Diagnostic methods, disease monitoring methods, and kits for endometriosis

Abstract

To provide methods for diagnosing endometriosis or determining whether or not a subject has endometriosis, methods for determining the degree of fibrosis or adhesions in the endometrium of a subject with endometriosis, methods for predicting pain in a subject with endometriosis, methods for monitoring the pathological condition of a subject with endometriosis, etc., and kits for performing these methods. [Solution] We found that there are multiple markers present in endometriosis patients that differ in quantity from those present in healthy individuals, and that by measuring the amount of these markers, it is possible to diagnose whether or not a subject has endometriosis, determine the degree of endometrial fibrosis and adhesions, predict pain in subjects with endometriosis, and monitor the pathological condition of subjects with endometriosis.

Description

[Technical Field]

[0001] This disclosure relates to methods for diagnosing endometriosis or determining whether or not a subject has endometriosis, methods for determining the degree of fibrosis or adhesions in the endometrium of a subject with endometriosis, methods for predicting pain in a subject with endometriosis, methods for monitoring the pathological condition of a subject with endometriosis, etc., and kits for performing these methods. [Background technology]

[0002] Endometriosis is an estrogen-dependent inflammatory disease affecting 6-10% of women of childbearing age, with infertility and pelvic pain occurring in over 50% of patients (Non-Patent Literature 1). Various hypotheses have been proposed regarding the pathogenesis of endometriosis (Non-Patent Literature 2), and associations with various factors such as inflammatory cytokines / chemokines, growth factors, and hormones have been reported (Non-Patent Literature 3). Furthermore, hormone-targeted drugs have already been developed, and the alleviation of pain and improvement of disease symptoms have been demonstrated with hormonal agents such as GnRH antagonists and progesterone preparations (Non-Patent Literature 4, 5). However, hormonal therapy has strong side effects, making long-term use difficult, which poses a therapeutic problem (Non-Patent Literature 6). In addition, drug development targeting inflammatory cytokines / chemokines is progressing, and anti-IL-8 antibodies have shown strong disease-improving effects in endometriosis monkey models (Patent Literature 1).

[0003] In diagnosing endometriosis, a definitive diagnosis can only be made through invasive methods such as endoscopic observation or open surgery. Furthermore, it is difficult for patients to distinguish between normal menstrual symptoms (such as pain) and the early symptoms of endometriosis. As a result, the hurdle to diagnosis for potential patients is high, and it is estimated that it takes an average of 7 to 11 years from the onset of symptoms to a definitive diagnosis. This delay in definitive diagnosis leads to a delay in the initiation of endometriosis treatment, which is a major clinical problem (Non-Patent Documents 7, 8). For this reason, there is a strong demand for non-invasive diagnostic methods that are less burdensome for patients, such as blood markers, but highly accurate diagnostic methods have not yet been established (Non-Patent Document 9). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO2018 / 025982 [Non-patent literature]

[0005] [Non-Patent Document 1] Giudice LC. Endometriosis. N Engl J Med 2010; 362:2389-98. [Non-Patent Document 2] Rogers PA et al. Research priorities for endometriosis. Reprod Sci 2017; 24:202-226. [Non-Patent Document 3] Beste MT et al. Molecular network analysis of endometriosis reveals a role for c-Jun-regulated macrophage activation. Sci Transl Med. 2014; 6:222ra216. [Non-Patent Document 4] Taylor HS et al. Treatment of Endometriosis-Associated Pain with Elagolix, an Oral GnRH Antagonist. N Engl J Med. 2017; 377:28-40. [Non-Patent Document 5] Kohler G et al. A dose-ranging study to determine the efficacy and safety of 1, 2, and 4mg of dienogest daily for endometriosis. Int J Gynaecol Obstet. 2010; 108:21-5. [Non-Patent Document 6] Treatment of Endometriosis-Associated Pain with Elagolix, an Oral GnRH Antagonist. [Non-Patent Document 7] Greene R et al. Diagnostic experience among 4,334 women reporting surgically diagnosed endometriosis. Fertility and Sterility, 2009; 91:32-39. [Non-Patent Document 8] Manderson L et al. Circuit breaking: Pathways of treatment seeking for women with endometriosis in Australia. Qualitative Health Research, 2008; 18:522-534. [Non-Patent Document 9] Fassbender A et al. Update on biomarkers for the detection of endometriosis. Biomed Res Int 2015; 2015:130854. [Overview of the project] [Problems that the invention aims to solve]

[0006] This disclosure is made in view of the circumstances described herein, and its purpose is to provide a method for diagnosing endometriosis or a method for determining whether or not a subject has endometriosis, a method for determining the degree of fibrosis or adhesions in the endometrium of a subject with endometriosis, a method for predicting pain in a subject with endometriosis, a method for monitoring the pathological condition of a subject with endometriosis, etc., and kits for performing these methods. [Means for solving the problem]

[0007] The inventors of this disclosure have diligently researched methods for diagnosing and monitoring the pathological condition of endometriosis and have found that there are several markers present in endometriosis patients that differ in quantity from those present in healthy individuals, and that by measuring the amount of these markers, it is possible to diagnose whether or not a subject has endometriosis, determine the degree of endometrial fibrosis and adhesions, predict pain in subjects with endometriosis, and monitor the pathological condition of subjects with endometriosis.

[0008] This disclosure is based on such findings and specifically relates to the following inventions. [1] A method for diagnosing endometriosis, A method comprising the step of measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B, in a sample obtained from a subject. [2] A method for determining whether or not a subject has endometriosis, A method comprising the step of measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B, in a sample obtained from a subject. [3] The method according to [1] or [2], further comprising the step of indicating that the subject from which the sample is derived has or may have endometriosis if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is high in the sample obtained from the subject, or if the abundance of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is low. [4] A method for determining the degree of endometrial fibrosis in a subject, A method comprising the step of measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B, in a sample obtained from a subject. [5] The method according to [4], further comprising the step of indicating that fibrosis in the endometrium has occurred or is likely to occur in the subject from which the sample originates if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is high in a sample obtained from a subject, or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is low. [6] A method for determining the degree of endometrial adhesion in a subject, A method comprising the step of measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B, in a sample obtained from a subject. [7] The method according to [6], further comprising the step of indicating that the subject from which the sample is derived has or may have adhesions in the endometrium if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is high in the sample obtained from the subject, or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is low. [8] A method for predicting the degree of pain caused by endometriosis in a person who has or is suspected of having endometriosis, A method comprising the step of measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B, in a sample obtained from a subject. [9] The method according to [8], further comprising the step of indicating that pain due to endometriosis occurs or is likely to occur in the subject from which the sample is derived if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is high in a sample obtained from a subject, or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is low.

[10] A method for determining the progression of endometriosis in a subject who has or is suspected of having endometriosis, A method comprising the step of measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B, in a sample obtained from a subject.

[11] The method according to

[10] , further comprising a step of indicating that endometriosis is progressing or may be progressing in the subject from which the sample was obtained, when the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A in the sample obtained from the subject is high, or when the abundance of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is low.

[12] The method according to [3], [5], [7], [9], or

[11] , wherein when the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is higher than the abundance of the same marker in a sample obtained from a healthy subject, it is determined that the abundance is high.

[13] The method according to [3], [5], [7], [9], or

[11] , wherein when the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, and markers shown in Table 5A is 2 times or more the abundance of the same marker in a sample obtained from a healthy subject, and / or when the abundance of at least one marker selected from the markers shown in Table 6A is 1.6 times or more the abundance of the same marker in a sample obtained from a healthy subject, it is determined that the abundance is high.

[14] The method according to [3], [5], [7], [9], or

[11] , wherein when the abundance of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is lower than the abundance of the same marker in a sample obtained from a healthy subject, it is determined that the abundance is low.

[15] The method according to [3], [5], [7], [9], or

[11] , wherein the abundance of at least one marker selected from the group consisting of the markers shown in Table 1B, the markers shown in Table 2B, and the markers shown in Table 5B is determined to be low if the abundance of at least one marker selected from the markers shown in Table 6B is 0.5 times or less the abundance of the same marker in a sample obtained from a healthy person, and / or if the abundance of at least one marker selected from the markers shown in Table 6B is 0.625 times or less the abundance of the same marker in a sample obtained from a healthy person.

[16] A method for monitoring the pathophysiology of endometriosis in subjects who have or are suspected of having endometriosis, A method comprising the step of measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B, in each of several samples taken from a subject at different time points.

[17] The method according to

[16] , further comprising the step of indicating that the pathophysiology of endometriosis is improving or potentially improving in a subject from which the sample originates if the amount of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the marker shown in Table 1A, the marker shown in Table 2A, the marker shown in Table 5A, and the marker shown in Table 6A decreases over time, or if the amount of at least one marker selected from the group consisting of the marker shown in Table 1B, the marker shown in Table 2B, the marker shown in Table 5B, and the marker shown in Table 6B increases over time.

[18] If the abundance of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 5A, and the markers shown in Table 6A increases over time, or if the abundance of at least one marker selected from the group consisting of the markers shown in Table 1B, the markers shown in Table 2B, the markers shown in Table 5B, and the markers shown in Table 6B decreases over time, the method according to

[16] , further comprising a step of indicating that the pathological condition of endometriosis is deteriorating or may be deteriorating in the subject from which the sample is derived.

[19] The method according to any one of [1] to

[18] , wherein the marker is measured as a polypeptide.

[20] The method according to any one of [1] to

[19] , wherein the sample is a blood sample.

[21] The method according to

[20] , wherein the abundance of the marker in the sample is the polypeptide concentration of the marker in the blood sample. [21-1] The method according to

[20] or

[21] , wherein the concentration of the type V collagen MMP degradation product in the blood sample is measured by specifically recognizing the peptide represented by SEQ ID NO: 4 or SEQ ID NO: 6.

[22] A kit for diagnosing endometriosis, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the marker shown in Table 1B, the marker shown in Table 2B, the markers shown in Table 5A, the markers shown in Table 6A, the markers shown in Table 5B, and the markers shown in Table 6B.

[23] A kit for determining the presence or absence of endometriosis in a subject, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the marker shown in Table 1B, the marker shown in Table 2B, the markers shown in Table 5A, the markers shown in Table 6A, the markers shown in Table 5B, and the markers shown in Table 6B.

[24] The kit according to

[22] or

[23] , further comprising instructions stating that if the sample obtained from a subject is of high abundance in at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A, or if it is of low abundance in at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B, it indicates that the subject from which the sample is derived has or may have endometriosis.

[25] A kit for determining the degree of endometrial fibrosis in a subject, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B.

[26] The kit according to

[25] , further comprising instructions stating that if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is high in a sample obtained from a subject, or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is low, it indicates that fibrosis in the endometrium is occurring or is likely to occur in the subject from which the sample originates.

[27] A kit for determining the degree of endometrial adhesion in a subject, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B.

[28] The kit according to

[27] , further comprising instructions stating that if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is high in a sample obtained from a subject, or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is low, it indicates that endometrial adhesions have occurred or are likely to occur in the subject from which the sample originates.

[29] A kit for predicting the degree of pain caused by endometriosis in subjects who have or are suspected of having endometriosis, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B.

[30] The kit according to

[29] , further comprising instructions stating that if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is high in a sample obtained from a subject, or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is low, it indicates that pain due to endometriosis occurs or is likely to occur in the subject from which the sample is derived.

[31] A kit for determining the progression of endometriosis in a subject who has or is suspected of having endometriosis, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B.

[32] The kit according to

[31] , further comprising instructions stating that if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is high in a sample obtained from a subject, or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is low, it indicates that endometriosis is progressing or is likely to be progressing in the subject from which the sample is derived.

[33] The kit according to

[24] ,

[26] ,

[28] ,

[30] , or

[32] , wherein the abundance of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the marker shown in Table 1A, the marker shown in Table 2A, the marker shown in Table 5A, and the marker shown in Table 6A is determined to be high if the abundance of the marker is higher than the abundance of the same marker in a sample obtained from a healthy person.

[34] The kit according to

[24] ,

[26] ,

[28] ,

[30] , or

[32] , wherein the abundance of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, and the markers shown in Table 5A is determined to be high if the abundance of at least one marker selected from the markers shown in Table 6A is 1.6 times or more the abundance of the marker in the sample obtained from a healthy person.

[35] The kit according to

[24] ,

[26] ,

[28] ,

[30] , or

[32] , wherein the abundance of at least one marker selected from the group consisting of the markers shown in Table 1B, Table 2B, Table 5B, and Table 6B is determined to be low if the abundance of that marker is lower than the abundance of the same marker in a sample obtained from a healthy person.

[36] The kit according to

[24] ,

[26] ,

[28] ,

[30] , or

[32] , wherein the abundance of at least one marker selected from the group consisting of the markers shown in Table 1B, the markers shown in Table 2B, and the markers shown in Table 5B is determined to be low if the abundance of at least one marker selected from the markers shown in Table 6B is 0.5 times or less the abundance of the same marker in a sample obtained from a healthy person, and / or if the abundance of at least one marker selected from the markers shown in Table 6B is 0.625 times or less the abundance of the same marker in a sample obtained from a healthy person.

[37] A kit for monitoring the pathogenesis of endometriosis in subjects who have or are suspected of having endometriosis, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B.

[38] The kit according to

[37] further includes instructions stating that the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5A, markers shown in Table 6A, markers shown in Table 5B, and markers shown in Table 6B is compared in each of several samples taken from the subject at different time points.

[39] The kit according to

[37] or

[38] , further comprising instructions stating that if the amount of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the marker shown in Table 1A, the marker shown in Table 2A, the marker shown in Table 5A, and the marker shown in Table 6A decreases over time, or if the amount of at least one marker selected from the group consisting of the marker shown in Table 1B, the marker shown in Table 2B, the marker shown in Table 5B, and the marker shown in Table 6B increases over time, it indicates that the pathophysiology of endometriosis is improving or is likely to improve in the subject from which the sample originates.

[40] The kit according to

[37] or

[38] , further comprising instructions stating that if the amount of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the marker shown in Table 1A, the marker shown in Table 2A, the marker shown in Table 5A, and the marker shown in Table 6A increases over time, or if the amount of at least one marker selected from the group consisting of the marker shown in Table 1B, the marker shown in Table 2B, the marker shown in Table 5B, and the marker shown in Table 6B decreases over time, it indicates that the condition of endometriosis is worsening or is likely to worsen in the subject from which the sample originates.

[41] A kit according to any one of

[22] to

[40] for measuring the marker as a polypeptide.

[42] The kit according to any one of

[22] to

[41] , wherein the sample is a blood sample.

[43] The amount of the marker present in the sample is the polypeptide concentration of the marker in the blood sample, as described in

[42] . [43-1] The kit described in

[42] or

[43] measures the concentration of type V collagen MMP degradation products in the blood sample by specifically recognizing the peptide indicated by SEQ ID NO: 4 or SEQ ID NO: 6.

[44] For use in the diagnosis of endometriosis, A reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B.

[45] For use in determining whether or not a subject has endometriosis, A reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B.

[46] For use in determining the degree of endometrial fibrosis in subjects, A reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B.

[47] For use in determining the degree of endometrial adhesion in subjects, A reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B.

[48] ​​For use in predicting the degree of pain caused by endometriosis in subjects who have or are suspected of having endometriosis, A reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B.

[49] For use in determining the progression of endometriosis in subjects who have or are suspected of having endometriosis, A reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B.

[50] For use in monitoring the pathogenesis of endometriosis in subjects who have or are suspected of having endometriosis, A reagent for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B.

[51] The reagent according to any one of

[44] to

[50] , for measuring the marker as a polypeptide.

[52] A reagent according to any one of items

[44] to

[51] , which measures the amount of the marker present by measuring the polypeptide concentration of the marker in a blood sample obtained from a subject.

[53] The reagent according to

[51] for measuring the concentration of type V collagen MMP degradation products in the blood sample by specifically recognizing the peptide indicated by SEQ ID NO: 4 or SEQ ID NO: 6 present in the blood sample. [Brief explanation of the drawing]

[0009] [Figure 1-1]Figures 1-1 to 1-10 show the results of LC-MS analysis of relative protein expression levels in healthy human plasma and endometriosis patient plasma. The title of each graph indicates the Gene Symbol:UniProt ID of the target protein, the X-axis is the sample name, and the Y-axis is the relative protein expression level. The bars in the graphs represent the standard deviation of the relative protein expression levels in the three datasets obtained from three analyses. Figure 1-1 shows the data for Gene Symbol:CALU, UniProt ID:O43852. [Figure 1-2] Figure 1-2 shows data for Gene Symbol:CAT and UniProt ID:P04040. [Figure 1-3] Figure 1-3 shows data for Gene Symbol: HBA1, UniProt ID: P69905. [Figure 1-4] Figure 1-4 shows data for Gene Symbol: HBB and UniProt ID: P68871. [Figure 1-5] Figure 1-5 shows data for Gene Symbol: HBD and UniProt ID: P02042. [Figure 1-6] Figure 1-6 shows data for Gene Symbol: PPIA, UniProt ID: P62937. [Figure 1-7] Figure 1-7 shows data for Gene Symbol:PRDX1;PRDX4, UniProt ID:Q06830. [Figure 1-8] Figure 1-8 shows data for Gene Symbol: S100A6 and UniProt ID: P06703. [Figure 1-9] Figure 1-9 shows data for Gene Symbol: TPI1 and UniProt ID: P60174. [Figure 1-10] Figure 1-10 shows data for Gene Symbol: UBE2V1; UBE2V2, UniProt ID: Q13404. [Figure 2-1]Figures 2-1 to 2-10 show the results of protein expression level analysis in healthy human plasma and endometriosis patient plasma using SOMAscan®. The title of each graph is the Gene Symbol:UniProt ID of the target protein, the X axis is the sample name, and the Y axis is the aptamer signal value of the target protein in SOMAscan®. Figure 2-1 shows the data for Gene Symbol:BGN, UniProt ID:P21810. [Figure 2-2] Figure 2-2 shows data for Gene Symbol:CDH12 and UniProt ID:P55289. [Figure 2-3] Figure 2-3 shows data for Gene Symbol: DDX19B and UniProt ID: Q9UMR2. [Figure 2-4] Figure 2-4 shows data for Gene Symbol: IL1B and UniProt ID: P01584. [Figure 2-5] Figure 2-5 shows data for Gene Symbol: IL2 and UniProt ID: P60568. [Figure 2-6] Figure 2-6 shows data for Gene Symbol: LAG3, UniProt ID: P18627. [Figure 2-7] Figure 2-7 shows the data for Gene Symbol: MFGE8 and UniProt ID: Q08431. [Figure 2-8] Figure 2-8 shows the data for Gene Symbol: PDE5A and UniProt ID: O76074. [Figure 2-9] Figure 2-9 shows the data for Gene Symbol: SERPINE2 and UniProt ID: P07093. [Figure 2-10] Figure 2-10 shows data for Gene Symbol: SMPDL3A and UniProt ID: Q92484. [Figure 3]Figure 3 shows the results of the analysis of type V collagen degradation product (C5M) expression levels in plasma from healthy individuals and endometriosis patients. The X-axis represents the sample name, and the Y-axis represents the concentration of the target degradation product in the plasma (ng / ml). [Modes for carrying out the invention]

[0010] marker In this disclosure, "marker" can also be rephrased as "biomarker," and refers to a specific endogenous chemical substance that can be objectively measured and evaluated as an indicator of normal biological processes, disease development, or pharmacological responsiveness to treatment. Markers are useful for evaluating the presence, progression, or susceptibility to disease, or for evaluating or predicting the efficacy, optimal dose, or safety of a drug, or for predicting prognosis. In this disclosure, markers are identified by protein names, protein fragment names, or gene names, and it is preferable to measure the marker gene as a polypeptide or polynucleotide (including DNA and mRNA forms), and it is preferable to measure the marker protein or protein fragment as a polypeptide.

[0011] Method for measuring marker abundance The amount of a marker can be measured using an appropriate method depending on the morphology of the marker or the type of sample to be measured. If the marker is a polypeptide, it can be measured using immunological methods with antibodies that specifically bind to the polypeptide. Examples of such methods include enzyme immunosorbent assays (ELISA, EIA), fluorescence immunoassays (FIA), radioimmunoassays (RIA), luminescence immunoassays (LIA), electrochemiluminescence (ECL), Western blotting, surface plasmon resonance, antibody arrays, immunohistochemistry, fluorescence-activated cell sequencing (FACS), immunochromatography, immunoprecipitation, immunoturbidimetric assays, and latex agglutination. If the marker is a polynucleotide, it can be measured using genetic engineering methods with oligonucleotides that specifically bind to the polynucleotide. Examples of such methods include polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), real-time quantitative PCR (Q-PCR), Northern blotting, and hybridization (including methods using oligonucleotide arrays such as DNA microarrays). When the marker to be measured is a protein expressed from a gene, the abundance can be rephrased as the expression level. In this disclosure, "abundance" includes the expression level of the protein, the gene expression level, and the concentration of the protein fragment.

[0012] In one aspect of this disclosure, the abundance of a marker may be a relative abundance. Relative abundance can be measured by comparing the level of a specific marker and other protein / metabolite levels in a sample obtained from a subject to a control. Relative abundance may also be measured by LC / MS (liquid chromatography / mass spectrometry).

[0013] Criteria for determining the amount of marker present In this disclosure, a high or large amount of a marker means that the measured value of the marker is higher or greater than a predetermined value (control level) set for that marker, and a low or small amount of a marker means that it is lower or less than a predetermined value (control level) set for that marker or is below the control level.

[0014] In this disclosure, "predetermined value" means a value predetermined based on some scientific evidence, but it can be any value as long as it can be used as a basis to determine whether or not endometriosis is present, to determine the degree of endometrial fibrosis in a subject, to determine the degree of endometrial adhesion in a subject, to predict the degree of pain in a subject with endometriosis, to determine the progression of endometriosis, or to monitor the pathological state of endometriosis. In this disclosure, the predetermined value may be set for each marker.

[0015] The predetermined values ​​in this disclosure can be set from marker measurement values ​​in healthy subjects, for example, healthy adults (control samples). This disclosure has shown that marker measurement values ​​in samples obtained from subjects with endometriosis are increased or decreased compared to marker measurement values ​​in samples obtained from healthy subjects. Therefore, one possible approach is to use the average value of marker measurement values ​​from samples obtained from multiple healthy subjects as the predetermined value. Alternatively, another possible approach is to use the average value of marker measurement values ​​from samples obtained from multiple healthy subjects plus a value of 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 times the standard deviation as the predetermined value. Therefore, in one aspect of this disclosure, it is shown that by comparing the amount of markers (control level) measured in a sample obtained from a healthy person (control sample) with the measured value of markers in a sample obtained from a test subject, it is possible to determine whether or not the test subject has endometriosis, determine the degree of endometrial fibrosis in the subject, determine the degree of endometrial adhesion in the subject, predict the degree of pain in a subject who has or is suspected of having endometriosis, determine the progression of endometriosis, and monitor the pathological state of endometriosis.

[0016] The marker measurements and predetermined values ​​in this disclosure may be numerical values ​​obtained by some method from the measurement results of the amount of marker present. In this disclosure, the marker measurements and predetermined values ​​may be the values ​​obtained from the measurement results (e.g., color intensity), or a positive control sample containing a known amount of marker may be prepared separately, and values ​​obtained by converting the measurement results by comparing them (e.g., concentration) may be used. Alternatively, values ​​obtained in the above manner may be divided into certain ranges and scored (e.g., grade 1, 2, 3) may be used.

[0017] sample In this disclosure, "sample" can also be rephrased as "biological sample," and refers to organs, tissues, cells, body fluids, or mixtures thereof contained within a living organism. Specific examples include skin, airways, intestines, urogenital tract, nerves, tumors, bone marrow, blood cells, blood (whole blood, plasma, serum), lymph, cerebrospinal fluid, intraperitoneal fluid, synovial fluid, pulmonary fluid, saliva, sputum, and urine. Samples obtained by washing these or by culturing them outside the body are also included in the scope of samples in this disclosure. A preferred sample in this disclosure is blood, and particularly preferred samples are plasma or serum.

[0018] In this disclosure, samples obtained from the subject may be processed by methods such as concentration, purification, extraction, isolation, or physical / chemical treatment before being subjected to measurement of the abundance of the marker. For example, blood cells or plasma components may be separated from a blood sample, or DNA or RNA may be extracted from a tissue / cell sample. Alternatively, unwanted components may be denatured / removed by heating or chemical reagents. Such processing is mainly performed to improve the sensitivity and specificity of the measurement of the abundance of the marker.

[0019] In the determination of the degree of endometrial fibrosis, the degree of endometrial adhesions, the prediction of the degree of pain due to endometriosis, the determination of the progression of endometriosis, and the monitoring of the pathology of endometriosis as described in this disclosure, the subjects from whom samples are obtained may be subjects who have already been diagnosed with endometriosis or subjects suspected of having endometriosis. Subjects with endometriosis may be any subject that has endometriosis. Subjects may be those who have not yet received treatment for endometriosis or those who have already received treatment. The subject matter of this disclosure is mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the subject matter is humans.

[0020] Markers in this disclosure Examples of markers in this disclosure include type V collagen MMP (Matrix metalloproteinase) degradation products (polypeptides derived from type V collagen, such as the protein fragments shown in SEQ ID NOs: 2 and 3, which contain the amino acid sequence described in SEQ ID NO: 4 at their N-terminus and / or the amino acid sequence described in SEQ ID NO: 6 at their C-terminus), the markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B.

[0021] [Table 1A] TIFF2026065204000003.tif233167TIFF2026065204000004.tif158166

[0022] [Table 1B]

[0023] [Table 2A] TIFF2026065204000007.tif255170TIFF2026065204000008.tif217170

[0024] [Table 2B] TIFF2026065204000010.tif252170TIFF2026065204000011.tif93168

[0025] [Table 5A]

[0026] [Table 5B]

[0027] [Table 6A]

[0028] [Table 6B]

[0029] The sequences of the markers (mRNA, protein) shown in these tables can be easily obtained from databases known to those skilled in the art (Uniprot: https: / / www.uniprot.org / ) based on the Uniprot IDs listed in the tables above. Specifically, it is possible to search for the corresponding Uniprot entry based on the Uniprot ID and obtain the sequence listed in the "sequences" section. Furthermore, it is also possible to obtain the ID / accession number of other databases (e.g., RefSeq, EMBL, GenBank, DDBJ, CCDS, PIR, UniGene, Ensemble, GeneID, KEGG, USCS) from the "sequences" section and obtain sequences from those databases as well. The sequences of each marker in this disclosure refer to the sequences or ID / accession numbers listed in the latest version of the corresponding Uniprot entry whose release number is prior to 2018-09 (e.g., 2018-08, 2018_07 if 2018-08 does not exist, 2018_06 if neither 2018_08 nor 2018_07 exists, and so on). Furthermore, the amino acid sequences of the markers listed in the table can also be obtained from the data contained in the directory "release-2018_08 / knowledgebase" (the "knowledgebase" directory included in "release-2018_08") in the FTP (ftp: / / ftp.uniprot.org / pub / databases / uniprot / previous_releases / ) where the Uniprot release data archive is stored, based on the Uniprot IDs listed in the table above.

[0030] The marker group listed in the table above includes markers indicating fibrosis (such as TGFB1 and SPARC). Furthermore, many of the markers listed in the table above are thought to be derived from platelets. For example, PPBP, THBS1, PF4, SERPINA1, TIMP3, APP, CALU, CASP3, MMRN1, SAA1, SRGN, and VWF are described in Uniprot as being involved in platelet degranulation. Zhang et al. showed that TGFb1 released from activated platelets in an in vitro experimental system using endometrial-derived cells induces epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT), leading to increased collagen production and fibrosis (Molecular and Cellular Endocrinology, 428, 1-16, 2016). This is consistent with the selection of TGFb1 and platelet-derived proteins as markers in this disclosure, suggesting that changes in these proteins may reflect changes such as platelet activation.

[0031] Type V collagen MMP degradation products, also known as MMP-mediated type V collagen degradation, are the products of degradation of the alpha 3 chain (Refseq: NP_056534.2, SEQ ID NO: 1) of type V collagen by MMPs. Specifically, type V collagen MMP degradation products are produced by cleavage between amino acids 1316 (G) and 1317 (H) of type V collagen, as shown in SEQ ID NO: 1 (Clin Biochem. 2012 May;45(7-8):541-6). Specifically, polypeptides derived from type V collagen that contain the amino acid sequence described in SEQ ID NO: 4 at the N-terminus and / or the amino acid sequence described in SEQ ID NO: 6 at the C-terminus are included in type V collagen MMP degradation products. Examples of type V collagen MMP degradation products include the protein fragments shown in SEQ ID NO: 2 or 3. Among type V collagen MMP degradation products, the peptide fragment of SEQ ID NO: 4 (HMGREGREGE) is sometimes referred to as C5M.

[0032] The detection of type V collagen MMP degradation products is limited to methods that can detect the type V collagen fragment after cleavage between amino acids 1316 (G) and 1317 (H) as described in SEQ ID NO: 1. In one embodiment, the amount of type V collagen MMP degradation products can be detected using an antibody that specifically recognizes the peptide HMGREGREGE (SEQ ID NO: 4) located at the N-terminus of the type V collagen fragment shown in SEQ ID NO: 2. In a more specific embodiment, the amount of type V collagen MMP degradation products can be detected using an antibody that can recognize the peptide HMGREGREGE (SEQ ID NO: 4) located at the N-terminus of the type V collagen fragment shown in SEQ ID NO: 2, but does not recognize GHMGREGREGE (SEQ ID NO: 5) (Clin Biochem. 2012 May;45(7-8):541-6. has established ELISA detection using such an antibody). In another embodiment, the amount of type V collagen MMP degradation products can be detected using an antibody that specifically recognizes the peptide GPPGKRGPSG (SEQ ID NO: 6) located at the C-terminus of the type V collagen fragment shown in SEQ ID NO: 3. In a more specific embodiment, the amount of type V collagen MMP degradation products can be detected using an antibody that can recognize the peptide GPPGKRGPSG (SEQ ID NO: 6) located at the C-terminus of the type V collagen fragment shown in SEQ ID NO: 3, but does not recognize GPPGKRGPSGH (SEQ ID NO: 7).

[0033] In this disclosure, a protein selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B may be used as a marker alone, or a combination of the selected proteins may be used as a marker. When multiple proteins are used as markers, the results may be combined after measuring each marker individually, or each marker may be measured simultaneously.

[0034] The abundance of the markers in this disclosure can be measured using the measurement methods described herein or using commercially available measuring reagents. The specific measured values ​​and predetermined values ​​for each marker described herein can be interpreted as values ​​measured using such measuring reagents.

[0035] The predetermined values ​​(control levels) set for the markers in this disclosure may vary depending on the type of sample to which the abundance of each marker is to be measured, but can be set, for example, from the range of 0.1 to 100 ng / mL. Alternatively, they can be set from ranges such as 0.2 to 90 ng / mL, 0.3 to 80 ng / mL, 0.4 to 70 ng / mL, 0.5 to 60 ng / mL, 1.0 to 50 ng / mL, 1.5 to 40 ng / mL, 2.0 to 30 ng / mL, 2.5 to 20 ng / mL, 3.0 to 10 ng / mL, etc., but are not limited to these.

[0036] Further, the predetermined values ​​set for each marker of the present disclosure are 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.7 ng / mL, 0.8 ng / mL, 0.9 ng / mL, 1.0 ng / mL, 1.5 ng / mL, 2.0 ng / mL, 2.5 ng / mL, 3.0 ng / mL, 3.5 ng / mL, 4.0 ng / mL, 4.5 ng / mL, 5.0 ng / mL, 5.5 ng / mL, 6.0 ng / mL, 6.5 ng / mL, 7.0 ng / mL, 7.5 ng / mL, 8.0 ng / mL, 8.5 ng / mL, 9.0 ng / mL, 9.5 ng / mL, 10 ng / mL, 15 ng / mL, 20 The value can be set from, but is not limited to, values ​​such as ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, and 100 ng / mL.

[0037] In this disclosure, a "high abundance" or "large abundance" can be determined if the abundance of each marker in this disclosure is greater than a predetermined value (control level). For example, in this disclosure, although not limited to these examples, if the abundance of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, and the markers shown in Table 5A is, for example, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more, 3.0 times or more, or greater than or equal to the abundance of the same marker in a sample obtained from a healthy person, it can be determined that a "high abundance" or "large abundance" is determined. Alternatively, although not limited to these, in this disclosure, if the amount of at least one marker selected from the markers shown in Table 6A is, for example, 1.10 times or more, 1.15 times or more, 1.20 times or more, 1.25 times or more, 1.30 times or more, 1.35 times or more, 1.40 times or more, 1.45 times or more, 1.50 times or more, 1.55 times or more, 1.60 times or more, 1.65 times or more, 1.70 times or more, 1.75 times or more, 1.80 times or more, 1.85 times or more, 1.90 times or more, 1.95 times or more, 2.00 times or more, or more than these amounts, it can be determined that the amount is "high" or "large".

[0038] In this disclosure, if the amount of each marker present is smaller than a predetermined value (control level), it can be determined that the amount is "low" or "small." For example, in this disclosure, but not limited to these examples, if the amount of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the markers shown in Table 1B, the markers shown in Table 2B, and the markers shown in Table 5B is, for example, 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, 0.1 times or less, or less than or equal to the amount of the same marker present in a sample obtained from a healthy person, it can be determined that the amount is "low" or "small." Alternatively, although not limited to these, in this disclosure, if the amount of at least one marker selected from the markers shown in Table 6B is, for example, 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, 0.1 times or less, or less than or equal to the amount of the same marker in a sample obtained from a healthy person, it can be determined that the amount is "low" or "small".

[0039] Diagnosis of endometriosis and determination of whether or not a subject has endometriosis. One aspect of this disclosure relates to a method for diagnosing endometriosis or determining the presence or absence of endometriosis in a subject. Another aspect of this disclosure relates to a method for detecting endometriosis or its markers. Another aspect of this disclosure relates to a method for assisting such diagnosis or determination. Another aspect of this disclosure relates to a method for providing instructions regarding such diagnosis or determination. Another aspect of this disclosure relates to reagents and kits for use in such methods.

[0040] Each of the above methods includes measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B, in a sample obtained from the subject. The method may also include a step of comparing the amount with a predetermined value (control level). The reagent described above is for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B. The kit described above includes the reagent.

[0041] Each of the above methods may include a step indicating that the subject from which the sample is derived suffers from or is likely to suffer from endometriosis if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A in the sample obtained from the subject is higher than a predetermined value (control level), or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B in the sample obtained from the subject is lower than a predetermined value (control level).

[0042] Each of the above methods can be performed in vivo or in vitro, but it is preferable to perform them in vitro. Furthermore, each of the above methods may include a step of obtaining a sample from the subject. Furthermore, each of the above methods may include a step of treating a subject who has been shown to have or be suspected of having endometriosis. Furthermore, each of the above methods may include a step of administering a known drug for treating endometriosis to a subject who has been shown to have or be suspected of having endometriosis. In other words, the present invention relates to a method for treating endometriosis in a subject who has been shown to have or be suspected of having endometriosis by each of the above methods. A variety of drugs for treating endometriosis are known to those skilled in the art, and are not limited to these, but examples include estrogen / progesterone combinations, progesterone preparations, GnRH agonists, GnRH antagonists, and danazol.

[0043] The above kit may further include instructions stating that if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is higher than a predetermined value (control level), or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is lower than a predetermined value (control level), it indicates that the subject from which the sample originates has or is likely to have endometriosis.

[0044] Determination of the degree of endometrial fibrosis in the subjects. One aspect of this disclosure relates to a method for determining the degree of endometrial fibrosis in a subject, a method for assisting in such determination, a method for providing instructions regarding such determination, and reagents and kits for use therein.

[0045] Each of the above methods includes measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B, in a sample obtained from the subject. The method may also include a step of comparing the amount with a predetermined value (control level). The reagent described above is for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B. The kit described above includes the reagent.

[0046] Each of the above methods may include a step indicating that fibrosis in the endometrium has occurred or is likely to occur in the subject from which the sample originates, if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A in the sample obtained from the subject is higher than a predetermined value (control level), or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B in the sample obtained from the subject is lower than a predetermined value (control level).

[0047] Each of the above methods can be performed in vivo or in vitro, but it is preferable to perform them in vitro. Furthermore, each of the above methods may include a step of obtaining a sample from the subject. Furthermore, each of the above methods may include a step of treating a subject who has been shown to have or be suspected of having fibrosis in the endometrium. Furthermore, each of the above methods may include a step of administering a known therapeutic agent for suppressing fibrosis in the endometrium to a subject who has been shown to have or be suspected of having fibrosis in the endometrium. In other words, the present invention relates to a method for suppressing fibrosis in the endometrium in a subject who has been shown to have or be suspected of having fibrosis in the endometrium by each of the above methods. Examples of therapeutic agents for suppressing fibrosis in the endometrium include the above-mentioned therapeutic agents for endometriosis.

[0048] The above kit may further include instructions stating that if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is higher than a predetermined value (control level), or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is lower than a predetermined value (control level), it indicates that fibrosis in the endometrium is occurring or is likely to occur in the subject from which the sample originates.

[0049] Determination of the degree of endometrial adhesion in the subject One aspect of this disclosure relates to a method for determining the degree of endometrial adhesion in a subject, a method for assisting in such determination, a method for providing instructions regarding such determination, and reagents and kits for use in such methods.

[0050] Each of the above methods includes measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B, in a sample obtained from the subject. The method may also include a step of comparing the amount with a predetermined value (control level). The reagent described above is for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B. The kit described above includes the reagent.

[0051] Each of the above methods may include a step indicating that endometrial adhesions have occurred or are likely to occur in the subject from which the sample originates, if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A in the sample obtained from the subject is higher than a predetermined value (control level), or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B in the sample obtained from the subject is lower than a predetermined value (control level).

[0052] Each of the above methods can be performed in vivo or in vitro, but it is preferable to perform them in vitro. Furthermore, each of the above methods may include a step of obtaining a sample from the subject. Furthermore, each of the above methods may include a step of treating a subject who has been shown to have or be suspected of having adhesions in the endometrium. Furthermore, each of the above methods may include a step of administering a known therapeutic agent for suppressing endometrial adhesions to a subject who has been shown to have or be suspected of having adhesions in the endometrium. In other words, the present invention relates to a method for suppressing endometrial adhesions in a subject who has been shown to have or be suspected of having adhesions in the endometrium by each of the above methods. Examples of therapeutic agents for suppressing endometrial adhesions include the above-mentioned therapeutic agents for endometriosis.

[0053] The above kit may further include instructions stating that if the concentration of type V collagen MMP degradation products in a sample obtained from a subject is higher than a predetermined value (control level) and the amount of at least one marker selected from the group consisting of markers shown in Table 1A, Table 2A, Table 5A, and Table 6A is lower than a predetermined value (control level), then it indicates that endometrial adhesions are present or likely to be present in the subject from which the sample originates.

[0054] Prediction of the severity of pain caused by endometriosis in individuals with or suspected of having endometriosis. One aspect of this disclosure relates to methods for predicting the degree of pain caused by endometriosis in subjects who have or are suspected of having endometriosis, methods for assisting such prediction, methods for providing instructions regarding such prediction, and reagents and kits for use in such methods.

[0055] Each of the above methods includes measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B, in a sample obtained from the subject. The method may also include a step of comparing the amount with a predetermined value (control level). The reagent described above is for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B. The kit described above includes the reagent.

[0056] Each of the above methods may include a step indicating that pain due to endometriosis occurs or is likely to occur in the subject from which the sample originates if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A in the sample obtained from the subject is higher than a predetermined value (control level), or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B in the sample obtained from the subject is lower than a predetermined value (control level).

[0057] Each of the above methods can be performed in vivo or in vitro, but it is preferable to perform them in vitro. Furthermore, each of the above methods may include a step of obtaining a sample from the subject. Furthermore, each of the above methods may include a step of alleviating pain caused by endometriosis in subjects who have been shown to be experiencing or potentially experiencing pain due to endometriosis. Furthermore, each of the above methods may include a step of administering a known therapeutic agent for suppressing pain caused by endometriosis to subjects who have been shown to be experiencing or potentially experiencing pain due to endometriosis. In other words, the present invention relates to a method for suppressing pain caused by endometriosis in subjects who have been shown to be experiencing or potentially experiencing pain due to endometriosis by each of the above methods. Examples of therapeutic agents for suppressing pain caused by endometriosis include the above-mentioned therapeutic agents for endometriosis.

[0058] The above kit may further include instructions stating that if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is higher than a predetermined value (control level), or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is lower than a predetermined value (control level), then pain due to endometriosis may occur or be likely to occur in the subject from which the sample originates.

[0059] Determining the progression of endometriosis One aspect of this disclosure relates to a method for determining the progression of endometriosis in a person who has or is suspected of having endometriosis, a method for assisting in such determination, a method for providing instructions regarding such determination, and reagents and kits for use in such methods.

[0060] Each of the above methods includes measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B, in a sample obtained from the subject. The method may also include a step of comparing the amount with a predetermined value (control level). The reagent described above is for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B. The kit described above includes the reagent.

[0061] Each of the above methods may include a step indicating that endometriosis is progressing or is likely to be progressing in the subject from which the sample originates if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A in the sample obtained from the subject is higher than a predetermined value (control level), or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B in the sample obtained from the subject is lower than a predetermined value (control level).

[0062] Each of the above methods can be performed in vivo or in vitro, but it is preferable to perform them in vitro. Furthermore, each of the above methods may include a step of obtaining a sample from the subject. Furthermore, each of the above methods may include a step of treating a subject who has been shown to have or may have advanced endometriosis. Furthermore, each of the above methods may include a step of administering a known therapeutic agent for inhibiting the progression of endometriosis to a subject who has been shown to have or may have advanced endometriosis. In other words, the present invention relates to a method for inhibiting the progression of endometriosis in a subject who has been shown to have or may have advanced endometriosis by each of the above methods. Examples of therapeutic agents for inhibiting the progression of endometriosis include the above-mentioned therapeutic agents for endometriosis.

[0063] The above kit may further include instructions stating that if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A is higher than a predetermined value (control level), or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B is lower than a predetermined value (control level), it indicates that endometriosis is progressing or is likely to be progressing in the subject from which the sample originated.

[0064] Endometriosis pathological monitoring One aspect of this disclosure relates to methods for monitoring the pathogenesis of endometriosis in subjects who have or are suspected of having endometriosis, methods for assisting such monitoring, methods for providing instructions for such monitoring, and reagents and kits for use in such methods. Monitoring in this disclosure includes determining the effectiveness of treatment and / or providing information about future treatments or treatment plans.

[0065] Each of the above methods includes measuring the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B, in the sample obtained from the subject. The reagent described above is for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B. The kit described above includes the reagent.

[0066] Each of the above methods may include a step of measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B, in each of several samples taken from the subject at different time points. It may also include a step of comparing the abundance of the markers in each of the several samples taken at different time points. In this disclosure, there are no particular limitations on the timing or number of sample collections, and samples can be collected at various points before, during, and after treatment as needed. Furthermore, samples can be collected at various times within each of the pre-treatment, during, and post-treatment periods. In this disclosure, "multiple" is not particularly limited, but for example, it could be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The above kit may include instructions stating that the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B is compared in each of multiple samples obtained from the subject at different time points.

[0067] Each of the above methods may include a step indicating that the pathological condition of endometriosis in a subject from which the sample originates is improving, or is likely to improve, if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A decreases over time, or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B increases over time. Each of the above methods may include a step indicating that, if the amount of at least one marker selected from the group consisting of type V collagen MMP degradation products, markers shown in Table 1A, markers shown in Table 2A, markers shown in Table 5A, and markers shown in Table 6A increases over time, or if the amount of at least one marker selected from the group consisting of markers shown in Table 1B, markers shown in Table 2B, markers shown in Table 5B, and markers shown in Table 6B decreases over time, it indicates that the pathological condition of endometriosis in the subject from which the sample originates is worsening or is likely to worsen.

[0068] Each of the above methods can be performed in vivo or in vitro, but it is preferable to perform them in vitro. Furthermore, each of the above methods may include a step of obtaining a sample from the subject. Furthermore, each of the above methods may include a step of treating a subject who has been shown to have an exacerbation of endometriosis or is likely to have such an exacerbation. Furthermore, each of the above methods may include a step of administering a known drug for treating endometriosis to a subject who has been shown to have an exacerbation of endometriosis or is likely to have such an exacerbation. In other words, the present invention relates to a method for treating endometriosis in a subject who has been shown to have an exacerbation of endometriosis or is likely to have such an exacerbation by each of the above methods. The drugs mentioned above can be used as drugs for treating endometriosis.

[0069] The above kit may include instructions stating that if the amount of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the markers shown in Table 1A, Table 2A, Table 5A, and Table 6A decreases over time, or if the amount of at least one marker selected from the group consisting of the markers shown in Table 1B, Table 2B, Table 5B, and Table 6B increases over time, it indicates that the pathological condition of endometriosis in the subject from which the sample originates is improving or is likely to improve. The above kit may include instructions stating that if the amount of at least one marker selected from the group consisting of the type V collagen MMP degradation product, the markers shown in Table 1A, Table 2A, Table 5A, and Table 6A increases over time, or if the amount of at least one marker selected from the group consisting of the markers shown in Table 1B, Table 2B, Table 5B, and Table 6B decreases over time, it indicates that the condition of endometriosis is worsening or is likely to worsen in the subject from which the sample originates.

[0070] Reagents and kits The reagents included in the kit of this disclosure are not particularly limited as long as they can measure the amount of the marker present, but reagents can be appropriately selected according to the form of the marker. If the marker is a polypeptide, a reagent containing an antibody that specifically binds to the polypeptide is preferred, and if the marker is a polynucleotide, a reagent containing an oligonucleotide that specifically binds to the polynucleotide is preferred. In this disclosure, the antibody or the polynucleotide itself may be the reagent. Polynucleotides in this disclosure include DNA and mRNA forms.

[0071] The antibodies in this disclosure may be chimeric antibodies, humanized antibodies, or human antibodies. In some embodiments, the antibodies may also be multispecific antibodies, such as bispecific antibodies. Alternatively, the antibodies in this disclosure may be antibody fragments or modifications thereof. Examples of antibody fragments include Fab, F(ab')2, Fv, or single-chain Fv (scFv) obtained by linking the H chain and L chain of Fv with an appropriate linker. These antibodies can be obtained by methods well known to those skilled in the art. Antibodies can be labeled by commonly known methods. Labeling substances known to those skilled in the art can include fluorescent dyes, enzymes, coenzymes, chemiluminescent substances, and radioactive materials.

[0072] The oligonucleotides in this disclosure may be any oligonucleotides that specifically hybridize to at least a portion of the polynucleotide or its complementary strand that is the marker in this disclosure. The nucleotide sequence of such an oligonucleotide for detecting the polynucleotide is selected from a nucleotide sequence complementary to the sense strand of the marker in this disclosure. The oligonucleotides typically have a chain length of at least 15 bp. The oligonucleotides in this disclosure can also be labeled and used by commonly known methods. The oligonucleotides in this disclosure can be prepared, for example, by a commercially available oligonucleotide synthesizer.

[0073] Furthermore, the kit of this disclosure preferably includes a positive control sample that serves as a reference when measuring the amount of the marker present. The positive control sample is not particularly limited as long as the amount of the marker contained therein is predetermined, but it can be appropriately prepared depending on the form of the marker measured by the kit. For example, if the form of the marker is a polypeptide, a sample containing isolated, purified, and quantified polypeptide of the same form as the marker is preferred as the positive control sample. In addition to these, the kit of the present invention may also contain, as necessary, sterile water, physiological saline, vegetable oil, surfactant, lipids, solubilizers, buffers, protein stabilizers (such as BSA or gelatin), preservatives, blocking solutions, reaction solutions, reaction stop solutions, reagents for processing samples, etc.

[0074] Also included in this disclosure are sets of type V collagen MMP degradation products, markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B. Such sets can be used for the diagnosis of endometriosis, determining whether or not a patient has endometriosis, determining the degree of fibrosis or adhesions in the endometrium of a patient with endometriosis, predicting pain in a patient with endometriosis, and monitoring the condition of a patient with endometriosis.

[0075] Furthermore, all prior art documents cited herein are incorporated herein by reference. [Examples]

[0076] This disclosure is further illustrated by the following embodiments, but is not limited to those embodiments.

[0077] (Example 1) Biomarker discovery by LC-MS analysis using plasma from healthy humans and endometriosis patients (1-1) Obtaining human plasma samples Plasma from healthy individuals and endometriosis patients was purchased from Proteogenex.

[0078] (1-2) LC-MS analysis of plasma proteins Large amounts of protein in each plasma sample were removed by binding to a Seppro IgY14 column (Sigma Aldrich) according to the manufacturer's recommended method. Proteins in the flow-through fraction that did not bind to the column were precipitated by the methanol / chloroform method and dissolved in an 8M urea / 400mM ammonium bicarbonate solution. The dissolved proteins were reduced, alkylated, and then digested with lysyl endopeptidase and trypsin to form peptides. This peptide solution was desalted using Monospin C18 (GL Science) according to the manufacturer's recommended method.

[0079] The peptide was labeled using the manufacturer's recommended method with TMT10plex Mass Tag Labeling Kits and Reagents (ThermoFisher Scientific) (hereinafter referred to as TMT 10-plex). The labeled peptide was then processed by Pierce TM The peptides were fractionated using the High pH Reversed-Phase Peptide Fractionation Kit (ThermoFisher Scientific) according to the manufacturer's recommended method. Each fraction was analyzed using an LC-MS analysis system consisting of a nano-LC system (Ultimate3000, Dionex) connected to an Orbitrap fusion Lumos (ThermoFisher Scientific). The analytical samples included seven types of peptides: peptides obtained from the plasma of three healthy individuals and three endometriosis patients, and a control sample (equal volumes of these six peptide solutions mixed together). Each sample was analyzed three times, and each analysis was later analyzed as an independent dataset (N=3, hereafter referred to as datasets TMT1, 2, 3).

[0080] Proteins in the samples were identified from raw data obtained from LC-MS analysis using MaxQuant software (http: / / www.biochem.mpg.de / 5111795 / maxquant), and their relative expression levels were analyzed. Database searches for protein identification were performed using the following parameters: Taxonomy: uniprot human, Fixed modification: carbamidomethylation (C), Variable modification: oxidation (M); deamidation (NQ), Acetyl (protein N-term), FDR (protein, peptide) <1%. Relative expression levels were calculated using the following formula: TIFF2026065204000016.tif16160

[0081] The target protein signal values ​​used in the above formula are obtained from the "Reporter intensity corrected" column in Protein groups.txt, one of the output files of MaxQunat. For each label of the TMT 10-plex, the sum of the intensities is divided by the sum of the intensities of the control sample using pipeline pilot (BIOVIA), resulting in 1x10 11 It is calculated by multiplying by [a certain factor].

[0082] (1-3) Selection of candidate biomarker molecules LC-MS results were analyzed for each of the TMT1, 2, and 3 datasets using the statistical programming environment R and Microsoft Excel. For each dataset, 58 proteins showing altered expression in endometriosis patients were selected based on the relative expression levels of each protein in each sample (3 healthy individuals and 3 endometriosis patients) according to the following criteria (Tables 3A and 3B). TIFF2026065204000017.tif11166- The relative expression levels of the protein in question in 3 healthy individuals and 3 endometriosis patients were p<0.05 in the Welch t-test.

[0083] [Table 3A] TIFF2026065204000019.tif237148TIFF2026065204000020.tif235125TIFF2026065204000021.tif236144

[0084] [Table 3B]

[0085] The selected proteins show promise as biomarkers for diagnosing endometriosis, and relative expression graphs of representative proteins are shown in Figures 1-1 to 1-10.

[0086] (Example 2) Biomarker discovery by aptamer-binding protein analysis (SOMAscan®) using plasma from healthy humans and endometriosis patients (2-1) Obtaining human plasma samples Plasma from healthy individuals and endometriosis patients was purchased from Proteogenex.

[0087] (2-2) Analysis of plasma proteins using SOMAscan® Low-expression proteins in plasma samples (3 healthy individuals and 3 endometriosis patients) were analyzed using SOMAscan (registered trademark, Somalogic). Specifically, aptamers that specifically bind to each of the 1310 proteins were first prepared, mixed with plasma, and bound to the proteins in the plasma. Next, the proteins bound to the aptamers were biotinylated, and the aptamer-protein complexes were purified using streptavidin beads. Subsequently, the aptamers were eluted and detected by microarray, allowing for the analysis of the expression levels of the plasma proteins bound to those aptamers.

[0088] (2-3) Selection of biomarker candidates SOMAscan® aptamer signal data were analyzed using the statistical programming environment R and Microsoft Excel. 200 proteins showing altered expression in endometriosis patients were selected based on the following criteria (Tables 4A and 4B). In the above calculation (TIFF2026065204000023.tif11165), aptamer signals with a maximum value of less than 1000 were considered below the detection limit and were excluded from the analysis.

[0089] [Table 4A] TIFF2026065204000025.tif233147TIFF2026065204000026.tif234149TIFF2026065204000027.tif234149TIFF2026065204000028.tif234115

[0090] [Table 4B] TIFF2026065204000030.tif234151TIFF2026065204000031.tif234151TIFF2026065204000032.tif234107

[0091] The selected proteins show promise as biomarkers for diagnosing endometriosis, and the expression level graphs of representative proteins are shown in Figures 2-1 to 2-10.

[0092] (Example 3) Biomarker discovery by analysis of extracellular matrix degradation products using plasma from healthy humans and endometriosis patients (3-1) Obtaining human plasma samples Plasma from healthy individuals and endometriosis patients was purchased from Proteogenex.

[0093] (3-2) Measurement of plasma proteins in the extracellular matrix Plasma samples (from 3 healthy individuals and 3 endometriosis patients) were analyzed using an ELISA system with antibodies specific to extracellular matrix cleavage sites to analyze the total amount of extracellular matrix and its degradation / synthesis state (Nordic Bioscience). Specifically, the expression levels of degradation products were analyzed using antibodies against the cleavage sites contained in the degradation products of type III, IV, V, and VI collagen, decorin, or nidogen. As a result, type V collagen MMP degradation product (C5M) was significantly elevated in 2 out of 3 patients (C5M concentrations were 10.2 ng / ml and 11.8 ng / ml in the plasma of the two elevated patients), while C5M concentrations in the plasma of healthy individuals were all below the detection limit, suggesting that C5M is a promising biomarker for endometriosis. A graph of plasma concentrations of type V collagen MMP degradation product (C5M) is shown in Figure 3.

[0094] (Example 4) Luminex xMAP using plasma from healthy humans and endometriosis patients TM Biomarker discovery using technology (4-1) Obtaining human plasma samples Plasma from healthy individuals (5 secretory phase plasma samples and 5 proliferative phase plasma samples) was purchased from Proteogenex. For endometriosis patients, a total of 111 plasma samples were obtained from 37 endometriosis patients, collected pre-operatively, 3 days after surgery, and 1 month after surgery.

[0095] (4-2) Luminex xMAP of plasma proteins TM Analysis using technology Plasma sample (the proteins in it are Luminex xMAP) TM Analysis was performed using Luminex xMAP (a trademark of Luminex). TM This technology involves staining macrobeads with two fluorescent dyes at various concentrations, using the amount of fluorescent dye as an identification code, and immobilizing substances that bind to individual analytes on each bead, thereby enabling simultaneous analysis of multiple parameters with small sample sizes. In this example, 152 types of proteins were selected and analyzed using a protein measurement panel. Specifically, for each panel, an antibody that binds to the target protein was immobilized on a bead. A plasma sample was reacted with the antibody on the bead, and then a reporter antibody (labeled with a fluorescent dye) against the target protein was reacted with it. Then, using flow cytometry technology, the fluorescence intensity was measured with two types of lasers to analyze the expression level of the target protein (using a Luminex100 instrument).

[0096] (4-3) Selection of biomarker candidates The plasma concentrations of the various proteins obtained were analyzed using Microsoft Excel. Twenty-four proteins showing altered expression in endometriosis patients were selected based on the following criteria (Tables 7A and 7B). Of the 24 proteins selected, 12 were found to be at the highest levels during surgery in endometriosis patients, decreased one month after surgery, and showed the lowest levels in healthy individuals.

[0097] [Table 7A] TIFF2026065204000035.tif80170 [Table 7B]

[0098] (Example 5) Biomarker discovery by LC-MS analysis using plasma from healthy humans and endometriosis patients (5-1) Obtaining human plasma samples Plasma from healthy individuals (5 secretory phase plasma samples and 5 proliferative phase plasma samples) was purchased from Proteogenex. For endometriosis patients, a total of 125 plasma samples were obtained from 39 endometriosis patients, collected pre-operatively, 3 days after surgery, and 1 month after surgery.

[0099] (5-2) LC-MS analysis of plasma proteins High Select TMThey were bound to and removed from the column using Top14 Abundant Protein Depletion Mini Spin Columns (Thermo) by the manufacturer's recommended method. The proteins in the flow-through fraction that did not bind to the column were precipitated by the methanol / chloroform method and dissolved in an 8M urea / 400 mM ammonium bicarbonate solution. The dissolved proteins were reduced, alkylated, and then digested with lysyl endopeptidase and trypsin to form peptides. This peptide solution was desalted using Monospin C18 (GL Science) by the manufacturer's recommended method.

[0100] The peptides were labeled with TMT10plex by the manufacturer's recommended method. The labeled peptides were fractionated using AssayMAP reversed phase (RP-S) cartridges (Agilent technologies) by the High pH Reversed-Phase method using triethylamine, and each fraction was analyzed using an LC-MS analysis system that connected an Orbitrap Fusion Lumos (ThermoFisher scientific) to a nano-LC system (EASY-nLC TM 1200 system, ThermoFisher Scientific). The analysis samples were peptides obtained from the plasma of 5 healthy individuals (a total of 10 samples in the secretory and proliferative phases respectively) and 39 endometriosis patients (a total of 125 samples before surgery, 3 days after surgery, and 1 month after surgery). For each analysis, a sample obtained by equally mixing 7 purchased healthy individual samples and 7 purchased endometriosis patient samples was used as a control for calibration between measurements, so that there were 10 samples for each analysis. Each sample was analyzed 3 times, and they were integrated during the analysis process.

[0101] The proteins in the sample were obtained from the Raw data of the LC-MS analysis using Thermo Scientific TM Proteome Discoverer TMThe proteins were identified using [specific method / tool], and their relative expression levels were analyzed. Database searches for protein identification were performed using the following parameters. Taxonomy: uniprot human Static modifications: TMT6plex / +229.163 Da (Any N-Terminus), Carbamidomethyl / +57.021 Da (C, C-Terminus), TMT6plex / +229.163 Da (K, C-Terminus) Dynamic Modifications: Oxidation / +15.995 Da (M), Acetyl / +42.011Da (N-Terminus) FDR: Target FDR (Strict) / 0.01, Target FDR (Relaxed) / 0.05 The relative expression levels of the target protein in preoperative plasma samples from endometriosis patients compared to plasma samples from healthy individuals were calculated using analysis software (Proteome Discoverer).

[0102] (5-3) Selection of candidate biomarker molecules LC-MS results were analyzed using Proteome Discoverer. Based on the relative expression levels of each protein in the samples (10 samples from 5 healthy individuals in the proliferative and secretory phases, and pre-operative samples from 39 endometriosis patients), 27 proteins showing altered expression in endometriosis patients were selected according to the following criteria (Tables 8A and 8B). - Abundance Ratio (ratio of presence) had a P-Value ≤ 0.5 in ANOVA and Tukey HSD post hoc. - Abundance Ratios ≤ 0.6250 or ≥ 1.6000 Of the 27 proteins selected, 16 were found to be at the highest levels during surgery in endometriosis patients, decreased one month after surgery, and showed the lowest levels in healthy individuals.

[0103] [Table 8A] TIFF2026065204000039.tif144170 [Table 8B] [Industrial applicability]

[0104] This disclosure demonstrates that it is possible to diagnose whether or not a subject suffers from endometriosis by measuring the concentration of type V collagen MMP degradation products in a sample obtained from the subject, or by measuring the amount of at least one marker selected from the group consisting of markers shown in Table 1A, Table 2A, Table 1B, Table 2B, Table 5A, Table 6A, Table 5B, and Table 6B. The invention of this disclosure makes it possible to diagnose endometriosis by a non-invasive method and is extremely useful for the diagnosis and treatment of the disease.

Claims

1. A method for diagnosing endometriosis, A method comprising the step of measuring the amount of at least one marker selected from the group consisting of transforming growth factor beta-1, latent-transforming growth factor beta-binding protein 1, type V collagen MMP degradation products, a marker indicated in group 1A, a marker indicated in group 2A, a marker indicated in group 1B, a marker indicated in group 2B, a marker indicated in group 3A, a marker indicated in group 4A, a marker indicated in group 3B, and a marker indicated in group 4B, in a sample obtained from a subject. [1A group] [Group 1B] [Group 2A] [Group 2B] [Group 3A] [Group 3B] [4A group] [Group 4B]

2. A method for determining whether or not a subject has endometriosis, A method comprising the step of measuring the amount of at least one marker selected from the markers described in claim 1 in a sample obtained from a subject.

3. A method for determining the degree of endometrial fibrosis in a subject, A method comprising the step of measuring the amount of at least one marker selected from the markers described in claim 1 in a sample obtained from a subject.

4. A method for determining the degree of endometrial adhesion in a subject, A method comprising the step of measuring the amount of at least one marker selected from the markers described in claim 1 in a sample obtained from a subject.

5. A method for predicting the degree of pain caused by endometriosis in a person who has or is suspected of having endometriosis, A method comprising the step of measuring the amount of at least one marker selected from the markers described in claim 1 in a sample obtained from a subject.

6. A method for determining the progression of endometriosis in a subject who has or is suspected of having endometriosis, A method comprising the step of measuring the amount of at least one marker selected from the markers described in claim 1 in a sample obtained from a subject.

7. A method for monitoring the pathological condition of endometriosis in subjects who have or are suspected of having endometriosis, A method comprising the step of measuring the amount of at least one marker selected from the markers described in claim 1 in each of several samples taken from a subject at different time points.

8. The method according to any one of claims 1 to 7, wherein the amount of the marker present is the polypeptide concentration of the marker in the blood sample.

9. This is a kit for diagnosing endometriosis. A kit comprising a reagent for measuring the abundance of at least one marker selected from the markers described in claim 1.

10. A kit for determining whether or not a subject has endometriosis, A kit comprising a reagent for measuring the abundance of at least one marker selected from the markers described in claim 1.

11. A kit for determining the degree of endometrial fibrosis in a subject, A kit comprising a reagent for measuring the abundance of at least one marker selected from the markers described in claim 1.

12. A kit for determining the degree of endometrial adhesion in a subject, A kit comprising a reagent for measuring the abundance of at least one marker selected from the markers described in claim 1.

13. A kit for predicting the degree of pain caused by endometriosis in subjects who have or are suspected of having endometriosis, A kit comprising a reagent for measuring the abundance of at least one marker selected from the markers described in claim 1.

14. A kit for determining the progression of endometriosis in subjects who have or are suspected of having endometriosis, A kit comprising a reagent for measuring the abundance of at least one marker selected from the markers described in claim 1.

15. A kit for monitoring the pathology of endometriosis in subjects who have or are suspected of having endometriosis, A kit comprising a reagent for measuring the abundance of at least one marker selected from the markers described in claim 1, and instructions for comparing the abundance of at least one marker selected from the markers described in claim 1 in each of a plurality of samples taken from a subject at different time points.

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