Endometriosis biomarkers

Abstract

To provide screening, diagnostic and prognostic methods of using biomarkers associated with endometriosis.SOLUTION: A method comprises the steps of: (a) assessing an expression level of at least one protein, selected from Table 1, 2 or 3 in a sample from a subject, where the at least one protein may be selected from the list consisting of 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.SELECTED DRAWING: None

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 the biomarkers. Furthermore, the present invention relates to methods for evaluating medical interventions for endometriosis and for identifying drug targets for endometriosis. [Background technology]

[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. The condition affects one in ten women of reproductive age, and its incidence and health burden rivals that of diabetes.

[0003] Endometriosis causes chronic pain and infertility, but is often difficult to diagnose because its symptoms are common to many other gynecological conditions. On average, it takes 8.5 years from the first symptoms for a woman to be diagnosed. Imaging scans and existing blood tests are inconclusive; therefore, the current gold standard for diagnosis is through direct visualization of the tissue with confirmation by histological analysis. This can only be achieved through an invasive laparoscopy / laparotomy under general anesthesia, in which a camera is inserted into the pelvis through a small incision in the abdominal wall.

[0004] In view of the above, there exists a need for improved endometriosis biomarkers and related methods of use thereof, including methods for diagnosing endometriosis. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention The present invention provides (a) assessing the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from the subject; (b) using the expression level to determine whether the subject has endometriosis; The present invention provides a method comprising:

[0006] The present invention also provides (a) 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; and We provide tests including:

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

[0008] The present invention also provides a method of evaluating an endometriosis intervention in a subject, comprising: (a) administering an intervention to a subject; (b) assessing the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from the subject; (c) using the expression level to determine the effect of an intervention on the subject; The present invention provides a method comprising:

[0009] The present invention also provides the use of at least one protein selected from Tables 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 invention, given by way of example and not intended to limit the invention to the particular embodiments described, can be read in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0011] [Figure 1] An overview of the study design for endometriosis biomarkers is shown (Note: isobaric (iTRAQ) markers are designated 114, 115, 116, 117). DETAILED DESCRIPTION OF THE INVENTION

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

[0013] [Table 1]

[0014] [Table 2]

[0015] For the purposes of the present invention, the term endometriosis includes the pathological proliferation of ectopic endometrium-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 proliferations of ectopic endometrium-like tissue in the uterine sacral ligament, the rectovaginal space, the upper part of the posterior vaginal wall, the intestine, and / or the urinary tract.

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

[0017] Preferably, the 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, hyaluronic acid-binding protein 2, protein disulfide isomerase A6, or coactosin-like protein.

[0018] Preferably, the 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, the at least one protein comprises at least one protein selected from Table 2.

[0020] [Table 3]

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

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

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

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

[0025] [Table 4]

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

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

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

[0029] According to another preferred embodiment of the present invention, the 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, the 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 assessing the expression level of at least one protein can include any suitable method for assessing protein expression. Preferably, step (a) includes at least one of spectroscopy, such as mass spectrometry, surface-enhanced Raman spectroscopy, flow cytometry, ELISA, protein arrays (including mass-detected BioCD protein arrays), protein microarrays, quantum dot-based detection, electrochemical immunoassays, gel electrophoresis, 9G DNA technology, nanoparticles including lanthanide chelates such as europium EuNPs and gold nanoparticles, immunoaffinity mass spectrometry, and immunocapture mass spectrometry.

[0032] When step (a) involves mass spectrometry, this may involve multiple reaction monitoring (MRM) mass spectrometry or selected reaction monitoring (SRM) mass spectrometry.

[0033] Preferably, step (a) comprises assessing the expression level of at least one protein by assessing the amount of a fragment or peptide of the 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 relative to the expression level of at least one protein in subjects without endometriosis.

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

[0037] The sample can include a biological sample and / or a sub-sample thereof. Preferably, the biological sample is a bodily 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 blood, ascites, cerebrospinal fluid, buccal sample, cavity washings, organ washings, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, female ejaculate, sweat, fecal material, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstrual fluid, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery stool, pancreatic juice, nasal lavage fluid, bronchopulmonary aspirate, or other lavage fluids. Biological samples may also include the blastocyl cavity, umbilical cord blood, or maternal circulation (which may be of fetal or maternal origin). Biological samples may also be tissue samples or biopsies. Subsamples include extracts from the sample, including protein extracts.

[0038] Preferably, the subject is a mammal such as a human.The subject may be a subject who has been previously diagnosed or identified as having endometriosis, and optionally has already undergone or is currently undergoing therapeutic intervention.Alternatively, the subject may also be a subject who has not previously been diagnosed or identified as having endometriosis.For example, the subject may be a subject who exhibits one or more risk factors for endometriosis, or a subject who does not exhibit any of these risk factors, or a subject who is asymptomatic for endometriosis.The subject may also be a subject who suffers from endometriosis or is at risk of developing endometriosis.

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

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

[0041] Preferably, step (b) comprises 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 can be a reference protein expression level from at least one second subject (wherein the reference protein expression level is known to be correlated with endometriosis).

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

[0044] Another use of the expression level from step (a) in step (b) is to compare it with other expression levels from the same subject obtained at different time points, allowing for comparison of expression levels over time in a subject, and therefore whether the subject has endometriosis.

[0045] Preferably, the methods of the present invention determine 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 methods of the present invention determine 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 methods of the present invention determine 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] The method can include the use of a control to better assess the expression level of at least one protein, and can use the control to determine whether the subject has endometriosis. Preferably, the control is a control protein, such as a control protein that is not specifically expressed in endometriosis.

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

[0050] According to a third aspect of the present invention, (a) 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; and Tests including:

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

[0052] Preferably, the test includes the device.

[0053] Preferably, the apparatus includes a spectrometer.

[0054] Preferably, the test comprises 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 determining whether a subject has endometriosis based on the expression level. The written instructions may include instructions for comparing protein expression and / or a predetermined value (e.g., a value for determining whether the expression level of the protein is indicative of endometriosis).

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

[0058] The proteins and combinations thereof of the present invention can be implemented in a variety of test systems. Typically, a test system includes a means for obtaining test results from a sample, a means for collecting, storing, processing, and / or tracking the test results for the sample, usually in a database, and a means for reporting the test results. The means for obtaining test results may include a module adapted for automated testing, utilizing one or more biochemical, immunological, and protein detection assays. Some test systems are capable of processing multiple samples and performing multiple tests on a given sample. The means for collecting, storing, processing, and / or tracking test results may include physical and / or electronic data storage, such as a hard drive or flash memory, or printing to paper. The means for reporting test results may include a visual display, a link to a data structure or database, or a printer. In this regard, the reporting means may simply be a data link adapted to send the results to another device, such as a database, a visual display, or a printer.

[0059] Typically, the test results from the systems of the present invention serve as input to a computer, or a microprocessor programmed with machine language, or software that obtains data related to the expression level 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 prognosis of endometriosis. The risk of having or developing endometriosis can be assessed by measuring the expression of one or more proteins in Tables 1, 2, or 3 and comparing the measured value with a reference or index value. Such comparisons can be performed using mathematical algorithms or formulas to combine information from the results of multiple individual proteins and other parameters into a single measurement or index. Subjects identified as being at high risk for endometriosis can optionally be selected to receive a treatment regimen, such as the administration of a prophylactic or therapeutic compound.

[0061] The expression level of at least one protein can be measured in a sample and compared to a reference or normal level using techniques such as a reference limit, discrimination limit, or risk-defining threshold to define a cutoff point, and an abnormal value for endometriosis. Normal control levels are levels of one or more proteins or a combined biomarker index typically found in subjects who do not suffer from endometriosis. Normal and abnormal levels and cutoff points can vary based on whether at least one protein is used alone or in a manner integrated with other biomarkers into a single index. Alternatively, normal or abnormal levels can be a database of biomarker patterns or "signatures" from previously tested subjects who have or have not developed endometriosis over a clinically relevant time range.

[0062] Thus, the expression level of at least one protein can be used to generate a profile or signature of a subject who (i) does not have and is unlikely to develop endometriosis, and / or (ii) is likely to have or develop such a condition. The subject's profile can be compared with a predetermined or reference biomarker profile to diagnose or identify subjects at risk of developing endometriosis, monitor the progression of endometriosis and the rate of endometriosis progression, and monitor the effectiveness of interventions. The profiles of the present invention are preferably contained in a machine-readable medium and are "raw" insofar as they can be updated with additional data obtained (thus improving the strength and clinical significance of the biomarker). Data regarding the level of at least one protein of the present invention can also be combined or correlated with other data or test results, such as, but not limited to, measurements of clinical parameters for endometriosis or other algorithms. The machine-readable medium can also include subject information such as medical history and any relevant family history.

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

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

[0065] Preferably, the expression level of at least one protein is assessed at least twice, whereby a change in expression level following an intervention can identify the intervention as an intervention for treating endometriosis.

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

[0067] Preferably, the intervention is selected from the list comprising hormone therapy, hormonal contraceptives, androgenic 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 Tables 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 detecting endometriosis. The disclosed method can aid in the early detection of endometriosis. The disclosed method can be useful for subjects with undiagnosed endometriosis. The disclosed method can reduce the false positive and false negative rates obtained from other techniques for assessing endometriosis, improving diagnostic accuracy.

[0070] General Those skilled in the art will recognize that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all of the steps and features referred to or shown in this specification, individually or collectively, and any or all combinations of any two or more of these steps and features.

[0071] Each document, reference, patent application, or patent cited in this text is expressly incorporated herein by reference in its entirety, meaning that it should be interpreted and considered by the reader as part of this text. It is for the sake of brevity only that the documents, references, patent applications, or patents cited in this text are not repeated within this text. However, none of the cited material or the information contained therein should be understood to be common general knowledge.

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

[0073] The invention described herein may include one or more ranges of values ​​(e.g., magnitudes, etc.) It will be understood that a range of values ​​includes all values ​​within the range, including the values ​​defining the range and any adjacent values ​​within the range that produce the same or substantially the same result as the values ​​immediately adjacent to the values ​​defining the range boundaries.

[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 a 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 herein may be referenced within the detailed description of the present invention and are applicable throughout. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [Example]

[0076] Example 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 provided informed consent. Blood samples from participants were collected in EDTA tubes. Plasma was separated by centrifugation (1000 g, 10 min) within 2 h of collection and stored at -80°C.

[0077] The study was conducted on two independent cohorts collected by Wesley Medical Research Institute. In the first cohort, 30 individuals were divided into three groups: endometriosis group (n=10, endometriosis) (where endometriosis was diagnosed by laparoscopy), symptom-only group (n=10, no diagnosis) (where patients had symptoms of endometriosis but had not been diagnosed by laparoscopy), and healthy control group without pelvic symptoms (n=10, control). In the second cohort, 26 individuals were divided into three groups: endometriosis group (n=12), no diagnosis group (n=5), and 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). Technical replicates for 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). Samples were first reduced, alkylated, and trypsin-digested. Peptide concentrations in the resulting samples were measured and normalized to provide equivalent amounts for labeling with isobaric tag (iTRAQ) reagents (Sciex) for relative and absolute quantification according to the manufacturer's instructions. An iTRAQ 4-plex kit was used, which allows simultaneous protein identification and quantification. Samples were labeled according to the scheme in Figure 1.

[0080] Peptides were desalted on a Strata-X 33 μm polymeric reverse-phase column (Phenomenex) and dissolved in a buffer containing 2% acetonitrile and 0.1% formic acid. High-pH separation was then performed on an Agilent 1100 HPLC system using a Zorbax C18 column (2.1 × 150 mm). Peptides were eluted with a linear gradient of 20 mM ammonium formate, 2% ACN, to 20 mM ammonium formate, 90% ACN at 0.2 ml / min. The 95 fractions were combined into 12 fractions and dried. Each fraction was analyzed by electrospray ionization mass spectrometry using a Thermo UltiMate 3000 nanoflow UHPLC system (Thermo Scientific) coupled to a Q Exactive HF mass spectrometer (Thermo Scientific). Peptides were 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). MS / MS spectra were matched against the human SwissProt database. Search parameters were as follows: sample type: iTRAQ 4plex (labeled peptides); Cys alkylation: MMTS; digestion: trypsin; instrument: Orbi MS and Orbi MS / MS; specificity factors: none; species: human (Homo sapiens); quantification tab checked; bias correction and background correction tab checked; ID focus: biological modification; 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 of >1.3 (corresponding to proteins identified with >95% confidence) and an overall false positive rate (FDR) of <0.1%, as determined at the protein level using the software's PSPEP algorithm. Proteins found to be differentially expressed in endometriosis-diagnosed versus healthy controls and / or in symptomatic, undiagnosed versus healthy controls were considered potential endometriosis biomarkers only if they were not differentially expressed in symptomatic, undiagnosed versus healthy controls. Specifically, if a protein was differentially expressed in symptomatic, undiagnosed versus healthy controls, it was not considered a biomarker for endometriosis.

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

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

[0085] result Using two independent cohorts, patients with laparoscopically diagnosed endometriosis were compared 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] By applying the selection criteria described in the data analysis section to the protein identification and quantification results and comparing protein expression in endometriosis diagnosed versus healthy controls, and in symptomatic versus undiagnosed endometriosis diagnosed, a list of candidate biomarkers was determined to be significant. Further analysis of these candidate biomarkers included the following: Averaging significant fold changes across replicates for each protein; A significant P value is indicated as less than the smallest significant P value for the protein across replicates. The number of data points used for averaging is included (out of four possible replicates). Biomarkers were ranked by P-value (if equal), then significant data points (if equal), then fold change

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

[0090] [Table 6]

[0091] [Table 7]

Claims

1. (a) assessing the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from a subject; (b) using the expression level to determine whether the subject has endometriosis; A method comprising:

2. 2. The method of claim 1, wherein the at least one protein is 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.

3. The method of claim 1 or 2, wherein the at least one protein comprises a plurality of proteins.

4. The method of claim 3 , wherein the plurality of proteins comprises two, three, four, or five proteins.

5. 2. The method of claim 1, wherein the 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 CAT, 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, hyaluronic acid-binding protein 2, or protein disulfide isomerase A6.

6. 6. The method of any one of claims 1 to 5, wherein step (a) comprises at least one of spectroscopy, such as mass spectrometry, surface-enhanced Raman spectroscopy, flow cytometry, ELISA, protein arrays including mass-detected BioCD protein arrays, protein microarrays, quantum dot-based detection, electrochemical immunoassays, gel electrophoresis, 9G DNA technology, nanoparticles including lanthanide chelates such as europium EuNPs and gold nanoparticles, immunoaffinity mass spectrometry, and immunocapture mass spectrometry.

7. 7. The method of claim 6, wherein step (a) comprises multiple reaction monitoring (MRM) mass spectrometry.

8. 8. The method of any one of claims 1 to 7, wherein step (a) comprises assessing the expression level of said at least one protein by assessing the amount of a fragment or peptide of said at least one protein.

9. The method of any one of claims 1 to 8, wherein step (a) comprises quantifying the expression level of the at least one protein.

10. 10. The method of any one of claims 1 to 9, wherein step (a) comprises quantifying the expression level of the at least one protein relative to the expression level of the at least one protein in a subject not having endometriosis.

11. The method of any one of claims 1 to 10, wherein step (a) comprises labeling said at least one protein.

12. The method of any one of claims 1 to 11, wherein the sample comprises a biological sample and / or a sub-sample thereof.

13. 13. The method of claim 12, wherein the biological sample is a bodily fluid such as blood, serum, plasma, urine, sweat, tears, saliva, sputum, or any combination or fraction thereof.

14. The method of any one of claims 1 to 13, wherein the sample comprises blood.

15. The method of any one of claims 1 to 14, wherein step (b) comprises comparing the expression level from step (a) with a reference value indicative of endometriosis.

16. 10. Use of at least one protein selected from Table 1, 2, or 3 to determine whether a subject has endometriosis.

17. (a) 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; and Inspection including.

18. 18. The inspection of claim 17, including a spectrometer.

19. The test of claim 17, comprising a kit.

20. 20. The test of claim 19, wherein the kit comprises reagents for detecting at least one protein.

21. 21. The test of claim 19 or 20, wherein the kit comprises any one or more of a detectable label, a standard, a sample buffer(s), and controls (positive and / or negative).

22. 10. Use of at least one protein selected from Tables 1, 2, or 3 as a biomarker for endometriosis.

23. 1. A method of evaluating an endometriosis intervention in a subject, comprising: (a) administering the intervention to the subject; (b) assessing the expression level of at least one protein selected from Table 1, 2, or 3 in a sample from the subject; (c) using the expression levels to determine the effect of the intervention on the subject; A method comprising:

24. 24. The method of claim 23, wherein the intervention is selected from the list comprising hormone therapy, hormonal contraceptives, androgenic agents gonadotropin-releasing hormone (Gn-RH) agonists and antagonists, progestin therapy, aromatase inhibitors, and surgery, including laser surgery.

25. 10. Use of at least one protein selected from Tables 1, 2, or 3 as a target for a therapeutic agent for endometriosis.

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