Biomarkers for endometriosis

Abstract

A method comprising the steps of: (a) assessing an expression level of at least one protein, selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, in a sample from a subject, and (b) using the expression level to determine whether the subject has endometriosis.

Description

[0001] Biomarkers for endometriosis FIELD OF THE INVENTION The invention relates to biomarkers associated with endometriosis. The invention also relates to screening, diagnostic and prognostic methods of using the biomarkers. Still further the invention relates to methods of assessing medical interventions for endometriosis and methods of identifying drug targets for endometriosis. BACKGROUND TO THE INVENTION Endometriosis occurs when the tissues that line the uterus spread outside of the uterine cavity and surround other organs, including in the peritoneum, ovaries, fallopian tubes, pleura and lungs. The condition affects one in ten women in their reproductive years and its incidence and health burden are comparable with diabetes. Endometriosis causes chronic pain and infertility but is often difficult to diagnose because the symptoms are shared by many other gynaecological conditions. On average, it takes 8.5 years for women to be diagnosed from their first symptoms. Imaging scans and existing blood tests are inconclusive, so the current gold standard for diagnosis is by direct visualisation of the tissue with confirmation by histological analysis. This can only be achieved by invasive laparoscopy / laparotomy under a general anaesthetic, where a camera is inserted into the pelvis through a small cut in the abdominal wall. With the above in mind there is a need for improved endometriosis biomarkers and associated methods of their use including methods for diagnosing endometriosis. SUMMARY OF THE INVENTION The present invention provides a method comprising the steps of: (a) assessing an expression level of at least one protein, selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, in a sample from a subject, and (b) using the expression level to determine whether the subject has endometriosis. The present invention also provides a test comprising: (a) means for obtaining an expression level of at least one protein selected from selected from the list consisting of hemoglobin subunit beta and inter-alpha- trypsin inhibitor light chain, in a sample from a subject; and (b) means for processing the expression level generated in step (a) to determine whether the subject has endometriosis. The present invention also provides a test system comprising: (a) a means for obtaining a test result from a sample from a subject indicative of an expression level of at least one protein, selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain; and (b) means for correlating the test result with a risk of the subject having endometriosis. The present invention also provides for the use of at least one protein selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain as a biomarker for endometriosis. The present invention also provides a method of treating endometriosis in a subject having an expression level of at least one protein indicative of endometriosis, wherein the at least one protein is selected the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, the method comprising the step of administering a therapeutically effective amount of a treatment for endometriosis to the subject. The present invention also provides a method of preventing endometriosis in a subject having an expression level of at least one protein indicative of an increased risk of developing endometriosis, wherein the at least one protein is selected the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, the method comprising the step of administering a therapeutically effective amount of a preventative treatment for endometriosis to the subject. BRIEF DESCRIPTION OF DRAWINGS The following Detailed Description of the Invention, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying Figures, in which: Figure 1 outlines the study design for biomarkers of endometriosis (Note: the isobaric (iTRAQ) labels are designated 114, 115, 116, 117); Figure 2A is a boxplot of the Mann–Whitney U test results in Table D for Hemoglobin subunit beta; Figure 2B is a boxplot of the Mann–Whitney U test results in Table D for inter- alpha-trypsin inhibitor light chain; Figure 3 depicts receiver operating characteristic curves for Models 1, 2, and 3. Model 1: all endometriosis n = 443 versus general population controls n = 147, Model 2: endometriosis (stages II–IV) n = 212 versus symptomatic controls n = 130, and Model 3: endometriosis (stage IV) n = 89 versus symptomatic controls n = 130. Only participants with complete data were included in each model; and Figure 4 depicts receiver operating characteristic curves for Model 3: endometriosis (stage IV) versus symptomatic controls applied to all rASRM stages. Stage I: n = 241, stage II: n = 65, stage III: n = 58, and stage IV: n = 89. Only participants with complete data were included in this modeling. DETAILED DESCRIPTION OF THE INVENTION According to a first aspect, the present invention provides a method comprising the steps of: (a) assessing an expression level of at least one protein, selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, in a sample from a subject, and (b) using the expression level to determine whether the subject has endometriosis. For the purposes of the present invention the term endometriosis includes the pathological growth of ectopic endometrial-like tissue outside of the uterine cavity. Preferably, the term endometriosis comprises one or more of peritoneal superficial endometriosis, ovarian endometriosis and deep infiltrating endometriosis. Deep infiltrating endometriosis may comprise one or more of pathological growth of ectopic endometrial-like tissue in the uterosacral ligaments, rectovaginal space, the upper third of the posterior vaginal wall, the bowel and / or the urinary tract. Preferably, the at least one protein comprises a plurality of proteins such as two, three or four proteins selected from the list consisting of: Neuropilin-1, Serum paraoxonase / arylesterase 1, hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain. Preferably, the at least one protein comprises neuropilin-1 and / or serum paraoxonase / arylesterase 1. Preferably, the at least one protein further comprises at least one protein selected from Table 1.

[0002] Even more preferably, the at least one protein selected from Table 1 comprises two, three, four or five proteins selected from Table 1. Even more preferably, the at least one protein selected from Table 1 comprises at least one protein selected from the group consisting of: hemoglobin subunit alpha and inter-alpha-trypsin inhibitor heavy chain H3, Vitamin K-dependent protein S, Beta-Ala- His dipeptidase, Apolipoprotein L1, Methanethiol oxidase, von Willebrand factor, Plasminogen, Selenoprotein P, Protein disulfide-isomerase A6, L-lactate dehydrogenase A chain, Beta-2-glycoprotein 1, Afamin, Clusterin, Prothrombin, Hepatocyte growth factor activator, Endoplasmic reticulum chaperone BiP, Peroxiredoxin-2, C4b binding protein alpha chain, Complement component C9, C4b binding protein beta chain, Coagulation factor XII, Bisphosphoglycerate mutase, Carbonic anhydrase 2, Coagulation factor X, Complement factor H-related protein 2, Hyaluronan-binding protein 2, Proteoglycan 4, Corticosteroid-binding globulin, Fibrillin-1, Heparin cofactor 2, Rho GDP-dissociation inhibitor 2, Sex hormone-binding globulin, serum paraoxonase / arylesterase 1 and neuropilin-1. Even more preferably, the at least one protein selected from Table 1 comprises two, three, four or five proteins from Table 1 or two, three, four or five proteins selected from the list comprising: hemoglobin subunit alpha and inter-alpha-trypsin inhibitor heavy chain H3, Vitamin K-dependent protein S, Beta-Ala-His dipeptidase, Apolipoprotein L1, Methanethiol oxidase, von Willebrand factor, Plasminogen, Selenoprotein P, Protein disulfide-isomerase A6, L-lactate dehydrogenase A chain, Beta-2-glycoprotein 1, Afamin, Clusterin, Prothrombin, Hepatocyte growth factor activator, Endoplasmic reticulum chaperone BiP, Peroxiredoxin-2, C4b binding protein alpha chain, Complement component C9, C4b binding protein beta chain, Coagulation factor XII, Bisphosphoglycerate mutase, Carbonic anhydrase 2, Coagulation factor X, Complement factor H-related protein 2, Hyaluronan-binding protein 2, Proteoglycan 4, Corticosteroid-binding globulin, Fibrillin-1, Heparin cofactor 2, Rho GDP-dissociation inhibitor 2, Sex hormone-binding globulin serum, paraoxonase / arylesterase 1 and neuropilin-1. The step (a) of assessing an expression level of at least one protein can comprise any suitable method for assessing protein expression. Preferably, step (a) comprises at least one of spectrometry such as mass spectrometry, surface enhanced Raman spectroscopy, flow cytometry, ELISA, protein arrays including mass-sensing BioCD protein array, protein micro-arrays, quantum dots based detection, electrochemical immunoassay, gel electrophoresis, 9G DNA technology, nanoparticles including lanthanide chelates such as europium EuNPs and gold nanoparticles, immune-affinity mass spectrometry and immune capture mass spectrometry. When step (a) comprises mass spectrometry it may comprise multiple reaction monitoring (MRM) mass spectrometry or selective reaction monitoring (SRM) mass spectrometry. Preferably, step (a) comprises assessing the expression level of the at least one protein by assessing the amount of a fragment or peptide of the at least one protein. Preferably, step (a) comprises quantifying the expression level of the at least one protein. Preferably, step (a) comprise quantifying the expression level of the at least one protein relative to the expression level of the at least one protein in a subject without endometriosis. Step (a) may also comprise labelling the at least one protein. Exemplary labels include protein labels e.g. biotin, active site probes, enzyme conjugates e.g. HRP, and fluorescent probes, isotopic labelling and isobaric labelling. The sample may comprise a biological sample and / or sub-samples 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 a biological sample include whole blood, peripheral blood, ascites, cerebrospinal fluid, buccal sample, cavity rinse, organ rinse, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, female ejaculate, sweat, faecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, or other lavage fluids. A biological sample can also include the blastocyl cavity, umbilical cord blood, or maternal circulation which can be of foetal or maternal origin. The biological sample can also be a tissue sample or biopsy. Sub-samples include extracts from the sample including protein extracts. Preferably, the subject is a mammal such as a human. A subject can be one who has been previously diagnosed or identified as having endometriosis, and optionally has already undergone, or is undergoing, a therapeutic intervention. Alternatively, a subject can also be one who has not been previously diagnosed or identified as having endometriosis. For example, a subject can be one who exhibits one or more risk factors for endometriosis, or a subject who does not exhibit any such risk factors or a subject who is asymptomatic for endometriosis. A subject can also be one who is suffering from or at risk of developing endometriosis. Step (b) comprises any use of the expression level from step (a) to determine whether the subject has endometriosis. 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) may be combined with a second measure to determine whether the subject has endometriosis. Preferably, step (b) comprises comparing the expression level from step (a) with a reference value indicative of endometriosis. 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, wherein the reference protein expression level is known to correlate with endometriosis. The at least one second subject can be a cohort or population of subjects. Another use, according to step (b) of the expression level from step (a) is comparing it with another expression level from the same subject taken at a different time. Such use allows for the comparison of expression levels, and hence whether the subject has endometriosis, over time in a subject. Preferably, the method of the present invention determines whether the 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%. Preferably, the method of the present invention determines whether the 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%. Preferably, the method of the present invention determines whether the 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%. The method may involve the use of a control to better assess the expression level of the at least one protein and use it to determine whether the subject has endometriosis. Preferably, the control is a control protein such as control protein that is not differentially expressed with respect to endometriosis. According to a second aspect the present invention provides for the use of at least one protein selected from the list consisting of: Neuropilin-1, Serum paraoxonase / arylesterase 1, hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, to determine whether a subject has endometriosis. Preferably, the use further comprises the use of at least one protein from Table 1. The other preferred features of the method described above in relation to the first aspect also form preferred features of this aspect of the invention. According to a third aspect of the present invention there is provided a test comprising: (a) means for obtaining an expression level of at least one protein selected from selected from the list consisting of: hemoglobin subunit beta and inter-alpha- trypsin inhibitor light chain, in a sample from a subject; and (b) means for processing the expression level generated in step (a) to determine whether the subject has endometriosis. Preferably said means for obtaining an expression level is also for obtaining an expression level of at least one protein from Table 1. The 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 comprise any suitable method for assessing expression of protein. Preferably the test comprises an apparatus. Preferably, the apparatus comprises a spectrometer. Preferably, the test comprises a kit. Preferably, the kit comprises a reagent for detecting the at least one protein. Preferably, the kit comprises written instructions for quantifying an expression level of the at least one protein and / or for 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 a protein is indicative of endometriosis. Preferably, the kit comprises any one or more of the following: a detectable label, standards, sample buffer(s) and controls (positive and / or negative). The proteins and combinations thereof of the present invention can be implemented in a range of test systems. Thus, the present invention also provides a test system comprising: (a) a means for obtaining a test result from a sample from a subject indicative of an expression level of at least one protein, selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain; and (b) means for correlating the test result with a risk of the subject having endometriosis. Preferably, the test system further comprises: (c) a means for collecting, storing, processing and / or tracking the test result. Preferably, the means for collecting, storing, processing and / or tracking the test result comprises a database. Preferably, the test system further comprises a means for reporting the test results. The means for obtaining test results can include a module adapted for automatic testing utilising one or more of biochemical, immunological and protein detection assays. Some test systems can process multiple samples and can run multiple tests on a given sample. The means for collecting, storing, processing and / or tracking test results may comprise a physical and / or electronic data storage device such as a hard drive or flash memory or paper print-outs. The means for reporting test results can include a visible display, a link to a data structure or database, or a printer. In this regard, the reporting means may simply be a data link that is adapted to send results to another device such as a database, visual display, or printer. Typically, the test results serve as inputs to a computer or microprocessor programmed with a machine code or software that takes the data relating to the expression level of the at least one protein described herein and determines the risk of developing or already having endometriosis. The 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 of the proteins selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, and optionally at least one protein from Table 1, and comparing the measured values to reference or index values. Such a comparison can be undertaken with mathematical algorithms or formula in order to combine information from results of multiple individual proteins and other parameters into a single measurement or index. Subjects identified as having an increased risk of endometriosis can optionally be selected to receive treatment regimens, such as administration of prophylactic or therapeutic compounds. The expression level of the at least one protein can be measured in the sample and compared to a reference or normal level, utilizing techniques such as reference limits, discrimination limits, or risk defining thresholds to define cut-off points and abnormal values for endometriosis. The normal control level is the level of one or more proteins or combined biomarker indices typically found in a subject not suffering from endometriosis. The normal and abnormal levels and cut-off points may vary based on whether the at least one protein is used alone or in a formula combined with other biomarkers into an index. Alternatively, the normal or abnormal level can be a database of biomarker patterns or “signatures” from previously tested subjects who did or did not develop endometriosis over a clinically relevant time horizon. Thus, the expression levels of the at least one protein can be used to generate a profile or signature of subjects: (i) who do not have and are not expected to develop endometriosis and / or (ii) who have or expected to develop such conditions. The profile of a subject can be compared to a predetermined or reference biomarker profile to diagnose or identify subjects at risk for developing endometriosis, to monitor the progression of the endometriosis, as well as the rate of progression of the endometriosis, and to monitor the effectiveness of interventions. Profiles of the present invention are preferably contained in a machine-readable medium and are “live” insofar as they can be updated with further data that comes to hand, thus improving the strength and clinical significance of the biomarkers. Data concerning the levels of the at least one protein of the present invention can also be combined or correlated with other data or test results, such as, without limitation, measurements of clinical parameters or other algorithms for endometriosis. The machine-readable media can also comprise subject information such as medical history and any relevant family history. The present invention also provides for the use of at least one protein selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, and optionally at least one protein from Table 1, as a biomarker for endometriosis. The methods of the present invention can also include assessing endometriosis intervention. Thus, according to another aspect the present invention provides a method of assessing an endometriosis intervention in a subject, the method comprising the steps of: (a) applying the intervention to the subject; (b) assessing an expression level of at least one protein selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, in a sample from the subject; and (c) using the expression level to determine the effect of the intervention on the subject. Preferably, the expression level of the at least one protein is assessed at least twice. In this regard, changes in the expression levels after the intervention may identify the intervention as an intervention for treating endometriosis. Preferably the expression level of the at least one protein is assessed before, during and / or after the intervention. 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. Preferably, the method further comprises the step of assessing the expression level of at least one protein selected from Table 1. The present invention also provides for the use of at least one protein selected from the list consisting of: hemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, and optionally at least one protein from Table 1, as a target for a therapeutic agent for endometriosis. In this regard, the proteins described herein may be useful as drug targets. According to a fourth aspect of the present invention there is provided a method of treating endometriosis in a subject having an expression level of at least one protein indicative of endometriosis, wherein the at least one protein is selected the list consisting of: haemoglobin subunit beta and inter-alpha-trypsin inhibitor light chain, the method comprising the step of administering a therapeutically effective amount of a treatment for endometriosis to the subject. The term “treating” as used herein includes reducing the occurrence of endometriosis or uterine fibroids in a subject as well as reducing the severity or frequency of a symptom of endometriosis or uterine fibroids such as one or more symptoms from the list consisting of pain, excessive menstrual cramps, pain during intercourse, abnormal or heavy menstrual flow, infertility, painful urination during menstrual periods, painful bowel movements during menstrual periods, gastrointestinal problems, diarrhea, constipation and nausea. The treatment for endometriosis may be selected from the list consisting of: an analgesic, a hormone, an antioxidant, an aramotase inhibitor, a surgical intervention, an alternate therapy. Preferably, the analgesic is selected from the list consisting of: NSAIDs, naproxen, ibuprofen, mefenamic acid and ketoprofen. Preferably, the hormone is selected from the list consisting of: a contraceptive, a progestin, dienogest, a progestogen, etonorgestrel, a gonadotropin-releasing hormone (“GnRH”) agonist, leuprolide, leuprorelin, triptorelin, goserelin, nafarelin, buserelin, a GnRH antagonist, elagolix, linzagolix, opigolix, relugolix, selective estrogen receptor modulators, raloxifene, aromatase inhibitors, letrozole, anastrozole, selective progesterone receptor modulators, tibolone, mifepristone, gestrinon and ulipristal. Preferably, the antioxidant is selected from the list consisting of: resveratrol and curcumin. Preferably, the aromatase inhibitor is puerarin. Preferably, the surgical intervention is selected from the list consisting of: a laparoscopy, excision of lesion(s), ablation of lesion(s), a laparotomy, a hysterectomy and a nerve interruption. Preferably, the alternate therapy is selected from the list consisting of: physiotherapy, yoga, virtual reality delivered exercise, psychotherapy, somatosensory stimulation, acupuncture, moxibustion, mindfulness, dietary supplements and cannabis based medicines. Preferably, the method of treating endometriosis further comprises the step of applying the method of the first aspect of the invention, a use according to the second aspect of the invention and / or a test system of the third aspect of the invention. According to a fifth aspect of the present invention there is provided a method of preventing endometriosis in a subject having an expression level of at least one protein indicative of an increased risk of developing endometriosis, wherein the at least one protein is selected the list consisting of: hemoglobin subunit beta and inter-alpha- trypsin inhibitor light chain, the method comprising the step of administering a therapeutically effective amount of a preventative treatment for endometriosis to the subject. The term “preventing” as used herein includes delaying the onset of endometriosis or uterine fibroids in a subject as well as delaying a symptom of endometriosis or uterine fibroids such as one or more symptoms from the list consisting of pain, excessive menstrual cramps, pain during intercourse, abnormal or heavy menstrual flow, infertility, painful urination during menstrual periods, painful bowel movements during menstrual periods, gastrointestinal problems, diarrhea, constipation and nausea. The preventative treatment for endometriosis may be selected from the list consisting of: a hormonal contraceptive such as a birth control pill, patch, or a vaginal ring that contains estrogen and progestin to help regulate or suppress menstruation; progestin therapy; anti-inflammatory diet including omega-3 rich fatty acids, fruit, vegetables and whole grains; and regular exercise. The various aspects of the present invention can provide, for example, a relatively economical, accurate, non-invasive, and easy to implement test for detection of endometriosis. Methods of the present disclosure can aid early detection and treatment or prevention of endometriosis. Methods of the present disclosure can be useful for subjects with undiagnosed endometriosis. Methods of the present disclosure can reduce the rate of false positives and false negatives obtained from other approaches to assessing endometriosis and can improve the accuracy of diagnosis. General Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features. For the purposes of the present invention it will be appreciated that reference to a particular protein herein, as a biomarker for endometriosis, includes: (i) any protein, polypeptide or fragments of the particular protein having at least about 85%, 90%, 95% or 99% amino acid identity to the amino acid sequence of the protein according to the UniProt Accession No that is referenced herein e.g. Neuropilin-1 (UniProt accession No: O14786), Serum paraoxonase / arylesterase 1 (UniProt accession No: P27169), Hemoglobin subunit beta (UniProt accession No: P68871) and inter-alpha- trypsin inhibitor light chain (UniProt accession No: P02670); and (ii) any protein, polypeptide or fragments the particular protein which binds an antibody that specifically binds to the particular protein biomarker. With respect to inter-alpha- trypsin inhibitor light chain, UniProt accession No: P02670 relates to Protein AMBP that is the pre-cursor / pre-protein to inter-alpha-trypsin inhibitor light chain. Thus, reference in this paragraph to amino acid sequences of a protein and antibodies that specifically bind to a protein in this paragraph, with respect to UniProt P02670, relate only to the portion of UniProt P02670 corresponding to inter-alpha-trypsin inhibitor light chain. Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. None of the cited material or the information contained in that material should, however be understood to be common general knowledge. The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products and methods are clearly within the scope of the invention as described herein. The invention described herein may include one or more range of values (e.g. size etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. 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. Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. EXAMPLES Example 1 – Identification of endometriosis biomarkers 1. Materials / Methods Study demographics The study was approved by the Belberry Human Research Ethics Committee and all participants gave informed consent. Blood samples from participants were collected in EDTA tubes. Plasma was separated by centrifugation (1000g, 10 min) within 2 hours of collection and stored at -80oC. The study was performed on two independent cohorts collected by the Wesley Medical Research Institute. In the first cohort, 30 individuals were divided into 3 groups as follows: an endometriosis group (n=10, endometriosis) where endometriosis had been diagnosed by laparoscopy, a symptoms only group (n=10, no diagnosis) where patients displayed symptoms of endometriosis but laparoscopy did not confirm the diagnosis, and a healthy control group with no pelvic symptoms (n=10, control). In the second cohort, 26 individuals were divided into 3 groups as follows: endometriosis group (n=12), a no diagnosis group (n=5), and a healthy control group (n=9). Plasma preparation and isobaric tag (iTRAQ) labelling Individual plasma samples were pooled for each group. The first cohort was analysed as a single replicate and the second cohort was analysed as 3 replicates (Figure 1). Technical replicates of the second cohort were generated by splitting each of the groups into three aliquots (Figure 1). Fourteen high-abundant proteins in plasma were immunodepleted using a MARS14 chromatography column (Agilent Technologies) before samples were desalted on Vivaspin® 610kDa centrifugal concentrators (Sartorius). Samples were first reduced, alkylated, and trypsin digested. The resulting sample peptide concentrations were measured and normalised to provide equivalent amounts for labelling with isobaric tags for relative and absolute quantitation (iTRAQ) reagents (Sciex) according to the manufacturer’s instructions. The iTRAQ 4-plex kit enabling simultaneous protein identification and quantitation was used. Samples were labelled according to the scheme in Figure 1. Peptides were desalted on a Strata-X 33 μm polymeric reversed phase columns (Phenomenex) and dissolved in a buffer containing 2% acetonitrile 0.1% formic acid before separation by high pH on an Agilent 1100 HPLC system using a Zorbax C18 column (2.1 x 150 mm). Peptides were eluted with a linear gradient of 20mM ammonium formate, 2% ACN to 20mM ammonium formate, 90% ACN at 0.2ml / min. Ninety five fractions were concatenated into 12 fractions and dried down. Each fraction was analysed by electrospray ionisation 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, 2μm particle size x 150mm (Thermo Scientific) and separated with a linear gradient of water / acetonitrile / 0.1% formic acid (v / v). Data Analysis Protein identification and quantification were performed using ProteinPilot™ 5.0 (Sciex). MS / MS spectra were searched against the human SwissProt database. Search parameters were: Sample type: iTRAQ 4plex (peptide labelled); Cys alkylation: MMTS; Digestion: Trypsin; Instrument: Orbi MS and Orbi MS / MS; Special factors: None; Species: Homo sapiens; Quantitate 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. All identified proteins had an Unused Protscore of > 1.3 (which corresponds to proteins identified with > 95% confidence) and a global false discovery rate (FDR) of < 0.1% determined at the protein level using the software’s PSPEP algorithm. Proteins found to be differentially expressed in either Endometriosis Diagnosis group vs Healthy Control group and / or Endometriosis Diagnosis group vs Symptoms, No Diagnosis group were considered as candidate biomarkers for endometriosis only if they were not differentially expressed in the Symptoms, No Diagnosis group vs Healthy Control group. To clarify, if proteins were differentially expressed between the Symptoms, No Diagnosis group vs Healthy Control group they were not considered as biomarkers for endometriosis. Primary selection criteria were established and applied where differentially expressed proteins were required to have at least two unique peptides with confidence >95% and have significantly different protein ratios (fold change of > 10%) in at least one replicate from both of the cohorts (P value of 0.05, as calculated by the software). Secondary selection criteria were also established and applied to select further biomarkers to widen the pool of potential candidates. To be considered as a secondary candidate a protein needed to fulfill 2 rules across both cohorts. Firstly, one of the two cohort data points must meet the primary selection criteria. The second data point from the other cohort must have either 2 fold change in protein abundance with at least 2 high confidence peptides (> 95%) or have a P Value of 0.1 with the fold change > 10% and with at least 2 high confidence peptides (> 95%). 2. Results Using two independent cohorts, patients with endometriosis diagnosed by laparoscopy were compared to a) patients with symptoms only, and b) a healthy control group with no pelvic symptoms. The proteome coverage for each of the four experiments outlined in Figure 1 is shown in Table A. Table A. Proteome Coverage (Proteins Identified) in four experiments Identifications are at the 95% confidence level. The selection criteria described in the data analysis section were applied to the protein identification and quantitation output and a list of candidate biomarkers were determined to be significant by comparing protein expression in the Endometriosis Diagnosis versus Healthy Controls and the Endometriosis Diagnosis versus Symptoms, No Diagnosis. Further analysis of these candidate biomarkers involved: averaging significant fold changes across replicates for each protein; Significant P values are shown as less than the least significant P value for that protein across the replicates. The number of data points used for the averaging has been included (out of 4 possible replicates). Biomarkers were then ranked by P value, (and if equal) then Significant data points, (and if equal) then Fold change. The outcome of this further analysis resulted in the biomarkers set out in the Tables B and C.

[0003] Table B. Endometriosis Diagnosis vs Healthy Controls Summary Data Table C. Endometriosis Diagnosis vs Symptoms (no Diagnosis) Summary Data Example 2 – Further characterisation of endometriosis biomarkers 1. Materials / Methods Analytical validation For analytical validation, targeted mass spectrometry assays using multiple reaction monitoring (MRM) were defined for each candidate biomarker from Example 1. Each assay measured changes in relative peptide abundances of individual plasma samples against an18O-labeled reference plasma to calculate peak area ratios for each of the biomarkers. These ratios were normalized to the median value for each peptide. The analytical targeted assay was designed utilizing the following method. Each plasma sample was immunodepleted (removal of top 14 abundant proteins) before diafiltration, reduction, alkylation, and digestion of the plasma proteins. The reverse phase desalted sample was then injected along with a fixed amount of the internal standard18O labelled reference plasma digest onto a microflow (5 μl / min) HPLC system and analyzed on a Sciex 6500 Triple Quad mass spectrometer (Sciex, USA). Assays were assessed for robustness with analytical validation considered successful if the MRM signal for each peptide was individually verified to be unique and where the signal to noise (S / N) was >3. Clinical validation In clinical validation, a cohort comprising individual samples (n = 464 endometriosis cases, n = 132 symptomatic controls, and n = 153 general population controls) was measured using the analytically validated targeted MRM mass spectrometry assay. Samples were randomized across plates before analysis to minimize batch effects and ensure consistency. Analysis of the mass spectrometry data was carried out in Skyline software (University of Washington, USA) with both unlabelled and18O labelled peptide peaks, integrated with peak areas exported to enable calculation of the relative peak ratios. Statistical and data analysis The peptide data presented reflect the relative concentration of a protein biomarker between samples. To maximize the likelihood of identifying biomarkers for the disease, changes in protein concentration were initially assessed at the extremes of the disease spectrum, for example, symptomatic controls versus severe endometriosis or general population controls versus endometriosis. To improve the normality of the data, a natural logarithmic transformation was applied to all measurements. Candidate biomarkers were confirmed in bivariate analysis by comparing medians between groups using the Mann–Whitney U test on Ln- transformed peptide ratio values. Both unadjusted and multiple testing-adjusted p- values are reported to account for potential false positives. Adjusted p-values were calculated using the Benjamini-Hochberg procedure to control the false discovery rate. To evaluate the diagnostic relationship between clinical characteristics, biomarker concentration, and clinical groups, elasticnet logistic regression modeling was employed (R Statistical Software, v4.2.2; R Core Team, 2021). Clinical variables for inclusion in the models were restricted to age and BMI due to practical usability and accessibility. Repeated or nested cross-validation was performed (glmnet package v4.1-6; caret v6.0-93; nestedcv.glmnet package v0.7.4). During the nested cross validation approach, variables were filtered using a Mann–Whitney U test with a significance threshold of 0.2. A series of multivariate logistic regression models containing both clinical factors and biomarker concentrations were developed to distinguish: (i) endometriosis cases from general population controls and (ii) endometriosis cases (stages II–IV) from symptomatic controls. To further evaluate the complex interactions and non-linear relationships between predictors, a random forest classifier was employed using the predictors identified during elastic-net logistic regression modeling. This third model was constructed by comparing stage IV endometriosis and symptomatic controls. The random Forest package v4.6-14 was used with 5-fold cross-validation and hyper-parameter tuning (mtry = 2, 3, 4, ntree = 100). Only participants with complete data were included in each model. To assess the discriminative performance of each model, the area under the receiver operating characteristic curve (AUC) was assessed. DeLong’s test was used to compare the AUC between biomarker models with and without clinical variables. The optimal predicted probability threshold was determined at the maximum Youden Index. Diagnostic performance metrics were computed based at this optimal threshold, including sensitivity (Sn) and specificity (Sp), and positive predictive value (PPV) and negative predictive value (NPV). 2. Results Biomarker measurement Targeted mass spectrometry assays were built against all biomarkers identified in Example 1, and well-defined assays were successfully developed for 39 biomarkers, plus 12 putative biomarkers taken from the literature. Analytical validation was successful if analytically acceptable levels of reproducibility and signal to noise were achieved. For the clinical validation phase, 51 protein biomarkers were analyzed. During two-way comparisons using a Mann–Whitney U-test, significant (P 0.05) differences were observed for 41 of the 51 candidate proteins across one or both clinical group comparisons. Ten protein biomarkers were found to be independently associated with endometriosis after adjusting for age and BMI. Specifically, the Mann– Whitney U test results for Hemoglobin subunit beta and Inter-alpha-trypsin inhibitor light chain are shown in Table D with associated Boxplots in Figures 2A and 2B. Table D For the data in Table D, non-parametric Mann–Whitney U tests were performed to compare Ln-transformed peptide ratio values between groups. Both unadjusted and multiple testing-adjusted p-values are reported to account for potential false positives. Adjusted p-values were calculated using the Benjamini-Hochberg procedure to adjust for multiple testing and reduce false-positive findings. These biomarkers were assessed for any correlation with the other available clinical information (e.g. menstrual cycle length), and no significant strong or moderate correlations were observed (maximum correlation coefficient of 0.26). Model development and validation Regression models were developed to discriminate between endometriosis cases and the general population (Model 1) or symptomatic controls (Model 2), as shown in Table E. Table E - Protein associations: bivariate analysis (Mann Whitney U test) versusmultivariate modeling (logistic regression) Model 1: endometriosis versus general population; Model 2, endometriosis (stages II-IV) versus symptomatic controls; Model 3, endometriosis (stage IV) versus symptomatic controls. A random forest model (Model 3) was subsequently developed using the same biomarkers as Model 2 and constructed by comparing severe endometriosis and symptomatic controls, before being applied to all stages of endometriosis. For each model, the predicted probabilities for an endometriosis diagnosis were significantly higher (P < 0.0001) in the endometriosis group compared to the general population and symptomatic control groups. Table F and the receiver operating characteristic (ROC) curves in Figure 3 compare the outcomes predicted by the models against the observed diagnosis of endometriosis, along with the performance metrics (AUC, Sn, Sp, PPV, NPV) for each model. Table F. Predicted versus observed diagnosis of endometriosis and performance metrics Three of the 10 protein biomarkers demonstrated excellent utility in distinguishing between the two clinical groups in Model 1 (AUC = 0.993, 95% CI 0.988–0.998) compared to age and BMI alone (P < 0.001). In Model 2, age and BMI were significant independent associates of endometriosis (stages II–IV) (AUC = 0.649, 95% CI 0.589– 0.709). After adjusting for age and BMI, the remaining seven biomarkers provided significant incremental value to Model 2 (AUC = 0.729, 95% CI 0.676–0.783, P < 0.01). The same seven biomarkers demonstrated significant diagnostic accuracy in Model 3, with an AUC of 0.997 (95% CI 0.994–1.000) for discriminating stage IV endometriosis from symptomatic controls. Critically for clinical usage, Model 3 also showed strong diagnostic performance when applied to all stages of endometriosis (AUC for stage I: 0.852 (95% CI 0.811–0.893); stage II: 0.903 (95% CI 0.853–0.953); stage III: 0.908 (95% CI 0.852–0.965); stage IV: 0.997 (95% CI 0.994–1.000), respectively) (Figure 4).

Claims

AMENDED CLAIMS received by the International Bureau onCLAI MS 14 November 2025 (14.11.2025)1 . A method comprising the steps of:(a) assessing an expression level of at least one protein in a sample from a subject, and(b) using the expression level to determine whether the subject has endometriosis; wherein the at least one protein comprises inter-alpha-trypsin inhibitor light chain.

2. The method according to claim 1 wherein the at least one protein further comprises at least one protein selected from the list consisting of: Hemoglobin subunit beta, Neuropilin-1 , Serum paraoxonase / arylesterase 1 , Complement factor H-related protein 2, Vitamin K-dependent protein S, Afamin, Selenoprotein P, Complement component C9, Coagulation factor XII, and Heparin cofactor 2.

3. The method according to claim 1 or 2 wherein the at least one protein comprises two, three, four or five proteins.

4. The method according to any one of the preceding claims wherein the step (a) comprises at least one of spectrometry, such as mass spectrometry, surface enhanced Raman spectroscopy, flow cytometry, ELISA, protein arrays including mass-sensing BioCD protein array, protein micro-arrays, quantum dots based detection, electrochemical immunoassay, gel electrophoresis, 9G DNA technology, nanoparticles including lanthanide chelates such as europium EuNPs and gold nanoparticles, immune-affinity mass spectrometry and immune capture mass spectrometry.

5. The method according to any one of the preceding claims wherein step (a) comprises multiple reaction monitoring (MRM) mass spectrometry or selective reaction monitoring (SRM) mass spectrometry.

6. The method according to any one of the preceding claims wherein step (a) comprises assessing the expression level of the at least one protein by assessing the amount of a fragment or peptide of the at least one protein.

7. The method according to any one of the preceding claims wherein step (a) comprises quantifying the expression level of the at least one protein.

8. The method according to any one of the preceding claims 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 without endometriosis.

9. The method according to any one of the preceding claims wherein step (a) comprises labelling the at least one protein.

10. The method according to any one of the preceding claims wherein the sample comprises a biological sample and / or sub-samples thereof.11 . The method according to claim 10 wherein the biological sample is a body fluid.

12. The method according to claim 11 wherein the body fluid is blood, serum, plasma, urine, sweat, tears, saliva, sputum, or any combination or fraction thereof.

13. The method according to any one of the preceding claims wherein step (b) comprises comparing the expression level from step (a) with a reference value indicative of endometriosis.

14. Use of inter-alpha-trypsin inhibitor light chain to determine whether a subject has endometriosis.

15. A test comprising:(a) means for obtaining an expression level of inter-alpha-trypsin inhibitor light chain, in a sample from a subject; and(b) means for processing the expression level generated in step (a) to determine whether the subject has endometriosis.

16. The test according to claim 15 wherein the means for obtaining an expression level of at least one protein is a mass spectrometer.

17. A test system comprising:(a) a means for obtaining a test result from a sample from a subject indicative of an expression level of inter-alpha-trypsin inhibitor light chain; and(b) means for correlating the test result with a risk of the subject having endometriosis.

18. The test system according to claim 17 further comprising:(c) a means for collecting, storing, processing and / or tracking the test result.

19. Use of inter-alpha-trypsin inhibitor light chain, and optionally at least one protein selected from the list consisting of Hemoglobin subunit beta, Neuropilin-1 , Serum paraoxonase / arylesterase 1 , Complement factor H-related protein 2, Vitamin K- dependent protein S, Afamin, Selenoprotein P, Complement component C9, Coagulation factor XII, and Heparin cofactor 2, as a biomarker for endometriosis.

20. A method of assessing an endometriosis intervention in a subject, the method comprising the steps of:(a) applying the intervention to the subject;(b) assessing an expression level of inter-alpha-trypsin inhibitor light chain, in a sample from the subject; and(c) using the expression level to determine the effect of the intervention on the subject.

21. Use of inter-alpha-trypsin inhibitor light chain, and optionally at least one protein selected from the list consisting of Hemoglobin subunit beta, Neuropilin-1 , Serum paraoxonase / arylesterase 1 , Complement factor H-related protein 2, Vitamin K- dependent protein S, Afamin, Selenoprotein P, Complement component C9, Coagulation factor XII, and Heparin cofactor 2, as a target for a therapeutic agent for endometriosis.

22. A method of treating endometriosis in a subject having an expression level of inter- alpha-trypsin inhibitor light chain indicative of endometriosis, the method comprising the step of administering a therapeutically effective amount of a treatment for endometriosis to the subject.

23. The method of claim 22 wherein the treatment for endometriosis is selected from the list consisting of: an analgesic, a hormone, an antioxidant, an aramotase inhibitor, a surgical intervention, an alternate therapy.

24. The method of claim 23 wherein the analgesic is selected from the list consisting of: NSAIDs, naproxen, ibuprofen, mefenamic acid and ketoprofen.

25. The method of claim 23 wherein the hormone is selected from the list consisting of: a contraceptive, a progestin, dienogest, a progestogen, etonorgestrel, a gonadotropin-releasing hormone (“GnRH”) agonist, leuprolide, leuprorelin, triptorelin, goserelin, nafarelin, buserelin, a GnRH antagonist, elagolix, linzagolix, opigolix, relugolix, selective estrogen receptor modulators, raloxifene, aromatase inhibitors, letrozole, anastrozole, selective progesterone receptor modulators, tibolone, mifepristone, gestrinon and ulipristal.

26. The method of claim 23 wherein the antioxidant is selected from the list consisting of: resveratrol and curcumin.

27. The method of claim 23 wherein the aromatase inhibitor is puerarin.

28. The method of claim 23 wherein the surgical intervention is selected from the list consisting of: a laparoscopy, excision of lesion(s), ablation of lesion(s), a laparotomy, a hysterectomy and a nerve interruption.

29. The method of claim 23 wherein the alternate therapy is selected from the list consisting of: physiotherapy, yoga, virtual reality delivered exercise, psychotherapy, somatosensory stimulation, acupuncture, moxibustion, mindfulness, dietary supplements and cannabis based medicines.

30. A method of preventing endometriosis in a subject having an expression level of inter-alpha-trypsin inhibitor light chain indicative of an increased risk of developing endometriosis, the method comprising the step of administering a therapeutically effective amount of a preventative treatment for endometriosis to the subject.31 . The method of claim 30 wherein the preventative treatment is selected from the list consisting of: a hormonal contraceptive such as a birth control pill, patch, or a vaginal ring that contains estrogen and progestin to help regulate or suppressmenstruation; progestin therapy; anti-inflammatory diet including omega-3 rich fatty acids, fruit, vegetables and whole grains; and regular exercise.

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