Diagnostic kit for chronic endometritis

Abstract

We provide a minimally invasive, painless diagnostic technique for chronic endometritis with excellent sensitivity and specificity. [Solution] A kit for diagnosing chronic endometritis, comprising a specific binding substance for one or more proteins, wherein the one or more proteins are selected from the group consisting of IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-12(p40), IL-12(p70), IL-14, IL-15, IL-18, IL-1RA, IL-10, IL-19, IFNγ, IP-10, MCP-1, MDC, MIG, MIP-1β, RANTES, TNFα, TNFβ, sCD40L, G-CSF, M-CSF, FGF-2, FLT-3L, PDGF-AA, PDGF-AB / BB, TGFα, and VEGF-A.

Description

Technical Field

[0001] The present invention relates to a kit for diagnosing chronic endometritis.

Background Art

[0002] Chronic endometritis is a disease accompanied by chronic inflammation of the endometrium. It has no subjective symptoms and can cause infertility and miscarriage. Conventionally, as a method for examining chronic endometritis, histological diagnosis by endometrial biopsy and findings of hyperemia in the uterine cavity by hysteroscopy have been used.

[0003] However, since only the biopsy site can be evaluated in endometrial biopsy, there is a risk of false negatives depending on the location of the lesion. In addition, there are problems of pain and complications associated with the invasive technique of biopsy. In addition, hysteroscopy can only be performed at a medical institution equipped with examination equipment. In addition, there is a large difference in the disease detection rate depending on the skill of the operator, and the sensitivity is about 50%.

[0004] By the way, in recent years, the results of mass spectrometry of endometrium-derived extracellular vesicles from patients with unexplained infertility have been reported (for example, see Non-Patent Document 1). However, no reports have been made on patients with chronic endometritis.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

[0006] The present invention aims to provide a minimally invasive, painless diagnostic technique for chronic endometritis with excellent sensitivity and specificity. [Means for solving the problem]

[0007] The present invention includes the following embodiments. [1] A diagnostic kit for chronic endometritis, comprising a specific binding substance for one or more proteins, wherein the one or more proteins are selected from the group consisting of proteins listed in Table 1 below. [Table 1] [2] The chronic endometritis diagnostic kit according to [1], wherein one or more of the proteins are selected from the group consisting of VEGF-A, TNFα, MIG, MDC, MIP-1β, RANTES, TGFα, FGF-2, IL-1α, IL-4, IL-5, IL-8 and M-CSF. [3] The chronic endometritis diagnostic kit according to [1] or [2], wherein the one or more proteins are a combination of VEGF-A, TNFα, and MIG. [4] A diagnostic kit for chronic endometritis according to [1], used to measure the amount of one or more of the proteins in an intrauterine lavage fluid sample derived from a subject, wherein a higher amount of any one of the one or more proteins compared to a predetermined threshold indicates a high probability that the subject is suffering from chronic endometritis. [5] The kit for diagnosing chronic endometritis according to [4], wherein one or more of the proteins are selected from the group consisting of VEGF-A, TNFα, MIG, MDC, MIP-1β, RANTES, TGFα, FGF-2, IL-1α, IL-4, IL-5, IL-8 and M-CSF. [6] The diagnostic kit for chronic endometritis according to [4] or [5], wherein the one or more proteins are a combination of VEGF-A, TNFα, and MIG. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a minimally invasive, painless diagnostic technique for chronic endometritis with excellent sensitivity and specificity. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the Receiver Operating Characteristic (ROC) curve created in Experimental Example 3. [Modes for carrying out the invention]

[0010] [Diagnostic kit for chronic endometritis] In one embodiment, the present invention provides a diagnostic kit for chronic endometritis, comprising a specific binding substance for one or more proteins, wherein the one or more proteins are selected from the group consisting of the proteins listed in Table 1 above.

[0011] The kit of this embodiment is preferably used to measure the amount of one or more of the aforementioned proteins in an intrauterine lavage fluid sample derived from a subject. A higher amount of any one of the aforementioned proteins compared to a predetermined threshold indicates a high probability that the subject is suffering from chronic endometritis.

[0012] As will be described later in the examples, the kit of this embodiment allows for the diagnosis of chronic endometritis with excellent sensitivity and specificity using intrauterine lavage fluid samples derived from the subject. Therefore, chronic endometritis can be diagnosed minimally invasively, without pain or complications. Furthermore, since the collected intrauterine lavage fluid can be analyzed by contract analysis, it is a test method that can be performed in general clinical practice without requiring special equipment or medical technology. Hereinafter, in this specification, the proteins listed in Table 1 above may be referred to as biomarkers.

[0013] In the kit of this embodiment, it is preferable that the one or more proteins are selected from the group consisting of VEGF-A, TNFα, MIG, MDC, MIP-1β, RANTES, TGFα, FGF-2, IL-1α, IL-4, IL-5, IL-8, and M-CSF.

[0014] As will be described later in the examples, if the amount of any one of these proteins is higher than a predetermined threshold, it indicates that the subject is likely to be suffering from chronic endometritis.

[0015] Here, a predetermined threshold can be empirically set, for example, by accumulating data on the amount of biomarker present in intrauterine lavage fluid samples from subjects suffering from chronic endometritis and the amount of biomarker present in intrauterine lavage fluid samples from subjects not suffering from chronic endometritis.

[0016] In the step of setting a predetermined threshold, it is preferable to determine whether or not a person has chronic endometritis by a method other than measuring the amount of biomarker present in the uterine lavage fluid sample. For example, as described later in the examples, it is possible to determine whether or not a person has chronic endometritis by collecting endometrial tissue from the subject, staining it with anti-CD138 antibody, and detecting plasma cells. For example, if the presence of 5 or more plasma cells is detected in 10 high-magnification fields, chronic endometritis can be diagnosed.

[0017] Alternatively, a predetermined threshold value may be set as follows. A threshold value is set between the abundance of a biomarker in the intrauterine lavage fluid sample of a subject not suffering from chronic endometritis and the abundance of the biomarker in the intrauterine lavage fluid sample of a subject suffering from chronic endometritis. It is preferable to set as the threshold value a value that can accurately distinguish subjects suffering from chronic endometritis. Here, a discriminant formula may be created by a statistical analysis method known in the art, and the threshold value may be calculated from the accumulated measurement values using this formula. Alternatively, using a machine learning method or the like, it may be automatically determined based on the accumulated measurement values whether a subject is suffering from chronic endometritis or not.

[0018] As a more specific threshold value, for example, the cut-off value shown in Table 2 below can be used. When the abundance of any of the biomarkers (cytokines) shown in Table 2 below in the intrauterine lavage fluid sample derived from a subject is higher compared to the cut-off value shown in Table 2 below, it can be determined that the subject is suffering from chronic endometritis.

[0019]

Table 2

[0020] In the kit of the present embodiment, the one or more proteins may be a combination of VEGF-A, TNFα, and MIG. As will be described later in the examples, when the abundance of any of the biomarkers of VEGF-A, TNFα, and MIG in the intrauterine lavage fluid sample derived from a subject is higher compared to a predetermined threshold value, it can be determined that the subject is suffering from chronic endometritis. In particular, the Area under curve (AUC) when using VEGF-A, TNFα, and MIG as biomarkers is 0.9099, indicating a high disease detection power.

[0021] In the kit of this embodiment, examples of specific binding substances include antibodies and aptamers. The antibody may be an antibody fragment. Examples of antibody fragments include F(ab')2, Fab', Fab, Fv, and scFv. Examples of aptamers include nucleic acid aptamers and peptide aptamers.

[0022] In the kit of this embodiment, the specific binding substance is used to measure a biomarker. The method for measuring the biomarker is not particularly limited and includes, for example, immunochromatography, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoblotting, and latex agglutination.

[0023] Immunochromatography is a method for detecting a target substance using antigen-antibody reactions. A sample is dropped onto a sample application area on a strip-shaped developing membrane, and the sample is slowly moved by capillary action while dissolving reagents. For example, a nitrocellulose membrane is used as the developing membrane. Biomarkers in the sample move along the developing membrane, forming complexes with specific binding substances, such as metal colloids, that are prepared in advance on the sample application area. The complexes are trapped on the detection line, causing it to change color, which is then determined by visual inspection or other means.

[0024] When measuring biomarkers by immunochromatography, it is preferable that the system be adjusted so that a band appears on the detection line when the amount of biomarker present exceeds a predetermined threshold.

[0025] The latex agglutination method is a method for detecting an antigen or antibody by immobilizing an antibody or antigen onto latex particles, agglutinating the latex particles in the presence of the antigen or antibody, and observing this agglutination. Examples of latex particles include styrene-based latex such as polystyrene latex, acrylic acid-based latex, various modified latex, colored latex, and fluorescent latex. The average particle size of the latex particles is not particularly limited, but examples include those in which the aggregates produced by the antigen-antibody reaction between a biomarker and a specific binding substance are large enough to be detected by the naked eye or optically. Specifically, for example, it is 100 to 500 nm. [Examples]

[0026] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0027] [Experimental Example 1] (Searching for biomarkers for chronic endometritis 1) Intrauterine lavage fluid and endometrial tissue were collected during the follicular phase from patients attending the infertility treatment outpatient clinic at Juntendo University Hospital and from healthy individuals with no history of infertility.

[0028] The intrauterine lavage solution was collected by inserting a uterine balloon catheter (HysCath®, SB Kawasumi Co., Ltd.) into the uterus through the cervix, slowly injecting 2 mL of distilled water into the uterine cavity with a syringe, and immediately collecting the injected solution with the syringe.

[0029] Furthermore, the collected endometrial tissue was stained with anti-CD138 antibody to detect plasma cells. Chronic endometritis was diagnosed when five or more plasma cells were detected in 10 high-magnification fields. The magnification of the high-magnification fields was 400x.

[0030] Among the infertile patients, 14 had chronic endometritis, and 14 did not. There were 12 healthy individuals. Table 3 below shows the patient characteristics.

[0031] In Table 3, "CE group" represents the group with chronic endometritis (CE), "CE- group" represents the group without chronic endometritis, and "control group" represents the healthy control group. Different letters (a, b) indicate a statistically significant difference. "*" indicates a comparison between the CE group and the CE- group.

[0032] [Table 3]

[0033] The concentrations of 54 cytokines in intrauterine lavage fluid were measured using a multiplex assay in the CE group, CE- group, and control group. Table 4 below shows the 54 cytokines that were measured.

[0034] [Table 4]

[0035] Table 5 below shows the cytokines measured that showed a significant difference in concentration between the CE group and the control group based on the Kruskal-Wallis test. Significant differences were observed in the concentrations of 32 types of cytokines.

[0036] [Table 5]

[0037] [Experimental Example 2] (Searching for biomarkers for chronic endometritis, part 2) In Experimental Example 1, Receiver Operating Characteristic (ROC) curve analysis was performed on 32 cytokines that showed a significant difference in concentration between the CE group and the control group, based on whether or not the patient had chronic endometritis. Area under curve (AUC) was also calculated from the ROC curves. Cutoff values ​​were calculated using the Youden Index. The results, useful biomarkers indicating chronic endometritis (AUC > 0.800), are shown in Table 6 below. Table 6 also shows the cutoff values, sensitivity, and specificity.

[0038] [Table 6]

[0039] [Experimental Example 3] (Searching for biomarkers for chronic endometritis, part 3) In Experimental Example 1, for 32 cytokines that showed a significant difference in concentration between the CE group and the control group, multiple logistic regression analysis was used to investigate combinations of cytokines that could detect whether or not a person had chronic endometritis with high sensitivity and specificity.

[0040] The results revealed that the combination of VEGF-A, TNFα, and MIG can detect whether or not a person has chronic endometritis with high sensitivity and specificity. In particular, when the amount of any one of VEGF-A, TNFα, or MIG was positive (above the threshold), chronic endometritis could be determined with a sensitivity of 92.9% and a specificity of 89.3%.

[0041] VEGF-A, TNFα, and MIG levels were not elevated in infertile patients without chronic endometritis, suggesting they are highly specific markers for chronic endometritis. Figure 1 shows the ROC curves for combinations of VEGF-A, TNFα, and MIG. The AUC was 0.9099, indicating that combining multiple proteins showed higher disease detection power than using a single protein. Therefore, it is crucial to combine multiple biomarkers when diagnosing chronic endometritis. [Industrial applicability]

[0042] According to the present invention, it is possible to provide a minimally invasive, painless diagnostic technique for chronic endometritis with excellent sensitivity and specificity.

Claims

1. A diagnostic kit for chronic endometritis, comprising a specific binding substance for one or more proteins, wherein the one or more proteins are selected from the group consisting of proteins listed in Table 1 below. Table 1

2. The chronic endometritis diagnostic kit according to claim 1, wherein the one or more proteins are selected from the group consisting of VEGF-A, TNFα, MIG, MDC, MIP-1β, RANTES, TGFα, FGF-2, IL-1α, IL-4, IL-5, IL-8, and M-CSF.

3. The chronic endometritis diagnostic kit according to claim 1, wherein the one or more proteins are a combination of VEGF-A, TNFα, and MIG.

4. Used to measure the amount of one or more of the aforementioned proteins in a sample of intrauterine lavage fluid derived from a subject, The chronic endometritis diagnostic kit according to claim 1, wherein the presence of any one of the one or more of the aforementioned proteins being higher than a predetermined threshold indicates a high probability that the subject is suffering from chronic endometritis.

5. The chronic endometritis diagnostic kit according to claim 4, wherein the one or more proteins are selected from the group consisting of VEGF-A, TNFα, MIG, MDC, MIP-1β, RANTES, TGFα, FGF-2, IL-1α, IL-4, IL-5, IL-8, and M-CSF.

6. The chronic endometritis diagnostic kit according to claim 4, wherein the one or more proteins are a combination of VEGF-A, TNFα, and MIG.

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