPath program. After determining whether the data exhibited parametric or non-parametric distributions, data showing parametric distribution were analyzed using the Student-t test compared to the control group. ANOVA and Mann-Whitney tests were used for non-parametric data. Each experiment was repeated at least 5 times. p<0.05 was considered statistically significant. (*P <0.05; ** P<0.01; *** P<0.001; **** P<0.0001). Experimental Results 25418.120 Our research on this invention was conducted in the molecular cell biology research laboratories of the Department of Genetics and Bioengineering at Yeditepe University. Healthy endometrial mesenchymal stem cells (cMSCs) and endometriosis mesenchymal stem cells (hMSCs) were isolated from endometrial tissues taken from individuals diagnosed with endometriosis (patient groups) and those not diagnosed with endometriosis (control groups) in cell culture medium. Our invention experiments were conducted using samples from five different patient groups and five different healthy groups. All experiments were performed once for each tissue sample, thus creating five replicate experimental sets. Flow cytometry analysis was applied for the characterization of eMSCs.In the characterization of eMKHs, the specified protocol was followed, and CD 146, PDGFR, W5C5, CD44, CD29, CD73, Integrin p-1, CD90, and CD 105 antibodies were used as mesenchymal markers, and CD31, CD34, and CD45 antibodies were used as hematopoietic markers (to serve as negative controls). Figure 1 shows the mean binding affinities of CD markers for n=5 cells from each group, presented with their standard deviations. In kMKH, the surface markers were determined as follows: 42.74% CD 146, 44.60% W5C5, 43.38% PDGFR, 92.03% CD44, 93.93% CD29, 92.20% Integrin-1, 91.86% CD73, 89.90% CD90, and 90.91% CD 105, respectively. On the other hand, the hematopoietic stem cell surface markers used as a negative control were found to be 2.64% CD14, 1.44% CD31, 2.87% CD34, and 1.58% CD45, respectively, according to flow cytometry experiments. In Figure 2, flow cytometry was applied to characterize hMKH samples, and the average binding affinities of CD markers for n=5 cells from each group were given as standard deviations. When the averages of CD surface marker analyses were taken in hMKHs, the following expressions were obtained in order: 75.52% CD146, 73.99% W5C5, 73.99% PDGFR, 94.08% CD44, 95.65% CD29, 97.93% Tntegrinp-l, 96.70% CD73, 97.74% CD90 and 87.43% CD105. 25418.120 was determined. On the other hand, flow cytometry experiments revealed that the hematopoietic stem cell surface markers used as negative controls were 1.40% CD14, 1.20% CD31, 1.60% CD34, and 0.96% CD45, respectively. Mean values ​​and standard deviations of eMCHs isolated from endometrial samples taken from five healthy volunteers and one patient with endometriosis are given in Table 2. (p significance level: P<0.0001) Table 2: Mean percentage of stem cell markers in five different kMSC and hMSC samples. A P<0.0001 value is symbolized by ****. kMKH hMKH CD Markers % ± SD % ± SD p Value CD 146 42.74 3.08 75.52 2.49 **** (p< 0.0001) W5C5 44.6 2.36 73.99 6.29 **** (p< 0.0001) PDGFR 43.38 4.1 73.99 2.29 **** (p< 0.0001) CD 44 92.03 4.29 94.08 4.07 ns CD 29 93.93 5.42 95.65 1.58 ns ITGB-l 92.2 7.59 97.93 0.8 ns CD 73 91.86 6.1 96.7 2.02 ns CD 90 94.9 7.04 97.74 1.96 ns CD 105 90.91 5.67 87.43 3.65 ns CD 14 1.76 0.93 1.4 0.37 ns CD 31 1.13 0.49 1.2 0.19 ns CD 34 1.6 0.78 1.6 0.39 ns CD 45 1.41 0.34 0.96 0.4 ns When we examine the average results of the five different kMKH and hMKH values ​​presented in Table 2, we see a striking difference, particularly in the CD 146, W5C5, and PDGFR 15 surface markers, respectively, in hMKH samples diagnosed with endometriosis compared to kMKH. Our flow cytometry experiments revealed that CD146 was expressed significantly higher (****, p<0.0001) by 1.77 times, W5C5 by 1.65 times, and PDGFR by 1.71 times in diseased cell samples compared to healthy cells. In this context, our experiments have shown that CD146, W5C5, and PDFGR are synthesized more in diseased cell samples compared to healthy cells, and that these biomarkers, individually or in combination, can be used as biomarkers for the diagnosis of endometriosis. In our study, the Western impregnation method was used to determine the amount of dTG in the total protein isolated from control and healthy cells. For this purpose, the protein isolated from five different control samples and five different patient samples was separated using polyacrylamide gel electrophoresis (SDS-PAJE) technique and transferred to a nitrocellulose membrane using the Western impregnation technique. The results shown in Figure 3 were then analyzed for the antibody that recognizes dTG protein. In addition, β-actin antibody was used in the entire Western impregnation technique to ensure accurate protein loading. Figure 3 shows that dTG levels in hMKH lysates isolated from five different patients were higher than in kMKH lysates isolated from healthy individuals. When comparing dTG protein levels in cells isolated from healthy and diseased individuals, respectively, it was observed that the dTG level in patient cells in group 1 was 7.4 times higher than in healthy cells. Patients in group 2 were found to have 6.1 times higher dTG protein levels compared to healthy cells. Similar results were observed in the protein samples of patient cells in groups 3, 4, and 5, and Image J analysis revealed that endometriosis patients had 6.5, 6.3, and 5.8 times more dTG protein, respectively, when compared to healthy cells. Figure 3.Figure 3.c presents the average dTG protein level for all groups, and the statistical results shown in the figure were evaluated using the Oneway ANOVA test in GraphPad Prism 6. The statistical analysis yielded a p < 0.0001 value, which is indicated by ****. In the average dTG protein level across all groups (25418.120), it was found that hMKH samples had an average dTG protein level 4.7 times higher than kMKH samples. In this part of our study, we performed intentional / controlled dTG silencing using shRNA technology to prove our hypothesis that the increased CD146, PDGFR, and W5C5 in hMKH samples, which enhances the unique value of our discovery, may be dTG-driven. Experimental samples consisted of untreated kMHK, kMHK (kMHK + SCR) and hMKH (hMKH + SCR) treated with scrambled shRNA, and finally hMKH treated with shRNA targeting dTG (hMKH + shRNA). First, to prove that intentional and controlled silencing of dTG using shRNA technology was successful, Western Implantation (Figure 4) and GZ-PCR methods were applied to kMHK, kMHK + SCR, hMKH, hMKH + SCR, and hMKH + shRNA samples. In Figure 4.a, changes in dTG protein levels in cells after application of dTG-targeted shRNA were determined by measuring the band intensities on the membrane after Western blot technique. The quantitative results of these band intensities were determined using the Image J software program and are presented in the graph in Figure 4.b. According to these results, SCR shRNA transduction in all groups (n=5) for kMKH did not cause any change in dTG expression (Figure 4.c). Transduction of hMKH with viral particles carrying dTG-targeting shRNA caused a 1.62-fold decrease in Group 1, while this value was determined as 1.62 for Group 2, 3.1 for Group 3, 3 for Group 4, and 2.37 for Group 5. The measured dTG protein expression after reducing dTG expression in hMKH with shRNA (hMKH + shRNA) was found to be similar when compared with kMKH and kMKH + SCR (Figure 4).c) Statistical analysis revealed no significant difference between them, and this is represented as "ns" in the graph. A similar comparison is shown on the same graph. A comparison was made between 25418.120 kMKH samples and hMKH samples, and it was observed that hMKH synthesized 2.6 times more dTG than hMKH + shRNA, and a significant p-value <0.00001 is indicated by *****. Following successful silencing of dTG with shRNA, reflow cytometry experiments were performed to detect CD146, PDGFR, and W5C5 surface markers in five different healthy and endometriotic eMKH samples. The results comparing kMHK and kMHK + SCR samples are presented in Figure 5, and the results comparing hMKH + SCR and hMKH + shRNA SCR samples are presented in Figure 106. Flow cytometry results showed that the control-treated scrambled virus particle had similar values ​​in kMKH + SCR samples compared to kMKH samples. The results, which include a statistical analysis of the mean values ​​of kMKH and kMKH + SCR samples, are presented in Table 3. 25418.120 Table 3: Mean percentage of stem cell markers in five different kMKH and kMKH + SCR samples and p-values ​​for which no statistically significant difference was observed, symbolized by “ns”. kMHK kMHK + SCR CD Markers % ± SD % ± SD p Value CD 146 46.15 3.2 45.89 3.14 ns W5C5 44.89 4.05 45.22 3.66 ns PDGFR 43.05 3.01 42.93 4.15 ns CD 44 89.38 5.16 90.49 5.31 ns CD 29 92.58 4.5 91.27 5.77 ns ITGB-l 94.2 2.79 94.12 2.91 ns CD 73 94.37 4.76 94.69 4.65 ns CD 90 92.69 1.76 91.15 1.22 ns CD 105 90.78 6.43 91.56 6.54 ns CD 14 2.18 1.06 1.94 1.09 ns CD 31 1.69 0.91 1.6 0.78 ns CD 34 2.12 0.76 2.09 0.95 ns CD 45 1.33 0.53 0.89 0.24 ns In line with the results presented in Table 3, our experiments have proven that the lentivirus we used as a carrier has no effect on the cells. In this part of our results, we aimed to demonstrate that the increased CD 146, PDGFR, and W5C5 in hMKH samples may be dTG-driven. Flow cytometry was applied to hMKH + SCR samples treated with scrambled control lentiviral particles and hMKH + shRNA samples treated with shRNA targeting dTG, and the results are presented in Figure 6. 25418.120 Flow cytometry results revealed that hMKH + SCR cell surface markers were similar to those in hMKH cells. However, in hMKH + shRNA cells treated with dTG-targeting shRNA, the expression of CD146, W5C5, and PDGFR surface markers, which are highly prevalent in hMKH, decreased to levels seen in healthy eMKH after silencing by dTG. The results, including the statistical analysis of the mean values ​​of hMKH + SCR and hMKH + shRNA samples, are presented in Table 4. Table 4: Mean percentage of stem cell markers in five different hMKH + SCR and hMKH + shRNA samples and statistically significant p-values ​​(p<0.0001) are symbolized with “***”. hMKH + SCR hMKH + shRNA CD Markörleri % ± SD % ± SD p Değeri CD 146 71.43 5.1 35.54 5.37 **** (p< 0.0001) W5C5 76.63 2.49 42.09 3.43 **** (p< 0.0001) PDGFR 78.54 0.5 34.8 4.04 **** (p< 0.0001) CD 44 89.06 6.57 90.13 7.64 ns CD 29 92.47 3.26 93.31 5.55 ns 1TGB-1 93.59 3.55 93.7 3.4 ns CD 73 90.84 6.75 89.65 7.36 ns CD 90 88.21 7.26 87.86 8.17 ns CD 105 92.97 5.36 95.77 4.51 ns CD 14 1.74 0.67 1.92 0.88 ns CD 31 1.66 0.4 2 0.26 ns CD 34 1.96 0.89 2.02 0.91 ns CD 45 1.72 0.36 1.67 0.27 ns 25418.120 Considering the results in Table 4, when comparing hMKH + SCR samples with hMKH + SCR samples treated with dTG-targeting shRNA, a statistically significant decrease in expression was observed for CD146 (2.1-fold), W5C5 (1.82-fold), and PDGFR (2.26-fold) (****, p<0.0001). These results are presented for the first time in the literature and provide proof that the increased CD146, PDGFR, and W5C5 in hMKH samples in the development of endometriosis are dTG-driven. To comparatively determine cell proliferation in dTG-guided hMCS, the WST-1 test was applied to kMCS, kMCS + SCR, hMCS, hMCS + SCR, and hMCS + shRNA samples at different time points for 24, 48, 72, and 96 hours, and the results are presented in Figure 7. According to the results in Figure 7, kMKH and kMKH + SCR cells proliferated more slowly than hMKH and hMKH + SCR cells at 24, 48, 72, and 96 hours. At 24 and 48 hours, hMKH and hMKH + SCR samples with high dTG expression had an average of 2.1 and 2.3 times more cells than kMKH, respectively, and at 72 and 96 hours, this ratio increased to 3.2 and 3.0 times, respectively. In hMKH + shRNA cell samples where dTG expression was silenced, the average number of cells was similar to that of kMKH and kMKH + SCR cells (p>0.05). Tissue samples taken from endometriosis foci were immunohistochemically stained with dTG, CD 146, PGDGR, SUSD2 (W5C5), and Integrin β-1 antibodies. Five images were then taken from different regions of each tissue sample for each antibody staining using the 40x objective of a Zeiss fluorescence microscope. Representative images for each antibody are shown in Figure 8. In all samples, Integrin β-1, dTG, CD 146, and PGDGR markers were found to be more intensely expressed in the glands of the endometriosis foci compared to the stroma. 25418.120 No staining for SUSD2 (W5C5) antibody was observed in any of the endometriosis focal tissue samples. Table 5. Percentage expression levels of CD146, PDGFR, and dTG markers in the stroma and glans of studied endometriosis focus tissues. CD 146 ITGB1 PDGFR TG2 Gland Stroma Gland Stroma Gland Stroma Gland Stroma 0 11.8 58.8 23.5 35.3 17.6 11.8 0.0 41.2 4- 11.8 17.6 17.6 11.8 23.5 47.1 47.1 11.8 ++ 29.4 23.5 52.9 47.1 17.6 17.6 17.6 23.5 +++ 47.1 0.0 5.9 5.9 41.2 23.5 35.3 23.5 All studied endometriosis focal tissues showed more than 50% or very high (+++) expression of CD 146, PDGFR and dTG markers in their glands (Table 5). Results and Applications of the Invention As mentioned in previous studies, endometriosis is the condition in which endometrial tissue, which should be found inside the uterus, is found outside the uterine boundaries, most commonly on the surface of the abdominal cavity, in the ovaries, or in the brain, which is a much more distant region

[78] . When its prevalence among women is examined in general, it has been found to be 6-10%

[79] ,

[80] ,

[81] . However, in women with pelvic pain or infertility, the diagnosis of endometriosis is important. It has been shown that the incidence rates are between 35% and 50%

[79] ,

[80] ,

[81] . The cost report for endometriosis diagnosis in the USA is $69.4 million annually

[82] . Laparoscopy constitutes a very large part of this expensive diagnosis

[82] . Although laparoscopy is an important diagnostic method for endometriosis, it involves surgical risks such as surgical problems that may occur during the operation, damage to the bowel, bladder, ovary and large vessels, and bleeding, as well as high cost and a long patient observation period [6],

[82] ,

[83] . The lack of an easy-to-apply, reliable and fast non-invasive diagnostic test causes the diagnosis of endometriosis in women to be delayed by an average of 7-11 years

[84] ,

[85] . Based on the information in the literature, in our invention, which initiated studies on a highly sensitive diagnostic test, we discovered that dTG, which could be a new biological marker for the diagnosis of endometriosis and offers a "quick application / result" opportunity without cost or surgical intervention, can be used in the diagnosis of endometriosis by targeting CD146, W5C5, ITGB1, and PDFGR, which are specific to endometrial MSCs. In this context, the importance of dTG in both the diagnosis and development of endometriosis has been demonstrated for the first time in the literature with the experiments we conducted within the scope of our invention. Flow cytometric analysis proved that cells isolated from the endometrium of healthy (Figure 1) and diseased (Figure 2) individuals have mesenchymal stem cell characteristics and is consistent with the eMSH characterization information in the literature

[86] ,

[87] ,

[88] ,

[89] . On the other hand, these results indicate that the endometrium is a high source of mesenchymal stem cells and that mesenchymal stem cells localized in the endometrium contribute to the renewal of the menstrual cycle, the repair of the endometrium and the maintenance of its dynamic structure

[90] ,

[91] . The application of CD 146, W5C5 and PDFGR, which are markers specific to endometrial-derived mesenchymal stem cells, to the same cell groups

[92] , in healthy It was determined by flow cytometry that CD146, W5C5, and PDFGR

[92] , which are specific to endometrial-derived mesenchymal stem cells, were synthesized in higher amounts in patient cell samples compared to 25418,120 cells. The higher synthesis of CD146, W5C5, and PDFGR in patient endometriotic cells may have given hMSH samples the potential for cell migration, adhesion, and invasion. Since CD146, PDGFR, and W5C5, which are known to play a role in cell migration, adhesion, and invasion, are high in patient cells, and PDGFR directly interacts with dTG, it was thought that the increased CD146, PDGFR, and W5C5 in hMSH samples may be dTG-driven. In a previous study by Zemskov et al., it was shown in different cell samples that cell surface dTG interacts with PDGFR, triggering dTG-driven PDGFR-dependent cell adhesion together with integrins [931, 194].On the other hand, in recent years, studies with central nervous system cells have shown that CD146 plays an essential role in PDGFR-β-mediated signal transduction and that dimeration of CD146 leads to the phosphorylation of PDGFR-β, triggering cell migration and adhesion

[95] . Although the relationship between CD146 and PDGFR has been explained in this study, its relationship with dTG and its role in endometriosis have not been clarified. In line with the flow results we obtained, the presence of dTG was investigated in kMKH and hMKH samples to determine the role of dTG in increasing CD146, PDGFR and W5C5 in hMKH samples. In this context, the amount of dTG protein and gene expression in kMKH and hMKH samples were examined with Western Implantation (Figure 3) and GZ-PCR experiments, and it was determined that both the protein and gene expression of dTG were higher in patient samples compared to controls. In this part of our investigation, in order to demonstrate whether the increased CD146, PDGFR, and W5C5 surface markers in hMCHs are controlled by dTG expression, as stated in our hypothesis and as we have determined in the experimental results we have presented so far, dTG expression was intentionally and controllably silenced in eMCHs by applying shRNA technology (Figure 4). While it was observed that the level of dTG protein decreased after the application of shRNA 25418.120 (Figure 4), it was observed that the control lentiviral particle-containing (scrambled) shRNA applied to hMCH and kMCH did not reduce the dTG protein level. As stated in the literature, while shRNA inhibits proteins that are the product of the target gene, it is expected that there will be no changes in the cells treated with scrambled because the control lentiviral particle-containing scrambled contains approximately 19-25 nucleotide sequences that do not correspond to any cellular message in the human genome

[96] , and this information is parallel with our results.In hMKH samples that synthesize and express dTG at high levels, successful shRNA application revealed changes in CD146, PDGFR, and W5C5 surface markers, as well as changes in the cells' proliferative potential, their capacity to migrate to regions outside the uterus, and their ability to invade these new sites. This allowed us to elucidate the molecular role of dTG in endometriosis. As is known, dTG, in addition to its Ca+2-linked cross-enzyme activity

[41] ,

[46] ,

[97] ,

[98] ,

[99] , can lose its cross-linking activity independently of Ca+2 and bind to GTP, hydrolyze it and take an active role in cell adhesion and migration as a G protein

[41] ,

[45]

[36] ,

[50] ,

[52] . In light of this information in the literature and as a result of the experiment we present within the scope of our invention, we investigated whether hMCH Terin acquired the ability to migrate to regions outside the uterus and to invade by adhering to these regions thanks to the dTG expression to a high degree. As presented in Figure 4, after controlled and deliberate silencing of dTG with shRNA, the CD 146, W5C5, and PDGFR surface markers in hMHK+ shRNA cells were reduced to the levels observed in kMHK and kMHK+ SCR cells (Figure 6, Table 3, and Table 4). These results show that CD 146, W5C5, and PDGFR, which are highly expressed in hMHK cells, were reduced to the levels observed in hMHK+ shRNA cells. 25418.120 PDFGRTn surface markers have been shown to control dTG gene expression. WST-1 tests performed at 24, 48, 72, and 96 hours to determine the effect of dTG expression on eMKH cell proliferation (Figure 7) showed that hMKH cells proliferated 1.3 times faster when compared to kMKH samples. With dTG silencing, the proliferation capacity of hMKH + shRNA cells was reduced to healthy kMHK levels. These results demonstrate that cell proliferation in endometriosis occurs under dTG control. In addition to our studies on CD 146, integrin β-1, PDGFR, dTG, and W5C5 markers in endometrial medulla obstetric tissues (EMTs), we also examined the expression levels of endometrial foci taken from different regions of 17 patients with different stages of endometriosis using immunohistobiochemical staining. Staining in the glands and stroma of the tissues was evaluated separately, and it was determined that all endometrial foci showed expression of CD 146, PDGFR, and dTG markers in the glands of the tissue, with expression levels greater than 50% (++) or very high (+++), similar to our findings in EMTs (Table 5). The expression of CD 146, PDGFR, and dTG in the stroma was found to be lower compared to the glands in the endometrial foci (Figure 8). Although integrin β-1 expression did not increase in eMCHs, it was expressed in endometriotic foci, while no change in expression was detected in eMCHs isolated from healthy and patient individuals.This suggests that integrin β-1 expression is increased in eMC cells during endometriosis focus formation. Our immunohistobiochemical studies with W5C5, conducted as part of a patent application, showed that, contrary to Gargett et al., W5C5 expression is suppressed in endometriosis focus tissue, while demonstrating for the first time in the literature that this marker is increased in eMC cells isolated from healthy and endometriotic endometrium. 25418.120 This application seeks a patent for the use of dTG, CD 146, PGDGR, SUSD2 (W5C5), and Integrin β-1 markers, individually or in combination, as biomarkers in the diagnosis of endometriosis. 25418.120 REFERENCES [1] . Rokitansky, C. 1860. “Über Uterusdrüsen-Neubil-dung in Uterus- und Ovarial-Sarcomen. (On the neoplasm of uterine glands on uterine and ovarian sarcomas)”, Zeitschr Ges Aerzte Wien 16(1)577-581. [2] . Sampson JA: Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927; 14: 422-469. 13]. Cramer DW, Missmer SA. The epidemiology of endometriosis. Ann N Y Acad Sci 2002;955:11-22. [4] . Tietjen GE, Bushnell CD, Herial NA, Utley C, White L, Hafeez F. Endometriosis is associated with prevalence of comorbid conditions in migraine. Headache 2007;47:1069-78. [5] . Pasoto SG, Abrao MS, Viana VS, Bueno C, Leon EP, Bonfa E. Endometriosis and systemic lupus erythematosus: a comparative evaluation of clinical manifestations and serological autoimmune phenomena. Am J Reprod Immunol 2005;53:85-93. [6] . Sinaii N, Cleary SD, Ballweg ML, Nieman LK, Stratton P. High rates of autoimmune and endocrine disorders, fibromyalgia, chronic fatigue syndrome and atopic diseases among women with endometriosis: a survey analysis. Hum Reprod 2002;17:2715-24. [7] . Lamb K, Nichols TR. Endometriosis: a comparison of associated disease histories. Am J Prev Med 1986;2:324-9. [8] . Mao AJ, Anastasi JK. Diagnosis And Management Of Endometriosis: The Role Of The Advanced Practice Nurce In Primary Care. Journal of the American Academy of Nurse Practitioners 2010; 22: 109-116. [9] . Somigliana E, Vercellini P, Vigano P, Benaglia L, Crosignani PG, Fedele L. Non-invasive diagnosis of endometriosis: the goal or own goal? Hum Reprod 2010;25:1863-8. 25418.120

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Claims

25418.120 REQUESTS 1. Tissue transglutaminase used as a biomarker for the diagnosis of endometriosis in endometrial mensenchymal stem cells obtained from endometrial biopsy and / or menstrual blood sources from endometriosis patients.

2. As a biomarker, tissue transglutaminase as described in request 1, used for the diagnosis of endometriosis in endometrial mensenchymal stem cells isolated from endometrial biopsy and / or menstrual blood sources from endometriosis patients, along with at least one marker selected from a group consisting of Cluster of differentiation 146, Sushi domain-containing protein 2, Integrin beta 1, and Platelet-derived growth factor receptor markers and combinations thereof.

3. An endometriosis diagnostic kit containing the biomarkers described in Claims 1 and 2, either alone or in combination, and used for measuring gene and / or protein levels in endometrial mesenchymal cells isolated from endometrial biopsy samples and / or menstrual blood.

4. A biomarker, such as in Claim 1 or 2, used to determine the success of therapeutic drugs by measuring gene and protein expression levels in mesenchymal cells isolated from menstrual blood.

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