METHOD FOR DIAGNOSING ENDOMETRIOSIS, DISEASE STATE MONITORING METHOD, AND KIT

TECHNICAL FIELD

The present disclosure relates to methods of diagnosing endometriosis or methods of determining whether or not a subject is affected by endometriosis, methods of determining the extent of endometrial fibrosis or adhesion in a subject affected by endometriosis, methods of predicting pain of a subject affected by endometriosis, methods of monitoring pathological conditions of a subject affected by endometriosis, and the like, and kits and the like for performing these methods.

BACKGROUND ART

Endometriosis is an estrogen-dependent inflammatory disease observed in 6-10% of women capable of pregnancy, and infertility or pelvic pain is observed in more than 50% of patients (NPL 1). Various hypotheses have been proposed for the pathogenic mechanism of endometriosis (NPL 2) and the relationship with various factors including inflammatory cytokines and chemokines, growth factors, and hormones has been reported (NPL 3). Drugs that target hormones have already been developed, and hormone agents such as GnRH antagonists and progesterone formulations have been shown to alleviate pain and improve pathological conditions (NPLs 4 and 5). However, the strong side effects and difficulty in long-term use of hormone agent treatment present therapeutic problems (NPL 6). Meanwhile, drugs targeting inflammatory cytokines and chemokines are under development and anti-IL-8 antibodies have shown a strong effect of alleviating pathological conditions in a simian endometriosis model (PTL 1).

In the diagnosis of endometriosis, a definitive diagnosis can only be carried out by invasive means such as endoscopic observation or laparotomy. Furthermore, it is difficult for patients to notice the difference between symptoms associated with regular menstruation (e.g., pain) and symptoms of early endometriosis. As such, the hurdle to diagnosing a group of candidate patients is high and it is considered to require 7 to 11 years on average from onset to definitive diagnosis. The delay in definitive diagnosis leads to a delay in the period to start treatment of endometriosis, which is a great clinical problem (NPLs 7 and 8). Therefore, non-invasive diagnostic methods with less burden for patients, such as blood markers, are strongly desired, but precise diagnostic methods have not been established (NPL 9).

CITATION LIST
Patent Literature

[PTL 1] WO2018/025982

Non-Patent Literature

[NPL 1] Giudice L C. Endometriosis. N Engl J Med 2010; 362:2389-98.

[NPL 2] Rogers P A et al. Research priorities for endometriosis. Reprod Sci 2017; 24:202-226.

[NPL 3] Beste M T et al. Molecular network analysis of endometriosis reveals a role for c-Jun-regulated macrophage activation. Sci Transl Med. 2014; 6:222ra216.

[NPL 4] Taylor H S et al. Treatment of Endometriosis-Associated Pain with Elagolix, an Oral GnRH Antagonist. N Engl J Med. 2017; 377:28-40.

[NPL 5] Kohler G et al. A dose-ranging study to determine the efficacy and safety of 1, 2, and 4 mg of dienogest daily for endometriosis. Int J Gynaecol Obstet. 2010; 108:21-5.

[NPL 6] Treatment of Endometriosis-Associated Pain with Elagolix, an Oral GnRH Antagonist.

[NPL 7] Greene R et al. Diagnostic experience among 4,334 women reporting surgically diagnosed endometriosis. Fertility and Sterility, 2009; 91:32-39.

[NPL 8] Manderson L et al. Circuit breaking: Pathways of treatment seeking for women with endometriosis in Australia. Qualitative Health Research, 2008; 18:522-534.

[NPL 9] Fassbender A et al. Update on biomarkers for the detection of endometriosis. Biomed Res Int 2015; 2015:130854.

SUMMARY OF INVENTION
Technical Problem

The present disclosure was achieved in view of the above circumstances. An objective of the present disclosure is to provide methods of diagnosing endometriosis or methods of determining whether or not a subject is affected by endometriosis, methods of determining the extent of endometrial fibrosis or adhesion in a subject affected by endometriosis, methods of predicting pain of a subject affected by endometriosis, methods of monitoring pathological conditions of a subject affected by endometriosis, and the like, and kits for performing these methods.

Solution to Problem

As a result of conducting dedicated research on methods of diagnosing and methods of monitoring pathological conditions of endometriosis, the inventors of the present disclosure have discovered that there are a number of markers whose abundance in patients with endometriosis is different from those in healthy individuals, and also discovered that by measuring the abundance of those markers, whether or not a subject is affected by endometriosis can be diagnosed, the extent of endometrial fibrosis or adhesion can be determined, pain of a subject affected by endometriosis can be predicted, and pathological conditions of a subject affected by endometriosis can be monitored.

The present disclosure is based on these findings and specifically relates to the following inventions:

- [1] A method of diagnosing endometriosis, comprising measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in a sample obtained from a subject.
- [2] A method of determining whether or not a subject is affected by endometriosis, comprising measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in a sample obtained from the subject.
- [3] The method of [1] or [2], further comprising a step in which 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 is high or 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 is low in the sample obtained from the subject, the subject that the sample is derived from is shown to be affected or potentially affected by endometriosis.
- [4] A method of determining the extent of endometrial fibrosis in a subject, comprising measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in a sample obtained from the subject.
- [5] The method of [4], further comprising a step in which 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 is high or 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 is low in the sample obtained from the subject, it is shown that endometrial fibrosis has occurred or potentially occurred in the subject that the sample is derived from.
- [6] A method of determining the extent of endometrial adhesion in a subject, comprising measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in a sample obtained from the subject.
- [7] The method of [6], further comprising a step in which 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 is high or 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 is low in the sample obtained from the subject, it is shown that endometrial adhesion has occurred or potentially occurred in the subject that the sample is derived from.
- [8] A method of predicting the degree of pain due to endometriosis in a subject affected or suspected of being affected by endometriosis, comprising measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in a sample obtained from the subject.
- [9] The method of [8], further comprising a step in which 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 is high or 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 is low in the sample obtained from the subject, the subject that the sample is derived from is shown to develop or potentially develop pain due to endometriosis.
- [10] A method of determining the degree of progression of endometriosis in a subject affected or suspected of being affected by endometriosis, comprising measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in a sample obtained from the subject.
- [11] The method of [10], further comprising a step in which 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 is high or 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 is low in the sample obtained from the subject, it is shown that endometriosis is progressing or potentially progressing in the subject that the sample is derived from.
- [12] The method of [3], [5], [7], [9], or [11], wherein when 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 is higher than the abundance of the same marker in a sample obtained from a healthy individual, the abundance is determined to be high.
- [13] The method of [3], [5], [7], [9], or [11], wherein when 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 two-fold or higher than the abundance of the same marker in a sample obtained from a healthy individual and/or when the abundance of at least one marker selected from the markers shown in Table 6A is 1.6-fold or higher than the abundance of the same marker in a sample obtained from a healthy individual, the abundance is determined to be high.
- [14] The method of [3], [5], [7], [9], or [11], wherein when 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 is lower than the abundance of the same marker in a sample obtained from a healthy individual, the abundance is determined to be low.
- [15] The method of [3], [5], [7], [9], or [11], wherein when 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 0.5-fold or lower than the abundance of the same marker in a sample obtained from a healthy individual and/or when the abundance of at least one marker selected from the markers shown in Table 6B is 0.625-fold or lower than the abundance of the same marker in a sample obtained from a healthy individual, the abundance is determined to be low.
- [16] A method of monitoring pathological conditions of endometriosis in a subject affected or suspected of being affected by endometriosis, comprising measuring each of the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in each of a plurality of samples collected from the subject at different points of time.
- [17] The method of [16], further comprising a step in which 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 decreases over time or 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 increases over time, it is shown that the pathological conditions of endometriosis are improving or potentially improving in the subject that the sample is derived from.
- [18] The method of [16], further comprising a step in which 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 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, it is shown that the pathological conditions of endometriosis are worsening or potentially worsening in the subject that the sample is derived from.
- [19] The method of any one of [1] to [18], wherein the marker is measured as a polypeptide.
- [20] The method of any one of [1] to [19], wherein the sample is a blood sample.
- [21] The method of [20], wherein the abundance of the marker in the sample is a concentration of a polypeptide of the marker in the blood sample.
- [21-1] The method of [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 a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 whether or not a subject is affected by endometriosis, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 of [22] or [23], further comprising instructions stating that 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 is high or 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 is low in a sample obtained from a subject, the subject that the sample is derived from is shown to be affected or potentially affected by endometriosis.
- [25] A kit for determining the extent of endometrial fibrosis in a subject, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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.
- [26] The kit of [25], further comprising instructions stating that 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 is high or 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 is low in a sample obtained from a subject, it is shown that endometrial fibrosis has occurred or potentially occurred in the subject that the sample is derived from.
- [27] A kit for determining the extent of endometrial adhesion in a subject, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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.
- [28] The kit of [27], further comprising instructions stating that 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 is high or 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 is low in a sample obtained from a subject, it is shown that endometrial adhesion has occurred or potentially occurred in the subject that the sample is derived from.
- [29] A kit for predicting the degree of pain due to endometriosis in a subject affected or suspected of being affected by endometriosis, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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.
- [30] The kit of [29], further comprising instructions stating that 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 is high or 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 is low in a sample obtained from a subject, the subject that the sample is derived from is shown to develop or potentially develop pain due to endometriosis.
- [31] A kit for determining the degree of progression of endometriosis in a subject affected or suspected of being affected by endometriosis, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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.
- [32] The kit of [31], further comprising instructions stating that 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 is high or 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 is low in a sample obtained from a subject, it is shown that endometriosis is progressing or potentially progressing in the subject that the sample is derived from.
- [33] The kit of [24], [26], [28], [30], or [32], wherein 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 is higher than the abundance of the same marker in a sample obtained from a healthy individual, the abundance is determined to be high.
- [34] The kit of [24], [26], [28], [30], or [32], wherein 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 two-fold or higher than the abundance of the same marker in a sample obtained from a healthy individual and/or if the abundance of at least one marker selected from the markers shown in Table 6A is 1.6-fold or higher than the abundance of the same marker in a sample obtained from a healthy individual, the abundance is determined to be high.
- [35] The kit of [24], [26], [28], [30], or [32], wherein 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 is lower than the abundance of the same marker in a sample obtained from a healthy individual, the abundance is determined to be low.
- [36] The kit of [24], [26], [28], [30], or [32], wherein 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, and the markers shown in Table 5B that is 0.5-fold or lower than the abundance of the same marker present in a sample obtained from a healthy individual and/or the abundance of at least one marker selected from the markers shown in Table 6B that is 0.625-fold or lower than the abundance of the same marker in a sample obtained from a healthy individual, the abundance is determined to be low.
- [37] A kit for monitoring pathological conditions of endometriosis in a subject affected or suspected of being affected by endometriosis, comprising a reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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.
- [38] The kit of [37], further comprising instructions stating that 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 1B, the markers 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 in each of a plurality of samples collected from the subject at different points of time is compared.
- [39] The kit of [37] or [38], further comprising instructions stating that 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 decreases over time or 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 increases over time, it is shown that the pathological conditions of endometriosis are improving or potentially improving in the subject that the sample is derived from.
- [40] The kit of [37] or [38], further comprising instructions stating that 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 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, it is shown that the pathological conditions of endometriosis are worsening or potentially worsening in the subject that the sample is derived from.
- [41] The kit of any one of [22] to [40], wherein the marker is measured as a polypeptide.
- [42] The kit of any one of [22] to [41], wherein the sample is a blood sample.
- [43] The kit of [42], wherein the abundance of the marker in the sample is a concentration of a polypeptide of the marker in the blood sample.
- [43-1] The kit of [42] or [43], 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.
- [44] A reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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, for use in the diagnosis of endometriosis.
- [45] A reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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, for use in the determination of whether or not a subject is affected by endometriosis.
- [46] A reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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, for use in the determination of the extent of endometrial fibrosis in a subject.
- [47] A reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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, for use in the determination of the extent of endometrial adhesion in a subject.
- [48] A reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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, for use in the prediction of the degree of pain due to endometriosis in a subject affected or suspected of being affected by endometriosis.
- [49] A reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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, for use in the determination of the degree of progression of endometriosis in a subject affected or suspected of being affected by endometriosis.
- [50] A reagent for measuring the abundance of at least one marker selected from the group consisting of a type V collagen MMP degradation product, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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, for use in the monitoring of pathological conditions of endometriosis in a subject affected or suspected of being affected by endometriosis.
- [51] The reagent of any one of [44] to [50], wherein the marker is measured as a polypeptide.
- [52] The reagent of any one of [44] to [51], wherein the abundance of the marker is measured by measuring a concentration of a polypeptide of the marker in a blood sample obtained from the subject.

[53] The reagent of [51], 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 present in the blood sample.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1-1 to FIG. 1-10 show the results of analyzing the relative expression levels of proteins in the plasma of healthy individuals/endometriosis patients by LC-MS. The title of each graph shows Gene Symbol:UniProt ID of the target protein. The X axis represents the name of the sample and the Y axis represents the relative expression level of the protein. The bars in the graph represent the standard deviation of the relative expression level of the protein in three data sets obtained by three analyses. FIG. 1-1 shows the data on Gene Symbol: CALU and UniProt ID: 043852.

FIG. 1-2 shows the data on Gene Symbol: CAT and UniProt ID: P04040.

FIG. 1-3 shows the data on Gene Symbol: HBA1 and UniProt ID: P69905.

FIG. 1-4 shows the data on Gene Symbol: HBB and UniProt ID: P68871.

FIG. 1-5 shows the data on Gene Symbol: HBD and UniProt ID: P02042.

FIG. 1-6 shows the data on Gene Symbol: PPIA and UniProt ID: P62937.

FIG. 1-7 shows the data on Gene Symbol: PRDX1;PRDX4 and UniProt ID: Q06830.

FIG. 1-8 shows the data on Gene Symbol: S100A6 and UniProt ID: P06703.

FIG. 1-9 shows the data on Gene Symbol: TPI1 and UniProt ID: P60174.

FIG. 1-10 shows the data on Gene Symbol: UBE2V1;UBE2V2 and UniProt ID: Q13404.

FIG. 2-1 to FIG. 2-10 show the results of analyzing the expression levels of proteins in the plasma of healthy individuals/endometriosis patients by SOMAscan®. The title of each graph shows Gene Symbol:UniProt ID of the target protein. The X axis represents the name of the sample and the Y axis represents the aptamer signal value of the target protein in SOMAscan®. FIG. 2-1 shows the data on Gene Symbol: BGN and UniProt ID: P21810.

FIG. 2-2 shows the data on Gene Symbol: CDH12 and UniProt ID: P55289.

FIG. 2-3 shows the data on Gene Symbol: DDX19B and UniProt ID: Q9UMR2.

FIG. 2-4 shows the data on Gene Symbol: IL1B and UniProt ID: P01584.

FIG. 2-5 shows the data on Gene Symbol: IL2 and UniProt ID: P60568.

FIG. 2-6 shows the data on Gene Symbol: LAG3 and UniProt ID: P18627.

FIG. 2-7 shows the data on Gene Symbol: MFGE8 and UniProt ID: Q08431.

FIG. 2-8 shows the data on Gene Symbol: PDE5A and UniProt ID: 076074.

FIG. 2-9 shows the data on Gene Symbol: SERPINE2 and UniProt ID: P07093.

FIG. 2-10 shows the data on Gene Symbol: SMPDL3A and UniProt ID: Q92484.

FIG. 3 shows the result of analyzing the expression level of a type V collagen degradation product (C5M) in the plasma of healthy individuals/endometriosis patients. The X axis represents the name of the sample and the Y axis represents the concentration of the target degradation product in the plasma (ng/ml).

DESCRIPTION OF EMBODIMENTS
Markers

Markers in the present disclosure can be reworded as biomarkers and refer to specific chemical substances in the body that can be measured and evaluated objectively as indices for normal biological processes, disease development processes, or pharmacological responsiveness to treatment. The markers are useful for evaluating the presence or absence of diseases, progress of diseases, or susceptibility to diseases; evaluating or predicting the effects, the optimal dose, or safety of pharmaceutical agents; predicting prognosis; or such. The markers in the present disclosure are specified by protein names, protein fragment names, or gene names Genes serving as markers are preferably measured as polypeptides or polynucleotides (including the form of DNA and the form of mRNA), and proteins or protein fragments serving as markers are preferably measured as polypeptides.

Methods of Measuring the Abundance of Markers

The abundance of a marker can be measured by selecting an appropriate method according to the form of the marker or the type of a sample in which the abundance of the marker is to be measured. When the marker is in the form of a polypeptide, it can be measured by immunological methods that use antibodies specifically binding to the polypeptide. Examples of such methods include enzyme immunoassays (ELISA, EIA), fluoroimmunoassays (FIA), radioimmunoassays (RIA), luminescent immunoassays (LIA), electrochemical luminescence (ECL) methods, Western blotting methods, surface plasmon resonance methods, methods that use antibody arrays, immunohistochemical staining methods, fluorescence activated cell sorting (FACS) methods, immunochromatography methods, immunoprecipitation methods, immunonephelometry methods, and latex agglutination methods. When the marker is in the form of a polynucleotide, it can be measured by genetic engineering methods that use oligonucleotides specifically binding to the polynucleotide. Examples of such methods include polymerase chain reaction (PCR) methods, reverse transcription PCR (RT-PCR) methods, real-time quantitative PCR (Q-PCR) methods, northern blotting, and hybridization methods (including methods that use oligonucleotide arrays such as DNA microarrays).

When a marker to be measured is a protein expressed from a gene, the abundance can be reworded as expression level. In the present disclosure, the “abundance” includes the expression level of a protein, the expression level of a gene, and the concentration of a protein fragment.

In an embodiment of the present disclosure, the abundance of a marker can be a relative abundance. A relative abundance can be measured by comparing the level of a specific marker and the level of other proteins/metabolites in a sample obtained from a subject with a control. A relative abundance can also be measured by LC/MS (liquid chromatography/mass spectrometry).

Criteria for the Abundance of Markers

In the present disclosure, “the abundance of a marker is high or large” means that the measured value of the marker is higher or larger than a predetermined value (control level) for the marker. “The abundance of a marker is low or small” means that it is lower or smaller than a predetermined value (control level) for the marker or not greater than the control level.

The predetermined value in the present disclosure means a value that is predetermined based on a certain scientific basis. It may be any value as long as it can be used as a reference to determine the presence or absence of endometriosis, determine the extent of endometrial fibrosis in a subject, determine the extent of endometrial adhesion in a subject, predict the degree of pain of a subject affected by endometriosis, determine the degree of progression of endometriosis, or monitor pathological conditions of endometriosis. The predetermined value in the present disclosure may be determined for each marker.

The predetermined value in the present disclosure can be determined from the measured value of a marker in a sample (control sample) obtained from a healthy subject, for example, a healthy adult. It has been found in the present disclosure that the measured value of a marker in a sample obtained from a subject affected by endometriosis is increased or decreased compared to the measured value of the marker in a sample obtained from a healthy subject. Thus, one possible approach may be to use as a predetermined value the average of the measured values of a marker in samples obtained from multiple healthy subjects. Another possible approach may be to use as a predetermined value the average of the measured values of a marker in 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. Accordingly, in one embodiment of the present disclosure, it is shown that determination of whether a test subject is affected by endometriosis, determination of the extent of endometrial fibrosis in a subject, determination of the extent of endometrial adhesion in a subject, prediction of the degree of pain of a subject affected or suspected of being affected by endometriosis, determination of the degree of progression of endometriosis, or monitoring of pathological conditions of endometriosis, is carried out by comparing the abundance of a marker (control level) measured in a sample (control sample) obtained from a healthy individual and the measured value of the marker in a sample obtained from a test subject.

In the present disclosure, the measured values and predetermined values of markers in the present disclosure may be measurement results of the abundance of the markers quantified by any method. In the present disclosure, values obtained as a result of measurement (e.g., color development intensity) may be directly used as measured values or predetermined values of markers, or values converted from measurement results (e.g., concentration) by comparing them with a separately prepared positive control sample that contains a known amount of the marker may be used. Alternatively, values given by, for example, grouping the values obtained as described above into certain intervals and scoring them (e.g., grades 1, 2, and 3) may be used.

Samples

Samples in the present disclosure can be reworded as biological samples and refer to organs, tissues, cells, body fluids, or mixtures thereof contained in living bodies. Specific examples include skin, respiratory tract, intestinal tract, urogenital tract, nerve, tumor, bone marrow, blood cells, blood (whole blood, plasma, serum), lymph, cerebrospinal fluid, intraperitoneal fluid, synovial fluid, intrapulmonary fluid, saliva, sputum, urine, and such. Samples obtained by washing these or obtained by culturing these ex vivo are also included in the samples of the present disclosure. A preferred sample in the present disclosure is blood, and a particularly preferred sample is plasma or serum.

In the present disclosure, samples obtained from subjects may be processed by methods such as concentration, purification, extraction, isolation, or physical/chemical treatment before subjected to measurement of the abundance of markers. For example, blood cells or plasma components may be isolated from blood samples, and DNA or RNA may be extracted from tissue/cell samples. Alternatively, unwanted components may be denatured/removed by heating or chemical reagents. Such processing is performed mainly for improving the sensitivity and specificity of measurement of the abundance of markers.

In determination of the extent of endometrial fibrosis, determination of the extent of endometrial adhesion, prediction of the degree of pain due to endometriosis, determination of the degree of progression of endometriosis, and monitoring of pathological conditions of endometriosis in the present disclosure, subjects from which samples are obtained may be subjects already diagnosed as being affected by endometriosis or subjects suspected of being affected by endometriosis. Subjects affected by endometriosis may be any subjects as long as they are affected by endometriosis. Subjects may be subjects that have not received, or have already been receiving, treatment for endometriosis.

Subjects in the present disclosure are mammals. Mammals include, but are not limited to, domesticated animals (for example, cows, sheep, cats, dogs, and horses), primates (for example, humans and non-human primates like monkeys), rabbits, and rodents (such as mice and rats). In a certain embodiment, subjects are humans.

Markers in the Present Disclosure

Markers in the present disclosure include type V collagen MMP (matrix metalloproteinase) degradation products (type V collagen-derived polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 4 at its N-terminus and/or comprising the amino acid sequence set forth in SEQ ID NO: 6 at its C-terminus, such as protein fragments set forth in SEQ ID NOs: 2 and 3), the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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.

TABLE 1A

UniProt ID
Protein name
Gene name

P02042
Hemoglobin subunit delta
HBD

P68871
Hemoglobin subunit beta;
HBB

LVV-hemorphin-7; Spinorphin

P55072
Transitional endoplasmic reticulum ATPase
VCP

P50395; P50395-2
Rab GDP dissociation inhibitor beta
GDI2

O43852-9; O43852-5; O43852-2;
Calumenin
CALU

O43852; O43852-4; O43852-3;

O43852-12; O43852-13; O43852-14;

O43852-7; O43852-15; O43852-11;

O43852-8; O43852-10; O43852-6

P04040
Catalase
CAT

Q06830; Q13162
Peroxiredoxin-1 ; Peroxiredoxin-4
PRDX1;

PRDX4

P69905
Hemoglobin subunit alpha
HBA1

Q13404-8; Q15819; Q13404;
Ubiquitin-conjugating enzyme E2
UBE2V1;

Q13404-7; Q13404-2; Q13404-1
variant 1; Ubiquitin-conjugating
UBE2V2

enzyme E2 variant 2

P06703
Protein S100-A6
S100A6

P60174-1; P60174; P60174-4
Triosephosphate isomerase
TPI1

P62937; P62937-2
Peptidyl-prolyl cis-trans
PPIA

isomerase A; Peptidyl-prolyl

cis-trans isomerase A, N-terminally

processed

P49746-2; P49746
Thrombospondin-3
THBS3

P09486
SPARC
SPARC

Q14766-3; Q14766-2; Q14766-5;
Latent-transforming growth factor
LTBP1

Q14766; Q14766-4
beta-binding protein 1

P01137
Transforming growth factor beta-1;
TGFB1

Latency-associated peptide

P61088; Q5JXB2
Ubiquitin-conjugating enzyme E2 N;
UBE2N;

Putative ubiquitin-conjugating
UBE2NL

enzyme E2 N-like

P00558-2; P00558
Phosphoglycerate kinase 1
PGK1

P07996; P07996-2
Thrombospondin-1
THBS1

P02776
Platelet factor 4; Platelet factor 4,
PF4

short form

P05067-7; P05067-11; P05067-8;
Amyloid beta A4 protein; N-APP;
APP

P05067-9; P05067; P05067-10;
Soluble APP-alpha; Soluble APP-beta;

P05067-3; P05067-4; P05067-5;
C99; Beta-amyloid protein 42;

P05067-6
Beta-amyloid protein 40; C83;

P3(42); P3(40); C80; Gamm

a-secretase C-terminal fragment 59;

Gamma-secretase C-terminal

fragment 57; Gamma-secretase

C-terminal fragment 50; C31

P00568
Adenylate kinase isoenzyme 1
AK1

P00918
Carbonic anhydrase 2
CA2

P32119
Peroxiredoxin-2
PRDX2

P16070-18; P16070-12; P16070-14;
CD44 antigen
CD44

P16070-13; P16070-11; P16070-10;

P16070-16; P16070-8; P16070-17;

P16070-6; P16070-4; P16070-3;

P16070-7; P16070-5; P16070;

P16070-15; P16070-9

Q13201
Multimerin-1; Platelet glycoprotein
MMRN1

la*; 155 kDa platelet multimerin

P27348
14-3-3 protein theta
YWHAQ

O00592-2; O00592
Podocalyxin
PODXL

P10599-2; P10599
Thioredoxin
TXN

P00915
Carbonic anhydrase 1
CA1

P62158
Calmodulin
CALM1

P58546
Myotrophin
MTPN

P62258; P62258-2
14-3-3 protein epsilon
YWHAE

O94919
Endonuclease domain-containing
ENDOD1

1 protein

P37837
Transaldolase
TALDO1

P07738
Bisphosphoglycerate mutase
BPGM

P30043
Flavin reductase (NADPH)
BLVRB

P02775
Platelet basic protein; Connective
PPBP

tissue-activating peptide III;

TC-2; Connective tissue-activating

peptide III(1-81); Beta-thromboglobulin;

Neutrophil-activating peptide 2(74);

Neutrophil-activating peptide 2(73);

Neutrophil-activating peptide 2; TC-1;

Neutrophil-activating peptide 2(1-66);

Neutrophil-activating peptide 2(1-63)

P15311
Ezrin
EZR

P0DJI8; P0DJI9-2
Serum amyloid A-1 protein;
SAA1;

Amyloid protein A;
SAA2

Serum amyloid protein A(2-104);

Serum amyloid protein A(3-104);

Serum amyloid protein A(2-103);

Serum amyloid protein A(2-102);

Serum amyloid protein A(4-101);

Serum amyloid A-2 protein

P10124
Serglycin
SRGN

P13798
Acylamino-acid-releasing enzyme
APEH

P23528
Cofilin-1
CFL1

P30041
Peroxiredoxin-6
PRDX6

Q8WUM4; Q8WUM4-2
Programmed cell death
PDCD6IP

6-interacting protein

P05090
Apolipoprotein D
APOD

Q99497
Protein deglycase DJ-1
PARK7

P35579; P35579-2
Myosin-9
MYH9

Q13228; Q13228-4;
Selenium-binding protein 1
SELENBP1

Q13228-2; Q13228-3

P13716; P13716-2
Delta-aminolevulinic acid
ALAD

dehydratase

Q9UL13-2; Q9UL13
Protein HEG homolog 1
HEG1

P07384
Calpain-1 catalytic subunit
CAPN1

P18065
Insulin-like growth factor-binding
IGFBP2

protein 2

P04083
Annexin A1
ANXA1

P0DMV8-2; P0DMV9; P0DMV8
Heat shock 70 kDa protein 1A;
HSPA1A;

Heat shock 70 kDa protein 1B
HSPA1B

P07911-3; P07911; P07911-5;
Uromodulin; Uromodulin, secreted
UMOD

P07911-4
form

P15924; P15924-2; P15924-3
Desmoplakin
DSP

TABLE 1B

UniProt ID
Protein name
Gene name

Q9C0H2-3
Protein tweety homolog 3
TTYH3

TABLE 2A

Entrez
Entrez Gene

Protein name
UniProt ID
Gene ID
Symbol

Pulmonary surfactant-associated
P35247
6441
SFTPD

protein D

Histone H1.2
P16403
3006
HIST1H1C

Sialic acid-binding Ig-like
Q9Y336
27180
SIGLEC9

lectin 9

Heterogeneous nuclear
P22626
3181
HNRNPA2B1

ribonucleoproteins A2/B1

Hexokinase-2
P52789
3099
HK2

High mobility group protein B1
P09429
3146
HMGB1

Thrombospondin-1
P07996
7057
THBS1

3-hydroxy-3-methylglutaryl-
P04035
3156
HMGCR

coenzyme A reductase

Platelet factor 4
P02776
5196
PF4

Protein S100-A9
P06702
6280
S100A9

40S ribosomal protein S3a
P61247
6189
RPS3A

Annexin A6
P08133
309
ANXA6

Inosine-5′-monophosphate
P20839
3614
IMPDH1

dehydrogenase 1

Tyrosine-protein kinase Fgr
P09769
2268
FGR

Serine/threonine-protein
O94768
9262
STK17B

kinase 17B

Neutrophil-activating peptide 2
P02775
5473
PPBP

Estradiol 17-beta-dehydrogenase 1
P14061
3292
HSD17B1

Connective tissue-activating
P02775
5473
PPBP

peptide III

Prostaglandin G/H synthase 2
P35354
5743
PTGS2

Amyloid beta A4 protein
P05067
351
APP

GTP-binding nuclear protein Ran
P62826
5901
RAN

NAD-dependent protein
Q8IXJ6
22933
SIRT2

deacetylase sirtuin-2

Protein S100-A12
P80511
6283
S100A12

Plasminogen activator inhibitor 1
P05121
5054
SERPINE1

SUMO-conjugating enzyme UBC9
P63279
7329
UBE2I

Bactericidal permeability-increasing
P17213
671
BPI

protein

6-phosphogluconate
P52209
5226
PGD

dehydrogenase, decarboxylating

Platelet-derived growth factor
P01127
5155
PDGFB

subunit B

Tyrosine-protein phosphatase
P29350
5777
PTPN6

non-receptor type 6

Metalloproteinase inhibitor 3
P35625
7078
TIMP3

NudC domain-containing protein 3
Q8IVD9
23386
NUDCD3

SPARC
P09486
6678
SPARC

Protein 4.1
P11171
2035
EPB41

Sex hormone-binding globulin
P04278
6462
SHBG

Death-associated protein kinase 2
Q9UIK4
23604
DAPK2

Hepatoma-derived growth
Q7Z4V5
84717
HDGFRP2

factor-related protein 2

Proto-oncogene vav
P15498
7409
VAV1

Serine/threonine-protein kinase
P11309
5292
PIM1

pim-1

Heterogeneous nuclear
Q99729
3182
HNRNPAB

ribonucleoprotein A/B

Proliferation-associated
Q9UQ80
5036
PA2G4

protein 2G4

Extracellular superoxide
P08294
6649
SOD3

dismutase [Cu-Zn]

Angiopoietin-1
Q15389
284
ANGPT1

Acid sphingomyelinase-like
Q92484
10924
SMPDL3A

phosphodiesterase 3a

Peroxiredoxin-6
P30041
9588
PRDX6

AMP Kinase (alpha1beta1gamma1)
Q13131
5562
PRKAA1

Q9Y478
5564
PRKAB1

P54619
5571
PRKAG1

Serum amyloid A-1 protein
P0DJI8
6288
SAA1

Insulin-like growth factor-binding
P18065
3485
IGFBP2

|protein 2

Histone H2B type 2-E
Q16778
8349
HIST2H2BE

Fibroblast growth factor 5
P12034
2250
FGF5

Non-histone chromosomal protein
P05114
3150
HMGN1

HMG-14

Calcium/calmodulin-dependent
Q13557
817
CAMK2D

protein kinase type II subunit delta

Interstitial collagenase
P03956
4312
MMP1

ICOS ligand
O75144
23308
ICOSLG

Calcium/calmodulin-dependent
Q13554
816
CAMK2B

protein kinase type II subunit beta

Inosine-5′-monophosphate
P12268
3615
IMPDH2

dehydrogenase 2

Dickkopf-related protein 4
Q9UBT3
27121
DKK4

Glia-derived nexin
P07093
5270
SERPINE2

14-3-3 protein beta/alpha
P31946
7529
YWHAB

Macrophage-capping protein
P40121
822
CAPG

ADP-ribosyl cyclase/cyclic
P28907
952
CD38

ADP-ribose hydrolase 1

SH2 domain-containing protein 1A
O60880
4068
SH2D1A

Vacuolar protein sorting-associated
Q9NP79
51534
VTA1

protein VTA1 homolog

Interleukin-1 beta
P01584
3553
IL1B

Chloride intracellular channel
O00299
1192
CLIC1

protein 1

MAP kinase-activated protein
Q16644
7867
MAPKAPK3

kinase 3

Mitogen-activated protein kinase 1
P28482
5594
MAPK1

Choline/ethanolamine kinase
Q9Y259
1120
CHKB

Creatine kinase B-type
P12277
1152
CKB

DNA topoisomerase 1
P11387
7150
TOP1

C-C motif chemokine 5
P13501
6352
CCL5

C3a anaphylatoxin
P01024
718
C3

Copine-1
Q99829
8904
CPNE1

Chymase
P23946
1215
CMA1

Mitogen-activated protein kinase
O43318
6885
MAP3K7

kinase kinase 7: TGF-beta-activated
Q15750
10454
TAB1

kinase 1 and MAP3K7-binding

protein 1 fusion

Dickkopf-related protein 1
O94907
22943
DKK1

Ephrin type-B receptor 4
P54760
2050
EPHB4

40S ribosomal protein S7
P62081
6201
RPS7

Intercellular adhesion molecule 1
P05362
3383
ICAM1

Ubiquitin + 1, truncated mutation
P62979
6233
RPS27A

for UbB

Ubiquitin-conjugating enzyme E2 N
P61088
7334
UBE2N

Moesin
P26038
4478
MSN

Small ubiquitin-related modifier 3
P55854
6613
SUMO3

G2/mitotic-specific cyclin-B1
P14635
891
CCNB1

ATP-dependent RNA helicase
Q9UMR2
11269
DDX19B

DDX19B

Tyrosine-protein kinase CSK
P41240
1445
CSK

Alpha-1-antitrypsin
P01009
5265
SERPINA1

Adenylate kinase isoenzyme 1
P00568
203
AK1

Mothers against decapentaplegic
P84022
4088
SMAD3

homolog 3

Brain-derived neurotrophic factor
P23560
627
BDNF

Atrial natriuretic factor
P01160
4878
NPPA

Annexin A1
P04083
301
ANXA1

Signal transducer and activator of
P42224
6772
STAT1

transcription 1-alpha/beta

Angiopoietin-related protein 4
Q9BY76
51129
ANGPTL4

Cadherin-12
P55289
1010
CDH12

Stress-induced-phosphoprotein 1
P31948
10963
STIP1

Glycylpeptide
P30419
4836
NMT1

N-tetradecanoyltransferase 1

Peroxiredoxin-1
Q06830
5052
PRDX1

Oxidized low-density lipoprotein
P78380
4973
OLR1

receptor 1

VPS10 domain-containing receptor
Q96PQ0
57537
SORCS2

SorCS2

FACT complex subunit SSRP1
Q08945
6749
SSRP1

Carbonic anhydrase 1
P00915
759
CA1

Mitogen-activated protein kinase 11
Q15759
5600
MAPK11

Triosephosphate isomerase
P60174
7167
TPI1

Neurexophilin-1
P58417
30010
NXPH1

Platelet-derived growth factor
P04085
5154
PDGFA

subunit A

lnterleukin-22 receptor subunit
Q8N6P7
58985
IL22RA1

alpha-1

TABLE 2B

Entrez
Entrez Gene

Protein name
UniProt ID
Gene ID
Symbol

Cystatin-SN
P01037
1469
CST1

lnterleukin-22
Q9GZX6
50616
IL22

Tyrosine-protein kinase Fyn
P06241
2534
FYN

Biglycan
P21810
633
BGN

Sonic hedgehog protein
Q15465
6469
SHH

Cathepsin L2
060911
1515
CTSV

Interferon alpha/beta receptor 1
P17181
3454
IFNAR1

Ectodysplasin-A, secreted form
Q92838
1896
EDA

Dynein light chain 1, cytoplasmic
P63167
8655
DYNLL1

Cytochrome c
P99999
54205
CYCS

Lactoperoxidase
P22079
4025
LPO

Tumor necrosis factor receptor
Q969Z4
84957
RELT

superfamily member 19L

Growth factor receptor-bound protein 2
P62993
2885
GRB2

Ectonucleotide pyrophosphatase/
Q6UWV6
339221
ENPP7

phosphodiesterase family member 7

Glucagon
P01275
2641
GCG

Eukaryotic translation initiation factor 4
P78344
1982
EIF4G2

gamma 2

C-X-C motif chemokine 10
P02778
3627
CXCL10

Platelet-derived growth factor receptor
P09619
5159
PDGFRB

beta

von Willebrand factor
P04275
7450
VWF

Iduronate 2-sulfatase
P22304
3423
IDS

cGMP-specific 3′,5′-cyclic
O76074
8654
PDE5A

phosphodiesterase

Small glutamine-rich tetratricopeptide
O43765
6449
SGTA

repeat-containing protein alpha

Interleukin-25
Q9H293
64806
IL25

MHC class I polypeptide-related
Q29980
4277
MICB

sequence B

C-C motif chemokine 7
P80098
6354
CCL7

Lactadherin
Q08431
4240
MFGE8

Macrophage metalloelastase
P39900
4321
MMP12

Ubiquitin-like protein ISG15
P05161
9636
ISG15

Fatty acid-binding protein, liver
P07148
2168
FABP1

Properdin
P27918
5199
CFP

Stromelysin-2
P09238
4319
MMP10

Protein FAM107B
Q9H098
83641
FAM107B

Myosin-binding protein C, slow-type
Q00872
4604
MYBPC1

Serine/threonine-protein kinase Chk2
O96017
11200
CHEK2

Xaa-Pro aminopeptidase 1
Q9NQW7
7511
XPNPEP1

NT-3 growth factor receptor
Q16288
4916
NTRK3

Interleukin-5 receptor subunit alpha
Q01344
3568
IL5RA

Ephrin type-A receptor 2
P29317
1969
EPHA2

Peptidyl-prolyl cis-trans isomerase F,
P30405
10105
PPIF

mitochondrial

B-cell lymphoma 6 protein
P41182
604
BCL6

Cell adhesion molecule 1
Q9BY67
23705
CADM1

Kinesin-like protein KIF23
Q02241
9493
KIF23

Netrin receptor UNC5D
Q6UXZ4
137970
UNC5D

Lymphocyte activation gene 3 protein
P18627
3902
LAG3

Serine protease HTRA2, mitochondrial
O43464
27429
HTRA2

lnterleukin-22 receptor subunit alpha-2
Q969J5
116379
IL22RA2

Caspase-3
P42574
836
CASP3

Platelet-derived growth factor receptor
P16234
5156
PDGFRA

alpha

Interleukin-20
Q9NYY1
50604
IL20

Ferritin
P02794
2495
FTH1

P02792
2512
FTL

Ephrin-A5
P52803
1946
EFNA5

Amphoterin-induced protein 2
Q86SJ2
347902
AMIGO2

Interleukin-2
P60568
3558
IL2

Phospholipase A2
P04054
5319
PLA2G1B

Protein kinase C alpha type
P17252
5578
PRKCA

Tumor necrosis factor ligand
P32971
944
TNFSF8

superfamily member 8

Endoglin
P17813
2022
ENG

Gamma-enolase
P09104
2026
ENO2

C-X-C motif chemokine 9
Q07325
4283
CXCL9

beta-nerve growth factor
P01138
4803
NGF

Hepcidin
P81172
57817
HAMP

Inorganic pyrophosphatase
Q15181
5464
PPA1

Kallikrein-8
O60269
11202
KLK8

Semaphorin-6B
Q9H3T3
10501
SEMA6B

Adapter molecule crk
P46108
1398
CRK

Phosphoglycerate mutase 1
P18669
5223
PGAM1

Proprotein convertase subtilisin/kexin
Q16549
9159
PCSK7

type 7

Pancreatic hormone
P01298
5539
PPY

Interleukin-1 receptor type 1
P14778
3554
IL1R1

C-X-C motif chemokine 11
O14625
6373
CXCL11

Secreted frizzled-related protein 1
Q8N474
6422
SFRP1

Inhibin beta A chain
P08476
3624
INHBA

Immunoglobulin D
P01880
3495
IGHD

50802
IGK@

3535
IGL@

Complement C3b
P01024
718
C3

Teratocarcinoma-derived growth factor 1
P13385
6997
TDGF1

Brother of CDO
Q9BWV1
91653
BOC

CD109 antigen
Q6YHK3
135228
CD109

Aminoacylase-1
Q03154
95
ACY1

60 kDa heat shock protein,
P10809
3329
HSPD1

mitochondrial

Importin subunit beta-1
Q14974
3837
KPNB1

C-reactive protein
P02741
1401
CRP

Ephrin type-A receptor 1
P21709
2041
EPHA1

Semaphorin-6A
Q9H2E6
57556
SEMA6A

SLIT and NTRK-like protein 5
O94991
26050
SLITRK5

Leucine-rich repeat transmembrane
Q9NZU0
23767
FLRT3

protein FLRT3

dCTP pyrophosphatase 1
Q9H773
79077
DCTPP1

Osteopontin
P10451
6696
SPP1

Formimidoyltransferase-cyclodeaminase
O95954
10841
FTCD

Ephrin-B2
P52799
1948
EFNB2

T-lymphocyte surface antigen Ly-9
Q9HBG7
4063
LY9

Sialic acid-binding Ig-like lectin 14
Q08ET2
100049587
SIGLEC14

N-acylethanolamine-hydrolyzing acid
Q02083
27163
NAAA

amidase

Endoplasmic reticulum aminopeptidase 1
Q9NZ08
51752
ERAP1

Low affinity immunoglobulin gamma Fc
P12318
2212
FCGR2A

region receptor II-a

TABLE 5A

UniProt ID
Protein name
Gene name

Q15389
Angiopoietin-1 (ANG-1)
ANGPT1

P23560
Brain-Derived Neurotrophic Factor (BDNF)
BDNF

O94907
Dickkopf-related protein 1 (DKK-1)
DKK1

P80511
S100-A12
S100A12

P42830
Epithelial-Derived Neutrophil-Activating
CXCL5

Protein 78 (ENA-78)

P02794
Ferritin (FRTN)
FTH1

P09341
Growth-Regulated alpha protein (GRO-alpha)
CXCL1

P01137
Latency-Associated Peptide of Transforming
TGFB1

Growth Factor beta 1 (LAP TGF-b1)

Q8WXL0
Luteinizing Hormone (LH)
LHB

G3CBL7
MHC class I chain-related protein A (MICA)
MICA

P02775
Platelet basic protein
PPBP

P01127
Platelet-Derived Growth Factor BB (PDGF-BB)
PDGFB

P01236
Prolactin (PRL)
PRL

PODJI8;
Serum amyloid A-1 protein; Amyloid protein A;
SAA1; SAA2

PODJI9-2
Serum amyloid protein A(2-104); Serum amyloid

protein A(3-104); Serum amyloid protein

A(2-103); Serum amyloid protein A(2-102);

Serum amyloid protein A(4-101); Serum

amyloid A-2 protein

P13501
T-Cell-Specific Protein RANTES (RANTES)
CCL5

TABLE 5B

UniProt ID
Protein name
Gene name

P10645
Chromogranin-A (CgA)
CHGA

Q9GZV9
Fibroblast growth factor 23 (FGF-23)
FGF23

P01266
Thyroglobul in (TG)
TG

TABLE 6A

UniProt ID
Protein name
Gene name

P07996
Thrombospondin-1 0S = Homo sapiens 0X = 9606
THBS1

GN = THBS1 PE = 1 SV = 2

P69905
Hemoglobin subunit alpha 0S = Homo sapiens 0X = 9606
HBA1

GN = HBA1 PE = 1 SV = 2

P32119
Peroxiredoxin-2 0S = Homo sapiens 0X = 9606 GN = PRDX2
PRDX2

PE = 1 SV = 5

P00918
Carbonic anhydrase 2 0S = Homo sapiens 0X = 9606
CA2

GN = CA2 PE = 1 SV = 2

P30043
Flavin reductase (NADPH) 0S = Homo sapiens 0X = 9606
BLVRB

GN = BLVRB PE = 1 SV = 3

O76011
Keratin, type I cuticular Ha4 0S = Homo sapiens
KRT34

0X = 9606 GN = KRT34 PE = 1 SV = 2

P13716
Delta-aminolevulinic acid dehydratase 0S =
ALAD

Homo sapiens 0X = 9606 GN = ALAD PE = 1 SV = 1

P00568
Adenylate kinase isoenzyme 1 0S = Homo sapiens
AK1

0X = 9606 GN = AK1 PE = 1 SV = 3

P07738
Bisphosphoglycerate mutase 0S = Homo sapiens
BPGM

0X = 9606 GN = BPGM PE = 1 SV = 2

P02775
Platelet basic protein 0S = Homo sapiens 0X =
PPBP

9606 GN = PPBP PE = 1 SV = 3

P22392
Nucleoside diphosphate kinase B 0S = Homo sapiens
NME2

0X = 9606 GN = NME2 PE = 1 SV = 1

P10124
Serglycin 0S = Homo sapiens 0X = 9606 GN = SRGN
SRGN

PE = 1 SV = 3

A0A0C4DH67
Immunoglobulin kappa variable 1-8 0S =
IGKV1-8

Homo sapiens 0X = 9606 GN = IGKV1-8 PE = 3 SV = 1

P63241
Eukaryotic translation initiation factor 5A-1
EIF5A

0S = Homo sapiens 0X = 9606 GN = EIF5A PE = 1 SV = 2

P02776
Platelet factor 4 0S = Homo sapiens 0X = 9606
PF4

GN = PF4 PE = 1 SV = 2

A0A075B6S2
Immunoglobulin kappa variable 2D-29 0S =
IGKV2D-29

Homo sapiens 0X = 9606 GN = IGKV2D-29 PE = 3 SV = 1

Q9UKU6
Thyrotropin-releasing hormone-degrading ectoenzyme
TRHDE

0S = Homo sapiens 0X = 9606 GN = TRHDE PE = 2 SV = 1

Q8WUM4
Programmed cell death 6-interacting protein 0S =
PDCD6IP

Homo sapiens 0X = 9606 GN = PDCD6IP PE = 1 SV = 1

P00441
Superoxide dismutase [Cu-Zn] 0S = Homo sapiens
SOD1

0X = 9606 GN = SOD1 PE = 1 SV = 2

TABLE 6B

UniProt ID
Protein name
Gene name

P36980
Complement factor H-related protein 2 0S =
CFHR2

Homo sapiens 0X = 9606 GN = CFHR2 PE = 1 SV = 1

PODOX3
Immunoglobulin delta heavy chain 0S = Homo sapiens
N/A

0X = 9606 PE = 1 SV = 1

P01880
Immunoglobulin heavy constant delta 0S =
IGHD

Homo sapiens 0X = 9606 GN = IGHD PE = 1 SV = 3

Q8N6C8
Leukocyte immunoglobulin-like receptor subfamily A
LILRA3

member 3 0S = Homo sapiens 0X = 9606 GN = LILRA3

PE = 1 SV = 3

P47929
Galectin-7 0S = Homo sapiens 0X = 9606 GN = LGALS7
LGALS7

PE = 1 SV = 2

O75339
Cartilage intermediate layer protein 1 0S = Homo sapiens
CILP

0X = 9606 GN = CILP PE = 1 SV = 4

O15389
Sialic acid-binding Ig-like lectin 5 0S = Homo sapiens
SIGLEC5

0X = 9606 GN = SIGLEC5 PE = 1 SV = 1

P35247
Pulmonary surfactant-associated protein D 0S =
SFTPD

Homo sapiens 0X = 9606 GN = SFTPD PE = 1 SV = 3

The sequences of the markers (mRNA and protein) listed in these tables can be easily retrieved from the database known to those skilled in the art (Uniprot: https://www.uniprot.org/) on the basis of the Uniprot IDs shown in the above tables. Specifically, a Uniprot ID can be used to search for the corresponding Uniprot entry to retrieve sequences provided in the “sequences” section. It is also possible to obtain IDs/accession numbers for other databases (for example, RefSeq, EMBL, GenBank, DDBJ, CCDS, PIR, UniGene, Ensemble, GeneID, KEGG, and USCS) from the description of the “sequences” section, and retrieve more sequences from those databases. In the present disclosure, the sequence of each marker refers to a sequence and an ID/accession number provided in the latest version of the corresponding Uniprot entry with a release number earlier than 2018-09 (for example, 2018-08; if 2018-08 does not exist, then 2018_07; if neither 2018_08 nor 2018_07 exists, then 2018_06; the same applies to numbers earlier than 2018_06).

Furthermore, in the FTP where the archives of Uniprot release data are stored (ftp://ftp.uniprot.org/pub/databases/uniprot/previous_releases/), the amino acid sequences of the markers in the above tables can also be retrieved from the data included in directory “release-2018_08/knowledgebase” (the “knowledgebase” directory included in “release-2018_08”) by using the Uniprot IDs shown in the tables.

The groups of markers listed in the above tables include markers indicating fibrosis (TGFB1, SPARC, etc.). Furthermore, the groups of markers listed in the above tables include many molecules considered to be derived from platelets. For example, uniprot says that PPBP, THBS1, PF4, SERPINA1, TIMP3, APP, CALU, CASP3, MMRN1, SAA1, SRGN, VWF, and others are involved in platelet degranulation and such. Zhang et al. demonstrated in an in vitro experimental system using endometrial-derived cells that TGFb1 released from activated platelets induced epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT), increased collagen production, and induced fibrosis (Molecular and Cellular Endocrinology, 428, 1-16, 2016). This is consistent with the fact that TGFb1 and platelet-derived proteins were selected as markers in the present disclosure, suggesting that changes in these proteins may reflect changes in platelet activation and such.

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

The presence of a type V collagen MMP degradation product is detected by any method that can detect a type V collagen fragment after cleavage between amino acids 1316 (G) and 1317 (H) set forth in SEQ ID NO: 1. In a certain embodiment, an antibody that specifically recognizes peptide HMGREGREGE (SEQ ID NO: 4) located at the N-terminus of the type V collagen fragment shown in SEQ ID NO: 2 can be used to detect the abundance of a type V collagen MMP degradation product. In a more specific embodiment, an antibody that can recognize peptide HMGREGREGE (SEQ ID NO: 4) located at the N-terminus of the type V collagen fragment shown in SEQ ID NO: 2 and that does not recognize GHMGREGREGE (SEQ ID NO: 5) can be used to detect the abundance of a type V collagen MMP degradation product (an ELISA detection method that uses such an antibody was established in Clin Biochem. 2012 May;45(7-8):541-6.). In another embodiment, an antibody that specifically recognizes peptide GPPGKRGPSG (SEQ ID NO: 6) located at the C-terminus of the type V collagen fragment set forth in SEQ ID NO: 3 can be used to detect the abundance of a type V collagen MMP degradation product. In a more specific embodiment, an antibody that can recognize peptide GPPGKRGPSG (SEQ ID NO: 6) located at the C-terminus of the type V collagen fragment shown in SEQ ID NO: 3 and that does not recognize GPPGKRGPSGH (SEQ ID NO: 7) can be used to detect the abundance of a type V collagen MMP degradation product.

In the present disclosure, a protein selected from the group consisting of type V collagen MMP degradation products, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers shown in Table 2B, the markers shown in Table 5A, the markers shown in in Table 6A, the markers shown in Table 5B, and the markers shown in Table 6B may be used alone as a marker, or a combination of selected proteins may be used as markers. When more than one protein is used as markers, each of the markers may be measured individually and then the results may be combined, or the markers may be measured together.

The abundance of the markers of the present disclosure can be measured using the measuring methods described in the present disclosure or using commercially available measuring reagents. Specific measured values and predetermined values of each marker described herein can be construed as values measured using the above-mentioned measuring reagents.

The predetermined values (control levels) determined for the markers of the present disclosure can vary depending on the type of a sample of a subject for which the abundance of each marker is measured, but can be determined, for example, from the range of 0.1 to 100 ng/mL. Alternatively, it can be determined from the range of 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, and so on, but is not limited thereto.

The predetermined value determined for each marker of the present disclosure can be determined from values such as 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 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, but is not limited thereto.

In the present disclosure, when the abundance of each marker of the present disclosure is greater than the predetermined value (control level), it can be decided that “the abundance is high” or “the abundance is large.” For example, in the present disclosure, it can be decided that “the abundance is high” or “the abundance is large” when, without limitation, the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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, or 3.0 times or more the abundance of the same marker in a sample obtained from a healthy individual, or more. Alternatively, in the present disclosure, it can be decided that “the abundance is high” or “the abundance is large” when, without limitation, the abundance 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, or 2.00 times or more the abundance of the same marker in a sample obtained from a healthy individual, or more.

In the present disclosure, when the abundance of each marker of the present disclosure is less than the predetermined value (control level), it can be decided that “the abundance is low” or “the abundance is small.” For example, in the present disclosure, it can be decided that “the abundance is low” or “the abundance is small” when, without limitation, the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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, or 0.1 times or less the abundance of the same marker in a sample obtained from a healthy individual, or less. Alternatively, in the present disclosure, it can be decided that “the abundance is low” or “the abundance is small” when, without limitation, the abundance 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, or 0.1 times or less the abundance of the same marker in a sample obtained from a healthy individual, or less.

Diagnosis of Endometriosis and Determination of Whether or Not a Subject is Affected by Endometriosis

One aspect of the present disclosure relates to methods of diagnosing endometriosis or methods of determining whether or not a subject is affected by endometriosis. One aspect of the present disclosure also relates to methods of detecting endometriosis or markers thereof. One aspect of the present disclosure also relates to methods of assisting the diagnosis or determination. One aspect of the present disclosure also relates to methods of providing instructions for the diagnosis or determination. One aspect of the present disclosure relates to reagents and kits for use in these methods.

The above respective methods comprise measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in a sample obtained from the subject. The methods can also comprise comparing the abundance with a predetermined value (control level).

The above reagents are reagents for measuring the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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. The above kits contain such reagents.

The above respective methods may comprise a step in which if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 in the sample obtained from the subject is higher than a predetermined value (control level) or 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 in the sample obtained from the subject is lower than a predetermined value (control level), the subject that the sample is derived from is shown to be affected or potentially affected by endometriosis.

The above respective methods can be performed both in vivo and in vitro, but are preferably performed in vitro.

Furthermore, the above respective methods may also comprise obtaining a sample from the subject.

Moreover, the above respective methods may also comprise treating a subject that has been shown to be affected or potentially affected by endometriosis. The above respective methods may also comprise administering a known therapeutic agent for endometriosis to a subject that has been shown to be affected or potentially affected by endometriosis. Thus, the present invention relates to methods of treating endometriosis in a subject that has been shown to be affected or potentially affected by endometriosis by each of the above methods. Various therapeutic agents for endometriosis are known to those skilled in the art, and examples thereof include, but are not limited to, estrogen/progesterone mixtures, progesterone preparations, GnRH agonists, GnRH antagonists, and danazol.

The above-mentioned kits may further comprise instructions stating that if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 in the sample obtained from the subject is higher than a predetermined value (control level) 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 is lower than a predetermined value (control level), the subject that the sample is derived from is shown to be affected or potentially affected by endometriosis.

Determination of the Extent of Endometrial Fibrosis in a Subject

One aspect of the present disclosure relates to methods of determining the extent of endometrial fibrosis in a subject, methods of assisting the determination, methods of providing instructions for the determination, and reagents and kits for use in these.

The above respective methods may comprise a step in which if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 in the sample obtained from the subject is higher than a predetermined value (control level) 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 in the sample obtained from the subject is lower than a predetermined value (control level), it is shown that endometrial fibrosis is occurring or potentially occurring in the subject that the sample is derived from.

The above respective methods can be performed both in vivo and in vitro, but is preferably performed in vitro.

Furthermore, the above respective methods may comprise obtaining a sample from the subject.

Moreover, the above respective methods may also comprise treating a subject in whom or which it has been shown that endometrial fibrosis has occurred or potentially occurred. The above respective methods may also comprise administering a known therapeutic agent for suppressing endometrial fibrosis to a subject in whom or which it has been shown that endometrial fibrosis has occurred or potentially occurred. Thus, the present invention relates to methods of suppressing fibrosis in a subject in whom or which it has been shown by the above respective methods that endometrial fibrosis has occurred or potentially occurred. Examples of therapeutic agents for suppressing endometrial fibrosis include the above-mentioned therapeutic agents for endometriosis.

The above kits may further comprise instructions stating that if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 is higher than a predetermined value (control level) or 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 is lower than a predetermined value (control level) in a sample obtained from the subject, it is shown that endometrial fibrosis has occurred or potentially occurred in the subject that the sample is derived from.

Determination of the Extent of Endometrial Adhesion in a Subject

One aspect of the present disclosure relates to methods of determining the extent of endometrial adhesion in a subject, methods of assisting the determination, methods of providing instructions for the determination, and reagents and kits for use in these methods.

The above respective methods may comprise a step in which if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 in a sample obtained from the subject is higher than a predetermined value (control level) 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 in a sample obtained from the subject is lower than a predetermined value (control level), it is shown that endometrial adhesion has occurred or potentially occurred in the subject that the sample is derived from.

The above respective methods can be performed both in vivo and in vitro, but are preferably performed in vitro.

Furthermore, The above respective methods may comprise obtaining a sample from the subject.

Moreover, the above respective methods may also comprise treating a subject in whom or which it has been shown that endometrial adhesion has occurred or potentially occurred. Each of the above methods may also comprise administering a known therapeutic agent for suppressing endometrial adhesion to a subject in whom or which it has been shown that endometrial adhesion has occurred or potentially occurred. Thus, the present invention relates to methods of suppressing endometrial adhesion in a subject in whom or which it has been shown by the above respective methods that endometrial adhesion has occurred or potentially occurred. Examples of therapeutic agents for suppressing endometrial adhesion include the above-mentioned therapeutic agents for endometriosis.

The above kits may further comprise instructions stating that if the abundance of at least one marker selected from the group consisting of the concentration of type V collagen MMP degradation products, 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 is higher than a predetermined value (control level) or 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 is lower than a predetermined value (control level) in a sample obtained from the subject, it is shown that endometrial adhesion has occurred or potentially occurred in the subject that the sample is derived from.

Prediction of the Degree of Pain Due to Endometriosis in a Subject Affected or Suspected of Being Affected by Endometriosis

One aspect of the present disclosure relates to methods of predicting the degree of pain due to endometriosis in a subject affected or suspected of being affected by endometriosis, methods of assisting the prediction, methods of providing instructions for the prediction, and reagents and kits for use in these methods.

The above respective methods may comprise a step in which if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 in a sample obtained from the subject is higher than a predetermined value (control level) 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 in a sample obtained from the subject is lower than a predetermined value (control level), the subject that the sample is derived from is shown to develop or potentially develop pain due to endometriosis.

The above respective methods can be performed both in vivo and in vitro, but is preferably performed in vitro.

Furthermore, the above respective methods may comprise obtaining a sample from the subject.

Moreover, the above respective methods may also comprise alleviating pain due to endometriosis in a subject that has been shown to develop or potentially develop pain due to endometriosis. The above respective methods may also comprise administering a known therapeutic agent for suppressing pain due to endometriosis to a subject that has been shown to develop or potentially develop pain due to endometriosis. Thus, the present invention relates to methods of suppressing pain due to endometriosis in a subject that has been shown to develop or potentially develop pain due to endometriosis by the above respective methods. Examples of therapeutic agents for suppressing pain due to endometriosis include the above-mentioned therapeutic agents for endometriosis.

The above kits may further comprise instructions stating that if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 is higher than a predetermined value (control level) or 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 is lower than a predetermined value (control level) in a sample obtained from the subject, the subject that the sample is derived from is shown to develop or potentially develop pain due to endometriosis.

Determination of the Degree of Progression of Endometriosis

One aspect of the present disclosure relates to methods of determining the degree of progression of endometriosis in a subject affected or suspected of being affected by endometriosis, methods of assisting the determination, methods of providing instructions for the determination, and reagents and kits for use in these methods.

The above respective methods may comprise a step in which if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 in a sample obtained from the subject is higher than a predetermined value (control level) 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 in a sample obtained from the subject is lower than a predetermined value (control level), it is shown that endometriosis is progressing or potentially progressing in the subject that the sample is derived from.

The above respective methods can be performed both in vivo and in vitro, but are preferably performed in vitro.

Furthermore, the above respective methods may comprise obtaining a sample from the subject.

Moreover, the above respective methods may also comprise treating a subject in whom or which it has been shown that endometriosis is progressing or potentially progressing. The above respective methods may also comprise administering a known therapeutic agent for suppressing progression of endometriosis to a subject in whom or which it has been shown that endometriosis is progressing or potentially progressing. Thus, the present invention relates to methods of suppressing progression of endometriosis in a subject in whom or which it has been shown by the above respective methods that endometriosis is progressing or potentially progressing. Examples of therapeutic agents for suppressing progression of endometriosis include the above-mentioned therapeutic agents for endometriosis.

The above kits may further comprise instructions stating that if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 is higher than a predetermined value (control level) or 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 is lower than a predetermined value (control level) in a sample obtained from the subject, it is shown that endometriosis is progressing or potentially progressing in the subject that the sample is derived from.

Monitoring of Pathological Conditions of Endometriosis

One aspect of the present disclosure relates to methods of monitoring pathological conditions of endometriosis in a subject affected or suspected of being affected by endometriosis, methods of assisting the monitoring, methods of providing instructions for the monitoring, and reagents and kits for use in these methods. The monitoring in the present disclosure includes evaluating a therapeutic effect and/or providing information on a future therapeutic regimen or therapeutic policy.

The above respective methods may comprise measuring each of the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in each of a plurality of samples collected from a subject at different points of time. The methods may also comprise comparing the abundance of the marker in each sample in a plurality of samples collected at different points of time. In the present disclosure, the timing and number of times of collecting samples are not limited, and samples can be collected at various points of time before treatment, during treatment, and after treatment as necessary. Samples can also be collected at various timings within each period of before treatment, during treatment, and after treatment. In the present disclosure, “plurality” is not particularly limited, and examples thereof include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The above kits may comprise instructions stating that the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 in each of a plurality of samples obtained from a subject collected at different points of time is compared.

The above respective methods may comprise a step in which if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 decreased 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 increased over time, it is shown that the pathological conditions of endometriosis are improving or potentially improving in the subject that the sample is derived from.

The above respective methods may comprise a step in which if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 increased 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 decreased over time, it is shown that the pathological conditions of endometriosis are worsening or potentially worsening in the subject the sample is derived from.

The above respective methods can be performed both in vivo and in vitro, but are preferably performed in vitro.

Furthermore, the above respective methods may comprise obtaining a sample from the subject.

Moreover, the above respective methods may comprise treating a subject in whom or which it has been shown that the pathological conditions of endometriosis are worsening or potentially worsening. The above respective methods may also comprise administering a known therapeutic agent for endometriosis to a subject in whom or which it has been shown that the pathological conditions of endometriosis are worsening or potentially worsening. Thus, the present invention relates to methods of treating endometriosis in a subject in whom or which it has been indicated by each of the above methods that the pathological conditions of endometriosis are worsening or potentially worsening. The above-mentioned agents can be used as therapeutic agents for endometriosis.

The above kits may comprise instructions stating that if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 decreased 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 increased over time, it is shown that the pathological conditions of endometriosis are improving or potentially improving in the subject that the sample is derived from.

The above kits may comprise instructions stating that if the abundance of at least one marker selected from the group consisting of type V collagen MMP degradation products, 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 increased 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 decreased over time, it is shown that the pathological conditions of endometriosis are worsening or potentially worsening in the subject that the sample is derived from.

Reagents/Kits

Reagents contained in the kits of the present disclosure are not particularly limited as long as the abundance of markers can be measured. Reagents can be appropriately selected depending on the form of markers. When the form of a marker is a polypeptide, reagents comprising an antibody that specifically binds to the polypeptide are preferable. When the form of a marker is a polynucleotide, reagents comprising an oligonucleotide that specifically binds to the polynucleotide are preferable. In the present disclosure, the antibody or the polynucleotide itself may be a reagent. Polynucleotides in the present disclosure include those in the form of DNA and in the form of mRNA.

Antibodies in the present disclosure may be chimeric antibodies, humanized antibodies, or human antibodies. In some embodiments, antibodies may be multispecific antibodies, e.g., bispecific antibodies. Alternatively, antibodies in the present disclosure may be antibody fragments or modified products thereof. For example, antibody fragments include Fab, F (ab′)2, Fv, or single chain Fv (scFv) in which an H chain Fv and an L chain Fv are linked 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, such as fluorescent dyes, enzymes, coenzymes, chemiluminescent substances, and radioactive substances, can be used.

Oligonucleotides in the present disclosure can be any oligonucleotide that specifically hybridizes to at least a portion of a polynucleotide that is a marker in the present disclosure or a complementary strand thereof. The nucleotide sequence of such an oligonucleotide for detecting a polynucleotide is selected from the nucleotide sequence complementary to the sense strand of a marker in the present disclosure. Oligonucleotides typically have a length of at least 15 bp or more.

Oligonucleotides in the present disclosure can also be used after being labeled by commonly known methods. Oligonucleotides in the present disclosure can be produced, for example, by commercially available oligonucleotide synthesizers.

Kits of the present disclosure preferably contain a positive control sample as a reference for measuring the abundance of markers. The positive control sample is not particularly limited as long as the amount of the markers contained therein has been determined in advance, and can be appropriately prepared depending on the form of markers measured by the kits. For example, when the form of a marker is a polypeptide, a positive control sample is preferably a sample comprising a polypeptide prepared by isolating, purifying, and quantifying the same polypeptide as the marker.

In addition to the above, kits of the present invention may include, for example, sterile water, saline, vegetable oil, surfactant, lipid, solubilizer, buffer, protein stabilizer (such as BSA and gelatin), preservative, blocking solution, reaction solution, reaction stop solution, and reagents for treating samples as necessary.

Sets of type V collagen MMP degradation products, the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers 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 are also included in the present disclosure. Such sets can be used for diagnosing endometriosis, determining the presence or absence of endometriosis, determining the extent of endometrial fibrosis or adhesion in patients affected by endometriosis, predicting pain of patients affected by endometriosis, monitoring pathological conditions of patients affected by endometriosis, and such.

All prior art documents cited herein are incorporated herein by reference.

EXAMPLES

The present disclosure will be further illustrated by the Examples below but is not limited thereto.

(Example 1) Search for Biomarkers by LC-MS Analysis Using Plasma of Healthy Human Individuals and Patients with Endometriosis
(1-1) Obtainment of Human Plasma Samples

Plasma from healthy individuals and endometriosis patients was purchased from Proteogenex.

(1-2) LC-MS Analysis of Plasma Proteins

With the Seppro IgY14 column (Sigma aldrich), highly abundant proteins in each plasma sample were bound to the column and removed by the method recommended by the manufacturer. The proteins in the flow-through fraction that did not bind to the column were precipitated by the methanol/chloroform method and dissolved in 8 M urea/400 mM ammonium bicarbonate solution. The dissolved proteins were reduced and alkylated, and then digested into peptides with lysyl endopeptidase and trypsin. The peptide solution was demineralized with Monospin C18 (GL Science) by the method recommended by the manufacturer.

The peptides were labeled with TMT10plex Mass Tag Labeling Kits and Reagents (ThermoFisher Scientific) (hereinafter, referred to as TMT 10-plex) by the method recommended by the manufacturer. The labeled peptides were fractionated with Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (ThermoFisher Scientific) by the method recommended by the manufacturer. Each fraction was analyzed by the LC-MS analysis system in which Orbitrap fusion Lumos (ThermoFisher scientific) was coupled to the nano-LC system (Ultimate3000, Dionex). The analysis samples included seven samples: the peptides obtained from the plasma of each of three healthy individuals and three endometriosis patients, and a sample (control) prepared by mixing an equal amount of these six peptide solutions. Each sample was analyzed three times, and an independent data set in each analysis was then analyzed (N=3, hereinafter, data set TMT1, 2, and 3).

The proteins in the samples were identified from the raw data of the LC-MS analysis by the MaxQuant software (http://www.biochem.mpg.de/5111795/maxquant), and their relative expression levels were analyzed. The database search for protein identification was performed with the following parameters. Taxonomy: uniprot human, Fixed modification: carbamidomethylation (C), Variable modification: oxidation (M); deamidation (NQ), Acetyl (protein N-term), and FDR (protein, peptide)<1%. The relative expression levels were calculated by the following formula:

$Relative expression level = \frac{\begin{matrix} Target protein signal in plasma samples \\ (from patients with endometriosis or healthy individuals) \end{matrix}}{Target protein signal in the control sample}$

The target protein signal value used in the above formula was calculated using the column of “Reporter intensity corrected” in Protein groups.txt, which is one of the output files of MaxQunat, where for each label of TMT 10-plex, the sum of the intensity was divided by the sum of the intensity of the control sample and multiplied by 1×10¹¹using pipeline pilot (BIOVIA).

(1-3) Selection of Biomarker Candidate Molecules

The LC-MS result data were analyzed for each data set of TMT1, 2, and 3 by statistical programming environment R and Microsoft Excel. From the relative expression level of each protein in each of the samples (three healthy individuals and three endometriosis patients), 58 proteins for which altered expression was found in endometriosis patients were selected according to the following criteria for each data set (Table 3A and Table 3B).

$- \frac{\begin{matrix} The average value of the relative expression level \\ of the protein in three patients with endometriosis \end{matrix}}{\begin{matrix} The average value of the relative expression \\ level of the protein in three healthy individuals \end{matrix}} ≧ 2 or ≦ 0.5$

- The relative expression level of the protein in three healthy individuals and three endometriosis patients was p<0.05 in the Welch t-test.

TABLE 3A

Relative expression level of protein:

Average value of 3 patients/

Average value of 3 healthy individuals

Data set
Data set
Data set

UniProt ID
Protein name
Gene name
TMT1
TMT2
TMT3

P02042
Hemoglobin subunit delta
HBD
4.848
4.12
4.27

P68871
Hemoglobin subunit beta;
HBB
3.662
4.58
4.153

LVV-hemorphin-7; Spinorphin

P55072
Transitional endoplasmic reticulum ATPase
VCP
2.915
3
2.306

P50395; P50395-2
Rab GDP dissociation inhibitor beta
GDI2
2.06
2.19
1.784

O43852-9; O43852-5; O43852-2;
Calumenin
CALU
6.081
5.24
4.619

O43852; O43852-4; O43852-3;

O43852-12; O43852-13; O43852-14;

O43852-7; O43852-15; O43852-11;

O43852-8; O43852-10; O43852-6

P04040
Catalase
CAT
3.085
3.36
3.258

Q06830; Q13162
Peroxiredoxin-1 ; Peroxiredoxin-4
PRDX1;
3.006
2.87
3.052

PRDX4

P69905
Hemoglobin subunit alpha
HBA1
4.877
5.88
5.135

Q13404-8; Q15819; Q13404;
Ubiquitin-conjugating enzyme E2
UBE2V1;
3.279
2.76
2.538

Q13404-7; Q13404-2; Q13404-1
variant 1; Ubiquitin-conjugating
UBE2V2

enzyme E2 variant 2

P06703
Protein S100-A6
S100A6
4.041
4.37
4.052

P60174-1; P60174; P60174-4
Triosephosphate isomerase
TPI1
2.433
2.71
2.574

P62937; P62937-2
Peptidyl-prolyl cis-trans
PPIA
2.957
2.84
2.631

isomerase A; Peptidyl-prolyl

cis-trans isomerase A, N-terminally

processed

P49746-2; P49746
Thrombospondin-3
THBS3
3.863
1.19
1.208

P09486
SPARC
SPARC
4.177
5.05
4.681

Q14766-3; Q14766-2; Q14766-5;
Latent-transforming growth factor
LTBP1
6.967
2.71
6.079

Q14766; Q14766-4
beta-binding protein 1

P01137
Transforming growth factor beta-1;
TGFB1
4.8
5.77
2.667

Latency-associated peptide

P61088; Q5JXB2
Ubiquitin-conjugating enzyme
UBE2N;
2.742
2.92
3.356

E2 N; Putative ubiquitin-conjugating
UBE2NL

enzyme E2 N-like

P00558-2; P00558
Phosphoglycerate kinase 1
PGK1
2.278
2.02
2.685

P07996; P07996-2
Thrombospondin-1
THBS1
8.012
8.27
6.402

P02776
Platelet factor 4; Platelet factor 4,
PF4
6.872
7.55
7.134

short form

P05067-7; P05067-11 ; P05067-8;
Amyloid beta A4 protein; N-APP;
APP
5.951
3.84
5.453

P05067-9; P05067; P05067-10;
Soluble APP-alpha; Soluble

P05067-3; P05067-4; P05067-5;
APP-beta; C99; Beta-amyloid

P05067-6
protein 42; Beta-amyloid protein

40; C83; P3(42); P3(40); C80;

Gamm a-secretase C-terminal

fragment 59; Gamma-secretase

C-terminal fragment 57;

Gamma-secretase C-terminal

fragment 50; C31

P00568
Adenylate kinase isoenzyme 1
AK1
2.762
3.62
2.942

P00918
Carbonic anhydrase 2
CA2
2.721
2.78
3.387

P32119
Peroxiredoxin-2
PRDX2
3.697
3.66
3.939

P16070-18; P16070-12; P16070-14;
CD44 antigen
CD44
2.047
1.83
1.814

P16070-13; P16070-11; P16070-10;

P16070-16; P16070-8; P16070-17;

P16070-6; P16070-4; P16070-3;

P16070-7; P16070-5; P16070;

P16070-15; P16070-9

Q13201
Multimerin-1 ; Platelet glycoprotein
MMRN1
4.569
4.27
3.898

la*; 155 kDa platelet multimerin

P27348
14-3-3 protein theta
YWHAQ
1.983
2.65
1.942

O00592-2; O00592
Podocalyxin
PODXL
1.953
2.03
2.009

P10599-2; P10599
Thioredoxin
TXN
2.744
1.94
2.597

P00915
Carbonic anhydrase 1
CA1
3.741
3.62
4.046

P62158
Calmodulin
CALM1
2.102
3.05
2.229

P58546
Myotrophin
MTPN
1.836
2.09
1.787

P62258; P62258-2
14-3-3 protein epsilon
YWHAE
2.002
1.16
1.448

O94919
Endonuclease domain-containing
ENDOD1
3.075
2.53
3.181

1 protein

P37837
Transaldolase
TALDO1
2.995
1.02
0.806

P07738
Bisphosphoglycerate mutase
BPGM
2.761
3.14
2.939

P30043
Flavin reductase (NADPH)
BLVRB
2.985
3.09
2.68

P02775
Platelet basic protein; Connective
PPBP
12.41
9.69
11.867

tissue-activating peptide III; TC-2;

Connective tissue-activating

peptide III(1-81); Beta-thromboglobulin;

Neutrophil-activating peptide 2(74);

Neutrophil-activating peptide 2(73);

Neutrophil-activating peptide 2;

TC-1; Neutrophil-activating peptide

2(1-66); Neutrophil-activating

peptide 2(1-63)

P15311
Ezrin
EZR
2.116
2.1
2.294

P0DJI8; P0DJI9-2
Serum amyloid A-1 protein;
SAA1; SAA2
3.374
4.17
3.645

Amyloid protein A; Serum amyloid

protein A(2-104); Serum amyloid protein

A(3-104); Serum amyloid protein

A(2-103); Serum amyloid protein

A(2-102); Serum amyloid protein

A(4-101); Serum amyloid A-2 protein

P10124
Serglycin
SRGN
3.41
5.68
4.664

P13798
Acylamino-acid-releasing enzyme
APEH
4.068
4.14
4.786

P23528
Cofilin-1
CFL1
2.704
2.79
2.566

P30041
Peroxiredoxin-6
PRDX6
2.111
2.01
1.81

Q8WUM4; Q8WUM4-2
Programmed cell death
PDCD6IP
2.152
2.36
2.291

6-interacting protein

P05090
Apolipoprotein D
APOD
2.213
2.51
2.381

Q99497
Protein deglycase DJ-1
PARK7
3.668
2.22
2.939

P35579; P35579-2
Myosin-9
MYH9
2.97
2.8
2.504

Q13228; Q13228-4; Q13228-2;
Selenium-binding protein 1
SELENBP1
2.032
2.33
1.843

Q13228-3

P13716; P13716-2
Delta-aminolevulinic acid
ALAD
1.983
2.28
2.524

dehydratase

Q9ULI3-2; Q9ULI3
Protein HEG homolog 1
HEG1
2.16
1.95
2.819

P07384
Calpain-1 catalytic subunit
CAPN1
1.752
0.97
2.358

P18065
Insulin-like growth factor-binding
IGFBP2
2.507
2.87
2.833

protein 2

P04083
Annexin A1
ANXA1
2.684
8.03
3.645

P0DMV8-2; P0DMV9; P0DMV8
Heat shock 70 kDa protein 1A;
HSPA1A; HSPA 1B
2.188
2.3
2.203

Heat shock 70 kDa protein 1B

P07911-3; P07911; P07911-5;
Uromodulin; Uromodulin, secreted form
UMOD
1.565
1.85
2.121

P07911-4

P15924; P15924-2; P15924-3
Desmoplakin
DSP
1.636
3.38
1.968

TABLE 3B

Relative expression level of protein:

Average value of 3 patients/

Average value of 3 healthy individuals

Data set
Data set
Data set

UniProt ID
Protein name
Gene name
TMT1
TMT2
TMT3

Q9C0H2-3
Protein tweety homolog 3
TTYH3
0.511
0.619
0.5

The selected proteins are promising as biomarkers for diagnosing endometriosis. FIGS. 1-1 to 1-10 show the graphs of relative expression levels of representative proteins among them.

(Example 2) Search for Biomarkers by Aptamer-Binding Protein Analysis (SOMAscan®) Using Plasma of Healthy Human Individuals and Patients with Endometriosis
(2-1) Obtainment of Human Plasma Samples

Plasma from healthy individuals and endometriosis patients was purchased from Proteogenex.

(2-2) SOMAscan® Analysis of Plasma Proteins

Low-expressed proteins in plasma samples (three healthy individuals and three endometriosis patients) were analyzed by SOMAscan (registered trademark, Somalogic). Specifically, aptamers that each specifically binds to 1310 proteins were prepared, mixed with plasma, and allowed to bind to proteins in the plasma. The proteins bound to the aptamers were then biotinylated, and the aptamer-protein complexes were purified with streptavidin beads. Next, the aptamers were eluted and detected by a microarray, thereby the expression levels of the proteins bound to the aptamers in the plasma were analyzed.

(2-3) Selection of Biomarker Candidates

The aptamer signal data of SOMAscan® were analyzed by statistical programming environment R and Microsoft Excel. According to the following criterion, 200 proteins for which altered expression was found in endometriosis patients were selected (Table 4A and Table 4B).

$- \frac{\begin{matrix} The average value of the aptamer signal of the target \\ protein in three patients with endometriosis \end{matrix}}{\begin{matrix} The average value of the aptamer signal of the target \\ protein in three healthy individuals \end{matrix}} ≧ 2 or ≦ 0.5$

In the above calculation, proteins with a maximum aptamer signal value of less than 1000 were considered to be below the detection limit and excluded from analysis.

TABLE 4A

Aptamer signal:

Entrez
Entrez Gene
Average value of 3 patients/

Protein name
UniProt ID
Gene ID
Symbol
Average value of 3 healthy individuals

Pulmonary surfactant-associated
P35247
6441
SFTPD
12.382

protein D

Histone H1.2
P16403
3006
HIST1H1C
11.014

Sialic acid-binding Ig-like lectin 9
Q9Y336
27180
SIGLEC9
9.473

Heterogeneous nuclear
P22626
3181
HNRNPA2B1
7.516

ribonucleoproteins A2/B1

Hexokinase-2
P52789
3099
HK2
6.671

High mobility group protein B1
P09429
3146
HMGB1
5.584

Thrombospondin-1
P07996
7057
THBS1
5.365

3-hydroxy-3-methylglutaryl-
P04035
3156
HMGCR
4.718

coenzyme A reductase

Platelet factor 4
P02776
5196
PF4
4.686

Protein S100-A9
P06702
6280
S100A9
4.681

40S ribosomal protein S3a
P61247
6189
RPS3A
4.5

Annexin A6
P08133
309
ANXA6
4.484

Inosine-5′-monophosphate
P20839
3614
IMPDH1
4.417

dehydrogenase 1

Tyrosine-protein kinase Fgr
P09769
2268
FGR
4.238

Serine/threonine-protein kinase 17B
O94768
9262
STK17B
3.957

Neutrophil-activating peptide 2
P02775
5473
PPBP
3.826

Estradiol 17-beta-dehydrogenase 1
P14061
3292
HSD17B1
3.787

Connective tissue-activating
P02775
5473
PPBP
3.746

peptide III

Prostaglandin G/H synthase 2
P35354
5743
PTGS2
3.686

Amyloid beta A4 protein
P05067
351
APP
3.667

GTP-binding nuclear protein Ran
P62826
5901
RAN
3.605

NAD-dependent protein
Q8IXJ6
22933
SIRT2
3.54

deacetylase sirtuin-2

Protein S100-A12
P80511
6283
S100A12
3.498

Plasminogen activator inhibitor 1
P05121
5054
SERPINE1
3.367

SUMO-conjugating enzyme UBC9
P63279
7329
UBE2I
3.336

Bactericidal permeability-increasing
P17213
671
BPI
3.321

protein

6-phosphogluconate
P52209
5226
PGD
3.204

dehydrogenase, decarboxylating

Platelet-derived growth factor
P01127
5155
PDGFB
3.158

subunit B

Tyrosine-protein phosphatase
P29350
5777
PTPN6
3.117

non-receptor type 6

Metalloproteinase inhibitor 3
P35625
7078
TIMP3
3.113

NudC domain-containing protein 3
Q8IVD9
23386
NUDCD3
3.112

SPARC
P09486
6678
SPARC
3.046

Protein 4.1
P11171
2035
EPB41
2.967

Sex hormone-binding globulin
P04278
6462
SHBG
2.905

Death-associated protein kinase 2
Q9UIK4
23604
DAPK2
2.834

Hepatoma-derived growth
Q7Z4V5
84717
HDGFRP2
2.777

factor-related protein 2

Proto-oncogene vav
P15498
7409
VAV1
2.723

Serine/threonine-protein kinase
P11309
5292
PIM1
2.694

pim-1

Heterogeneous nuclear
Q99729
3182
HNRNPAB
2.685

ribonucleoprotein A/B

Proliferation-associated
Q9UQ80
5036
PA2G4
2.654

protein 2G4

Extracellular superoxide
P08294
6649
SOD3
2.654

dismutase [Cu-Zn]

Angiopoietin-1
Q15389
284
ANGPT1
2.643

Acid sphingomyelinase-like
Q92484
10924
SMPDL3A
2.631

phosphodiesterase 3a

Peroxiredoxin-6
P30041
9588
PRDX6
2.624

AMP Kinase (alpha1beta1gamma1)
Q13131
5562
PRKAA1
2.577

Q9Y478
5564
PRKAB1

P54619
5571
PRKAG1

Serum amyloid A-1 protein
P0DJI8
6288
SAA1
2.556

Insulin-like growth factor-binding
P18065
3485
IGFBP2
2.52

protein 2

Histone H2B type 2-E
Q16778
8349
HIST2H2BE
2.491

Fibroblast growth factor 5
P12034
2250
FGF5
2.491

Non-histone chromosomal protein
P05114
3150
HMGN1
2.467

HMG-14

Calcium/calmodulin-dependent
Q13557
817
CAMK2D
2.464

protein kinase type II subunit delta

Interstitial collagenase
P03956
4312
MMP1
2.454

ICOS ligand
O75144
23308
ICOSLG
2.444

Calcium/calmodulin-dependent
Q13554
816
CAMK2B
2.425

protein kinase type II subunit beta

Inosine-5′-monophosphate dehydrogenase 2
P12268
3615
IMPDH2
2.398

Dickkopf-related protein 4
Q9UBT3
27121
DKK4
2.375

Glia-derived nexin
P07093
5270
SERPINE2
2.365

14-3-3 protein beta/alpha
P31946
7529
YWHAB
2.357

Macrophage-capping protein
P40121
822
CAPG
2.352

ADP-ribosyl cyclase/cyclic
P28907
952
CD38
2.349

ADP-ribose hydrolase 1

SH2 domain-containing protein 1A
O60880
4068
SH2D1A
2.324

Vacuolar protein sorting-associated
Q9NP79
51534
VTA1
2.319

protein VTA1 homolog

Interleukin-1 beta
P01584
3553
IL1B
2.312

Chloride intracellular channel
O00299
1192
CLIC1
2.306

protein 1

MAP kinase-activated protein
Q16644
7867
MAPKAPK3
2.298

kinase 3

Mitogen-activated protein kinase 1
P28482
5594
MAPK1
2.295

Choline/ethanolamine kinase
Q9Y259
1120
CHKB
2.293

Creatine kinase B-type
P12277
1152
CKB
2.287

DNA topoisomerase 1
P11387
7150
TOP1
2.286

C-C motif chemokine 5
P13501
6352
CCL5
2.28

C3a anaphylatoxin
P01024
718
C3
2.276

Copine-1
Q99829
8904
CPNE1
2.274

Chymase
P23946
1215
CMA1
2.27

Mitogen-activated protein kinase
O43318
6885
MAP3K7
2.27

kinase kinase 7: TGF-beta-activated
Q15750
10454
TAB1

kinase 1 and MAP3K7-binding

protein 1 fusion

Dickkopf-related protein 1
O94907
22943
DKK1
2.266

Ephrin type-B receptor 4
P54760
2050
EPHB4
2.261

40S ribosomal protein S7
P62081
6201
RPS7
2.253

Intercellular adhesion molecule 1
P05362
3383
ICAM1
2.248

Ubiquitin + 1, truncated mutation
P62979
6233
RPS27A
2.222

for UbB

Ubiquitin-conjugating enzyme E2 N
P61088
7334
UBE2N
2.219

Moesin
P26038
4478
MSN
2.201

Small ubiquitin-related modifier 3
P55854
6613
SUMO3
2.196

G2/mitotic-specific cyclin-B1
P14635
891
CCNB1
2.19

ATP-dependent RNA helicase
Q9UMR2
11269
DDX19B
2.178

DDX19B

Tyrosine-protein kinase CSK
P41240
1445
CSK
2.17

Alpha-1-antitrypsin
P01009
5265
SERPINA1
2.162

Adenylate kinase isoenzyme 1
P00568
203
AK1
2.145

Mothers against decapentaplegic
P84022
4088
SMAD3
2.137

homolog 3

Brain-derived neurotrophic factor
P23560
627
BDNF
2.136

Atrial natriuretic factor
P01160
4878
NPPA
2.131

Annexin A1
P04083
301
ANXA1
2.13

Signal transducer and activator of
P42224
6772
STAT1
2.12

transcription 1-alpha/beta

Angiopoietin-related protein 4
Q9BY76
51129
ANGPTL4
2.104

Cadherin-12
P55289
1010
CDH12
2.098

Stress-induced-phosphoprotein 1
P31948
10963
STIP1
2.096

Glycylpeptide
P30419
4836
NMT1
2.091

N-tetradecanoyltransferase 1

Peroxiredoxin-1
Q06830
5052
PRDX1
2.077

Oxidized low-density lipoprotein
P78380
4973
OLR1
2.075

receptor 1

VPS10 domain-containing receptor
Q96PQ0
57537
SORCS2
2.059

SorCS2

FACT complex subunit SSRP1
Q08945
6749
SSRP1
2.049

Carbonic anhydrase 1
P00915
759
CA1
2.035

Mitogen-activated protein kinase 11
Q15759
5600
MAPK11
2.034

Triosephosphate isomerase
P60174
7167
TPI1
2.029

Neurexophilin-1
P58417
30010
NXPH1
2.018

Platelet-derived growth factor
P04085
5154
PDGFA
2.017

subunit A

Interleukin-22 receptor subunit
Q8N6P7
58985
IL22RA1
2.004

alpha-1

TABLE 4B

Aptamer signal:

Average value of

3 patients/Average

Entrez
Entrez
value of 3 healthy

Protein name
UniProt ID
Gene ID
Gene Symbol
individuals

Cystatin-SN
P01037
1469
CST1
0.499

Interleukin-22
Q9GZX6
50616
IL22
0.497

Tyrosine-protein kinase Fyn
P06241
2534
FYN
0.497

Biglycan
P21810
633
BGN
0.495

Sonic hedgehog protein
Q15465
6469
SHH
0.492

Cathepsin L2
O60911
1515
CTSV
0.49

Interferon alpha/beta receptor 1
P17181
3454
IFNAR1
0.486

Ectodysplasin-A, secreted form
Q92838
1896
EDA
0.485

Dynein light chain 1, cytoplasmic
P63167
8655
DYNLL1
0.483

Cytochrome c
P99999
54205
CYCS
0.482

Lactoperoxidase
P22079
4025
LPO
0.48

Tumor necrosis factor receptor
Q969Z4
84957
RELT
0.48

superfamily member 19L

Growth factor receptor-bound protein 2
P62993
2885
GRB2
0.467

Ectonucleotide pyrophosphatase/
Q6UWV6
339221
ENPP7
0.466

phosphodiesterase family member 7

Glucagon
P01275
2641
GCG
0.466

Eukaryotic translation initiation factor 4
P78344
1982
EIF4G2
0.465

gamma 2

C-X-C motif chemokine 10
P02778
3627
CXCL10
0.457

Platelet-derived growth factor receptor
P09619
5159
PDGFRB
0.453

beta

von Willebrand factor
P04275
7450
VWF
0.452

Iduronate 2-sulfatase
P22304
3423
IDS
0.443

cGMP-specific 3′,5′-cyclic
O76074
8654
PDE5A
0.443

phosphodiesterase

Small glutamine-rich tetratricopeptide
O43765
6449
SGTA
0.441

repeat-containing protein alpha

Interleukin-25
Q9H293
64806
IL25
0.433

MHC class I polypeptide-related
Q29980
4277
MICB
0.433

sequence B

C-C motif chemokine 7
P80098
6354
CCL7
0.429

Lactadherin
Q08431
4240
MFGE8
0.427

Macrophage metalloelastase
P39900
4321
MMP12
0.425

Ubiquitin-like protein ISG15
P05161
9636
ISG15
0.423

Fatty acid-binding protein, liver
P07148
2168
FABP1
0.418

Properdin
P27918
5199
CFP
0.417

Stromelysin-2
P09238
4319
MMP10
0.415

Protein FAM107B
Q9H098
83641
FAM107B
0.413

Myosin-binding protein C, slow-type
Q00872
4604
MYBPC1
0.413

Serine/threonine-protein kinase Chk2
O96017
11200
CHEK2
0.41

Xaa-Pro aminopeptidase 1
Q9NQW7
7511
XPNPEP1
0.395

NT-3 growth factor receptor
Q16288
4916
NTRK3
0.394

Interleukin-5 receptor subunit alpha
Q01344
3568
IL5RA
0.392

Ephrin type-A receptor 2
P29317
1969
EPHA2
0.391

Peptidyl-prolyl cis-trans isomerase F,
P30405
10105
PPIF
0.387

mitochondrial

B-cell lymphoma 6 protein
P41182
604
BCL6
0.384

Cell adhesion molecule 1
Q9BY67
23705
CADM1
0.384

Kinesin-like protein KIF23
Q02241
9493
KIF23
0.383

Netrin receptor UNC5D
Q6UXZ4
137970
UNC5D
0.381

Lymphocyte activation gene 3 protein
P18627
3902
LAG3
0.377

Serine protease HTRA2, mitochondrial
O43464
27429
HTRA2
0.376

Interleukin-22 receptor subunit alpha-2
Q969J5
116379
IL22RA2
0.375

Caspase-3
P42574
836
CASP3
0.374

Platelet-derived growth factor receptor
P16234
5156
PDGFRA
0.372

alpha

Interleukin-20
Q9NYY1
50604
IL20
0.368

Ferritin
P02794 P02792
2495 2512
FTH1 FTL
0.367

Ephrin-A5
P52803
1946
EFNA5
0.361

Amphoterin-induced protein 2
Q86SJ2
347902
AMIGO2
0.353

Interleukin-2
P60568
3558
IL2
0.351

Phospholipase A2
P04054
5319
PLA2G1B
0.346

Protein kinase C alpha type
P17252
5578
PRKCA
0.345

Tumor necrosis factor ligand
P32971
944
TNFSF8
0.343

superfamily member 8

Endoglin
P17813
2022
ENG
0.342

Gamma-enolase
P09104
2026
ENO2
0.332

C-X-C motif chemokine 9
Q07325
4283
CXCL9
0.329

beta-nerve growth factor
P01138
4803
NGF
0.327

Hepcidin
P81172
57817
HAMP
0.323

Inorganic pyrophosphatase
Q15181
5464
PPA1
0.319

Kallikrein-8
O60259
11202
KLK8
0.314

Semaphorin-6B
Q9H3T3
10501
SEMA6B
0.312

Adapter molecule crk
P46108
1398
CRK
0.307

Phosphoglycerate mutase 1
P18669
5223
PGAM1
0.298

Proprotein convertase subtilisin/
Q16549
9159
PCSK7
0.298

kexin type 7

Pancreatic hormone
P01298
5539
PPY
0.297

Interleukin-1 receptor type 1
P14778
3554
IL1R1
0.296

C-X-C motif chemokine 11
O14625
6373
CXCL11
0.293

Secreted frizzled-related protein 1
Q8N474
6422
SFRP1
0.288

Inhibin beta A chain
P08476
3624
INHBA
0.284

Immunoglobulin D
P01880
3495 50802 3535
IGHD IGK@
0.277

IGL@

Complement C3b
P01024
718
C3
0.272

Teratocarcinoma-derived growth
P13385
6997
TDGF1
0.27

factor 1

Brother of CDO
Q9BWV1
91653
BOC
0.269

CD109 antigen
Q6YHK3
135228
CD109
0.26

Aminoacylase-1
Q03154
95
ACY1
0.259

60 kDa heat shock protein,
P10809
3329
HSPD1
0.255

mitochondrial

Importin subunit beta-1
Q14974
3837
KPNB1
0.253

C-reactive protein
P02741
1401
CRP
0.251

Ephrin type-A receptor 1
P21709
2041
EPHA1
0.242

Semaphorin-6A
Q9H2E6
57556
SEMA6A
0.24

SLIT and NTRK-like protein 5
O94991
26050
SLITRK5
0.233

Leucine-rich repeat transmembrane
Q9NZU0
23767
FLRT3
0.217

protein FLRT3

dCTP pyrophosphatase 1
Q9H773
79077
DCTPP1
0.21

Osteopontin
P10451
6696
SPP1
0.206

Formimidoyltransferase-cyclodeaminase
O95954
10841
FTCD
0.191

Ephrin-B2
P52799
1948
EFNB2
0.175

T-lymphocyte surface antigen Ly-9
Q9HBG7
4063
LY9
0.17

Sialic acid-binding Ig-like lectin 14
Q08ET2
100049587
SIGLEC14
0.157

N-acylethanolamine-hydrolyzing acid
Q02083
27163
NAAA
0.146

amidase

Endoplasmic reticulum aminopeptidase 1
Q9NZ08
51752
ERAP1
0.118

Low affinity immunoglobulin gamma Fc
P12318
2212
FCGR2A
0.053

region receptor II-a

The selected proteins are promising as biomarkers for diagnosing endometriosis. FIGS. 2-1 to 2-10 show the graphs of expression levels of representative proteins among them.

(Example 3) Search for Biomarkers by Extracellular Matrix Degradation Product Analysis Using Plasma of Healthy Human Individuals and Patients with Endometriosis
(3-1) Obtainment of Human Plasma Samples

Plasma from healthy individuals and endometriosis patients was purchased from Proteogenex.

(3-2) Extracellular Matrix Measurement of Plasma Proteins

Plasma samples (three healthy individuals and three endometriosis patients) were analyzed by the ELISA system with an antibody specific to the cleavage site of the extracellular matrix, and the total amount of extracellular matrix and the degradation/synthesis state of extracellular matrix in the samples were analyzed (Nordic Bioscience). Specifically, with antibodies against the cleavage site contained in degradation products when type III, IV, V, or VI collagen, decorin, or nidogen was degraded, the expression level of each degradation product was analyzed. The result showed that the type V collagen MMP degradation product (C5M) was remarkably increased in two of the three patients (the C5M concentration was 10.2 ng/ml and 11.8 ng/ml in the plasma of the two patients with increased C5M), while the plasma C5M concentration was below the detection limit in all healthy individuals. Thus, it was suggested that C5M is promising as a biomarker for endometriosis. FIG. 3 shows the graph of the plasma concentration of the type V collagen MMP degradation product (C5M).

(Example 4) Search for Biomarkers by Luminex xMAP™ Technology Using Plasma of Healthy Human Individuals and Patients with Endometriosis
(4-1) Obtainment of Human Plasma Samples

Plasma of healthy individuals (five plasma samples at secretory stage and five plasma samples at proliferative stage) was purchased from Proteogenex. As plasma of endometriosis patients, a total of 111 plasma samples were obtained from 37 endometriosis patients, each before surgery, at 3 days after surgery, and at 1 month after surgery.

(4-2) Analysis of Plasma Proteins by Luminex xMAP™ Technology

Proteins in plasma samples were analyzed by Luminex xMAP™ (a trademark of Luminex). The Luminex xMAP™ technology is a technology of staining macrobeads with two fluorescent dyes combined at various concentrations and immobilizing onto each bead a substance that binds to each analysis target, whereby multiple items can be simultaneously analyzed with a small amount of samples using the content of the fluorescent dyes as an identification code.

In this Example, 152 proteins were selected and analyzed using protein measurement panels. Specifically, an antibody that binds to a target protein was immobilized on beads for each panel, a plasma sample was reacted with the antibody on the beads, a reporter antibody (labeled with a fluorescent dye) against the target protein was further reacted, and the expression level of the target protein was analyzed by measuring the fluorescence intensity with two types of lasers by flow cytometry technology (using the Luminex100 device).

(4-3) Selection of Biomarker Candidates

The plasma concentrations of the obtained various proteins were analyzed by Microsoft Excel. According to the following criterion, 24 proteins for which altered expression was found in endometriosis patients were selected (Table 7A and Table 7B).

$\frac{\begin{matrix} The average value of the plasma concentration of the protein \\ in 37 patients with endometriosis before surgery \end{matrix}}{\begin{matrix} \begin{matrix} The average value of the plasma concentration of the protein \\ in 10 samples from 5 healthy individuals at \end{matrix} \\ secretory and proliferative stages \end{matrix}} ≧ 2 or ≦ 0.5$

Of the 24 proteins selected, 12 proteins showed the highest value in endometriosis patients at the time of surgery and a decreased value one month after surgery, and also showed the lowest value in healthy individuals.

TABLE 7A

Patients with endometriosis

(before surgery) > Patients

with endometriosis (1

Abundance
month after surgery) >

UniProt ID
Protein name
Gene name
ratio
Healthy individuals

Q15389
Angiopoietin-1 (ANG-1)
ANGPT1
2.96
∘

P23560
Brain-Derived Neurotrophic Factor (BDNF)
BDNF
4.03
∘

O94907
Dickkopf-related protein 1 (DKK-1)
DKK1
2.11
∘

P80511
S100-A12
S100A12
4.67
∘

P42830
Epithelial-Derived Neutrophil-Activating
CXCL5
2.52
∘

Protein 78 (ENA-78)

P02794
Ferritin (FRTN)
FTH1
2.99

P09341
Growth-Regulated alpha protein (GRO-alpha)
CXCL1
2.23
∘

P01137
Latency-Associated Peptide of Transforming
TGFB1
2.55
∘

Growth Factor beta 1 (LAPTGF-b1)

Q8WXL0
Luteinizing Hormone (LH)
LHB
2.26
∘

G3CBL7
MHC class I chain-related protein A (MICA)
MICA
2.29

P02775
Platelet basic protein
PPBP
2.92
∘

P01127
Platelet-Derived Growth Factor BB (PDGF-BB)
PDGFB
4.25
∘

P01236
Prolactin (PRL)
PRL
3.76
∘

P0DJI8; P0DJI9-2
Serum amyloid A-1 protein; Amyloid protein
SAA1; SAA2
3.01

A; Serum amyloid protein A(2-104); Serum

amyloid protein A(3-104); Serum amyloid

protein A(2-103); Serum amyloid protein

A(2-102); Serum amyloid protein A(4-

101); Serum amyloid A-2 protein

P13501
T-Cell-Specific Protein RANTES (RANTES)
CCL5
2.18
∘

TABLE 7B

UniProt ID
Protein name
Gene name
Abundance ratio

P10645
Chromogranin-A (CgA)
CHGA
0.38

Q9GZV9
Fibroblast growth factor 23 (FGF-23)
FGF23
0.22

P01266
Thyroglobulin (TG)
TG
0.47

(Example 5) Search for Biomarkers by LC-MS Analysis Using Plasma of Healthy Human Individuals and Patients with Endometriosis
(5-1) Obtainment of Human Plasma Samples

Plasma of healthy individuals (five plasma samples at secretory stage and five plasma samples at proliferative stage) was purchased from Proteogenex. As plasma of endometriosis patients, a total of 125 plasma samples were obtained from 39 endometriosis patients, each before surgery, at 3 days after surgery, and at 1 month after surgery.

(5-2) LC-MS Analysis of Plasma Proteins

With High Select™ Top14 Abundant Protein Depletion Mini Spin Columns (Thermo), highly abundant proteins in each plasma sample were bound to the column and removed by the method recommended by the manufacturer. The proteins in the flow-through fraction that did not bind to the column were precipitated by the methanol/chloroform method and dissolved in 8 M urea/400 mM ammonium bicarbonate solution. The dissolved proteins were reduced and alkylated, and then digested into peptides with lysyl endopeptidase and trypsin. The peptide solution was demineralized with Monospin C18 (GL Science) by the method recommended by the manufacturer.

The peptides were labeled with TMT10plex by the method recommended by the manufacturer. With AssayMAP reversed phase (RP-S) cartridges (Agilent technologies), the labeled peptides were fractionated by the High pH Reversed-Phase method with triethylamine Each fraction was analyzed by the LC-MS analysis system in which Orbitrap Fusion Lumos (ThermoFisher scientific) was coupled to the nano-LC system (EASY-nLC™ 1200 system, ThermoFisher Scientific). The analysis samples were peptides obtained from the plasma of each of five healthy individuals (each at secretory stage and proliferative stage; a total of 10 samples) and 39 endometriosis patients (before surgery, at 3 days after surgery, and at 1 month after surgery; a total of 125 samples). The sample prepared by mixing an equal amount of seven samples from healthy individuals and seven samples from endometriosis patients, which were purchased for correction between measurements, was used as a control in each analysis, and 10 samples were used for each analysis. Each sample was analyzed three times, and they were integrated during analysis.

The proteins in the samples were identified from the raw data of the LC-MS analysis by Thermo Scientific™ Proteome Discoverer™, and their relative expression levels were analyzed. The database search for protein identification was performed with 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/+42.011 Da (N-Terminus)
FDR: Target FDR (Strict)/0.01, Target FDR (Relaxed)/0.05

The relative expression levels of the target proteins in the plasma samples from endometriosis patients before surgery relative to the target proteins in the plasma samples from healthy individuals were calculated by the analysis software (Proteome Discoverer).

(5-3) Selection of Biomarker Candidate Molecules

The LC-MS result data were analyzed by Proteome Discoverer. From the relative expression level of each protein in the samples (10 samples from five healthy individuals at secretory stage and proliferative stage and samples from 39 endometriosis patients before surgery), 27 proteins for which altered expression was found in endometriosis patients were selected according to the following criteria (Table 8A and Table 8B).

- For the abundance ratio, P-Value≤0.5 in ANOVA and tukey HSD post hoc; and
- Abundance ratios≤0.6250 or ≥1.6000

$Abundance ratio = \frac{\begin{matrix} The median value of the relative expression level of the protein \\ in 39 patients with edometriosis before surgery \end{matrix}}{\begin{matrix} The median value of the relative expression level of the protein \\ in 10 samples from 5 healthy individuals at \\ secretory and proliferative stages \end{matrix}}$

Of the 27 proteins selected, 16 proteins showed the highest value in endometriosis patients at the time of surgery and a decreased value one month after surgery, and also showed the lowest value in healthy individuals.

TABLE 8A

Patients with endometriosis

(before surgery) > Patients

with endometriosis (1

Abundance
month after surgery) >

UniProt ID
Protein name
Gene name
ratio
Healthy individuals

P07996
Thrombospondin-1 OS = Homo sapiens
THBS1
3.149
∘

OX = 9606 GN = THBS1 PE = 1 SV = 2

P69905
Hemoglobin subunit alpha OS = Homo sapiens
HBA1
1.717
∘

OX = 9606 GN = HBA1 PE = 1 SV = 2

P32119
Peroxiredoxin-2 OS = Homo sapiens
PRDX2
1.772
∘

OX = 9606 GN = PRDX2 PE = 1 SV = 5

P00918
Carbonic anhydrase 2 OS = Homo sapiens
CA2
1.821
∘

OX = 9606 GN = CA2 PE = 1 SV = 2

P30043
Flavin reductase (NADPH) OS = Homo sapiens
BLVRB
1.787
∘

OX = 9606 GN = BLVRB PE = 1 SV = 3

O76011
Keratin, type I cuticular Ha4 OS = Homo sapiens
KRT34
2.091
∘

OX = 9606 GN = KRT34 PE = 1 SV = 2

P13716
Delta-aminolevulinic acid dehydratase OS =
ALAD
1.867
∘

Homo sapiens OX = 9606 GN = ALAD PE = 1 SV = 1

P00568
Adenylate kinase isoenzyme 1 OS = Homo sapiens
AK1
1.786
∘

OX = 9606 GN = AK1 PE = 1 SV = 3

P07738
Bisphosphoglycerate mutase OS = Homo sapiens
BPGM
1.735
∘

OX = 9606 GN = BPGM PE = 1 SV = 2

P02775
Platelet basic protein OS = Homo sapiens
PPBP
3.628
∘

OX = 9606 GN = PPBP PE = 1 SV = 3

P22392
Nucleoside diphosphate kinase B OS =
NME2
1.779
∘

Homo sapiens OX = 9606 GN = NME2 PE = 1 SV = 1

P10124
Serglycin OS = Homo sapiens
SRGN
3.181
∘

OX = 9606 GN = SRGN PE = 1 SV = 3

A0A0C4DH67
Immunoglobulin kappa variable 1-8 OS =
IGKV1-8
1.615
x

Homo sapiens OX = 9606 GN = IGKV1-8 PE = 3 SV = 1

P63241
Eukaryotic translation initiation factor 5A-1 OS =
EIF5A
1.874
∘

Homo sapiens OX = 9606 GN = EIF5A PE = 1 SV = 2

P02776
Platelet factor 4 OS = Homo sapiens
PF4
3.438
∘

OX = 9606 GN = PF4 PE = 1 SV = 2

A0A075B6S2
Immunoglobulin kappa variable 2D-29 OS =
IGKV2D-29
2.174
x

Homo sapiens OX = 9606 GN = IGKV2D-29 PE = 3 SV = 1

Q9UKU6
Thyrotropin-releasing hormone-degrading ectoenzyme
TRHDE
1.879
∘

OS = Homo sapiens OX = 9606 GN = TRHDE PE = 2 SV = 1

Q8WUM4
Programmed cell death 6-interacting protein OS =
PDCD6IP
1.644
x

Homo sapiens OX = 9606 GN = PDCD6IP PE = 1 SV = 1

P00441
Superoxide dismutase [Cu—Zn] OS = Homo sapiens
SOD1
2.01
∘

OX = 9606 GN = SOD1 PE = 1 SV = 2

TABLE 8B

UniProt ID
Protein name
Gene name
Abundance ratio

P36980
Complement factor H-related protein 2 OS = Homo sapiens
CFHR2
0.443

OX = 9606 GN = CFHR2 PE = 1 SV = 1

P0D0X3
Immunoglobulin delta heavy chain OS = Homo sapiens
N/A
0.518

OX = 9606 PE = 1 SV = 1

P01880
Immunoglobulin heavy constant delta OS = Homo sapiens
IGHD
0.456

OX = 9606 GN = IGHD PE = 1 SV = 3

Q8N6C8
Leukocyte immunoglobulin-like receptor subfamily A member
LILRA3
0.318

3 OS = Homo sapiens OX = 9606 GN = LILRA3 PE = 1 SV = 3

P47929
Galectin-7 OS = Homo sapiens
LGALS7
0.533

OX = 9606 GN = LGALS7 PE = 1 SV = 2

O75339
Cartilage intermediate layer protein 1 OS = Homo sapiens
CILP
0.614

OX = 9606 GN = CILP PE = 1 SV = 4

O15389
Sialic acid-binding Ig-like lectin 5 OS = Homo sapiens
SIGLEC5
0.546

OX = 9606 GN = SIGLEC5 PE = 1 SV = 1

P35247
Pulmonary surfactant-associated protein D OS =
SFTPD
0.605

Homo sapiens OX = 9606 GN = SFTPD PE = 1 SV = 3

INDUSTRIAL APPLICABILITY

The present disclosure proved that by measuring the concentration of a type V collagen MMP degradation product or the abundance of at least one marker selected from the group consisting of the markers shown in Table 1A, the markers shown in Table 2A, the markers shown in Table 1B, the markers shown in Table 2B, the markers shown in Table 5A, and the markers shown in Table 6A, the markers shown in Table 5B, and the markers shown in Table 6B in a sample obtained from a subject, whether or not the subject is affected by endometriosis can be diagnosed. The invention of the present disclosure allows for the diagnosis of endometriosis by non-invasive means and is highly useful in the diagnosis and treatment of the disease.

METHOD FOR DIAGNOSING ENDOMETRIOSIS, DISEASE STATE MONITORING METHOD, AND KIT

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