Serum EphA1 as biomarker of endometriosis

Abstract

The present invention relates to a method of diagnosing whether a subject has endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or associated with uterine / pelvic pathology; methods related to determining the therapeutic effect of a treatment regimen for endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology; and a method of monitoring the progression of endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject by determining the amount or concentration of EphA1 in a sample of the subject and comparing the determined level to a reference value.

Description

[0001] The present invention relates to a method of diagnosing endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject; a method of assessing the risk of endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject; a method relating to determining the effect of a treatment on a treatment regimen for endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology; and a method of monitoring the progression of endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject, by determining the amount or concentration of EphAl in a sample of the subject and comparing the determined level to a reference value. The present invention particularly relates to the early stage of endometriosis. BACKGROUND

[0002] Endometriosis is a chronic condition defined by the growth of endometrial glands and stromal-like lesions outside the uterus (Liu et al., 2011). The lesions can be peritoneal lesions, superficial implants or cysts on the ovary, or deep infiltrating disease. It originates from eutopic endometrial cells characterized by increased proliferation and adhesion properties (Liu et al., 2011). The increase in cell viability in eutopic endometrium is the result of a decrease in apoptosis and an increase in cell proliferation (Johnson et al., 2005). Endometriosis affects 5-8% of all women of reproductive age and 70% of women with chronic pelvic pain. It is estimated that 176 million women worldwide suffer from endometriosis (Adamson et al. J Endometr. 2010; 2: 3-6). For many of these women, the diagnosis of endometriosis is often delayed, resulting in unnecessary suffering and a decrease in quality of life. There is a 7-10 year delay in the diagnosis of endometriosis in patients aged 18-45. Since most women with endometriosis report the onset of symptoms during adolescence, early referral, diagnosis, disease identification and treatment can reduce pain, while it is also possible to prevent disease progression. Barriers to early diagnosis include the invasiveness of current diagnostic techniques such as laparoscopy, the high cost of diagnosis and treatment of adolescent patients, and the presentation of mixed symptoms such as cyclic and non-cyclic pain (Parasar et al. Curr Obstet Gynecol Rep. 2017; 6: 34-41).

[0003] The gold standard for diagnosing endometriosis is laparoscopic visualization and subsequent histological confirmation. To date, there is no universal non-invasive method for diagnosing endometriosis, in particular early, mild and minimal endometriosis (revised American Society for Reproductive Medicine rASRM stages I-II; Hsu et al. Clin Obstet Gynecol 2010: 53: 413-419). During diagnostic laparoscopy, gynecologists who have received training and skills in laparoscopic surgery for endometriosis should perform a systematic examination of the pelvis (NICE guideline NG73, 2017, ESHRE Guideline Endometriosis Human Reproduction Open, 2022). Surgical visualization requires good expertise, training and skills to make a reliable diagnosis. The fact that laparoscopic surgery is required for diagnosis is something that physicians would like to avoid as much as possible and is a major cause of diagnostic delay of 7-10 years. The lack of non-invasive diagnostic tests significantly contributes to the long delay between the onset of symptoms and the definitive diagnosis of endometriosis (Signorile and Baldi. J Cell Physiol 2014; 229: 1731-1735). Therefore, there is an unmet medical need for non-invasive tests for diagnosing endometriosis, in particular for diagnosing early, mild and minimal endometriosis (revised American Society for Reproductive Medicine rASRM stages I-II).

[0004] Non-invasive diagnosis of endometriosis would allow earlier diagnosis and treatment, potentially improving quality of life and reducing the social costs associated with endometriosis and was therefore selected as a research priority by the World Endometriosis Society (WES) and the World Endometriosis Research Foundation (WERF) (Fassbender et al., Springer, Peripheral Blood Biomarkers for Endometriosis. 2017). Therefore, non-invasive tools for diagnosing endometriosis could facilitate earlier diagnosis and intervention, ultimately improving quality of life and preserving fertility (Parasar et al. Curr Obstet Gynecol Rep. 2017; 6: 34-41).

[0005] Blood-based biomarkers are crucial to help reduce time delay in diagnosis of endometriosis requiring laparoscopy. CA-125 is one of the most commonly used blood biomarker, however, its diagnostic utility is limited to endometriosis rASRM stage III and IV (Nisenblat et al., Cochrane Database of Systematic Reviews. 2016; 5: CD012179). Therefore, the use of CA-125 is not recommended in clinical guidelines for the diagnosis of endometriosis (ESHRE Guideline Endometriosis Human Reproduction Open, 2022).

[0006] The same applies to similar conditions, such as uterine and pelvic pathologies and neuropathic pain associated with endometriosis and / or uterine / pelvic pathologies.

[0007] Neuropathic pain associated with endometriosis and / or uterine / pelvic pathologies is a component of endometriosis-associated pain (Coxon et al., Is there a Neuropathic-Like Component to Endometriosis-Associated Pain? Results From a Large Cohort Questionnaire Study, Front Pain Res (Lausanne), 2021; 2: 743812; doi: 10.3389 / fpain.2021.743812).

[0008] The Ephrin (Eph) receptor family includes the largest family of receptor tyrosine kinases and can be divided into two groups based on their structure and receptor-ligand specificity. EphA is composed of nine type A Eph receptors (EphAl-8 and EphAlO) and five type B Eph receptors (EphBl-4, 6). Eph receptors bind to Ephrin ligands, which are also divided into Ephrin-A (Ephrin-Al-6) and Ephrin-B (Ephrin-B1-3) based on their structure. Binding of Ephrins to Eph receptors activates a signaling cascade that modulates several biological processes such as cell proliferation, differentiation, migration, angiogenesis, and vascular remodeling. There is evidence that Ephrins and Eph receptors are involved in the regulation of folliculogenesis, ovulation, embryo transport, implantation, and placenta formation (Adu-Gyamfi et al., Biology of Reproduction, 2021).

[0009] In particular, Ephrin type-A receptor 1 (EphAl) is a receptor tyrosine kinase that binds to Ephrin-A family ligands residing on adjacent cells. Both EphAl and Ephrin-A undergo membrane binding, where their binding occurs through direct cell-cell interactions, resulting in contact-dependent bidirectional signaling into the adjacent cells. The forward signaling occurs in the cells expressing the Eph receptor, while the reverse signaling occurs in the cells expressing the Ephrin.

[0010] Dysregulated activation of EphA family members is found in various human cancers, such as lung cancer, gastric cancer, hepatocellular carcinoma, esophageal squamous cell carcinoma, and prostate cancer. There is evidence to indicate that EphA receptors are involved in regulating tumor growth, invasiveness, angiogenesis, and metastasis by altering cell proliferation, motility, invasion, and migration (Zhang X. Front Oncol. 2021; 11: 619949).

[0011] Fujii et al. (Hum Reprod 2011; 26: 299-306) have shown by RT-PCR that mRNA for EPHA1 is expressed in endometrial epithelial cells in part during the proliferative and secretory phases of the menstrual cycle. It was demonstrated by immunohistochemistry that the EphAl protein is expressed on the luminal surface and glandular epithelial cells of the endometrium.

[0012] The levels / presence of biomarkers can differ when measured in tissue or serum. For example, complement component C7 and complement component C4 were shown to be overexpressed in ectopic endometrium of women with endometriosis compared to eutopic endometrium of control women without endometriosis (Ahn et al. Fertil Steril 2016; Eyster et al. Fertil Steril 2007). However, neither serum complement component C7 protein nor complement component C4 protein was found to be increased in circulating blood (Hever et al. PNAS 2007). Brain-derived neurotrophic factor (BDNF) mRNA expression levels were higher in ovarian endometriotic lesions than in eutopic endometrium (Wang et al. Journal of Ovarian Research 2022). However, serum BDNF was not significantly different in women with endometriosis compared to control women without endometriosis (Perricos et al. Exp Biol Med (Maywood) 2018). Also, in breast cancer, while molecular markers such as CEA (O), ERp, CK19 and c-Myc have been observed to be significantly different between normal humans and patients’ blood, these markers were not significantly different in tissue samples.

[0013] Therefore, locally altered expression of biomarkers (mRNA or protein expression) in tissue does not translate 1 : 1 into significantly different levels of these biomarkers in circulating blood.

[0014] There is a strong need for non-invasive diagnosis of endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology by using biomarkers, which allows for a reliable and early risk assessment and / or identification of women showing signs and symptoms of endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0015] Therefore, the present application provides tools and methods that meet these needs. SUMMARY

[0016] In a first aspect, the present application relates to a method for assessing endometriosis in a subject, the method comprising the steps of:

[0017] a) determining the level of the extracellular protein fraction of EphAl in a biological fluid sample from the subject,

[0018] b) comparing the level of the extracellular protein part of EphAl to at least one appropriate reference value for the level of the extracellular protein part of EphAl,

[0019] c) identifying the subject as having endometriosis if the comparison in step b) indicates that the subject has a decreased level of the extracellular protein part of EphAl compared to the appropriate reference value,

[0020] wherein the stage of endometriosis of the subject is classified as stage I or stage II endometriosis according to the revised American Society for Reproductive Medicine scoring system (r-ASRM).

[0021] In a second aspect, the present application relates to a method for assessing a uterine / pelvic pathology in a subject, the method comprising the following steps:

[0022] a) determining the level of the extracellular protein part of EphAl in a biological fluid sample from the subject,

[0023] b) comparing the level of the extracellular protein part of EphAl to at least one appropriate reference value for the level of the extracellular protein part of EphAl,

[0024] c) identifying the subject as having a uterine / pelvic pathology if the comparison in step b) indicates that the subject has a decreased level of the extracellular protein part of EphAl compared to the appropriate reference value,

[0025] In a third aspect, the present application relates to a method for assessing a endometriosis-related and / or uterine / pelvic pathology-related neuropathic pain in a subject, the method comprising the following steps:

[0026] a) determining the level of the extracellular protein part of EphAl in a biological fluid sample from the subject,

[0027] b) comparing the level of the extracellular protein part of EphAl to at least one appropriate reference value for the level of the extracellular protein part of EphAl,

[0028] c) identifying the subject as having a endometriosis-related and / or uterine / pelvic pathology-related neuropathic pain if the comparison in step b) indicates that the subject has a decreased level of the extracellular protein part of EphAl compared to the appropriate reference value,

[0029] wherein the stage of endometriosis in the subject is classified as stage I or stage II endometriosis according to the revised American Society for Reproductive Medicine scoring system (r-ASRM).

[0030] In a fourth aspect, the present application relates to a method for monitoring the progression of endometriosis, the progression of uterine / pelvic pathology, and / or the progression of neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject, the method comprising the following steps:

[0031] i. determining the level of the extracellular protein fraction of EphAl in a biological fluid sample from the subject according to the method steps a) to b) of the first aspect,

[0032] ii. repeating step i. using a biological fluid sample obtained from the subject during or after the treatment for a specific time interval; and

[0033] iii. comparing the level of the extracellular protein fraction of EphAl identified in i. with the level of the extracellular protein fraction of EphAl identified in ii., wherein a change in the level of the extracellular protein fraction of EphAl from i. to ii. is indicative of a change in the progression of endometriosis, the progression of uterine / pelvic pathology, and / or the progression of neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in the subject,

[0034] wherein the stage of endometriosis in the subject is classified as stage I or stage II endometriosis according to the revised American Society for Reproductive Medicine scoring system (r-ASRM).

[0035] In a fifth aspect, the present application relates to a method for determining the effect of a treatment on a treatment regimen for endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject, the method comprising the following steps:

[0036] i. determining the level of the extracellular protein fraction of EphAl in a biological fluid sample from the subject according to the method steps a) to b) of the first aspect,

[0037] ii. repeating step i. using a biological fluid sample obtained from the subject during or after the treatment for a specific time interval; and

[0038] iii. comparing the level of the extracellular protein part of EphAl identified in i. with the level of the extracellular protein part of EphAl identified in ii. and identifying the treatment regimen as having a therapeutic effect if the level of the extracellular protein part of EphAl is increased after the treatment,

[0039] wherein the subject's endometriosis is classified as stage I or stage II endometriosis according to the revised American Society for Reproductive Medicine scoring system (r-ASRM).

[0040] In a sixth aspect, the present application relates to a computer-implemented method for assessing a patient suspected of having endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology, the computer-implemented method comprising the following steps:

[0041] a) receiving a value for the level of a first biomarker in a biological fluid sample of a subject, the first biomarker being the extracellular protein part of EphAl,

[0042] b) receiving a value for the level of a second biomarker in the sample of the subject, wherein the second biomarker is CA125,

[0043] c) receiving a value for the level of dysmenorrhea according to VAS and / or lower abdominal pain according to VAS,

[0044] d) comparing the value for the level of steps (a) to (c) to reference values for the biomarkers and the amount of dysmenorrhea and / or calculating a score for assessing a patient suspected of having endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology based on the levels of the biomarkers and the amount of dysmenorrhea; and

[0045] e) assessing the patient based on the comparison and / or calculation made in step (d),

[0046] wherein the subject's endometriosis is classified as stage I or stage II endometriosis according to the revised American Society for Reproductive Medicine scoring system (r-ASRM). BRIEF DESCRIPTION OF DRAWINGS

[0047] Embodiments of the present application will be further described below with reference to the accompanying drawings.

[0048] Box plot analysis EphAl: EphAl in serum samples of control and endometriosis cases (rASRM stage I endometriosis, rASRM stage II endometriosis, rASRM stage III endometriosis and rASRM stage IV endometriosis). Serum EphAl peptide levels were measured using advanced mass spectrometry-based proteomics techniques.

[0049] Receiver operating characteristic (ROC) analysis is shown, which describes the area under the curve (AUC) of the ROC analysis and the associated 95% confidence interval. N indicates the number of samples tested (cases plus controls, the number in each group varies depending on the analyte).

[0050] Figure 1 : Box plot analysis: reduced serum EphAl levels in cases of symptomatic women with endometriosis and in women without endometriosis and with other uterine and pelvic pathologies (such as myoma, adenomyosis only, ovarian cysts requiring endometriosis, cancer) (“cases SOF”; SOF = symptomatic other findings) compared to controls without endometriosis and without other uterine and pelvic pathologies (“Ctrl SNF”; SNF = symptomatic no findings).

[0051] Figures 2a and 2b: Box plot analysis (a) and ROC analysis (b): reduced EphAl in serum samples of women with other uterine and pelvic pathologies (such as myoma, adenomyosis only, ovarian cysts requiring surgery, cancer) (“cases SOF”; SOF = symptomatic other findings) compared to control women without endometriosis and without uterine and pelvic pathologies (“Ctrl”).

[0052] Figures 3a and 3b: Box plot analysis (a) and ROC analysis (b): reduced EphAl in serum samples of women with early stage endometriosis rASRM I / II (“cases”) compared to control women without endometriosis and without uterine and pelvic pathologies (“Ctrl”).

[0053] Figures 4a and 4b: Box plot analysis (a) and ROC analysis (b): EphAl reduction in serum samples of women "cases" with early endometriosis rASRM stage I compared to women without endometriosis and without uterine and pelvic pathology ("Ctrl").

[0054] Figures 5a and 5b: Box plot analysis (a) and ROC analysis (b): EphAl reduction in serum samples of women "cases" with early endometriosis rASRM stage II compared to women without endometriosis and without uterine and pelvic pathology ("Ctrl").

[0055] Figures 6a and 6b: Box plot analysis (a) and ROC analysis (b): EphAl reduction in serum samples of women "cases" with endometriosis stage III compared to control women without endometriosis and without uterine and pelvic pathology ("Ctrl").

[0056] Figures 7a and 7b: Box plot analysis (a) and ROC analysis (b): EphAl reduction in serum samples of women "cases" with endometriosis rASRM stage IV compared to controls ("Ctrl") without endometriosis and without uterine and pelvic pathology.

[0057] Figures 8a and 8b: Box plot analysis (a) and ROC analysis (b) of CA-125. Comparison with CA-125 levels measured in the same sample set as for EphAl using Roche Elecsys CA-125 immunoassay method. CA-125 in serum samples of women "cases" with early endometriosis rASRM stage I compared to controls ("Ctrl") without endometriosis and without uterine and pelvic pathology.

[0058] Figures 9a and 9b: Box plot analysis (a) and ROC analysis (b) of CA-125 in serum samples of women "cases" with early endometriosis rASRM stage II compared to controls ("Ctrl") without endometriosis and without uterine and pelvic pathology.

[0059] Figure 10: Structure of the EphA1 protein. The extracellular domain contains ephrin binding domains, a cysteine-rich region (including Sushi domain and EGF-like domains) and fibronectin type III repeats (FN1 and FN2). The transmembrane domain (TM) connects the extracellular and intracellular domains. The intracellular domain consists of a juxtamembrane domain, a kinase domain and a SAM domain. DETAILED DESCRIPTION

[0060] Unbiased proteomic findings were performed in serum samples of women with endometriosis or other uterine pelvic pathologies and symptoms (pain symptoms such as dysmenorrhea, pelvic pain, dyspareunia, dysuria, dyschezia, other menstrual cycle dependent pain symptoms) as well as women without endometriosis (controls). Details of the unbiased proteomic findings using advanced mass spectrometry (MS) based proteomics, including an automated sample preparation procedure, are described in Example 4 - Materials and Methods.

[0061] The present invention is based on the surprising finding that reduced levels of EphA1 in serum, in particular of the extracellular protein portion of EphA1, are associated with endometriosis, uterine / pelvic pathologies, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathologies. Using serum from women with endometriosis (cases) and controls (without endometriosis), peptides of the detected extracellular protein portion of EphA1 were identified using advanced mass spectrometry proteomics technology. In total, seven peptides of EphA1 were detected using this mass spectrometry technology, all of which belong to the extracellular protein portion of EphA1 (Table 1). The structure of the EphA1 protein is shown in Figure 10. EphA1 levels were reduced in all rASRM stages I, II, III and IV, which confers diagnostic potential of serum EphA1 for early detection of endometriosis.

[0062] The present inventors show for the first time that measured EphA1 in serum is reduced in women with endometriosis, uterine / pelvic pathologies, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathologies compared to controls.

[0063] There is an unmet medical need for a reliable, non-invasive test for the diagnosis and / or classification of endometriosis (particularly early endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with and / or uterine / pelvic pathology). EphA1 offers the advantage of a non-invasive, blood-based test that identifies women with early endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with and / or uterine / pelvic pathology.

[0064] The inventors have investigated the levels of EphA1 in serum obtained from women with endometriosis. Surprisingly, they found that reduced EphA1 levels could be detected in serum samples from women with endometriosis. In particular, the fact that reduced EphA1 levels were detected in women with early-stage endometriosis makes this biomarker a useful tool for the early diagnosis of endometriosis. Therefore, the ability to determine the levels of EphA1 in such biofluids may be useful for endometriosis risk stratification, diagnosis, prognosis, and patient stratification for treatment.

[0065] The data presented in this article demonstrate that determining serum EphA1 levels, particularly the extracellular protein portion of EphA1, provides a means of diagnosing endometriosis and risk stratification for those with endometriosis. This also allows for monitoring endometriosis progression and / or evaluating treatment options. In particular, the data suggest the potential for early diagnosis of endometriosis by determining EphA1 levels.

[0066] The data presented in this article also show that determining serum EphA1 levels provides a more accurate means of detecting early-stage endometriosis and control samples than CA-125.

[0067] The inventors have investigated the levels of EphA1 in serum obtained from women with uterine / pelvic pathology. Surprisingly, they found reduced EphA1 levels in serum samples from women with uterine / pelvic pathology. This makes this biomarker a useful tool for the early diagnosis of uterine / pelvic pathology. Therefore, the ability to determine the level of EphA1 in such biofluids may be useful for risk stratification, diagnosis, prognosis, and patient stratification for treatment of uterine / pelvic pathology.

[0068] The data presented in this article demonstrate that determining serum EphA1 levels, particularly the extracellular protein fraction of EphA1, provides a means of diagnosing uterine / pelvic pathology and risk stratification for those with uterine / pelvic pathology. This also allows for monitoring the progression of uterine / pelvic pathology and / or evaluating treatment options.

[0069] Definitions

[0070] The word “comprise” and its variants such as “comprises” and “comprising” should be understood to imply the inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0071] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” “the,” and “the” include the plural referents.

[0072] Concentration, level, amount, and other numerical data may be expressed or presented in the “range” format herein. It should be understood that this range format is used solely for convenience and brevity, and therefore should be flexibly interpreted to include not only the values ​​explicitly listed as range limits, but also all individual values ​​or subranges covered by the range, as if each value and subrange were explicitly listed. For example, the numerical range “150 mg to 600 mg” should be interpreted to include not only the explicitly listed value of 150 mg to 600 mg, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 150, 160, 170, 180, 190, ... 580, 590, 600 mg and subranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. The same principle applies to ranges that cite only one value. Furthermore, this interpretation applies regardless of the breadth of the range or characteristic stated.

[0073] When used in conjunction with numerical values, the term “about” means to cover a range of values ​​that has a lower limit of 5% less than the indicated value and an upper limit of 5% greater than the indicated value.

[0074] Generally, the described method is an in vitro method using a sample already obtained from the subject (i.e., providing a sample for the method, and the steps taken to obtain the sample from the subject are not included as part of the method). Therefore, the method may include the step of providing a biofluid sample from the subject.

[0075] As used herein, “providing,” “obtaining,” or “acquiring” can mean any means of acquiring a sample by “direct” or “indirect” means. Directly obtaining a sample means obtaining it through processing (e.g., performing physical methods such as extraction). Indirectly obtaining a sample means receiving it from another party or source (e.g., a third-party laboratory that directly obtained the sample).

[0076] The methods described herein include providing biological fluid samples (e.g., blood samples) from subjects. Samples tested in the methods described herein are also referred to as "test samples".

[0077] As used herein, the terms “biological (fluid) sample,” “test sample,” and “sample” are used interchangeably, and variations thereof refer to a sample obtained from or derived from a subject. For the purposes described herein, a sample is or includes a biological fluid (also referred to herein as a body fluid) sample.

[0078] Examples of samples include, but are not limited to, fluid samples such as blood, serum, plasma, synovial fluid, interstitial fluid, capillary blood, peritoneal fluid, menstrual fluid, urine, saliva, and lymph. Sample analysis can be performed on a chemical basis. Chemical analysis includes, but is not limited to, detecting the presence or absence of a specific indicator or changes in the amount, concentration, or level of a specific indicator.

[0079] The samples are in vitro samples, which will be analyzed in vitro and will not be transferred back into the body.

[0080] Blood samples can be whole blood samples or processed blood samples, such as serum, plasma, etc. Methods for obtaining biological fluid samples (e.g., whole blood, serum, plasma, etc.) from subjects are well known in the art. For example, methods for obtaining blood samples from subjects are well known and include established techniques for venipuncture. The obtained blood samples can be further processed using standard techniques to obtain, for example, serum or plasma samples. Advantageously, methods for obtaining biological fluid samples from subjects are generally low-invasive or non-invasive.

[0081] A whole blood sample is defined as a blood sample drawn from the body without (substantially) removing any components such as platelets or plasma. In other words, the relative proportions of the components in a whole blood sample are substantially the same as those in the blood within the body. In this context, "substantially the same" allows for very small variations in the relative proportions of whole blood components, such as at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, etc. Whole blood comprises both the cellular and fluid components of blood. Therefore, a whole blood sample can also be defined as a blood sample containing (substantially) all of its cellular components in plasma, where the cellular components (i.e., the blood includes at least the essential white blood cells, red blood cells, and platelets) are intact.

[0082] In a preferred example, the biofluid sample is serum.

[0083] Methods for analyzing (and optionally isolating, enriching or extracting) protein biomarkers from blood, plasma, serum, saliva and urine samples have been previously described, see, for example, Heitzer, E., Haque, IS, Roberts, CES et al. Current and future perspectives of liquid biopsies ingenomics-driven oncology. Nat Rev Genet 20, 71–88 (2019).

[0084] In the context of this invention, the term "biomarker" refers to a substance within a biological system that serves as an indicator of the biological state of said system. In the art, the term "biomarker" is sometimes also applicable to means of detecting said endogenous substance (e.g., antibodies, nucleic acid probes, imaging systems). In the context of this invention, the term "biomarker" should be applied only to substances, not to detection means. Therefore, a biomarker can be any kind of molecule present in a living organism, such as nucleic acids (DNA, mRNA, miRNA, rRNA, etc.), proteins (cell surface receptors, cytoplasmic proteins, etc.), metabolites or hormones (glucose, insulin, estrogen, etc.), a molecular characteristic of a modified form of another molecule (e.g., a sugar moiety or phosphoryl residue on a protein, a methyl residue on genomic DNA), or a substance already in vivo or a metabolite of such a substance. A biomarker is an organic biomolecule (e.g., protein, polypeptide, peptide, its isoforms, its immunologically detectable fragments, the corresponding nucleic acid molecule (e.g., mRNA, cDNA, etc.)) that is differentially present in samples taken from subjects with a disease compared to subjects without the disease. If the mean or median levels of a biomarker in different groups are calculated to be statistically relevant, then the biomarker is differentially represented. Common tests for statistical significance include, in particular, the t-test (e.g., Student's t-test), ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney, recipient operating characteristics (ROC curve), accuracy, and odds ratio. Biomarkers, alone or in combination, provide a measure of the relative risk of a subject belonging to one phenotypic state or another.

[0085] Therefore, they can be used as biomarkers for disease (diagnosis), drug treatment efficacy, and drug toxicity.

[0086] Typically, the biomarkers mentioned in this article are measured at the protein level.

[0087] The terms "EphA1," EPH receptor A1, or hepatocyte ligand A receptor 1, refer to the protein encoded by the EPHA1 gene in humans. This gene belongs to the hepatocyte ligand receptor subfamily of the protein-tyrosine kinase family. EPH and EPH-associated receptors have been involved in mediating developmental events, particularly in the nervous system. Receptors in the EPH subfamily typically have extracellular, transmembrane, and cytoplasmic portions. The extracellular portion contains a ligand-binding domain (hepatocyte ligand-binding domain), a cysteine-rich EGF-like motif, and two fibronectin type III repeat sequences. The cytoplasmic portion contains a protein kinase domain, a SAM domain, and a PDZ-binding domain. Based on the similarity of the extracellular domain sequences of hepatin receptors and their affinity for binding hepatin-A and hepatin-B ligands, hepatin receptors are divided into two groups (EphA and EphB receptors) (Adu-Gyamfi et al., Biology of Reproduction 2021, Darling and Lamb, Frontiers in Immunology 2019). The amino acid sequence of human EphA1 is accessible via UniProt (see UniProtKB – P21709·EPHA1_HUMAN). Three isoforms are described for EphA1 using UniProtKB – P21709-1, UniProtKB – P21709-2, and UniProtKB – P21709-3.

[0088] EphA1 is a receptor tyrosine kinase that binds to hepatin-A family ligands residing on neighboring cells. Both EphA1 and hepatin-A are membrane-bound, and their binding occurs through direct cell-cell interactions, resulting in contact-dependent bidirectional signaling to neighboring cells. Forward signaling occurs in cells expressing the Eph receptor, while inverse signaling occurs in cells expressing hepatin.

[0089] Using serum from women with endometriosis (cases) and controls (without endometriosis), advanced mass spectrometry proteomics was used to identify seven peptides of the detected EphA1 protein. The seven identified peptides described in Table 1 are all part of the extracellular portion of the EphA1 protein, comprising the ligand-binding domain (hepatin-binding domain) and fibronectin type III repeat sequence. The EphA1 protein consists of extracellular, transmembrane, and intracellular domains (Figure 10). The extracellular portion of the Eph receptor can be cleaved by proteolytic hydrolysis and released into the circulating blood (shedding of the extracellular portion of the Eph receptor). The role of soluble circulating EphA1 in immune cell transport was discussed.

[0090] Therefore, the determined level of the extracellular protein fraction of EphA1 represents the level of EphA1 in the sample. The terms “EphA1 level”, “level of the extracellular protein fraction of EphA1”, and “level of the extracellular protein fraction of EphA1” are used interchangeably in this document.

[0091] The methods described herein refer to "determining" the levels of one or more proteins. As will be apparent to those skilled in the art, the levels of one or more proteins are typically "determined" by measuring the levels of the protein in a sample. Therefore, the term "determined" may be replaced herein by the terms "measured" or "determined by measurement."

[0092] As used herein, the terms “determine” or “assess” also refer to assessing / determining whether a woman has endometriosis. Therefore, assessment / determination as used herein includes diagnosing endometriosis by determining the amount or concentration of EphA1 in a patient sample and comparing the determined amount or concentration to a reference; assessing the risk of a subject having endometriosis; selecting treatment for endometriosis; and monitoring patients with endometriosis or those undergoing treatment for endometriosis. Generally, assessment as referred to in this invention is an assessment of the presence of endometriosis.

[0093] Typically, the assessment referred to in this invention is the diagnosis of endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in the subject.

[0094] Typically, the assessment referred to in this invention is a stratification of the risk of a subject having endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0095] The terms “measurement,” “measuring,” or “determining” preferably include qualitative, semi-quantitative, or quantitative measurements.

[0096] Conventional "deterministic" methods may include sending clinical samples to a commercial laboratory to measure biomarker levels in biological fluid samples, or using commercially available assay kits to measure biomarker levels in biological fluid samples. Exemplary kits and suppliers will be readily apparent to those skilled in the art. In various instances, ELISA assays or lateral flow devices may be used to determine, detect, and / or quantify biomarkers, such as for field care use and for random colorimetric testing.

[0097] As used herein, the terms “level” or “amount” encompass the absolute amount of a biomarker as mentioned herein, the relative amount or concentration of said biomarker, and any value or parameter associated with or derived therefrom. Such values ​​or parameters include intensity signal values ​​derived from all specific physical or chemical properties obtained by direct measurement of the peptide, such as intensity values ​​in mass spectrometry or NMR spectra. Furthermore, all values ​​or parameters obtained by indirect measurement specified elsewhere in this specification are also covered, such as the response amount measured from a bioreading system in response to the peptide or the intensity signal obtained from a specifically bound ligand. It should be understood that values ​​associated with the foregoing amounts or parameters can also be obtained through all standard mathematical operations.

[0098] The level of a biomarker present in a biofluid sample can be determined, for example, by measuring the amount of a protein biomarker present in the sample. Measurements for determining the amount of a specific protein are well known in the art and include both direct and indirect measurements. The level of a protein biomarker in a sample can also be determined by determining the level of activity of the protein biomarker in the sample. Therefore, the term "protein level" encompasses both the amount of the protein itself and its activity level.

[0099] For example, the level of protein biomarkers in a biofluid sample can be determined (e.g., measured) by any suitable method and material known in the art, including processes selected from the group consisting of: mass spectrometry, immunoassays, enzyme assays, spectrophotometry, colorimetric assays, fluorescence assays, bacterial assays, protein microarrays, compound separation techniques, or other known techniques for determining the presence and / or quantity of an analyte. Examples of related techniques include enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, and lateral flow (using, for example, a lateral flow device (LFD) utilizing membrane-bound antibodies specific to protein biomarkers).

[0100] Preferably, the levels of protein biomarkers in the biofluid sample are measured by ELISA or lateral flow.

[0101] CA-125, or carbohydrate antigen 125, sometimes also called cancer antigen 125 or tumor antigen 125, is a mucin-type glycoprotein produced by the MUC16 gene and associated with the cell membrane. CA-125 is a biomarker for epithelial ovarian cancer, originating from coelomic epithelium, including the endometrium, fallopian tubes, ovaries, and peritoneum. The diagnostic use of CA-125 is limited to stage III and IV endometriosis (moderate to severe endometriosis) with moderate sensitivity.

[0102] The “symptoms” of a disease refer to the noticeable effects of the disease on the affected tissue, organ, or organism, and include, but are not limited to, pain, weakness, tenderness, strain, stiffness, and spasms in the tissue, organ, or individual. The “signs” or “symptoms” of a disease include, but are not limited to, changes or alterations, the presence, absence, increase, decrease, or decline of specific indicators such as biomarkers or molecular markers, or the development, presence, or worsening of symptoms. Symptoms of pain include, but are not limited to, unpleasant sensations that may be persistent or of varying degrees, such as burning, throbbing, itching, or stinging.

[0103] The terms “disease” and “illness” are used interchangeably in this text, referring to an abnormal condition, particularly an abnormal medical condition, such as illness or injury, in which an tissue, organ, or individual is no longer able to perform its function effectively. Usually, but not always, a disease is associated with specific symptoms or signs that indicate its presence. Therefore, the presence of such symptoms or signs can indicate an affected tissue, organ, or individual. Changes in these symptoms or signs may indicate the progression of the disease. The progression of a disease is usually characterized by an increase or decrease in such symptoms or signs, which can indicate a “worsening” or “improving” condition. A “worsening” condition is characterized by a decline in the ability of an tissue, organ, or organism to perform its function effectively, while a “improving” condition is usually characterized by an increase in the ability of an tissue, organ, or organism to perform its function effectively. An tissue, organ, or individual at “risk of developing a disease” is in a healthy state but shows signs of potential disease development. Typically, the risk of developing a disease is associated with early or mild signs or symptoms of that disease. In such cases, the onset of the disease can still be prevented through treatment. Examples of diseases include, but are not limited to, inflammatory diseases, infectious diseases, skin diseases, endocrine diseases, intestinal diseases, nervous system disorders, joint diseases, genetic diseases, autoimmune diseases, traumatic diseases, and various types of cancer.

[0104] Endometriosis is a chronic, hormone-dependent, inflammatory disease characterized by the presence of endometrial-like tissue outside the uterus. The clinical manifestations of endometriosis vary significantly from patient to patient. Common symptoms include intermenstrual bleeding, menstrual pain (dysmenorrhea), dyspareunia (painful intercourse), painful defecation (difficulty defecating), and painful urination (difficulty urinating). Pelvic pain caused by endometriosis is usually chronic (lasting ≥ 6 months) and is accompanied by dysmenorrhea (50-90% of cases), dyspareunia, deep pelvic pain, and lower abdominal pain, with or without back and lower back pain. The pain can occur unpredictably and intermittently throughout the menstrual cycle, or it can be continuous, and can be dull, throbbing, or severe, and can be exacerbated by physical activity. Bladder and bowel-related symptoms (nausea, bloating, and early satiety) are usually cyclical. The pain typically worsens over time and its characteristics can change; in rare cases, women report burning sensations or hypersensitivity, symptoms suggesting a neuropathic component. Endometriosis is often asymptomatic and may only come to the clinician's attention during infertility assessments (Sinaii et al. Fertil Steril. 2008; 89(3): 538-545). In women with endometriosis, monthly fertility is reduced (2-10%) compared to fertile couples (15-20%). Although endometriosis impairs fertility, it does not usually prevent conception entirely (Fadhlaoui et al., Front Surg. 2014; 1: 24).

[0105] The most common sites affected by endometriosis are the pelvic organs and peritoneum, but other parts of the body, such as the lungs, are occasionally affected. The severity of the disease ranges from a few small lesions in other normal pelvic organs to large ovarian endometriotic cysts (endometriomas) and / or extensive fibrosis and adhesion formation that significantly distorts the pelvic anatomy. Based on location, endometriotic lesions can be classified as peritoneal endometriosis, ovarian endometriotic cysts (endometriomas), and deep nodules (deep invasive endometriosis; Kennedy et al. Hum Reprod. 2005;20(10): 2698-2704). Deep infiltrative endometriosis is considered any manifestation of endometriosis located outside the superficial tissues of the rectovaginal septum and vaginal fornix, pelvic wall, parametrial tissue, intestines, uterus, or bladder (Halis et al. (2010). Deutsches Ärzteblatt International, 107(25), 446). Endometriosis can also involve the diaphragm (diaphragmatic endometriosis) or the thoracic cavity (thoracic endometriosis) (Nezhat et al. JSLS 2019).

[0106] As used herein, the terms “uterine / pelvic pathology” and “uterine and pelvic pathology” are used interchangeably and include adenomyosis, uterine fibroids, ovarian cysts requiring surgery, and uterine / pelvic cancers (such as ovarian cancer or endometrial cancer).

[0107] As used in this article, “neuropathic pain associated with endometriosis and / or uterine / pelvic pathology” refers to neuropathic pain as defined by the International Association for the Study of Pain as “pain caused by a disease or lesion of the somatosensory nervous system,” in contrast to nociceptive pain, which is defined as “pain caused by actual or threatening damage to non-nervous tissue and resulting from activation of nociceptors.” Neuropathic pain can be expected in the context of endometriosis for a variety of reasons. (Coxon et al., Is there a Neuropathic-Like Component to Endometriosis-Associated Pain? Results From a Large Cohort Questionnaire Study, Front PainRes (Lausanne), 2021; 2: 743812; doi: 10.3389 / fpain.2021.743812).

[0108] The term "rASRM stage" or "rASRM staging" refers to a revised classification system established by the American Society for Reproductive Medicine (ASRM) that describes the severity of endometriosis based on surgical (laparoscopic) findings. This classification is based on the morphology of peritoneal and pelvic implants, such as red, white, and black lesions, and should include the percentage of each lesion involved. The number, size, and location of endometrial implants, plaques, endometriomas, and adhesions should be noted. Endometriosis in the intestines, urinary tract, fallopian tubes, vagina, cervix, skin, or other locations should be documented according to ASRM guidelines. According to ASRM guidelines, endometriosis is classified into stages I, II, III, and IV based on a scoring system, corresponding to mild, moderate, and severe endometriosis, respectively. rASRM Stage I and II endometriosis (mild to mild endometriosis) are defined as: superficial peritoneal endometriosis, possibly with small deep lesions, without endometriomas and / or mild membranous adhesions. rASRM Stage III and IV endometriosis (moderate to severe endometriosis) are defined as: superficial peritoneal endometriosis, deep invasive endometriosis with moderate to extensive adhesions between the uterus and intestines, and / or endometrial cysts with moderate to extensive adhesions (involving the ovaries and fallopian tubes).

[0109] The term “VAS,” or Visual Analogue Scale, is a tool for assessing pain intensity. The VAS consists of a 10 cm horizontal line marked “no pain” and “most imaginable pain” at its two ends. Each patient marks her pain level on this line and measures the distance from the leftmost “no pain” mark to the checkmark in centimeters, resulting in a pain score from 0 to 10. “No pain” corresponds to a pain score of 0, and “most imaginable pain” corresponds to a pain score of 10. In women with endometriosis, dysmenorrhea is associated with the highest perceived pain, with a mean VAS score of approximately 6 (Cozzolino et al. Rev Bras Ginecol Obstet 2019; 41(3): 170-175).

[0110] Subjects may be referred to herein as patients. The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to animals, preferably mammals, and more generally humans. Patients are preferably human females. Diagnosis of endometriosis needs to be made at a young age, as it begins at the onset of menstruation. Therefore, patients are preferably young or adolescent human females aged 12-24 years. In one embodiment of the invention, the patient is a young or adolescent human female. Subjects may be symptomatic (e.g., the subject exhibits symptoms associated with endometriosis) or asymptomatic (e.g., the subject does not exhibit symptoms associated with endometriosis). Subjects may be diagnosed with, at risk of developing, or exhibiting symptoms of: endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology. Subjects may have or be suspected of having (e.g., exhibiting symptoms or a history that indicate or suggest they have) endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0111] Therefore, in some embodiments, the subject suffers from endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology (and the method diagnoses, identifies, (or detects) that the subject suffers from endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology). In this context, the terms "diagnose," "identify," and "detect" may be used interchangeably.

[0112] In a specific instance, the subject had early-stage (stage I or II) endometriosis.

[0113] Patients studied using the method of this invention should be those suspected of having endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0114] As used herein, the term "suspected endometriosis" means that the patient should exhibit clinical parameters, signs, and / or symptoms of endometriosis. Therefore, patients according to the present invention are typically those with endometriosis or suspected endometriosis. Patients suspected of having endometriosis exhibit the following signs and symptoms: dysmenorrhea (painful menstruation), dysuria (painful urination), dysdefecation (difficulty or pain during defecation), dyspareunia (pain during or after intercourse), and chronic abdominal / pelvic pain unrelated to the menstrual cycle, heavy menstrual bleeding, prolonged menstrual cycles, infertility, fatigue, recurring lung problems (pneumothorax), recurring cough, chest pain or hemoptysis (hemoptysis), shoulder pain, painful rectal bleeding or hematuria (hematuria), and recurring scar swelling and pain (EHRE Information on Endometriosis, 2022 www.eshre.eu / guidelines).

[0115] Alternatively, EphA1 levels can be routinely determined as part of a screening test without suspecting endometriosis, but can be used to detect asymptomatic endometriosis at an early stage.

[0116] By detecting a decrease in EphA1 levels in a subject, suspicion of endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology will be confirmed, and the subject will have a high risk of developing neuropathic pain related to endometriosis, uterine / pelvic pathology, and / or uterine / pelvic pathology. Specifically, in cases where a subject already exhibits clinical parameters, signs, and / or symptoms of neuropathic pain related to endometriosis, uterine / pelvic pathology, and / or uterine / pelvic pathology, the determination of a decrease in EphA1 levels will confirm the presence of neuropathic pain related to uterine / pelvic pathology and / or uterine / pelvic pathology.

[0117] As used herein, the term "comparison" means comparing the amount / level of a biomarker in a sample from a subject with a reference amount or reference value for the biomarker specified elsewhere in this specification. It should be understood that, as used herein, comparison generally refers to a comparison of corresponding parameters or values, such as comparing an absolute amount with an absolute reference amount, or a concentration with a reference concentration, or comparing an intensity signal obtained from a biomarker in a sample with an intensity signal of the same type obtained from a reference sample. Comparisons can be performed manually or computer-assisted. Therefore, comparisons can be made by a computing device. For example, the value of a measured or detected amount of a biomarker in a sample from a subject can be compared to a reference amount, and such comparisons can be performed automatically by a computer program that performs the comparison algorithm. The computer program performing the assessment will provide the desired evaluation in an appropriate output format. For computer-assisted comparisons, the value of a measured amount can be compared to a value stored in a database by the computer program that corresponds to an appropriate reference. The computer program can further evaluate the results of the comparison, i.e., automatically provide the desired evaluation in an appropriate output format. For computer-assisted comparisons, the value of a measured amount can be compared to a value stored in a database by the computer program that corresponds to an appropriate reference. Computer programs can further evaluate the results of the comparison, that is, automatically provide the required assessments in a suitable output format.

[0118] As used herein, the terms "(appropriate) reference value," "reference sample," or "control (sample)" refer to a sample that is analyzed in substantially the same manner as the target sample and whose information is compared with that of the target sample. In one embodiment, the "(appropriate) reference value" is a predetermined reference. Thus, a reference sample provides a standard for evaluating information obtained from a target sample. A control sample may be derived from bodily fluids of a healthy individual, particularly serum or plasma used for noninvasive testing, thereby providing a standard for the health status of a tissue, organ, or individual. The difference between the status of a normal reference sample and the status of the target sample can indicate the presence or further progression of such a disease or condition. A control sample may be derived from abnormal or diseased tissue, organ, or individual, thereby providing a standard for the diseased status of the tissue, organ, or individual. The difference between the status of a normal or abnormal reference sample and the status of the target sample can indicate the absence or improvement of such a disease or condition.

[0119] Reference samples can also originate from the same tissue, organ, or individual as the target sample, but were collected at an earlier time point. The difference between the state of the earlier collected reference sample and the state of the target sample can indicate the progression of the disease, i.e., whether the disease improves or worsens over time.

[0120] The determined value can be compared with more than one (appropriate) reference value, which can be of different kinds. For example, the determined value can be compared with one or more values ​​obtained from the same subject at an earlier time point, and in parallel, it can be compared with one or more values ​​obtained from other subjects (with known stages of endometriosis).

[0121] Control samples can be internal or external. Internal control samples are used by assessing biomarker levels in the test sample and in one or more other samples taken from the same subject to determine if there are any changes in the biomarker levels. External control samples compare the presence or amount of the biomarker in a sample derived from an individual to its presence or amount in an individual known to have the given condition, known to be at risk of having the given condition, or known not to have the given condition (i.e., a “normal individual”).

[0122] Those skilled in the art will understand that such external control samples can be obtained from a single individual or from an age-matched reference population free of confounding diseases. Typically, a sample of 100 well-characterized individuals from an appropriate reference population is used to establish a “reference value.” However, a reference population of 20, 30, 50, 200, 500, or 1000 individuals may also be selected. Healthy individuals represent the preferred reference population for establishing control values.

[0123] For example, the concentration of a biomarker in a patient sample can be compared to concentrations known to be associated with a specific course of a disease. For instance, it can be compared to concentrations known to be associated with a particular stage of endometriosis. Typically, the biomarker concentration in a sample is directly or indirectly related to the diagnosis, and the biomarker concentration is used, for example, to determine whether an individual is at risk of having the disease. Alternatively, the biomarker concentration can be compared to concentrations obtained from the same subject at an earlier time point. Alternatively, in assessing the risk of disease progression or in patient follow-up, the biomarker concentration in a sample can be compared, for example, to concentrations known to be associated with treatment response to a disease, diagnosis of a disease, assessment of the severity of a disease, or guidance for selecting appropriate medication for a disease. Depending on the intended diagnostic use, an appropriate control sample is selected, and a control or reference value for the biomarker is established therein. It is also clear to those skilled in the art that the absolute biomarker value established in the control sample will depend on the assay used.

[0124] For the methods described herein, the most common control samples and / or reference values ​​derived therefrom are obtained from, but not limited to, “symptomatic controls” (also known as: SNF = Symptomatic No Detection). The corresponding subjects from whom these samples were obtained are “symptomatic subjects”.

[0125] A “symptomatic control” (also known as SNF = symptomatic no-detection) refers to a control sample of subjects who have symptoms commonly associated with endometriosis (e.g., menstrual / abdominal pain, infertility, etc.), but endometriosis can be ruled out based on laparoscopy and no tissue changes (e.g., adenomyosis, uterine / ovarian cysts, uterine fibroids, or uterine / pelvic cancers (such as ovarian cancer, endometrial cancer)) can be observed. In other words, the symptomatic control does not have endometriosis or any other uterine / pelvic pathology (e.g., adenomyosis, uterine / ovarian cysts, uterine fibroids, uterine / pelvic cancers (such as ovarian cancer and endometrial cancer)).

[0126] Women with other “uterine / pelvic pathologies” (SOF = Other Findings with Symptoms) refer to a sample of subjects who have tissue changes (e.g., adenomyosis, uterine fibroids, surgically induced uterine / ovarian cysts, and uterine / pelvic cancer) that are not similar to endometriosis. Furthermore, these subjects most frequently present with symptoms (e.g., menstrual / abdominal pain, infertility, etc.).

[0127] Control samples can be measured at the same time as, before or after, alone or simultaneously with, the test samples. The control value used for comparison with the test samples can be a value calculated as the average or median of more than one control sample (e.g., two or more, five or more, ten or more, a group, etc.). Alternatively, control samples can be samples (i.e., a mixture thereof) derived from more than one (e.g., two or more, five or more, ten or more, a group, etc.) individual who does not have endometriosis (or a "symptomatic control") and does not have other uterine / pelvic pathologies.

[0128] In one instance, the control sample was obtained from “symptomatic control” subjects who did not have endometriosis or other uterine / pelvic pathologies (adenomyosis, uterine fibroids, ovarian cysts requiring surgery, uterine / pelvic cancer).

[0129] Alternatively, the levels of biomarkers (e.g., proteins) in the biofluid sample can be compared to predetermined reference levels for the target biomarker. As used herein, a “predetermined reference level” refers to a biomarker level obtained from a reference database that can be used to generate a predetermined cutoff value, i.e., a score that is statistically significant for predicting endometriosis. In one instance, the predetermined reference level is the mean or median level of the biomarker in at least one individual from the same species who does not have endometriosis. In one instance, the predetermined reference value can be calculated as the mean or median taken from a group or population of individuals who do not have endometriosis. For example, the predetermined reference value can be calculated as the mean or median taken from a group or population of individuals serving as a “symptomatic control.” Individuals or groups of individuals may be the same age or in the same health status or condition as the subjects from whom the test sample was obtained.

[0130] In one instance, the predetermined reference level is therefore the average level of biomarkers in control subjects who do not have endometriosis. In a further instance, the predetermined reference level is the average level of biomarkers in subjects serving as “symptomatic controls.”

[0131] Typically, in methods used to diagnose endometriosis in a subject, a control sample or predetermined reference is obtained from an individual or group of individuals different from the subject being tested (i.e., the subject from whom the test sample was obtained / provided). In such instances, the control or predetermined reference is used as a baseline to determine whether the subject being tested has endometriosis.

[0132] In alternative instances, controls or predetermined reference values ​​may be obtained from the same individuals as the test sample, but at an earlier time point. This is particularly relevant to the method described herein, which determines the subject's progress, the effectiveness of treatment regimens for endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology, and / or adherence to prescribed treatment regimens for endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology. For this purpose, samples are taken from the same biological fluid from the same subject, wherein the biological fluid is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, preferably serum.

[0133] In such instances, control samples or predetermined reference levels are used to determine any changes in the levels of biomarkers in the same subject over a time interval. Therefore, the predetermined reference level or control sample may be derived from the same subject from whom the test sample was obtained, for example, at an earlier time point. This earlier time point could be prior to their diagnosis of endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0134] The predetermined level can be a single cutoff value, such as the median or mean. It can be a range of cutoff values ​​(or thresholds), such as confidence intervals. It can be established based on comparison groups, such as the risk in one defined group being a factor of two (e.g., approximately 2, 4, 8, 16, or more) higher or lower than the risk in another defined group. For example, it can be a range in which the subject population (e.g., control subjects) is divided equally (or unequally) into groups, such as low-risk, intermediate-risk, and high-risk groups, or into quartiles, with the lowest quartile representing the subject with the lowest risk and the highest quartile representing the subject with the highest risk, or into n quartiles (i.e., an interval of n regular intervals), with the lowest of the n quartiles representing the subject with the lowest risk and the highest of the n quartiles representing the subject with the highest risk. Furthermore, the reference can be a calculation of the relative or absolute amounts of the biomarker in the individual population of subjects to be studied, most preferably the mean or median. How to calculate a suitable reference value, preferably the mean or median, is well known in the art.

[0135] Therefore, in some cases, the level of a protein biomarker in a subject being less than or equal to the level of a biomarker in a control sample or a predetermined reference level indicates a clinical condition (e.g., indicating endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology).

[0136] Generally, but not necessarily, a greater than or less than condition sufficient to distinguish a subject from a control subject is statistically significant. In cases where the level of a biomarker in a subject is equal to the level of a biomarker in a control subject indicating endometriosis (stage), “equal” means approximately equal (e.g., no statistical difference).

[0137] Predetermined values ​​can be dependent on the specific subject population selected (e.g., human subjects). For example, a clearly healthy population will have different “normal” ranges for protein biomarkers than a subject population that has or may have endometriosis. Therefore, the selected predetermined values ​​can take into account the category to which the subject (e.g., human subject) belongs (e.g., healthy, ill, disease stage).

[0138] Those skilled in the art can select the appropriate range and category through routine experiments.

[0139] Appropriately, the level of a specific biomarker detected in a sample (e.g., a test sample, a control sample, etc.) can be normalized by adjusting the measured level (amount or activity) of the biomarker using the level of a reference protein in the same sample, where the reference protein itself is not a biomarker (it is, for example, a constitutively expressed protein). This normalization allows comparison of the biomarker level in one sample to another, or between samples from different sources. The normalized level can then optionally be compared to a reference or control. For example, when measuring a protein biomarker in a whole blood sample, the biomarker can be expressed as an absolute concentration, or, alternatively, normalized to the concentration of a known constitutively expressed protein in whole blood, such as albumin, immunoglobulin, or plasma protein.

[0140] For example, when measuring protein biomarkers in a serum (or plasma) sample, the biomarkers can be expressed as absolute concentrations, or alternatively, they can be normalized to known constitutively expressed proteins in the serum (or plasma).

[0141] The levels of biomarkers in a test sample can be compared with the levels of the same biomarkers in a control sample or with a predetermined reference level for the same biomarker to identify increases or decreases in the levels of one or more biomarkers in a subject's sample.

[0142] In the method described herein, a subject may be identified as having endometriosis if a comparison (between the biomarker levels in the control sample / predetermined reference value and the subject's test sample) indicates that the subject has a reduced level of EphA1 compared to the control sample or predetermined reference level.

[0143] Furthermore, it should be understood that if the risk of health deterioration is being predicted, predictions are typically made within a 6-month and 2-year prediction window. More typically, for non-invasive tests that depend on symptoms such as pelvic pain, the prediction window is a time window of approximately 6 to 12 months.

[0144] As those skilled in the art will understand, while assessments made according to the present invention are preferred, they may not be correct for 100% of the subjects studied. This term generally requires the ability to correctly assess the statistically significant subset of subjects. Those skilled in the art can effortlessly determine whether a subset is statistically significant using a variety of well-known statistical assessment tools (e.g., determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc.). See Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983 for more details. Confidence intervals are typically assumed to be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. p-values ​​are typically 0.2, 0.1, or 0.05.

[0145] The term "decreased" or "reduced" level of an indicator refers to a reduction in the level of such indicator in a sample compared to a reference (value) or a reference sample. The terms "decreased," "reduced," "reduced," "downgraded," or "decreased" are generally used herein to refer to a statistically significant reduction in quantity. However, for the avoidance of doubt, “reduced,” “decreased,” “lowered,” or “lowered” means a reduction of at least 10% compared to the reference level / control, such as a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at most and including a 100% reduction (i.e., no level compared to the reference / control sample), or any reduction between 10% and 100% compared to the reference level / control, or a reduction of at least about 0.5 times, or at least about 1.0 times, or at least about 1.2 times, or at least about 1.5 times, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times or at least about 10 times compared to the reference level / control, or any reduction between 1.0 times and 10 times or greater.

[0146] The term "elevated" or "increased" level of an indicator / (bio)marker refers to a higher level of such an indicator in a sample compared to a reference (value) or reference sample. For example, an elevated level of protein may be detected in a fluid sample of an individual suffering from the given disease compared to the same fluid sample of an individual who does not have the disease. The terms “increased,” “up,” or “upgraded,” “higher” are generally used herein to mean a statically significant increase in quantity; for the avoidance of any doubt, the terms “increased” or “up” mean an increase of at least 10% compared to the reference level / control, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at most and including 100% increase, or any increase between 10% and 100% compared to the reference level / control, or an increase of at least about 0.5 times, or at least about 1.0 times, or at least about 1.2 times, or at least about 1.5 times, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 1.0 times and 10 times or greater.

[0147] The term "decreased" or "reduced" level of an indicator / (bio)marker refers to a decrease in the level of such an indicator in a sample compared to a reference (value) or reference sample. For example, a decreased level is the detection of a lower amount of protein in a fluid sample of an individual suffering from the given disease compared to the same fluid sample of an individual who does not have the disease. The terms “smaller,” “reduced,” or “downgraded,” “lower” are generally used herein to mean a statistically significant reduction in quantity; to avoid any ambiguity, the terms “reduced” or “lower” mean a reduction of at least 10% compared to the reference level / control, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at most and including 100% reduction, or any reduction between 10% and 100% compared to the reference level / control, or any reduction between 1.0 and 10 times or greater compared to the reference level / control.

[0148] As used herein, the term "immunoglobulin (Ig)" refers to the immunity conferred by glycoproteins of the immunoglobulin superfamily. "Surface immunoglobulins" attach to the membrane of effector cells via their transmembrane regions and encompass molecules such as, but not limited to, B cell receptors, T cell receptors, major histocompatibility complex (MHC) proteins of classes I and II, β-2 microglobulin (approximately 2M), CD3, CD4, and CDS.

[0149] Generally, as used herein, the term "antibody" refers to secreted immunoglobulins that lack a transmembrane region and are therefore released into the bloodstream and body cavities. Human antibodies are grouped into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, represented by the Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of antibodies, i.e., these chains are present in IgA, IgD, IgE, IgG, and IgM antibodies, each playing a different role and guiding an appropriate immune response against different types of antigens. Different heavy chains differ in size and composition; and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is present in mucosal regions, such as the intestines, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, preventing pathogen colonization (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389-417). IgD primarily functions as an antigen receptor on B cells unexposed to antigens and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437; Chen et al. (2009) Nat. Immunol. 10:889-898). IgE participates in allergic reactions by binding to allergens and triggering the release of histamine from mast cells and basophils. IgE also participates in parasite protection (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides most of the antibody-based immunity against invading pathogens and is the only antibody isotype capable of crossing the placenta to provide passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four distinct IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum. IgG1 is the most abundant (approximately 66%), followed by IgG2 (approximately 23%), IgG3 (approximately 7%), and IgG (approximately 4%). The biological characteristics of different IgG classes are determined by the structure of their respective hinge regions. IgM is expressed on the surface of B cells in both monomeric and secretory pentamer forms, exhibiting very high affinity.Before sufficient IgG is produced, IgM participates in the elimination of pathogens in the early stages of B cell-mediated (humoral) immunity (Geisberger et al. (2006) Immunology 118:429-437). Antibodies exist not only as monomers but are also known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM from bony fish), or pentamers of five Ig units (e.g., mammalian IgM). Antibodies typically consist of four polypeptide chains, including two identical heavy chains and two identical light chains linked by disulfide bonds and resembling a "Y"-shaped macromolecule. Each chain contains numerous immunoglobulin domains, some of which are constant domains and others are variable domains. The immunoglobulin domains consist of a two-layer sandwich structure, in which seven to nine antiparallel chains are arranged in two sheets. Typically, the heavy chain of an antibody contains four Ig domains, three of which are constant (CH domains: CHI, CH2, CH3) and one is a variable domain (VH). The light chain typically contains one constant Ig domain (CL) and one variable Ig domain (VL). For example, the human IgG heavy chain consists of four linked Ig domains in the sequence VwCH1-CH2-CH3 (also known as VwCyl-Cy2-Cy3) from the N-terminus to the C-terminus, while the human IgG light chain consists of two linked immunoglobulin domains in the sequence VL-CL from the N-terminus to the C-terminus, which are either κ- or λ-type (VK-CK or VA-CA). For example, the constant chain of human IgG contains 447 amino acids. In this specification and claims, the amino acid positions of immunoglobulins are numbered using the “EU Index” numbers, as in the following literature: Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS, and Foeller, C., (1991) Sequences of proteins of immune interest, No. Version 5. US Department of Health and Human Service, National Institutes of Health, Bethesda, MD. "EU index as in Kabat" refers to the residue number of the human IgG 1EU antibody. Therefore, the CH domains in the IgG context are as follows: "CH1" refers to amino acid positions 118-220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index as in Kabat; and "CH3" refers to amino acid positions 341-447 according to the EU index as in Kabat.

[0150] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein and refer to antibodies in their substantially complete form rather than antibody fragments as defined below. Specifically, the term refers to antibodies having a heavy chain containing an Fc region.

[0151] Antibody digestion with papain produces two identical antigen-binding fragments, called "Fab fragments" (also known as "Fab moieties" or "Fab regions"), each with a single antigen-binding site, and a residual "Fe fragment" (also known as "Fe moieties" or "Fe regions"), the name reflecting its ease of crystallization. The crystal structure of the Fe region of human IgG has been determined (Deisenhofer (1981) Biochemistry 20:2361-2370). In the IgG, IgA, and IgD isotypes, the Fe region consists of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of the antibody; in the IgM and IgE isotypes, the Fe region contains three heavy chain constant domains (CH2-4) in each polypeptide chain. Furthermore, smaller immunoglobulin molecules are either naturally occurring or artificially constructed. The term "Fab' fragment" refers to a Fab fragment that additionally includes the hinge region of the Ig molecule, while "F(ab')2 fragment" should be understood to include two Fab' fragments linked chemically or via disulfide bonds. While "single-domain antibodies (sdAbs)" (Desmyter et al. (1996) Nat. Structure Biol. 3:803-811) and "nanobodies" consist of only a single VH domain, "single-chain Fv (scFv)" fragments include a heavy-chain variable domain linked to a light-chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs (scFvA-scFvB). This can be achieved by generating a single peptide chain with two VH and two VL regions, resulting in a “tandem scFv” (VHA-VLA-VHB-VLB). Another possibility is to create an scFv with a linker that is too short for the two variable regions to fold together, thus forcing the scFv to dimerize. Typically, a linker of 5 residues in length is used to generate these dimers. This type is called a “bispecific antibody.” Even shorter linkers (one or two amino acids) between the VH and VL domains lead to the formation of monospecific trimers, known as “tribodies.” Bispecific bispecific antibodies are formed by expressing chains with arrangements of VHA-VLB and VHB-VLA, or VLA-VHB and VLB-VHA, respectively.Single-chain bispecific antibodies (scDb) comprise VHA-VLB and VHB-VLA fragments linked by a 12-20 amino acid, preferably 14 amino acid, adaptor peptide (P) (VHA-VLB-P-VHB-VLA). “Bispecific T-cell adaptors (BiTEs)” are fusion proteins composed of two scFvs of different antibodies, one of which binds to T cells via the CD3 receptor, and the other binds to tumor cells via a tumor-specific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238-244). Biaffinity-based targeted molecules (“DART” molecules) are bispecific antibodies stabilized by a C-terminal disulfide bond.

[0152] Therefore, the term "antibody fragment" refers to a portion of a complete antibody, preferably including its antigen-binding region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments; bisomatic antibodies; sdAb, nanobodies, scFv, di-scFv, tandem scFv, tripois antibodies, bisomatic antibodies, scDb, BiTE, and DART.

[0153] The term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by methods commonly known in the art, including but not limited to surface plasmon resonance-based assays (such as the BIAcore assay described in PCT application publication WO2005 / 012359); enzyme-linked immunosorbent assays (ELISA); and competitive assays (e.g., RIA). Low-affinity antibodies typically bind antigens slowly and tend to dissociate readily, while high-affinity antibodies typically bind antigens rapidly and tend to maintain a longer binding time. Various methods for measuring binding affinity are known in the art, any of which may be used for the purposes of this invention.

[0154] Sandwich immunoassays are widely used to detect target analytes. In this assay, the analyte is sandwiched between a primary antibody and a secondary antibody. Typically, sandwich assays require the capture and detection of different non-covered epitopes on the target analyte by the antibody. This sandwich complex is measured and the analyte is quantified by means of an appropriate method. In a typical sandwich assay, a primary antibody bound to or capable of binding to a solid phase and a detectably labeled secondary antibody each bind to a different non-covered epitope of the analyte. A primary analyte-specific binder (e.g., an antibody) is covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, most commonly cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support can take the form of a tube, magnetic beads, a microplate tray, or any other surface suitable for immunoassays. Binding methods are well known in the art and typically consist of cross-linking covalent binding or physisorption, with the polymer-antibody complex washed during test sample preparation. Aliquots of the sample to be tested are then added to the solid-phase complex and incubated for a sufficient period of time (e.g., 2–40 minutes or overnight (if more convenient)) under suitable conditions (e.g., from room temperature to 40°C, such as between 25°C and 37°C) to allow binding of the first or capture antibody to the corresponding antigen. After this incubation period, the solid phase, which includes the first or capture antibody and the antigen bound thereto, is washed and incubated with a secondary antibody or labeled antibody bound to another epitope on the antigen. The second antibody is linked to a reporter molecule that indicates the binding between the second antibody and the first antibody-target antigen complex.

[0155] A widely used alternative sandwich assay involves using a solid phase coated with a first partner of the binding pair, such as paramagnetic streptavidin-coated microparticles. These microparticles are mixed with and incubated with: an analyte-specific binder (e.g., a biotinylated antibody) bound to a second partner of the binding pair; a sample suspected of containing or including the analyte, wherein the second partner of the binding pair binds to the analyte-specific binder; and a second analyte-specific binder that is detectably labeled. As will be apparent to those skilled in the art, the components are incubated under appropriate conditions for a sufficient period of time to allow the labeled antibody (passing through the analyte), the analyte-specific binder (bound to the second partner of the binding pair), and the first partner of the binding pair to bind to the solid-phase microparticles. Depending on the application, the assay may include one or more washing steps.

[0156] The term "detectably marked" covers markings that can be detected directly or indirectly.

[0157] Directly detectable labels provide a detectable signal or interact with a second label to modify the detectable signal provided by the first or second label, for example, to induce FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al., "Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058) provide a detectable signal and are generally suitable for labeling. In one embodiment, detectably labeled refers to a label that provides or induces a detectable signal, i.e., a fluorescent label, a luminescent label (e.g., a chemiluminescent label or an electrochemiluminescent label), a radioactive label, or a metal chelate-based label, respectively.

[0158] The large number of available markers (also known as dyes) can generally be divided into the following categories, the total of all categories and each of the categories representing embodiments as described in this disclosure:

[0159] (a) Fluorescent dyes

[0160] Fluorescent dyes, for example, were developed by Briggs et al. ("Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058).

[0161] Fluorescent labels or fluorophores include rare earth chelates (europium chelates); fluorescein-type labels, including FITC, 5-carboxyfluorescein, and 6-carboxyfluorescein; rhodamine labels, including TAMRA; dansyl; lissamine; anthocyanins; phycoerythrin; Texas Red; and analogues. Using the techniques disclosed herein, fluorescent labels can be conjugated to aldehyde groups contained in target molecules. Fluorescent dyes and fluorescent labeling reagents include those available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0162] (b) Luminescent dyes

[0163] Luminescent dyes or markers can be further classified into the following subcategories: chemiluminescent dyes and electrochemiluminescent dyes.

[0164] Different categories of chemiluminescent labels include luminol, acridine compounds, coenzymes and analogues, dioxane, peroxate-based systems and their derivatives. For immunodiagnostic procedures, acridine-based labels are mainly used (a detailed review is given in Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0165] The main relevant labels used for electrochemiluminescence (ECL) labeling are ruthenium-based and iridium-based ECL complexes. ECL has proven to be a highly sensitive and selective method useful in analytical applications. This method combines the analytical advantages of chemiluminescence analysis (no background light signal) with the more convenient control of the reaction through the use of electrode potentials. Typically, ruthenium complexes, especially [Ru(Bpy)3]2+ regenerated with TPA (tripropylamine) in the liquid phase or at the liquid-solid interface (which releases photons at approximately 620 nm), are used as ECL labels.

[0166] Electrochemiluminescence (ECL) assays provide sensitive and accurate measurements of the presence and concentration of target analytes. The technique utilizes labels or other reactants that are induced to emit light upon electrochemical oxidation or reduction under appropriate chemical conditions. This electrochemiluminescence is triggered by a voltage applied to a working electrode at a specific time and in a specific manner. The light emitted by the label, once measured, indicates the presence or quantity of the analyte. To provide a more comprehensive description of this type of ECL technology, the following references are cited: U.S. Patent No. 5,221,605, U.S. Patent No. 5,591,581, U.S. Patent No. 5,597,910, PCT Application WO90 / 05296, PCT Application WO92 / 14139, PCT Application WO90 / 05301, PCT Application WO96 / 24690, PCT Application US95 / 03190, PCT Application US97 / 16942, PCT Application US96 / 06763, PCT Application WO95 / 08644, PCT Application WO96 / 06946, PCT Application WO96 / 33411, PCT Application WO87 / 06706, PCT Application WO96 / 39534, PCT... Published applications WO96 / 41175, PCT published applications WO96 / 40978, PCT / US97 / 03653, and U.S. Patent Application 08 / 437,348 (U.S. Patent No. 5,679,519) are cited. Also cited is Knight et al.'s 1994 review of ECL analytical applications (Analyst, 1994, 119: 879-890) and the literature cited in that article. In one embodiment, the method according to this description is implemented using an electrochemiluminescence label.

[0167] Recently, iridium-based ECL markings have also been described (WO2012107419).

[0168] (c) Radioactive labeling uses radioactive isotopes (radionuclides), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At or 131Bi.

[0169] (d) Metal chelate complexes suitable as markers for imaging and therapeutic purposes are well known in the art (US 2010 / 0111861; US ​​5,342,606; US 5,428,155; US 5,316,757; US 5,480,990; US 5,462,725; US 5,428,139; US 5,385,893; US 5,739,294; US 5,750,660; US 5,834,461; Hnatowich et al., J. Immunol. Methods 65 (1983) 147-157; Meares et al., Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al., Bioconjugate Chem. 1 (1990)). 59-65; Meares et al., J. Cancer (1990), Supplement 10:21-26; Izard et al., Bioconjugate Chem. 3 (1992) 346-350; Nikula et al., Nucl. Med. Biol. 22 (1995) 387-90; Camera et al., Nucl. Med. Biol. 20 (1993) 955-62; Kukis et al., J. Nucl. Med. 39 (1998) 2105-2110; Verel et al., J. Nucl. Med. 44 (2003) 1663-1670; Camera et al., J. Nucl. Med. 21 (1994) 640-646; Ruegg et al., Cancer Res. 50 (1990) 4221-4226; Verel et al., J. Nucl. Med. 44 (2003) 1663-1670; Lee et al., Cancer Res. 61 (2001) 4474-4482; Mitchell et al., J. Nucl. Med. 44 (2003) 1105-1112; Kobayashi et al., BioconjugateChem. 10 (1999) 103-111; Miederer et al., J. Nucl. Med.45 (2004) 129-137; DeNardo et al., Clinical Cancer Research 4 (1998) 2483-90; Blend et al., Cancer Biotherapy & Radiopharmaceuticals 18 (2003) 355-363; Nikula et al., J. Nucl. Med. 40 (1999) 166-76; Kobayashi et al., J. Nucl. Med. 39 (1998) 829-36; Mardirossian et al., Nucl. Med. Biol. 20 (1993) 65-74; Roselli et al., Cancer Biotherapy & Radiopharmaceuticals, 14 (1999) 209-20.

[0170] The methods described herein may further include selecting and optionally administering a treatment regimen to the subject based on a diagnosis (i.e., based on a comparison of biomarker levels with reference values / levels / controls). Treatment may include, for example, surgery, and in some cases, therapies, or combinations thereof. However, in some cases, immediate treatment may not be necessary, and active monitoring of the subject may be optional.

[0171] As used in this article, the terms “active surveillance,” “monitoring,” and “watchful waiting” are used interchangeably to refer to closely monitoring a patient’s condition without administering any treatment until symptoms appear or change.

[0172] As used herein, the terms “treat,” “treating,” and “treatment” are considered to include interventions aimed at altering the pathology of a condition, disorder, or symptom (i.e., in this case, endometriosis). Therefore, “treatment” refers to therapeutic treatment in which the aim is to alleviate (reduce) the target condition, disorder, or symptom. Thus, “treatment” encompasses reducing, alleviating, or suppressing the symptoms of endometriosis, for example, by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to pre-treatment symptoms. In the case of endometriosis, appropriate treatment may include analgesics, hormonal therapy (such as hormonal contraceptives), gonadotropin-releasing hormone (GnRH) agonists, and / or surgery. (Longo, DL et al. 2020. Sc.D. N Engl J Med, 382, ​​pp. 1244-56).

[0173] As used in this article, the term "surgery" refers to surgical procedures performed to remove endometrial tissue, such as laparoscopy or nerve-preserving surgery.

[0174] As used herein, the term "therapy" includes drug-based therapies, radiation therapy, hormone therapy, cryosurgery, chemotherapy, immunotherapy, biotherapy, and high-intensity focused ultrasound. Drug-based therapies for endometriosis may, for example, involve inhibiting or targeting neurogenic inflammation and / or pain relief and / or hormone therapy.

[0175] The type of treatment will vary depending on the specific form and / or stage of neuropathic pain that the subject has or is suspected of having, including endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0176] Examples

[0177] The inventors have surprisingly identified a novel protein biomarker, EphA1, which is reduced in the biological fluid, particularly in the serum, of women with endometriosis, especially those in the early stages of endometriosis.

[0178] Compared with controls (e.g., non-pathological subjects or symptomatic subjects), the biomarker EphA1 can be used to diagnose endometriosis in subjects.

[0179] Specifically, serum EphA1 can be used as a blood biomarker for the early diagnosis and risk stratification of endometriosis. Furthermore, serum EphA1 can be used to select patients with stage I and II endometriosis for early medical management. Therefore, it can significantly reduce diagnostic delays in endometriosis, improve patients' lives, and alleviate their economic burden.

[0180] Biomarkers can be advantageously used in any of the methods, kits, assays or uses described herein.

[0181] Methods for assessing endometriosis in a subject

[0182] In a first aspect, the present invention relates to a method for assessing endometriosis in a subject, the method comprising the following steps:

[0183] a) Determine the level of the extracellular protein fraction of EphA1 in biofluid samples from subjects.

[0184] b) Compare the level of the extracellular protein fraction of EphA1 to at least one appropriate reference value for the level of the extracellular protein fraction of EphA1.

[0185] c) If the comparison in step b) indicates that the subject has a reduced level of the extracellular protein portion of EphA1 compared to an appropriate reference value, then the subject is identified as having endometriosis.

[0186] According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

[0187] In this embodiment, a decreased level, amount, or concentration of EphA1 in a subject's fluid sample indicates the presence of endometriosis in the subject. Specifically, if the amount or concentration of EphA1 in a subject's fluid sample is less than the amount or concentration of EphA1 according to a reference value, then the amount or concentration of EphA1 in the subject's fluid sample indicates the presence of endometriosis in the subject.

[0188] In one embodiment, the method is an in vitro method.

[0189] In a particular embodiment, at least one appropriate reference value is

[0190] i. The level of EphA1 in SNF or SOF subjects, or for

[0191] ii. The average EphA1 level in a group of SNF subjects, a group of SOF subjects, or a combination thereof.

[0192] Or at least one of the appropriate reference values ​​is

[0193] iii. The predetermined level of EphA1 in SNF or SOF subjects, or...

[0194] iv. The predetermined mean level of EphA1 in a group of SNF subjects or a group of SOF subjects or a combination thereof.

[0195] In a particular embodiment, the inventors were able to detect a decrease in serum EphA1 in both early and late stages of endometriosis compared to control levels.

[0196] Specifically, a decrease in EphA1 levels of 15% or more, 20% or more, 30% or more, 40% or more, or 50% or more indicates the presence of endometriosis or a risk of developing endometriosis. Specifically, a decrease in EphA1 levels of 100% or more indicates the presence of endometriosis. Specifically, a decrease in EphA1 levels of 150% or more indicates the presence of endometriosis. Specifically, a decrease in EphA1 levels of 200% or more indicates endometriosis.

[0197] In a particular embodiment, a decrease of 10% or more in EphA1 indicates the presence of endometriosis or a risk of having or developing endometriosis.

[0198] Appropriately, the biological fluid sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, preferably serum.

[0199] In one embodiment, the biofluid sample is serum.

[0200] In this embodiment, the sample is an in vitro sample, meaning it will be analyzed in vitro and not transferred back into the subject's body. In this embodiment, the method of the present invention is an in vitro method.

[0201] In a particular embodiment, the subject is a human subject. In a particular embodiment, the patient is a female human subject. In a particular embodiment, the subject is a young or adolescent human woman. Specifically, the subject is a subject who is susceptible to endometriosis due to a physical condition.

[0202] Specifically, assessment is performed without laparoscopy. Specifically, assessment is performed without using laparoscopy and / or rASRM staging to determine the presence or severity of endometriosis in the patient.

[0203] According to the present invention, endometriosis is early-stage endometriosis, particularly stage I endometriosis according to rASRM staging or stage II endometriosis according to rASRM staging.

[0204] In embodiments of the invention, the protein level of EphA1 is optionally determined using a method selected from the following: ELISA assay, Western blotting, lateral flow assay, protein microarray, and mass spectrometry.

[0205] Preferably, the level of EphA1 is determined by detecting the extracellular protein portion of EphA1 using ELISA or ECLIA.

[0206] In a particular embodiment, the extracellular protein moiety of EphA1 is detected by identifying at least one of the following peptides: WEPPADTGGR (SEQ ID NO: 1), AQGELGWLLDPPK (SEQ ID NO: 2), MHCSPDGEWLVPVGR (SEQ ID NO: 3), GLYLAFHNPGACVALVSVR (SEQ ID NO: 4), APGEGPQVACTGPPSAPR (SEQ ID NO: 5), VTTVAADQSFTIR (SEQ ID NO: 6), or VHVELQFTVR (SEQ ID NO: 7). Details are shown in Table 1.

[0207] Table 1: Identification of peptides of the extracellular protein moiety of EphA1 in serum samples from women with endometriosis (cases) and women without endometriosis (controls) using advanced mass spectrometry proteomics technology.

[0208]

[0209] In the embodiments, antibodies, particularly monoclonal antibodies, are used to determine the amount of EphA1. In the embodiments, step a) of determining the amount of EphA1 in a patient's sample includes performing an immunoassay. In the embodiments, the immunoassay is performed directly or indirectly. In the embodiments, such immunoassays are selected from the group consisting of: enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0210] In a particular embodiment, step a) of determining the level of EphA1 in a subject's sample includes the following steps:

[0211] i) Incubating the subject's sample with one or more antibodies that specifically bind to EphA1, thereby forming a complex between the antibody and EphA1, and

[0212] ii) Quantify the complex formed in step i) to quantify the amount of EphA1 in the subject's sample.

[0213] In a particular embodiment, in step i), the sample is incubated with two antibodies that specifically bind to EphA1. It will be apparent to those skilled in the art that the sample can be contacted with the first and second antibodies in any desired order, i.e., firstly with the first antibody and then with the second antibody; or firstly with the second antibody and then with the first antibody; or simultaneously with both antibodies, to form a first anti-EphA1 antibody / EphA1 / second anti-EphA1 antibody complex under sufficient time and conditions. As will be readily understood by those skilled in the art, this is merely a routine experiment to set suitable or sufficient time and conditions for the formation of a complex of a specific anti-EphA1 antibody and an EphA1 antigen / analyte (= anti-EphA1 complex), or a secondary complex or sandwich complex comprising a first antibody against EphA1, EphA1 (analyte), and a second anti-EphA1 antibody (= anti-EphA1 antibody / EphA1 / second anti-EphA1 antibody complex).

[0214] The detection of anti-EphA1 antibody / EphA1 complexes can be performed by any suitable means. The detection of first anti-EphA1 antibody / EphA1 / second anti-EphA1 antibody complexes can be performed by any suitable means. Those skilled in the art are well familiar with the described methods.

[0215] In some embodiments, a sandwich comprising a first antibody against EphA1, EphA1 (analyte), and a second antibody against EphA1 will be formed, wherein the second antibody is detectably labeled.

[0216] In one embodiment, a sandwich comprising a first antibody against EphA1, EphA1 (analyte), and a second antibody against EphA1 is formed, wherein the second antibody is detectably labeled and wherein the first anti-EphA1 antibody is capable of binding to or conjugating to a solid phase.

[0217] In the embodiments, the second antibody is detectably labeled directly or indirectly. In a particular embodiment, the second antibody is detectably labeled with a luminescent dye, particularly a chemiluminescent or electrochemiluminescent dye.

[0218] In this embodiment, the assessed endometriosis is selected from the group consisting of: peritoneal endometriosis, endometrioma, and deep invasive endometriosis (DIE).

[0219] In a particular embodiment, the diagnosed endometriosis is peritoneal endometriosis. In other particular embodiments, the diagnosed endometriosis is stage I or II peritoneal endometriosis according to the rASRM staging.

[0220] In another embodiment, the method further includes selecting a treatment regimen for the subject based on a comparison of EphA1 levels with a control sample or a predetermined reference level. In a particular embodiment, the method further includes administering the selected treatment regimen to the subject, optionally wherein the selected treatment regimen includes drug-based therapy and / or surgical treatment (laparoscopy). Drug-based therapy for endometriosis may be performed, for example, by analgesics, hormone therapy, and / or surgery.

[0221] Based on whether the diagnosis suggests a serious stage of disease, those skilled in the art will clearly know how to select the most appropriate and promising treatment option.

[0222] In an embodiment, the method further includes assessing the presence of dysmenorrhea and / or lower abdominal pain in the patient. In an embodiment, the presence of dysmenorrhea and / or lower abdominal pain is assessed according to the VAS scale. In an embodiment, a VAS score of 4 or higher indicates moderate or severe dysmenorrhea. In an embodiment, a score of 3 or lower indicates no dysmenorrhea or mild dysmenorrhea.

[0223] In an embodiment, the method further includes determining the level of CA-125 in a biofluid sample from the subject.

[0224] In one embodiment, when determining the levels of EphA1 and CA-125, the method includes calculating

[0225] ●The ratio of the amount or concentration of the extracellular protein fraction of EphA1 to the amount or concentration of CA-125, or

[0226] ●The ratio of the amount or concentration of the extracellular protein fraction of EphA1 to dysmenorrhea, or

[0227] ●The amount or concentration of the extracellular protein fraction of EphA1 relative to the ratio of the amount or concentration of CA-125 to dysmenorrhea, or

[0228] ● The ratio of the amount or concentration of the extracellular protein portion of EphA1 to lower abdominal pain according to the VAS scale.

[0229] Methods for assessing uterine / pelvic pathology in a subject

[0230] The inventors have surprisingly identified a novel protein biomarker, EphA1, which is reduced in the biofluids, particularly the serum, of women with uterine / pelvic pathology.

[0231] Compared to controls (e.g., SNF or SOF), the biomarker EphA1 can be used to diagnose uterine / pelvic pathology in subjects.

[0232] Specifically, serum EphA1 can be used as a blood biomarker for the early diagnosis and risk stratification of uterine / pelvic pathology. Furthermore, serum EphA1 can be used to select patients for early medical management of uterine / pelvic pathology. Therefore, it can significantly reduce diagnostic delays in uterine / pelvic pathology, improve patients' lives, and alleviate their economic burden.

[0233] Therefore, in a second aspect, the present invention relates to a method for assessing uterine / pelvic pathology in a subject, the method comprising the following steps:

[0234] a) Determine the level of the extracellular protein fraction of EphA1 in biofluid samples from subjects.

[0235] b) Compare the level of the extracellular protein fraction of EphA1 to at least one appropriate reference value for the level of the extracellular protein fraction of EphA1.

[0236] c) If the comparison in step b) indicates that the subject has a reduced level of the extracellular protein portion of EphA1 compared to the appropriate reference value, then the subject is identified as having a uterine / pelvic pathology.

[0237] In one embodiment, the method is an in vitro method.

[0238] In a particular embodiment, at least one appropriate reference value is

[0239] i. The level of EphA1 in SNF or SOF subjects, or for

[0240] ii. The average EphA1 level in a group of SNF subjects, a group of SOF subjects, or a combination thereof.

[0241] Or at least one of the appropriate reference values ​​is

[0242] iii. The predetermined level of EphA1 in SNF or SOF subjects, or...

[0243] iv. The predetermined mean level of EphA1 in a group of SNF subjects or a group of SOF subjects or a combination thereof.

[0244] Specifically, a decrease in EphA1 level of 15% or more, 20% or more, 30% or more, 40% or more, or 50% or more indicates the presence of uterine / pelvic pathology or a risk of developing uterine / pelvic pathology. Specifically, a decrease in EphA1 level of 100% or more indicates the presence of uterine / pelvic pathology. Specifically, a decrease in EphA1 level of 150% or more indicates the presence of uterine / pelvic pathology. Specifically, a decrease in EphA1 level of 200% or more indicates uterine / pelvic pathology.

[0245] In certain embodiments, a decrease of 10% or more in EphA1 indicates the presence of uterine / pelvic pathology or a risk of having or developing uterine / pelvic pathology.

[0246] Appropriately, the biological fluid sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, preferably serum.

[0247] In one embodiment, the biofluid sample is serum.

[0248] In embodiments of the invention, the protein level of EphA1 is optionally determined using a method selected from the following: ELISA assay, Western blotting, lateral flow assay, protein microarray, and mass spectrometry.

[0249] Preferably, the level of EphA1 is determined by detecting the extracellular protein portion of EphA1 using ELISA or ECLIA.

[0250] In a particular embodiment, the extracellular protein moiety of EphA1 is detected by identifying at least one of the following peptides: WEPPADTGGR (SEQ ID NO: 1), AQGELGWLLDPPK (SEQ ID NO: 2), MHCSPDGEWLVPVGR (SEQ ID NO: 3), GLYLAFHNPGACVALVSVR (SEQ ID NO: 4), APGEGPQVACTGPPSAPR (SEQ ID NO: 5), VTTVAADQSFTIR (SEQ ID NO: 6), or VHVELQFTVR (SEQ ID NO: 7). Details are shown in Table 1.

[0251] Statements made elsewhere in this document regarding the determination of EphA1 levels (e.g., in the section concerning the assessment of endometriosis) also apply here.

[0252] In the embodiments, the uterine / pelvic pathology is selected from the group consisting of: adenomyosis, uterine fibroids, ovarian cysts requiring surgery, and uterine / pelvic cancer.

[0253] In another embodiment, the method further includes selecting a treatment regimen for the subject based on a comparison of EphA1 levels with a control sample or a predetermined reference level. In a particular embodiment, the method further includes administering the selected treatment regimen to the subject, optionally wherein the selected treatment regimen includes drug-based therapy and / or surgical treatment (laparoscopy).

[0254] Detailed information on biomarkers, combinations, samples, methodological procedures, subjects, types of endometriosis, treatments, reference values, etc., is available elsewhere and applies equally to this and all other aspects.

[0255] Methods for assessing endometriosis-associated and / or uterine / pelvic pathology-associated neuropathic pain in a subject Methods for assessing pain

[0256] According to a third aspect, the present invention relates to a method for assessing neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject, the method comprising the following steps:

[0257] a) Determine the level of the extracellular protein fraction of EphA1 in biofluid samples from subjects.

[0258] b) Compare the level of the extracellular protein fraction of EphA1 to at least one appropriate reference value for the level of the extracellular protein fraction of EphA1.

[0259] c) If the comparison in step b) indicates that the subject has a reduced level of the extracellular protein portion of EphA1 compared to an appropriate reference value, then the subject is identified as having neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0260] According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

[0261] In one embodiment, the method is an in vitro method.

[0262] In a particular embodiment, at least one appropriate reference value is

[0263] i. The level of EphA1 in SNF or SOF subjects, or for

[0264] ii. The average EphA1 level in a group of SNF subjects, a group of SOF subjects, or a combination thereof.

[0265] Or at least one of the appropriate reference values ​​is

[0266] iii. The predetermined level of EphA1 in SNF or SOF subjects, or...

[0267] iv. The predetermined mean level of EphA1 in a group of SNF subjects or a group of SOF subjects or a combination thereof.

[0268] Specifically, a decrease in EphA1 level of 15% or more, 20% or more, 30% or more, 40% or more, or 50% or more indicates the presence of neuropathic pain associated with endometriosis and / or uterine / pelvic pathology, or a risk of developing neuropathic pain associated with endometriosis and / or uterine / pelvic pathology. Specifically, a decrease in EphA1 level of 100% or more indicates the presence of neuropathic pain associated with endometriosis and / or uterine / pelvic pathology. Specifically, a decrease in EphA1 level of 150% or more indicates the presence of neuropathic pain associated with endometriosis and / or uterine / pelvic pathology. Specifically, a decrease in EphA1 level of 200% or more indicates neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0269] In certain embodiments, a decrease of 10% or more in EphA1 indicates the presence of neuropathic pain associated with endometriosis and / or uterine / pelvic pathology, or a risk of having or developing neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0270] Appropriately, the biological fluid sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid, or menstrual fluid, preferably serum.

[0271] In one embodiment, the biofluid sample is serum.

[0272] In embodiments of the invention, the protein level of EphA1 is optionally determined using a method selected from the following: ELISA assay, Western blotting, lateral flow assay, protein microarray, and mass spectrometry.

[0273] Preferably, the level of EphA1 is determined by detecting the extracellular protein portion of EphA1 using ELISA or ECLIA.

[0274] In a particular embodiment, the extracellular protein moiety of EphA1 is detected by identifying at least one of the following peptides: WEPPADTGGR (SEQ ID NO: 1), AQGELGWLLDPPK (SEQ ID NO: 2), MHCSPDGEWLVPVGR (SEQ ID NO: 3), GLYLAFHNPGACVALVSVR (SEQ ID NO: 4), APGEGPQVACTGPPSAPR (SEQ ID NO: 5), VTTVAADQSFTIR (SEQ ID NO: 6), or VHVELQFTVR (SEQ ID NO: 7). Details are shown in Table 1.

[0275] Statements made elsewhere in this document regarding the determination of EphA1 levels (e.g., in the section concerning the assessment of endometriosis) also apply here.

[0276] In another embodiment, the method further includes selecting a treatment regimen for the subject based on a comparison of EphA1 levels with a control sample or a predetermined reference level. In a particular embodiment, the method further includes administering the selected treatment regimen to the subject, optionally wherein the selected treatment regimen includes drug-based therapy and / or surgical treatment (laparoscopy).

[0277] Detailed information on biomarkers, combinations, samples, methodological procedures, subjects, types of endometriosis, treatments, reference values, etc., is available elsewhere and applies equally to this and all other aspects.

[0278] Methods for monitoring progression of endometriosis, uterine / pelvic pathology, and / or endometriosis-associated and / or uterine / pelvic pathology-associated neuropathic pain in a subject Methods for determining the effectiveness of a treatment regimen for endometriosis, uterine / pelvic pathology, or endometriosis-associated and / or uterine / pelvic pathology-associated neuropathic pain in a subject

[0279] In a fourth aspect, the present invention relates to a method for monitoring the progression of endometriosis, uterine / pelvic pathology, and / or the progression of neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject, the method comprising the following steps:

[0280] i. Determine the level of the extracellular protein fraction of EphA1 in the biofluid sample from the subject according to steps a) to b) of the method described above in this paper.

[0281] ii. Repeat step i. for a specific time interval using a biofluid sample obtained from the subject during or after treatment; and

[0282] iii. The levels of the extracellular protein fraction of EphA1 identified in i. will be compared with the levels of the extracellular protein fraction of EphA1 identified in ii., wherein changes in the level of the extracellular protein fraction of EphA1 from i. to ii. indicate changes in the progression of endometriosis, uterine / pelvic pathology, and / or endometriosis-related and / or uterine / pelvic pathology-related neuropathic pain in the subject.

[0283] According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

[0284] In this embodiment, patients with endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology are monitored to determine whether the amount or concentration of EphA1 in the patient's sample changes over time. Specifically, patients with endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology are monitored to determine whether the amount or concentration of EphA1 increases, decreases, or remains unchanged over time. In this embodiment, if a decrease in the amount of EphA1 in the patient's sample is determined, patients with endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology are monitored.

[0285] In one embodiment, the method is an in vitro method.

[0286] In one embodiment, the biofluid sample is serum.

[0287] This method can be used to monitor the progression of any type of endometriosis described in this article.

[0288] This method can be used to monitor the progression of any type of uterine / pelvic pathology described in this article.

[0289] Typically, this type of monitoring is performed on subjects who have not yet received treatment for endometriosis (i.e., they have not previously received endometriosis treatment (therapies or surgery)), for uterine / pelvic pathology, or for neuropathic pain associated with endometriosis and / or uterine / pelvic pathology. Such subjects are described in this document as “untreated” subjects.

[0290] However, this type of monitoring also includes methods for subjects who have already received treatment (e.g., for endometriosis).

[0291] Monitoring the progression of endometriosis in a subject over time helps to identify disease progression (e.g., worsening of disease status or symptoms) as early as possible. This monitoring naturally involves repeated sample collection over time. Therefore, this method can be repeated for a specific subject at one or more time intervals, and the results compared to monitor the development, progression, or improvement of that subject's endometriosis over time, where changes in the amount of biomarkers tested in the biological fluid sample (e.g., serum) indicate changes in the subject's endometriosis progression.

[0292] It is also applicable to monitoring neuropathic pain associated with uterine / pelvic pathology, or endometriosis and / or uterine / pelvic pathology.

[0293] When comparing results from two or more time intervals on the same subject, disease progression can be indicated by a decrease in the level of EphA1 detected over time. A “decrease” in the level of EphA1 encompasses the absence of EphA1 in later time intervals when it was detected previously (i.e., at an earlier time interval) on the same subject (and an equivalent type of biofluid sample).

[0294] In other words, if this method is performed multiple times, disease progression can be indicated when the level of EphA1 detected at a later time interval is equal to or lower than the level of EphA1 detected at an earlier time interval.

[0295] The appropriate time interval for monitoring disease progression can be readily determined by those skilled in the art and will depend on the specific form of endometriosis being monitored. As a non-limiting example, the method may be repeated at least weekly, monthly, every six months, or at least annually, or whenever clinically necessary, i.e., when there is a significant change in endometriosis symptoms.

[0296] Detailed information on biomarkers, combinations, samples, methodological procedures, subjects, types of endometriosis, treatments, reference values, etc., is available elsewhere and applies equally to this and all other aspects.

[0297] Computer-implemented methods for assessing a patient having endometriosis, uterine / pelvic pathology, or endometriosis-associated and / or uterine / pelvic pathology-associated neuropathic pain In combination with CA-125, symptoms, or clinical data

[0298] In a fifth aspect, the present invention relates to a method for determining the therapeutic effect of a treatment regimen for a subject with endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology, the method comprising the following steps:

[0299] i. Determine the level of the extracellular protein fraction of EphA1 in the biofluid sample from the subject according to steps a) to b) of the method described above in this paper.

[0300] ii. Repeat step i. for a specific time interval using a biofluid sample obtained from the subject during or after treatment; and

[0301] iii. The levels of the extracellular protein fraction of EphA1 identified in i. will be compared with the levels of the extracellular protein fraction of EphA1 identified in ii., and the treatment regimen will be considered effective if the level of the extracellular protein fraction of EphA1 increases after treatment.

[0302] According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

[0303] In one instance, the change in EphA1 levels, which indicates the therapeutic effect, is an increase in EphA1 levels after treatment. An “increase” in EphA1 levels encompasses the detection of EphA1 at a later time interval than when it was previously (i.e., at an earlier time interval) on the same subject (and an equivalent biofluid sample type).

[0304] In one embodiment, the method is an in vitro method.

[0305] In one embodiment, the biofluid sample is serum.

[0306] Step i. may be performed first, according to the method, using a biofluid sample obtained from the subject at a time point prior to the initiation of a treatment regimen for endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology. Alternatively, step i. may be performed first, using a biofluid sample obtained from the subject at the same time as the start of the treatment regimen or at a time point after the start of a treatment regimen for neuropathic pain associated with endometriosis, uterine / pelvic pathology, or endometriosis-related and / or uterine / pelvic pathology. Therefore, this method can be used to determine the therapeutic effect of a treatment regimen for endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology, either at the start of the treatment regimen (i.e., from the start of the regimen) or at a point in time after the treatment regimen has started (i.e., determining the therapeutic effect of a treatment regimen for endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology during the treatment regimen itself).

[0307] In this embodiment, an unchanged or decreased amount or concentration of EphA1 in the sample of a subject being treated for endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology indicates treatment ineffectiveness; that is, an unchanged or decreased amount or concentration of EphA1 in the sample of a subject being treated for endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology indicates persistent or recurrent endometriosis. Specifically, if the amount of EphA1 decreases to 50% or less, treatment for endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology is ineffective. In particular, if the level of EphA1 decreases to 100%, treatment for neuropathic pain associated with endometriosis, uterine / pelvic pathology, or endometriosis-related and / or uterine / pelvic pathology-related pain will be ineffective.

[0308] Alternatively, in cases of endometriosis, unchanged EphA1 levels may indicate disease stagnation or progression from stage I to stage II, III, or IV, where EphA1 levels are comparable to those in stage I.

[0309] Improvement in disease state or symptoms (e.g., during treatment) can also be indicated by stable levels of EphA1 over time (compared to EphA1 levels observed in the absence of treatment over the same time period or to equivalent controls). In other cases, EphA1 levels will increase if the disease itself improves; this may also encompass improvements in symptoms such as pelvic pain or neuropathic pain associated with endometriosis or other uterine / pelvic pathologies.

[0310] If a treatment regimen results in a delay in disease progression or symptom development (e.g., during treatment), it can be identified as having a therapeutic effect.

[0311] A treatment regimen can also be identified as having a therapeutic effect if it leads to an improvement in the disease state or symptoms (e.g., during treatment). Methods for determining whether a treatment regimen has a therapeutic effect are well known in the art.

[0312] The treatment period refers to the time interval between treatments (e.g., 1 month, 3 months, 6 months, 1 year, 2 years, etc.).

[0313] As will be clear to those skilled in the art, the direction of change in EphA1 levels, which indicate treatment efficacy, can depend on the subject’s disease status before treatment and the control / reference used.

[0314] Changes in EphA1 levels can also indicate adherence to or compliance with prescribed treatment after treatment.

[0315] The trend used to identify whether a subject has adhered to or followed the prescribed treatment regimen is equivalent to those described above in detail regarding the determination of the treatment efficacy of a treatment regimen for endometriosis. This is because a “prescribed treatment regimen” is a recommended treatment regimen and therefore usually has a therapeutic effect (and therefore, observation of the therapeutic effect at biomarker levels is an indicator of subject adherence or compliance with the prescribed treatment regimen).

[0316] In some embodiments, subjects are monitored several times at different time points. In some embodiments, patients are monitored several times over a period of weeks, months, or years. In certain embodiments, subjects are monitored monthly or annually. In some embodiments, subjects suffering from endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology are monitored monthly or annually after a diagnosis of endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology. In some embodiments, subjects are monitored once after treatment, particularly once after surgical treatment, for subjects undergoing treatment for neuropathic pain associated with endometriosis, uterine / pelvic pathology, or endometriosis and / or uterine / pelvic pathology. Specifically, subjects undergoing treatment for endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology should be monitored monthly or annually to determine the efficacy of the treatment and / or the recurrence of neuropathic pain associated with endometriosis, uterine / pelvic pathology, or endometriosis-related and / or uterine / pelvic pathology.

[0317] This method can also be used as a screening tool to determine whether a specific regimen or treatment is effective for endometriosis, uterine / pelvic pathology, or neuropathic pain associated with and / or uterine / pelvic pathology. The regimen or treatment tested can be novel, modified, or a known regimen or treatment requiring further testing. In this context, a treatment is, for example, a drug / medicament that is available or suspected of being available for the treatment of endometriosis, uterine / pelvic pathology, or neuropathic pain associated with and / or uterine / pelvic pathology.

[0318] In embodiments, the treatment for endometriosis is selected from the group consisting of: drug-based therapies or surgical therapies. In embodiments, treatment options include surgical therapies, radiation therapy, immunotherapy, hormone therapy, ultrasound therapy, or combinations thereof. In a preferred embodiment, surgical treatment for endometriosis is laparoscopy or nerve-sparing surgery. In embodiments, drug-based therapies for endometriosis are the inhibition or targeting of neurogenic inflammation and / or analgesics and / or hormone therapy.

[0319] It also applies to neuropathic pain associated with uterine / pelvic pathology, or endometriosis, and / or uterine / pelvic pathology.

[0320] In a particular embodiment, a therapy is appropriate if an unchanged or reduced amount or concentration of EphA1 is determined in a sample of a patient being treated for endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0321] Detailed information on biomarkers, combinations, samples, methodological procedures, subjects, types of endometriosis, treatments, etc., is available elsewhere and applies equally to this matter.

[0322] Therefore, all aspects described above in detail regarding methods for assessing and / or monitoring neuropathic pain associated with endometriosis, uterine / pelvic pathology, or endometriosis-related and / or uterine / pelvic pathology-related pain are equally applicable here.

[0323] Kits and devices Data storage aspects

[0324] In another aspect, the present invention relates to a computer-implemented method for assessing neuropathic pain in patients suspected of having endometriosis, uterine / pelvic pathology, or endometriosis-related and / or uterine / pelvic pathology-related conditions, the computer-implemented method comprising the following steps:

[0325] a) Receive the value of the level of a first biomarker in a biological fluid sample of the subject, wherein the first biomarker is an extracellular protein portion of EphA1;

[0326] b) Receive the value of the level of a second biomarker in the sample of the subject, wherein the second biomarker is CA125.

[0327] c) Receive values ​​for the level of dysmenorrhea and / or lower abdominal pain according to VAS.

[0328] d) Compare the value for that level from steps (a) to (c) with a reference for the biomarker and the amount of dysmenorrhea, and / or calculate a score for assessing subjects suspected of having endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology based on the biomarker level and the amount of dysmenorrhea; and

[0329] e) Assess the subject based on the comparisons and / or calculations performed in step (d).

[0330] According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

[0331] As used herein, the term "computer implementation" means that the method is executed in an automated manner on a data processing unit, which is typically included in a computer or similar data processing apparatus. The data processing unit should receive values ​​for the quantity of the biomarker. Such values ​​may be quantities, relative quantities, or any other calculated values ​​reflecting quantities as described in detail elsewhere herein. Therefore, it should be understood that the above method does not require determining the quantity of the biomarker, but rather uses values ​​for a predetermined quantity.

[0332] In principle, the present invention also contemplates a computer program, a computer program product, or a computer-readable storage medium having the computer program tangibly embedded therein, wherein the computer program includes instructions that, when executed on a data processing apparatus or computer, perform the methods described above. Specifically, this disclosure further covers:

[0333] - A computer or computer network, comprising at least one processor, wherein the processor is adapted to perform a method according to one of the embodiments described in this specification.

[0334] - A computer-loadable data structure, adapted to perform one of the methods described in this specification when the data structure is executed on the computer.

[0335] - A computer script, wherein the computer program is adapted to perform, when executed on a computer, a method according to one of the embodiments described in this specification.

[0336] - A computer program, including program means for performing a method according to one of the embodiments described herein when the computer program is executed on a computer or on a computer network.

[0337] - A computer program, comprising program means according to the foregoing embodiments, wherein the program means is stored on a computer-readable storage medium.

[0338] - A storage medium on which a data structure is stored and wherein the data structure is adapted to perform a method according to one of the embodiments described in this specification after being loaded into the main storage device and / or working storage device of a computer or computer network.

[0339] - A computer program product having program code tools, wherein these program code tools can be stored or stored on a storage medium for performing a method according to one of the embodiments described in this specification, when these program code tools are executed on a computer or on a computer network.

[0340] - Data stream signals, typically encrypted, include data containing parameters defined elsewhere in this document, and

[0341] - The data stream signal, which is typically encrypted, includes an evaluation provided by the method of this invention.

[0342] Uses

[0343] The method according to the invention can be combined with other tests, biomarkers, clinical data or other information that can be used to diagnose or classify endometriosis in order to obtain the most reliable results.

[0344] Although CA-125 has rather weak diagnostic properties, it is still routinely used as a biomarker for endometriosis. Therefore, in the context of the methods described herein, it may be advantageous to combine the determination of EphA1 levels with CA-125 obtained from the subject.

[0345] In addition to CA-125, other symptoms or clinical data used to diagnose or classify endometriosis can also be used in conjunction with the determination of EphA1 levels. Such symptoms or clinical data may include, but are not limited to, age, dysmenorrhea, abdominal pain, or other biomarkers.

[0346] Companion diagnostics

[0347] On the other hand, kits are provided for diagnosing or staging endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in subjects. The kits include reagents suitable for determining the levels of multiple analytes in the test sample (e.g., reagents suitable for determining the levels of biomarkers disclosed herein).

[0348] The kits described in this article typically include detectably labeled agents that specifically bind to the EphA1 protein.

[0349] This kit may additionally include a detectably labeled agent that specifically binds to CA-125.

[0350] The kits described herein can take many forms. Typically, the kits will include reagents suitable for determining the levels of multiple biomarkers (e.g., EphA1 and optionally CA-125) in a sample.

[0351] Optionally, the kit may contain one or more control samples or references. Typically, the comparison between the level of a biomarker in a subject and the level of the biomarker in a control sample indicates a clinical status (e.g., a diagnosis of endometriosis). Additionally, in some cases, the kit will include written information (labels) providing a reference (e.g., predetermined values), wherein the comparison between the level of a biomarker in a subject and the reference (predicted values) indicates a clinical status (e.g., a diagnosis of endometriosis). In some cases, the kit includes software that can be used to compare biomarker levels or occurrences with a reference (e.g., a predictive model). Typically, the software will be provided in a computer-readable format (such as a CD), but may also be available for download via the Internet. However, the kit is not limited to this, and other variations will be apparent to those skilled in the art.

[0352] The kit components can be contained in containers suitable for transport. Details regarding biomarkers are given above and apply here as well. Appropriately, biomarkers may be proteins.

[0353] In some instances, the kit includes a detectably labeled reagent on a continuous (e.g., solid) surface, such as a sideflow surface. Alternatively, in instances including more than one detectably labeled reagent, the detectably labeled reagent may be located in different (i.e., spatially separated) areas on a (e.g., solid) surface, such as a multi-walled microtiter plate (e.g., for ELISA assays). Other suitable surfaces and containers well known in the art may also form part of the kits described herein.

[0354] In one example, the kit further includes one or more reagents for detecting a detectably labeled pharmaceutical agent. Suitable reagents are well known in the art and include, but are not limited to, standard reagents and buffers (and are well known in the art) required to perform any suitable detection method that can be used.

[0355] In one instance, the kit includes one or more of the following: a multi-well plate, ball bearings, extraction buffer, extraction bottle, and a side-flow device for measuring flow.

[0356] It also provides a measuring device for diagnosing endometriosis, uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in subjects.

[0357] Typically, the device includes a surface having at least one detectably labeled agent that specifically binds to the EphA1 protein.

[0358] This device may additionally include a detectably labeled agent that specifically binds to CA-125.

[0359] If two detectably labeled agents are used, they can be located in different regions of the surface. In other words, two detectably labeled agents can be located in different (i.e., spatially separated) regions on a (e.g., solid) surface (such as a porous microtiter plate). Detectably labeled agents that specifically bind to a target biomarker are described in detail elsewhere in this document.

[0360] The measuring apparatus includes a surface on which a detectably labeled reagent is located. Suitable surfaces include continuous (e.g., solid) surfaces, such as lateral flow surfaces, dot-printed surfaces, test paper surfaces, or surfaces suitable for surface plasmon resonance. Other suitable surfaces include microtiter plates, well plates, etc. Other suitable surfaces well known in the art may also form part of the measuring apparatus described herein.

[0361] Therefore, suitable measurement device forms include, but are not limited to, those suitable for performing any of the side flow, dot blot, ELISA, or surface plasmon resonance assays to detect the presence, level, or absence of the target biomarker.

[0362] Figure 2

[0363] Biomarker levels and / or reference levels can be stored in a suitable data storage medium (e.g., a database) and are therefore also available for future diagnosis. This also allows for the efficient diagnosis of disease prevalence, as appropriate reference results can be identified in the database once it is confirmed that a subject from whom a corresponding reference sample is obtained (in the future) does indeed have endometriosis. As used herein, "database" includes data collected on a suitable storage medium (e.g., analyte and / or reference level information and / or patient information). Furthermore, the database may further include a database management system. Preferably, the database management system is a web-based, hierarchical, or object-oriented database management system. Additionally, the database can be a federated or integrated database. More preferably, the database will be implemented as a distributed (federated) system, for example, as a client-server system. More preferably, the database is configured to allow a search algorithm to compare the test dataset with datasets included in the dataset. Specifically, by using this algorithm, similar or identical datasets indicative of endometriosis can be searched in the database (e.g., query search). Thus, if identical or similar datasets can be identified in the dataset, the test dataset is associated with endometriosis. Therefore, the information obtained from the dataset can be used to diagnose endometriosis or based on test datasets obtained from subjects. More preferably, the dataset includes the characteristic values ​​of all analytes included in any of the above groups.

[0364] The methods described herein may further include communicating results or diagnoses (or both) to, for example, technicians, physicians, or patients. In some instances, computers will be used to communicate results or diagnoses (or both) to relevant parties (e.g., physicians and their patients).

[0365] In some instances, the results or diagnosis (or both) are communicated to the subject as quickly as possible after diagnosis is obtained. The results or diagnosis (or both) may be communicated to the subject by their attending physician. Alternatively, the results or diagnosis (or both) may be sent to the subject by email or by telephone. Computers can be used to communicate results or diagnoses via email or telephone. In some instances, a combination of computer hardware and software familiar to those skilled in the telecommunications field may be used to automatically generate and deliver messages containing results or diagnoses to the subject.

[0366] Figure 3

[0367] This article also provides the use of the biomarker EphA1 as a biofluid biomarker for endometriosis (especially early stage), uterine / pelvic pathology, or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology.

[0368] In preferred embodiments, EphA1 is commonly used as a biomarker for endometriosis. In this document, "endometriosis" generally refers to all forms of endometriosis, including but not limited to peritoneal endometriosis, endometriomas, and deep invasive endometriosis.

[0369] EphA1 can also be used in conjunction with CA-125.

[0370] In a preferred embodiment, EphA1 can be used as a biomarker for neuropathic pain associated with uterine / pelvic pathology, or endometriosis and / or uterine / pelvic pathology.

[0371] Detailed information on biomarkers, samples, methods, subjects, types of endometriosis, etc., is available elsewhere and applies equally to this matter.

[0372] Figure 4

[0373] The methods, kits, assay devices, and uses described herein can be used as part of companion diagnostics, such as as part of a medical device (typically an in vitro device), which provides the information necessary for the safe and effective use of the corresponding drug or biological product for the treatment or prevention of endometriosis.

[0374] In other embodiments, the invention relates to the aspect in which the subject's endometriosis is classified as stage I or stage II endometriosis according to the American Society for Reproductive Medicine's revised scoring system (r-ASRM):

[0375] 1. An in vitro method for diagnosing endometriosis in a subject, the method comprising the following steps:

[0376] a) Determine the level of the extracellular protein fraction of EphA1 in biofluid samples from the subject.

[0377] b) Compare the level of the extracellular protein fraction of EphA1 to at least one appropriate reference value for the level of the extracellular protein fraction of EphA1.

[0378] c) If the comparison in step b) indicates that the subject has a reduced level of the extracellular protein portion of EphA1 compared to the appropriate reference value, then the subject is identified as having endometriosis.

[0379] 2. The method according to aspect 1, wherein the at least one suitable reference value is

[0380] i. The level of EphA1 in non-pathological or symptomatic subjects, or...

[0381] ii. The average level of EphA1 in a group of non-pathological subjects, a group of symptomatic subjects, or a combination thereof.

[0382] Or at least one of the appropriate reference values ​​is

[0383] iii. Predicted levels of EphA1 in non-pathological or symptomatic subjects, or...

[0384] iv. The predetermined mean level of EphA1 in a group of non-pathological subjects, a group of symptomatic subjects, or a combination thereof.

[0385] 3. The method according to aspect 1 or 2, wherein the at least one suitable reference value is

[0386] ●According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the level of EphA1 in non-pathological subjects, symptomatic subjects, or subjects with stage I, II, III, or IV endometriosis, or...

[0387] ●According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the average EphA1 level in a group of subjects who are non-pathological, symptomatic, or a combination thereof, or in a group of subjects with stage I, II, III, or IV endometriosis,

[0388] Or at least one of the appropriate reference values ​​is

[0389] ●According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the predetermined value of EphA1 level in non-pathological subjects, or in symptomatic subjects, or in subjects with stage I, II, III, or IV endometriosis, or...

[0390] ● The predetermined mean level of EphA1 in a cohort of non-pathological subjects, symptomatic subjects, or a combination thereof, or in a cohort of subjects with stage I, II, III, or IV endometriosis, according to the American Society for Reproductive Medicine's revised scoring system (r-ASRM).

[0391] 4. The method according to any of the preceding claims, wherein the biofluid sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, preferably the biofluid sample is serum.

[0392] 5. The method according to any of the foregoing aspects, wherein the subject is a human, preferably a female human.

[0393] 6. The method according to any of the foregoing aspects, wherein the protein level of EphA1 (particularly its extracellular protein fraction) is optionally determined using a method selected from the following: ELISA assay, Western blotting, lateral flow assay, protein microarray, and mass spectrometry.

[0394] 7. The method according to any of the foregoing aspects, wherein the extracellular protein portion of EphA1 is detected by mass spectrometry.

[0395] 8. The method according to any of the foregoing aspects, wherein the extracellular protein moiety of EphA1 is detected by identifying at least one of the following peptides: WEPPADTGGR (SEQ ID NO: 1), AQGELGWLLDPPK (SEQ ID NO: 2), MHCSPDGEWLVPVGR (SEQ ID NO: 3), GLYLAFHNPGACVALVSVR (SEQ ID NO: 4), APGEGPQVACTGPPSAPR (SEQ ID NO: 5), VTTVAADQSFTIR (SEQ ID NO: 6) or VHVELQFTVR (SEQ ID NO: 7).

[0396] 9. The method according to any of the foregoing aspects, wherein the endometriosis is selected from the group consisting of: peritoneal endometriosis, endometrioma, and deep invasive endometriosis.

[0397] 10. The method according to any of the foregoing aspects further includes selecting a treatment regimen for the subject based on a comparison of the level of the extracellular protein fraction of EphA1 with a control sample or a predetermined reference level.

[0398] 11. The method according to aspect 10, further comprising administering a selected treatment regimen to the subject, optionally wherein the selected treatment regimen includes a drug-based therapy and / or surgical treatment (laparoscopy) or a combination thereof.

[0399] 12. An in vitro method for monitoring the progression of endometriosis in a subject, the method comprising the following steps:

[0400] i. Determining the level of the extracellular protein fraction of EphA1 in a biofluid sample from the subject, according to method steps a) to b) of any one of aspects 1 to 13.

[0401] ii. Repeat step i. for a specific time interval using a biofluid sample obtained from the subject during or after treatment; and

[0402] iii. The levels of the extracellular protein fraction of EphA1 identified in i. are compared with the levels of the extracellular protein fraction of EphA1 identified in ii., wherein changes in the levels of the extracellular protein fraction of EphA1 from i. to ii. indicate changes in the progression of endometriosis in the subject.

[0403] 13. An in vitro method for determining the therapeutic effect of a treatment regimen for endometriosis in a subject, the method comprising the following steps:

[0404] i. Determining the level of the extracellular protein fraction of EphA1 in a biofluid sample from the subject, according to method steps a) to b) of any one of aspects 1 to 13.

[0405] ii. Repeat step i. for a specific time interval using a biofluid sample obtained from the subject during or after treatment; and

[0406] iii. The level of the extracellular protein fraction of EphA1 identified in i. is compared with the level of the extracellular protein fraction of EphA1 identified in ii., and if the level of the extracellular protein fraction of EphA1 increases after treatment, the treatment regimen is deemed to have therapeutic effect.

[0407] 14. The method according to any one of the preceding claims, further comprising assessing dysmenorrhea and / or assessing lower abdominal pain according to the Visual Analogue Scale (VAS).

[0408] 15. The method according to any one of the foregoing aspects, further comprising determining the level of CA-125 in the biofluid sample from the subject.

[0409] 16. The method according to aspect 15, comprising calculating

[0410] ●The ratio of the amount or concentration of the extracellular protein fraction of EphA1 to the amount or concentration of CA-125, or

[0411] ●The ratio of the amount or concentration of the extracellular protein fraction of EphA1 to dysmenorrhea, or

[0412] ●The amount or concentration of the extracellular protein fraction of EphA1 relative to the ratio of the amount or concentration of CA-125 to dysmenorrhea, or

[0413] ● The ratio of the amount or concentration of the extracellular protein portion of EphA1 to lower abdominal pain according to the VAS scale.

[0414] 17. A computer-implemented method for assessing a patient suspected of having endometriosis, the computer-implemented method comprising the following steps:

[0415] a) Receive the value of the level of a first biomarker in a biological fluid sample of the subject, wherein the first biomarker is an extracellular protein portion of EphA1;

[0416] b) Receive the value of the level of a second biomarker in the sample of the subject, wherein the second biomarker is CA125.

[0417] c) Receive values ​​for the level of dysmenorrhea and / or lower abdominal pain according to VAS.

[0418] d) Compare the values ​​for the level from steps (a) to (c) with a reference for the biomarker and the amount of dysmenorrhea, and / or calculate a score for assessing the subject suspected of having endometriosis based on the level of the biomarker and the amount of dysmenorrhea; and

[0419] e) Assess the subject based on the comparisons and / or calculations performed in step (d).

[0420] 18. Use of elevated EphA1 levels in biological fluid samples as a biomarker for endometriosis.

[0421] 19. The use according to aspect 18, wherein the biological fluid sample is blood or blood-derived, preferably serum.

[0422] 20. The use as described in aspects 18 and 19, wherein said use is for the diagnosis and / or classification of endometriosis.

[0423] 21. A kit for diagnosing and / or classifying endometriosis in a subject, comprising at least one detectably labeled agent that specifically binds to the EphA1 protein.

[0424] 22. The kit according to aspect 21, further comprising one or more reagents for detecting the detectably labeled pharmaceutical agent.

[0425] 23. An assay device for diagnosing and / or classifying endometriosis in a subject, the device comprising a surface having at least one detectably labeled agent specifically binding to an EphA1 protein located thereon.

[0426] The invention is illustrated by the following non-limiting examples. The following examples and drawings are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the described procedures without departing from the spirit of the invention.

[0427] Example

[0428] Example 1: Diagnostic performance of the biomarker EphA1 in women with endometriosis

[0429] For the measurements, a total of 164 serum samples from human women were analyzed (see the corresponding section of this article for clinical data). Assay concentrations were determined using advanced mass spectrometry-based proteomics techniques. The case group consisted of patients diagnosed with endometriosis (peritoneal endometriosis (with or without endometriomas) and / or deep invasive endometriosis; rASRM stages I-IV) or other uterine / pelvic pathologies diagnosed by laparoscopy (e.g., adenomyosis, uterine fibroids, etc.) and subsequently histologically confirmed, while the control group included symptomatic women without endometriosis or other uterine / pelvic pathologies.

[0430] Advanced mass spectrometry-based proteomics techniques were used to determine the concentration of EphA1 in human serum.

[0431] The results are shown in Figure 1.

[0432] Serum EphA1 levels were decreased in stages I through IV (and in other uterine / pelvic pathological findings; case SOF) compared to the control group (symptomatic control without uterine and pelvic pathology, “Ctrl SNF”; SNF = symptomatic no findings).

[0433] like Figure 5 As shown in a and 2b, serum EphA1 was decreased in women with other uterine / pelvic pathological findings (case SOF) compared to symptomatic control women without endometriosis and without uterine and pelvic pathology (“Ctrl SNF”; SNF = symptomatic no finding).

[0434] like Figure 6As shown in a and 3b, serum EphA1 levels were lower in “cases” of women with early-stage rASRM I / II endometriosis compared to control women (“Ctrl”) without endometriosis and without uterine and pelvic pathology.

[0435] like Figure 7 As shown in a and 4b, serum EphA1 levels were lower in “cases” of women with early-stage rASRM stage I endometriosis compared to women without endometriosis and without uterine and pelvic pathology (“Ctrl”).

[0436] like Figure 9 As shown in a and 5b, serum EphA1 levels were lower in “cases” of women with early-stage rASRM stage II endometriosis compared to control women (“Ctrl”) without endometriosis and without uterine and pelvic pathology.

[0437] like ​ As shown in a and 6b, serum samples from women with stage III endometriosis (“cases”) showed decreased EphA1 levels compared to control women (“Ctrl”) without endometriosis and without uterine and pelvic pathology.

[0438] like ​ As shown in a and 7b, serum samples from women with rASRM stage IV endometriosis showed decreased EphA1 levels compared to controls (“Ctrl”) without endometriosis and without uterine and pelvic pathology.

[0439] In Table 2 below, model performance is determined by examining the area under the curve (AUC). The best possible AUC is 1, while the lowest is 0.5. The optimal critical value is selected using the Youden index (the maximum sum of sensitivity and specificity - 1).

[0440] Table 2 shows the diagnostic performance of the EphA1 biomarker in distinguishing between women with histologically confirmed endometriosis and women without endometriosis (control) using recipient operating characteristic (ROC) analysis. The area under the curve (AUC) and associated 95% confidence intervals for the ROC analysis are described. N represents the number of samples tested (cases plus controls, with the number in each group varying depending on the analyte).

[0441]

[0442] Serum EphA1 can be used as a blood biomarker for the early diagnosis and risk stratification of endometriosis and other uterine / pelvic pathologies such as adenomyosis, uterine fibroids, ovarian cysts requiring surgery, and uterine / pelvic cancers such as ovarian cancer or endometrial cancer.

[0443] Serum EphA1 can be used to select patients with stage I and II rASRM disease for early medical management of endometriosis. Therefore, it can significantly reduce diagnostic delays of endometriosis in women with signs and symptoms of endometriosis (such as dysmenorrhea, pelvic pain, and infertility), improve patients' lives, and alleviate their economic burden.

[0444] Serum EphA1 can be used as a potential companion diagnostic biomarker for therapy selection and monitoring.

[0445] Example 2: Diagnostic performance of biomarker EphA1 versus CA-125 in women with endometriosis

[0446] In further experiments, the performance of the biomarker EphA1 was compared with that of the current standard biomarker CA-125.

[0447] The concentration of CA-125 was determined using a cobas e 601 analyzer. CA 125 II detection using the cobas e 601 analyzer is based on Elecsys® Electro-Chemical Luminescence (ECL) technology. In short, biotin-labeled and ruthenium-labeled antibodies are bound to the corresponding amounts of undiluted sample and incubated on the analyzer. Subsequently, streptavidin-coated magnetic microparticles are added to the instrument and incubated to promote the binding of the biotin-labeled immune complex. After this incubation step, the reaction mixture is transferred to the measuring cell, where the magnetic beads are magnetically trapped on the electrode surface. ProCell M buffer containing tripropylamine (TPA) for subsequent ECL reactions is then introduced into the measuring cell to separate the bound immunoassay complex from the free remaining particles. Voltage sensing between the working and counter electrodes then initiates a reaction, causing the ruthenium complex and TPA to emit photons. The resulting electrochemiluminescence signal is recorded by a photomultiplier tube and converted into a numerical value indicating the concentration level of the corresponding analyte.

[0448] The results are shown in Figures 8a and 8b, and Figures 9a and 9b, and in Table 3. Note that for CA-125, slightly more sample measurements were available in some subgroups.

[0449]

[0450] Figures 8a and 8b, and Figures 9a and 9b, show the differences in early-stage rASRM stage I (Figure 8) and stage II (Figure 9) compared to a control group (“Ctrl”) without endometriosis and without uterine and pelvic pathology. ​ CA-125 in serum samples from female “cases”.

[0451] Box plots for CA-125 were generated using a cobas e 601 analyzer based on Elecsys® electrochemiluminescence (ECL) technology for controls and for stages I and II of endometriosis, as shown in Figures 8a and 8b, and Figures 9a and 9b. Data were presented using box plots and whisker plots, including the median (middle quartile), quartile range (representing the middle 50% of the scores in the group), upper quartile (75% of scores are below the upper quartile), and lower quartile (25% of scores are below the lower quartile). The 5th and 95th percentiles are also shown.

[0452] As can be seen from the comparison in Tables 2 and 3, serum EphA1 shows better diagnostic performance for early-stage (stage I and II) endometriosis compared to the reference biomarker CA-125.

[0453] In Table 4 below, the performance of EphA1 as a biomarker in combination with CA-125 was determined by examining the area under the curve (AUC) for early endometriosis (stages I and II), late endometriosis (stages III and IV), and all stages (stages I to IV).

[0454] Table 4: Diagnostic performance of serum EphA1 biomarker combined with biomarker CA-125 in women with endometriosis and controls. CA-125 was measured using a cobas e 601 analyzer based on Elecsys® electrochemiluminescence (ECL) technology. Due to the low sample size, bivariate analyses for individual stages (I, II, III, IV, SOF) were not performed. Only samples with both EphA1 and CA-125 measurements available were used.

[0455]

[0456] Combined serum EphA1 and serum CA-125 can be used as blood biomarkers for (early) diagnosis and risk stratification of endometriosis or other uterine / pelvic pathologies, particularly for early diagnosis of rASRM stage 1 and 2 endometriosis.

[0457] Compared to the CA-125 reference biomarker alone, the combined serum EphA1 and serum CA-125 showed better identification for all cases (endometriosis and other uterine / pelvic pathologies).

[0458] Combined serum EphA1 and serum CA-125 can be used to select patients for early medical management of endometriosis (medical therapy or surgery). Therefore, it can significantly reduce diagnostic delays in endometriosis, improve patients' lives, and alleviate their economic burden.

[0459] As potential companion diagnostic biomarkers, combined serum EphA1 and serum CA-125 can be used for therapy selection and monitoring.

[0460] Example 3: Clinical data in cohorts (cases and controls):

[0461] 1) Cases of endometriosis (or other uterine pathologies):

[0462] Total number of patients in the queue: 116

[0463] a) Hormonal contraceptive pills / devices:

[0464]

[0465] No contraceptive hormones / devices: 54.31%

[0466] Oral contraceptive hormones / devices: 45.69%

[0467] b) Endometriosis staging / other uterine / pelvic pathological findings:

[0468]

[0469] 2) Control: No findings, severe pain ("Symptoms present, no endometriosis and no uterine or pelvic pathology")

[0470] a) Total number of patients in the cohort: 48; Hormonal contraceptives / devices:

[0471]

[0472] No contraceptive hormones / devices: 58.33%

[0473] Taking contraceptive hormones / devices: 37.50%

[0474] Example 4:

[0475] Materials and methods:

[0476] Proteomics discovery methods for detecting EphA1

[0477] Advanced mass spectrometry (MS)-based proteomics, including automated sample preparation procedures, was used to analyze human serum samples from women with pain symptoms and endometriosis or other uterine / pelvic pathologies (such as adenomyosis and uterine fibroids) (cases) and symptomatic women without endometriosis (symptom controls). Serum samples were blinded for clinical information and randomized for proteomic analysis.

[0478] Samples were digested with trypsin / endopeptide LysC to generate proteolytic peptides, which were then subjected to high-resolution liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a data-independent acquisition (DIA) method. Based on batch design, an in-depth spectral library of 50 representatively selected samples spanning all six depleted batches was generated using advanced neural network-based data analysis tools.

[0479] For basic quality control, the number of identified proteomes for each sample and the principal component analysis (PCA) performed are determined, which does not show obvious separation of the samples.

[0480] To address batch effects related to sample preparation, protein strength data were corrected for accurate sample comparison. Furthermore, serum contamination analysis was performed and compared with a previously established hemolysis scale based on appearance.

[0481] 4.1 Sample Preparation

[0482] Serum samples were transported by Roche on dry ice and stored at -80°C. For experimental processing, samples were thawed on ice and centrifuged at 2,000 xg for 15 min at 4°C. For recording, a hemolysis score was determined before transferring the supernatant to a new tube. Following the manufacturer's instructions, 7 μl of serum from each sample was subjected to depletion of 14 high-abundance proteins using a highly selective Top14 high-abundance protein removal microcentrifuge column (Thermo Scientific). Depleted lyophilized plasma protein samples were subjected to enzymatic digestion and peptide removal using an MS sample preparation kit, following the manufacturer's instructions. The workflow was performed in 96-well plates using an Agilent BRAVO automated liquid handling platform. Serum control samples were used in each sample preparation batch and were treated identically.

[0483] 4.2 Mass Spectrometry Analysis

[0484] LC-MS / MS analysis was performed on an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) equipped with an Easy n-LC 1200 UHPLC system. The peptide was separated by a 125-min gradient before being sprayed directly into the mass spectrometer using a nanoelectrospray ionization source (Proxeon Biosystems). For each analysis, the mass spectrometer was operated in data-independent mode (DIA).

[0485] 4.3 Data Processing

[0486] All raw files acquired in this study were processed using the DIA-NN software suite (version 1.8.0) for peptide / protein identification and quantification using a select Uniprot database (Swissprot and varsplic, including protein isoforms, version 2020_03). The false discovery rate (FDR) for protein identification was set to 1%. A spectral library (DIA Speclib) was generated from 50 single DIA files using the "FASTA digest for library free search" option, and "Deep learning" was enabled for RT estimation. "Unrelated runs" were enabled to individually determine the quality accuracy and retention time scan window for each run.

[0487] Proteome inference from DiaNN outputs was performed using a custom algorithm based on Nesvizhskii & Aebersold. For protein quantification, the MaxLFQ algorithm from the DIA-NN R package (https: / / github.com / vdemichev / diann-rpackage) was applied, with log10 intensity subjected to median scaling. Protein intensity data were corrected for experimental batch effects (“depletion batches”) related to sample preparation, allowing for proper comparison of samples using the removeBatchEffect function from the LIMMA R-package (10.18129 / B9.bioc.limma). For principal component analysis (PCA), only proteins without missing values ​​were considered.

[0488] 4.4 Calculation of Serum Contamination Index

[0489] The red blood cell and platelet contamination indices were calculated based on a list of gene names provided by Geyer et al., 2019. For each sample, the corrected log10 average protein intensity of the proteins expressed in all samples from the list was calculated.

[0490] Example 5: Structure of the EphA1 protein

[0491] The EphA1 protein consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains a hepatin-binding domain or a hepatin receptor-ligand-binding domain (LBD), cysteine-rich regions (including the Sushi domain and an EGF-like domain), and fibronectin type III repeat sequences (FN1 and FN2). The transmembrane domain (TM) connects the extracellular and intracellular domains. The intracellular domain consists of a juxtamembrane domain, a kinase domain, and a SAM domain. Figure 10 shows the structure of the EphA1 protein (Figure adapted from OwensHE. Corrected Thesis. Characterisation of EphA1 and its potential role in Alzheimer's Disease. 2019).

Claims

1. A method for assessing endometriosis in a subject, the method comprising the following steps: a) Determine the level of the extracellular protein fraction of EphA1 in biofluid samples from the subject. b) Compare the level of the extracellular protein fraction of EphA1 to at least one appropriate reference value for the level of the extracellular protein fraction of EphA1. c) If the comparison in step b) indicates that the subject has a reduced level of the extracellular protein portion of EphA1 compared to the appropriate reference value, then the subject is identified as having endometriosis. According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

2. The method according to claim 1, wherein the endometriosis is selected from the group consisting of: peritoneal endometriosis, endometrioma, and deep invasive endometriosis.

3. A method for assessing uterine / pelvic pathology in a subject, the method comprising the following steps: a) Determine the level of the extracellular protein fraction of EphA1 in biofluid samples from the subject. b) Compare the level of the extracellular protein fraction of EphA1 to at least one appropriate reference value for the level of the extracellular protein fraction of EphA1. c) If the comparison in step b) indicates that the subject has a reduced level of the extracellular protein portion of EphA1 compared to the appropriate reference value, then the subject is identified as having a uterine / pelvic pathology.

4. The method of claim 3, wherein the uterine / pelvic pathology is selected from the group consisting of: adenomyosis, uterine fibroids, ovarian cysts requiring surgery, and uterine / pelvic cancer.

5. A method for assessing neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject, the method comprising the following steps: a) Determine the level of the extracellular protein fraction of EphA1 in biofluid samples from the subject. b) Compare the level of the extracellular protein fraction of EphA1 to at least one appropriate reference value for the level of the extracellular protein fraction of EphA1. c) If the comparison in step b) indicates that the subject has a reduced level of the extracellular protein portion of EphA1 compared to the appropriate reference value, then the subject is identified as suffering from neuropathic pain associated with endometriosis and / or uterine / pelvic pathology. According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

6. The method according to any of the preceding claims, wherein the biofluid sample is blood, serum, plasma, capillary blood, interstitial fluid, peritoneal fluid or menstrual fluid, preferably serum.

7. The method according to any of the preceding claims, further comprising selecting a treatment regimen for the subject based on a comparison of the level of the extracellular protein fraction of EphA1 with a control sample or a predetermined reference level.

8. The method according to any of the preceding claims, further comprising administering a selected treatment regimen to the subject, wherein the selected treatment regimen optionally includes a drug-based therapy and / or surgical treatment (laparoscopy).

9. A method for monitoring the progression of endometriosis, uterine / pelvic pathology, and / or the progression of neuropathic pain associated with endometriosis and / or uterine / pelvic pathology in a subject, the method comprising the steps of: i. Method steps a) to b) according to any one of claims 1 to 8, determining the level of the extracellular protein fraction of EphA1 in a biofluid sample from the subject, ii. Repeat step i for a specific time interval using a biofluid sample obtained from the subject during or after treatment; as well as iii. The levels of the extracellular protein fraction of EphA1 identified in i. will be compared with the levels of the extracellular protein fraction of EphA1 identified in ii., wherein changes in the levels of the extracellular protein fraction of EphA1 from i. to ii. indicate changes in the progression of endometriosis, uterine / pelvic pathology, and / or endometriosis-related and / or uterine / pelvic pathology-related neuropathic pain in the subject. According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

10. A method for determining the therapeutic effect of a treatment regimen for a subject with endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology, the method comprising the steps of: i. Method steps a) to b) according to any one of claims 1 to 8, determining the level of the extracellular protein fraction of EphA1 in a biofluid sample from the subject, ii. Repeat step i for a specific time interval using a biofluid sample obtained from the subject during or after treatment; as well as iii. The levels of the extracellular protein fraction of EphA1 identified in i. are compared with the levels of the extracellular protein fraction of EphA1 identified in ii., and if the levels of the extracellular protein fraction of EphA1 increase after treatment, the treatment regimen is deemed to have a therapeutic effect. According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

11. The method according to any one of the preceding claims, further comprising assessing dysmenorrhea and / or assessing lower abdominal pain according to the Visual Analogue Scale (VAS).

12. The method according to any one of the preceding claims, further comprising determining the level of CA-125 in the biofluid sample from the subject.

13. The method of claim 12, further comprising calculating ●The ratio of the amount or concentration of the extracellular protein fraction of EphA1 to the amount or concentration of CA-125, or ●The ratio of the amount or concentration of the extracellular protein fraction of EphA1 to dysmenorrhea, or ●The amount or concentration of the extracellular protein fraction of EphA1 relative to the ratio of the amount or concentration of CA-125 to dysmenorrhea, or ● The ratio of the amount or concentration of the extracellular protein portion of EphA1 to lower abdominal pain according to the VAS scale.

14. A computer-implemented method for assessing neuropathic pain in patients suspected of having endometriosis, uterine / pelvic pathology, and / or endometriosis-related and / or uterine / pelvic pathology-related pain, the computer-implemented method comprising the steps of: a) Receive the value of the level of a first biomarker in a biological fluid sample of the subject, wherein the first biomarker is an extracellular protein portion of EphA1; b) Receive the value of the level of a second biomarker in the sample of the subject, wherein the second biomarker is CA125. c) Receive values ​​for the level of dysmenorrhea and / or lower abdominal pain according to VAS. d) Compare the values ​​for the level from steps (a) to (c) with a reference for the biomarker and the amount of dysmenorrhea, and / or calculate a score for the subject to assess suspected endometriosis, uterine / pelvic pathology, and / or neuropathic pain associated with endometriosis and / or uterine / pelvic pathology based on the level of the biomarker and the amount of dysmenorrhea; as well as e) Assess the subject based on the comparisons and / or calculations performed in step (d). According to the American Society for Reproductive Medicine's revised scoring system (r-ASRM), the subjects' endometriosis was classified as stage I or stage II endometriosis.

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