Endometriosis Biomarker

Abstract

1. A method comprising: (a) assessing the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from a subject (wherein the at least one protein can be selected from the list including β-Ala-His dipeptidase, apolipoprotein L1, methanethiol oxidase, vitamin K-dependent protein S, von Willebrand factor, plasminogen, selenoprotein P, protein disulfide isomerase A6, and inter-α-trypsin inhibitor heavy chain H3); and (b) using the expression level to determine whether the subject has endometriosis.

Description

Technical Field

[0001] Field of the Invention The present invention relates to biomarkers associated with endometriosis. The present invention also relates to screening, diagnostic, and prognostic methods using such biomarkers. Further, the present invention relates to methods for evaluating medical interventions for endometriosis and methods for identifying drug targets for endometriosis.

Background Art

[0002] Background of the Invention Endometriosis occurs when the tissue lining the uterus spreads outside the uterine cavity and surrounds other organs (including the peritoneum, ovaries, fallopian tubes, pleura, and lungs). This condition affects 1 in 10 women of reproductive age, and its incidence and health burden are comparable to those of diabetes.

[0003] Endometriosis causes chronic pain and infertility, but is often difficult to diagnose because the symptoms are common to many other gynecological diseases. On average, it takes 8.5 years from the first symptoms for a woman to be diagnosed. Imaging scans and existing blood tests are not conclusive, i.e., the current gold standard for diagnosis is by direct visualization of the tissue with confirmation by histological analysis. This can only be achieved by invasive laparoscopy / laparotomy under general anesthesia, where a camera is inserted into the pelvis through a small incision in the abdominal wall.

[0004] Considering the above, there is a need for improved endometriosis biomarkers and related methods of their use, including methods for diagnosing endometriosis.

Summary of the Invention

Means for Solving the Problems

[0005] Summary of the Invention The present invention is (a) Evaluating the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from a subject; (b) Using this expression level to determine whether the subject has endometriosis; A method comprising the above is provided.

[0006] The present invention also provides (a) Means for obtaining the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from a subject; and (b) Means for processing the expression level obtained in step (a) to determine whether the subject has endometriosis. An assay comprising the above is provided.

[0007] The present invention also provides the use of at least one protein selected from Table 1, 2, or 3 as a biomarker for endometriosis.

[0008] The present invention also provides a method for evaluating endometriosis intervention in a subject, comprising: (a) Applying an intervention to the subject; and (b) Evaluating the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from the subject; and (c) Using this expression level to determine the effect of the intervention on the subject. A method comprising the above is provided.

[0009] The present invention also provides the use of at least one protein selected from Table 1, 2, or 3 as a target for a therapeutic agent for endometriosis.

[0010] Brief Description of the Drawings The following detailed description of the present invention, given by way of example and not intended to limit the invention to the specific embodiments described, can be interpreted in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0011]

Figure 1

Mode for Carrying Out the Invention

[0012] Detailed Description of the Invention According to a first aspect, the present invention provides a method comprising: (a) evaluating the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from a subject; and (b) using the expression level to determine whether the subject has endometriosis.

[0013]

Table 1

[0014]

Table 2

[0015] For the purposes of the present invention, the term endometriosis includes the pathological growth of ectopic endometrial-like tissue outside the uterine cavity. Preferably, the term endometriosis includes one or more of peritoneal superficial endometriosis, ovarian endometriosis, and deep endometriosis. Deep endometriosis can include one or more pathological growths of ectopic endometrial-like tissue in the sacrouterine ligament, rectovaginal space, upper posterior vaginal wall, intestine, and / or urinary tract.

[0016] Preferably, the at least one protein includes a plurality of proteins, such as two, three, four, or five proteins in Table 1.

[0017] ​Preferably, at least one protein comprises complement factor H-related protein 2, β-Ala-His dipeptidase, sex hormone-binding globulin, corticosteroid-binding globulin, apolipoprotein L1, catalase, C4b-binding protein α chain, carbonic anhydrase 2, superoxide dismutase [Cu-Zn], peroxiredoxin-1, annexin A1, methanethiol oxidase, bisphosphoglycerate mutase, profilin-1, afamin, von Willebrand factor, L-lactate dehydrogenase A chain, plasminogen, selenoprotein P, proteoglycan 4, hyaluronan-binding protein 2, protein disulfide isomerase A6, or coactosin-like protein.

[0018] Preferably, at least one protein comprises at least two, three, or four of complement factor H-related protein 2, β-Ala-His dipeptidase, sex hormone-binding globulin, corticosteroid-binding globulin, apolipoprotein L1, catalase, C4b-binding protein α chain, carbonic anhydrase 2, superoxide dismutase [Cu-Zn], peroxiredoxin-1, annexin A1, methanethiol oxidase, bisphosphoglycerate mutase, profilin-1, afamin, von Willebrand factor, L-lactate dehydrogenase A chain, plasminogen, selenoprotein P, proteoglycan 4, hyaluronan-binding protein 2, protein disulfide isomerase A6, or coactosin-like protein.

[0019] Preferably, at least one protein comprises at least one protein selected from Table 2.

[0020]

Table 3

[0021] Even more preferably, at least one protein comprises two, three, four, or five proteins selected from Table 2.

[0022] Preferably, at least one protein comprises at least one protein selected from the list consisting of β-Ala-His dipeptidase, apolipoprotein L1, methanethiol oxidase, and vitamin K-dependent protein S, and is included in Table 2.

[0023] Even more preferably, at least one protein comprises two, three, four, or five proteins selected from the list consisting of β-Ala-His dipeptidase, apolipoprotein L1, methanethiol oxidase, and vitamin K-dependent protein S, and is included in Table 2.

[0024] Preferably, at least one protein comprises at least one protein selected from Table 3.

[0025]

Table 4

[0026] Even more preferably, at least one protein comprises two, three, four, or five proteins selected from Table 3.

[0027] Preferably, at least one protein comprises at least one protein selected from the list consisting of von Willebrand factor, plasminogen, selenoprotein P, protein disulfide isomerase A6, and inter-α-trypsin inhibitor heavy chain H3, and is included in Table 3.

[0028] Even more preferably, at least one protein comprises two, three, four, or five proteins selected from the list consisting of von Willebrand factor, plasminogen, selenoprotein P, protein disulfide isomerase A6, and inter-α-trypsin inhibitor heavy chain H3, and is included in Table 3.

[0029] According to another preferred embodiment of the present invention, at least one protein comprises at least one protein selected from the list comprising β-Ala-His dipeptidase, apolipoprotein L1, methanethiol oxidase, vitamin K-dependent protein S, von Willebrand factor, plasminogen, selenoprotein P, protein disulfide isomerase A6, and inter-α-trypsin inhibitor heavy chain H3.

[0030] Even more preferably, at least one protein comprises two, three, four, or five proteins selected from the list comprising β-Ala-His dipeptidase, apolipoprotein L1, methanethiol oxidase, vitamin K-dependent protein S, von Willebrand factor, plasminogen, selenoprotein P, protein disulfide isomerase A6, and inter-α-trypsin inhibitor heavy chain H3.

[0031] Step (a) of evaluating the expression level of at least one protein can include any suitable method for evaluating protein expression. Preferably, step (a) includes at least one of spectrometry, such as mass spectrometry, surface-enhanced Raman spectroscopy, flow cytometry, ELISA, protein array (including mass detection BioCD protein array), protein microarray, quantum dot-based detection, electrochemical immunoassay, gel electrophoresis, 9G DNA technology, nanoparticles containing lanthanide chelates such as europium EuNP and gold nanoparticles, immunoaffinity mass spectrometry, and immunocapture mass spectrometry.

[0032] When step (a) includes mass spectrometry, this can include multiple reaction monitoring (MRM) mass spectrometry or selected reaction monitoring (SRM) mass spectrometry.

[0033] Preferably, step (a) includes evaluating the expression level of at least one protein by evaluating the amount of a fragment or peptide of at least one protein.

[0034] Preferably, step (a) comprises quantifying the expression level of at least one protein.

[0035] Preferably, step (a) comprises quantifying the expression level of at least one protein by comparing it with the expression level of at least one protein in a subject without endometriosis.

[0036] Step (a) can also comprise labeling at least one protein. Exemplary labels include protein labels such as biotin, active site probes, enzyme complexes such as HRP, and fluorescent probes, isotope labeling, and isobaric labeling.

[0037] The sample can include a biological sample and / or a secondary sample thereof. Preferably, the biological sample is a body fluid such as blood, serum, plasma, urine, sweat, tears, saliva, sputum, or any combination or fraction thereof. Other non-limiting examples of biological samples include whole blood, peripheral peripheral blood, ascites, cerebrospinal fluid, buccal samples, cavity washings, organ washings, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural effusion and ascites, pericardial fluid, lymph, atherosclerotic fluid, chyle, bile, interstitial fluid, menstrual secretions, pus, sebum, vomitus, vaginal secretions, mucosal secretions, watery stools, pancreatic juice, washings from the nasal cavity, bronchoalveolar aspirates, or other washings. The biological sample can also include a blastocyst cavity, cord blood, or maternal circulation (which can be of fetal or maternal origin). The biological sample can also be a tissue sample or a biopsy material. The secondary sample includes extracts from samples containing protein extracts.

[0038] Preferably, the subject is a mammal such as a human. The subject may be one previously diagnosed or identified as having endometriosis and, optionally, who has already received or is currently receiving a therapeutic intervention. Alternatively, the subject may also be one not previously diagnosed or identified as having endometriosis. For example, the subject may be one presenting one or more risk factors for endometriosis, or one presenting no such risk factors, or one asymptomatic for endometriosis. The subject may also be one suffering from or at risk of developing endometriosis.

[0039] Step (b) includes any use of the expression level from step (a) to determine whether the subject has endometriosis.

[0040] Preferably, the expression level from step (a) alone determines whether the subject has endometriosis. However, the expression level from step (a) may partially determine whether the subject has endometriosis. In this regard, the expression level from step (a) can be combined with a second measure to determine whether the subject has endometriosis.

[0041] Preferably, step (b) includes comparing the expression level from step (a) with a reference value indicative of endometriosis.

[0042] Preferably, the reference value is a protein expression level. For example, the reference value may be a reference protein expression level from at least one second subject (where the reference protein expression level has been found to correlate with endometriosis).

[0043] The at least one second subject may be a cohort or population of the subject.

[0044] Another use of the expression level by step (a), by step (b), is to compare this with another expression level from the same subject obtained at another time point. Such use enables comparison of the expression level over time in the subject, and thus whether the subject has endometriosis.

[0045] Preferably, the method of the present invention determines whether a subject has endometriosis with a sensitivity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98%, 99%, 99.5%, or about 100%.

[0046] Preferably, the method of the present invention determines whether a subject has endometriosis with a specificity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98%, 99%, 99.5%, or about 100%.

[0047] Preferably, the method of the present invention determines whether a subject has endometriosis with an accuracy of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98%, 99%, 99.5%, or about 100%.

[0048] This method includes the use of a control for more appropriately evaluating the expression level of at least one protein, and the control can be used to determine whether the subject has endometriosis. Preferably, the control is a control protein such as a control protein that is not specifically expressed with respect to endometriosis.

[0049] According to a second aspect, the present invention provides the use of at least one protein selected from Table 1, 2, or 3 for determining whether a subject has endometriosis. Other preferred features of the method described above with respect to the first aspect also form the preferred features of this aspect of the present invention.

[0050] According to a third aspect of the present invention, (a) Means for obtaining the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from a subject; (b) Means for processing the expression level obtained in step (a) to determine whether the subject has endometriosis An assay comprising is provided.

[0051] Other preferred features of the method described above according to the first aspect also form preferred features of this aspect of the invention. For example, the means for obtaining the expression level can include any suitable method for evaluating protein expression.

[0052] Preferably, the assay includes an apparatus.

[0053] Preferably, the apparatus includes a spectrometer.

[0054] Preferably, the assay includes a kit.

[0055] Preferably, the kit includes reagents for detecting at least one protein.

[0056] Preferably, the kit includes written instructions for quantifying the expression level of at least one protein and / or for determining whether the subject has endometriosis based on its expression level. The written instructions may include instructions for comparing protein expression and / or a predefined value (e.g., a value for determining whether the expression level of a protein indicates endometriosis).

[0057] Preferably, the kit includes one or more of the following: a detectable label, a standard, a sample buffer, and a control (positive and / or negative).

[0058] The proteins of the present invention and combinations thereof can be implemented in a variety of assay systems. Typically, an assay system includes means for obtaining assay results from a sample, usually means for collecting, storing, processing, and / or tracking assay results for the sample in a database, and means for reporting the assay results. The means for obtaining assay results can include modules adapted for automated assays that utilize one or more of biochemical, immunological, and protein detection assays. Some assay systems can process multiple samples and can perform multiple assays on a given sample. The means for collecting, storing, processing, and / or tracking assay results can include physical and / or electronic data storage devices such as hard drives or flash memories or printing on paper. The means for reporting assay results can include a visual display, a link to a data structure or database, or a printer. In this regard, the reporting means can simply be a data link adapted to send the results to another device such as a database, visual display, or printer.

[0059] Typically, assay results from the system of the present invention are used as input to a computer, or a microprocessor programmed in machine language, or software that obtains data related to the expression levels of at least one protein described herein and determines the risk of developing or already having endometriosis.

[0060] The present invention provides improved diagnosis and prediction of endometriosis. The risk of having or developing endometriosis can be evaluated by measuring the expression of one or more proteins in Tables 1, 2, or 3 and comparing the measured values to a reference or index value. Such comparisons can be made using mathematical algorithms or formulas to integrate information from the results of multiple individual proteins with other parameters into a single measurement or index. Subjects identified as having a high risk of endometriosis can optionally be selected to receive a treatment regimen such as administration of a prophylactic or therapeutic compound.

[0061] The expression level of at least one protein is measured in a sample and compared to a reference or normal level using techniques such as a reference limit, discrimination limit, or risk definition threshold for defining a cut-off point, and abnormal values for endometriosis. Normal control levels are the levels of one or more proteins or combined biomarker indicators typically found in subjects without endometriosis. The normal and abnormal levels, as well as the cut-off point, can vary depending on whether at least one protein is used alone or integrated into one indicator together with other biomarkers. Alternatively, the normal or abnormal levels can be a database of biomarker patterns or "signatures" from previously tested subjects with or without endometriosis over a clinically relevant time range.

[0062] Accordingly, the expression level of at least one protein can be used to yield a profile or signature of a subject who (i) does not have endometriosis and has no likelihood of developing it, and / or (ii) has or is expected to develop such a condition. By comparing the profile of a subject to a predefined or reference biomarker profile, subjects at risk of developing endometriosis can be diagnosed or identified, the progression of endometriosis and the rate of progression of endometriosis can be monitored, and the effectiveness of an intervention can also be monitored. The profile of the present invention is preferably contained on a machine-readable medium and can be updated with additional data obtained (and thus be "live" to the extent that the strength and clinical significance of the biomarker is improved). Data regarding the level of at least one protein of the present invention can also be combined with or correlated to other data or test results, including but not limited to measurements of clinical parameters for endometriosis or other algorithms. The machine-readable medium can also include information about the subject, such as medical history and any applicable family history.

[0063] The present invention also provides the use of at least one protein selected from Table 1, 2, or 3 as a biomarker for endometriosis.

[0064] The method of the present invention can also include evaluating endometriosis intervention. Thus, according to another aspect, the present invention is a method for evaluating endometriosis intervention in a subject, comprising: (a) applying an intervention to the subject; and (b) evaluating the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from the subject; and (c) using this expression level to determine the effect of the intervention on the subject and provides a method comprising.

[0065] Preferably, the expression level of at least one protein is evaluated at least twice. In this regard, the change in the expression level after the intervention can identify the intervention as an intervention for treating endometriosis.

[0066] Preferably, the expression level of at least one protein is evaluated before, during, and / or after the intervention.

[0067] Preferably, the intervention is selected from the list including hormonal therapy, hormonal contraceptives, androgen agents, gonadotropin-releasing hormone (Gn-RH) agonists and antagonists, progestin therapy, aromatase inhibitors, and surgery including laser surgery.

[0068] The present invention also provides the use of at least one protein selected from Table 1, 2, or 3 as a target for a therapeutic agent for endometriosis. In this regard, the proteins described herein may be useful as drug targets.

[0069] Various aspects of the present invention can provide, for example, a relatively economical, accurate, non-invasive, and easy-to-perform test for the detection of endometriosis. The methods of the present disclosure can assist in the early detection of endometriosis. The methods of the present disclosure can be useful for subjects having undiagnosed endometriosis. The methods of the present disclosure can reduce the false positive and false negative rates obtained from other techniques for evaluating endometriosis and can improve the accuracy of diagnosis.

[0070] General Those skilled in the art will understand that the invention described herein is subject to changes and modifications other than those specifically described. The present invention includes all such changes and modifications. The present invention also includes, individually or collectively, all of the steps and features referred to or shown herein, and any combination or any two or more of these steps and features.

[0071] Each document, reference, patent application, or patent cited in this text is hereby expressly incorporated by reference in its entirety, which means that it should be construed and regarded by the reader as part of this text. The fact that a document, reference, patent application, or patent cited in this text is not repeated within this text is for simplicity only. However, none of the cited subject matter or the information contained therein should be understood as being of ordinary general knowledge.

[0072] The present invention should not be limited to the scope of any of the specific embodiments described herein. These embodiments are for illustrative purposes only. Functionally equivalent products and methods are clearly within the scope of the present invention described herein.

[0073] The present invention described in this specification can include ranges of one or more values (such as size, etc.). It will be understood that a range of values includes all values within the range, including the values that define the range and the values adjacent to the range that give the same or substantially the same result as the values immediately adjacent to the values that define the boundaries of the range.

[0074] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0075] Other definitions for selected terms used in this specification can be found within the detailed description of the present invention and apply throughout. Unless otherwise defined, all technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

Examples

[0076] Examples Example 1 - Identification of Endometriosis Biomarkers 1. Materials / Methods Study Demographics This study was approved by the Belberry Human Research Ethics Committee and all participants gave informed consent. Blood samples from the participants were collected into EDTA tubes. Plasma was separated by centrifugation (1000 g, 10 minutes) within 2 hours of collection and stored at -80°C.

[0077] The study was conducted on two independent cohorts collected by the Wesley Medical Research Institute. In the first cohort, 30 individuals were divided into three groups as follows: the endometriosis group (n = 10, endometriosis) (here, endometriosis was diagnosed by laparoscopy), the symptoms-only group (n = 10, no diagnosis) (here, the patients presented with symptoms of endometriosis but the diagnosis was not confirmed by laparoscopy), and a healthy control group without pelvic symptoms (n = 10, control). In the second cohort, 26 individuals were divided into three groups as follows: the endometriosis group (n = 12), the no-diagnosis group (n = 5), and a healthy control group (n = 9).

[0078] Plasma preparation and isobaric tag (iTRAQ) labeling Individual plasma samples were pooled for each group. The first cohort was analyzed as a single replicate, and the second cohort was analyzed as three replicates (Figure 1). The technical replicates of the second cohort were generated by dividing each group into three aliquots (Figure 1).

[0079] Fourteen abundant proteins in plasma were immunodepleted using a MARS14 chromatography column (Agilent Technologies), and then the samples were desalted using a Vivaspin® 6 10 kDa centrifugal concentrator (Sartorius). The samples were first reduced, alkylated, and digested with trypsin. The peptide concentration of the resulting samples was measured and normalized to provide an equivalent amount for labeling with isobaric tags (iTRAQ) reagents (Sciex) for relative and absolute quantification according to the manufacturer's instructions. An iTRAQ 4-plex kit that enables simultaneous protein identification and quantification was used. The samples were labeled according to the scheme in Figure 1.

[0080] The peptide was desalted on a Strata-X 33 μm polymeric reversed-phase column (Phenomenex), dissolved in a buffer containing 2% acetonitrile and 0.1% formic acid, and then separated at high pH on an Agilent 1100 HPLC system using a Zorbax C18 column (2.1×150 mm). The peptide was eluted with a linear gradient of 20 mM ammonium formate, 2% ACN to 20 mM ammonium formate, 90% ACN at 0.2 ml / min. 95 fractions were pooled into 12 fractions and dried. Each fraction was analyzed by electrospray ionization mass spectrometry using a Thermo UltiMate 3000 nanoflow UHPLC system (Thermo Scientific) connected to a Q Exactive HF mass spectrometer (Thermo Scientific). The peptide was loaded onto an Acclaim(™) PepMap(™) 100 C18 LC column, 2 μm particle size × 150 mm (Thermo Scientific) and separated with a linear gradient of water / acetonitrile / 0.1% formic acid (v / v).

[0081] Data analysis Protein identification and quantification were performed using ProteinPilot(™) 5.0 (Sciex). The MS / MS spectra were searched against the human SwissProt database. The search parameters were as follows: sample type: iTRAQ 4plex (labeled peptides); Cys alkylation: MMTS; digestion: trypsin; instrument: Orbi MS and Orbi MS / MS; special factors: none; species: human (Homo sapiens); quantification tab checked; bias correction and background correction tabs checked; ID focus: biological modifications; search effort: thorough; detected protein threshold [Unused ProtScore(Conf)]: 0.05 (10.0%); FDR analysis tab checked.

[0082] All identified proteins had an Unused ProtScore > 1.3 (corresponding to proteins identified with > 95% confidence) and an overall false discovery rate (FDR) < 0.1%, determined at the protein level using the software's PSPEP algorithm. Proteins that were specifically expressed in the endometriosis diagnosis group compared to the healthy control group and / or in the symptomatic, undiagnosed group compared to the endometriosis diagnosis group were considered biomarker candidates for endometriosis only if they were not specifically expressed in the symptomatic, undiagnosed group compared to the healthy control group. Specifically, if a protein was specifically expressed in the symptomatic, undiagnosed group compared to the healthy control group, it was not considered a biomarker for endometriosis.

[0083] The first selection criterion was established and applied, where specifically expressed proteins were required to have at least two unique peptides with > 95% confidence in at least one replicate measurement from both cohorts and a significantly different protein ratio (> 10-fold change) (P-value ≤ 0.05, as calculated by the software).

[0084] To select additional biomarkers and expand the pool of potential candidates, a second selection criterion was also established and applied. For a protein to be considered a second candidate, it was required to meet two criteria across both cohorts. First, one of the two cohort data points had to meet the first selection criterion. The second data point from the other cohort had to have a ≥ 2-fold change in protein abundance for a protein with at least two high-confidence peptides (> 95%), or a P-value ≤ 0.1 with a fold change > 10% and at least two high-confidence peptides (> 95%).

[0085] Results Two independent cohorts were used to compare patients with endometriosis diagnosed by laparoscopy with a) patients with symptoms only, and b) healthy controls without pelvic symptoms.

[0086] The proteome coverage for each of the four experiments outlined in Figure 1 is shown in Table A.

[0087] [Table 5]

[0088] The selection criteria described in the data analysis section were applied to the protein identification and quantification results to compare protein expression in endometriosis diagnosis against healthy controls and in endometriosis diagnosis against symptomatic, undiagnosed individuals, and a list of candidate biomarkers was determined to be significant. Further analysis of these candidate biomarkers included: · Averaging significant fold changes across replicate measurements for each protein; · A significant P-value was shown as less than the smallest significant P-value for the protein across replicate measurements. · The number of data points used for averaging was included (from four possible replicate measurements). · Biomarkers were ranked in order of P-value, then (if equal) significant data points, then (if equal) fold change

[0089] The results of this further analysis led to the biomarkers described in Tables B and C.

[0090] [Table 6]

[0091] [Table 7]

Claims

**Claim 1** (a) means for obtaining the expression level of at least one protein selected from Table 1 (split into boxed [Table 1] and [Table 2]) in a blood sample or plasma sample from a subject; and (b) means for processing the expression level to determine whether the subject has endometriosis or is at risk of developing endometriosis comprising wherein the at least one protein comprises vitamin K-dependent protein S, an inspection system. 【Table 1】 【Table 2】 **Claim 2** The inspection system according to claim 1, wherein the at least one protein further comprises one or more proteins selected from the list consisting of β-Ala-His dipeptidase, apolipoprotein L1, methanethiol oxidase, von Willebrand factor, plasminogen, selenoprotein P, protein disulfide isomerase A6, inter-α-trypsin inhibitor heavy chain H3, L-lactate dehydrogenase A chain, β-2-glycoprotein 1, afamin, clusterin, prothrombin, hepatocyte growth factor activator, endoplasmic reticulum chaperone BiP, peroxiredoxin-2, C4b-binding protein α chain, complement component C9, C4b-binding protein β chain, coagulation factor XII, bisphosphoglycerate mutase, carbonic anhydrase 2, coagulation factor X, complement factor H-related protein 2, hyaluronic acid-binding protein 2, proteoglycan 4, corticosteroid-binding globulin, fibrillin-1, heparin cofactor 2, Rho GDP dissociation inhibitor 2 and sex hormone-binding globulin. **Claim 3** The inspection system according to claim 1 or 2, wherein the at least one protein comprises two, three, four, or five proteins. **Claim 4** The test system according to any one of claims 1 to 3, further comprising two or more proteins selected from the list consisting of β-Ala-His dipeptidase, apolipoprotein L1, methanethiol oxidase, von Willebrand factor, plasminogen, selenoprotein P, protein disulfide isomerase A6, inter-α-trypsin inhibitor heavy chain H3, L-lactate dehydrogenase A chain, β-2-glycoprotein 1, afamin, clusterin, prothrombin, hepatocyte growth factor activator, endoplasmic reticulum chaperone BiP, peroxiredoxin-2, C4b-binding protein α chain, complement component C9, C4b-binding protein β chain, coagulation factor XII, bisphosphoglycerate mutase, carbonic anhydrase 2, coagulation factor X, complement factor H-related protein 2, hyaluronic acid-binding protein 2, proteoglycan 4, corticosteroid-binding globulin, fibrillin-1, heparin cofactor 2, Rho GDP dissociation inhibitor 2, and sex hormone-binding globulin.

5. The test system according to any one of claims 1 to 4, wherein the means for obtaining the expression level comprises at least one means selected from the list consisting of spectroscopy, mass spectrometry, surface-enhanced Raman spectroscopy, flow cytometry, ELISA, protein array, mass detection BioCD protein array, protein microarray, quantum dot-based detection, electrochemical immunoassay, gel electrophoresis, 9G DNA technology, immunoaffinity mass spectrometry, and immunocapture mass spectrometry.

6. The test system according to any one of claims 1 to 4, wherein the means for obtaining the expression level comprises multiple reaction monitoring (MRM) mass spectrometry.

7. The test system according to any one of claims 1 to 6, wherein the means for obtaining the expression level is adapted to evaluate the expression level of the at least one protein by evaluating the amount of a fragment or peptide of the at least one protein.

8. The test system according to any one of claims 1 to 7, wherein the means for obtaining the expression level is adapted to quantify the expression level of the at least one protein.

9. The means for obtaining the expression level is adapted to quantify the expression level of the at least one protein by comparing it with the expression level of the at least one protein in a subject without endometriosis. The test system according to any one of claims 1 to 8.

10. The test system according to any one of claims 1 to 9, wherein the at least one protein further comprises a detectable label.

11. The means for processing the expression level is adapted to compare the expression level with a reference value indicating endometriosis. The test system according to any one of claims 1 to 10.

12. The test system according to any one of claims 1 to 11, comprising a kit.

13. The test system according to claim 12, wherein the kit comprises a reagent for detecting the at least one protein.

14. The test system according to claim 12 or 13, wherein the kit comprises any one or more of a detectable label, a standard substance, a sample buffer (one or more), and a control (positive and / or negative).

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