Method for assessing endometrial immune microenvironment, and use thereof

Abstract

Provided in the present application is a method for assessing the endometrial immune microenvironment. The assessment method assesses the endometrial immune microenvironment on the basis of the aggregation state of immune cells on the endometrium, thereby distinguishing and determining the health status of the endometrium. Compared with the prior art, the present application has the following advantages: the method for assessing the endometrial immune microenvironment and the use thereof provided in the present application can efficiently and accurately assess the endometrial immune microenvironment by means of counting the proportion of immune cell aggregation foci on the endometrium, thereby effectively improving early-stage research and assessment of endometrium-related diseases such as chronic endometritis and pregnancy outcomes.

Description

Assessment methods and applications of the endometrial immune microenvironment Technical Field

[0001] This application relates to the field of reproductive medicine technology, specifically to methods for assessing the immune microenvironment of the endometrium and their applications. Background Technology

[0002] The endometrium is a layer of tissue in the female reproductive organs, located within the uterine cavity, and is a crucial structure for human conception and pregnancy. During the menstrual cycle, the endometrium periodically grows and sheds, participating in embryo implantation after the implantation window opens, supporting placental formation and fetal development after implantation. The immune microenvironment of the endometrium, mainly composed of immune cells, is precisely and complexly regulated, identifying high-quality embryos through highly adaptive regulation and ensuring successful implantation and pregnancy maintenance.

[0003] The immune microenvironment of the endometrium refers to the local "ecological" environment of the endometrial tissue, composed of various cells (especially immune cells), immune factors, and cytokines. Abnormalities in the endometrial immune microenvironment can interfere with the endometrium's ability to readily accept the embryo, hindering the establishment and maintenance of immune homeostasis during pregnancy, and ultimately leading to implantation failure or miscarriage.

[0004] In clinical practice, endometrial immune microenvironment dysregulation is closely related to the occurrence and development of common reproductive disorders such as chronic endometritis, endometrial fibrosis, endometriosis, and intrauterine adhesions. A common characteristic of the endometrial microenvironment considered "undesirable" in clinical practice is the presence of immune disturbances in the endometrium. With a deeper understanding of the endometrial immune microenvironment, it has become possible to identify and quantify these immune disturbances by developing specific targets.

[0005] Lymphocytic aggregates refer to localized clusters of lymphocytes in tissues. These aggregates are typically composed of immune cells such as lymphocytes, plasma cells, and dendritic cells, and are often accompanied by other immune response-related cells and molecules. Lymphocytic aggregates have multiple clinical applications: First, they can serve as markers of immune inflammatory responses, commonly seen in infections, autoimmune diseases, and tumors. Second, lymphocytic aggregates are also associated with the development and progression of diseases such as organ transplant rejection and inflammatory bowel disease. Furthermore, the number and distribution patterns of lymphocytic aggregates are of significant value in pathological diagnosis and prognostic assessment. Therefore, studying the immune cell composition and distribution patterns of lymphocytic aggregates can provide crucial information about disease mechanisms and immune response status, offering a basis for clinical treatment and prevention.

[0006] However, in the field of reproductive medicine technology, there is no standardized procedure and kit (including sample acquisition, detection methods, result interpretation, etc.) for qualitative or quantitative fluorescence staining of lymphocyte aggregation foci in the endometrial microenvironment. There is also a lack of standards, verification and evaluation methods for assessing the inflammatory status of the endometrium by lymphocyte aggregation foci. This greatly limits the clinical assessment and prediction of the endometrium's ability to accept embryos and the success rate of assisted reproduction. Summary of the Invention

[0007] To address the aforementioned technical limitations, this application proposes an assessment method for the endometrial immune microenvironment and its application, which overcomes the deficiencies and defects mentioned in the background art.

[0008] To achieve the above objectives, this application adopts the following technical solution:

[0009] The invention of this application provides a method for assessing the immune microenvironment of the endometrium, wherein the assessment method evaluates the health status of the endometrium by assessing the aggregation status of immune cells.

[0010] Optionally, in the above-mentioned assessment method for the endometrial immune microenvironment, the aggregation state refers to the proportion of immune cell aggregation foci per unit area of ​​the endometrium.

[0011] Optionally, in the above-mentioned method for assessing the endometrial immune microenvironment, the immune cell aggregation focus refers to an immune cell count greater than or equal to 20 and / or an immune cell density greater than or equal to 20 cells / 3000 μm. 2 An aggregate of immune cells; preferably 30 or more and with an immune cell density of 30 or more per 3000 μm. 2 An aggregate of immune cells.

[0012] Optionally, the above-mentioned assessment method for the endometrial immune microenvironment specifically includes:

[0013] S1. Sampling is performed at least at one point on the endometrium (sampling method: scraping around the uterine cavity to ensure multiple sampling points of the endometrium in the uterine cavity); the sampling usually uses a negative pressure tube to take the functional layer of the endometrial surface tissue, avoiding the basal tissue and blood (the sample is cylindrical or strip-shaped).

[0014] S2. Detect and statistically analyze the immune cell aggregation foci in the samples obtained in step S1;

[0015] S3. Calculate the proportion of immune cell aggregation foci in the unit area of ​​the endometrial sample in step S2;

[0016] S4. Assess the endometrial immune microenvironment using the percentage values ​​obtained in step S3.

[0017] Optionally, in the above-described method for assessing the endometrial immune microenvironment, in step S1, at least three sampling sites are selected; the sample volume is selected to be greater than or equal to 8 mm. 3 .

[0018] Optionally, in the above-described method for assessing the immune microenvironment of the endometrium, the detection method for immune cell aggregation foci in step S2 is selected as either multicolor immunofluorescence assay or H&E staining; preferably, multicolor immunofluorescence assay.

[0019] Optionally, in the above-described method for assessing the immune microenvironment of the endometrium, in step S3, the ratio of immune cell aggregation foci within a unit area of ​​the endometrial sample is selected as either the ratio of aggregation foci area or the ratio of aggregation foci number.

[0020] Optionally, in the above-described method for assessing the endometrial immune microenvironment, step S4 involves comparing the percentage value with a standard value to assess the endometrial immune microenvironment.

[0021] Optionally, in the above-described method for assessing the immune microenvironment of the endometrium, the immune cells are selected as one or more of lymphocytes and cells involved in the immune response; the lymphocytes are selected as one or more of T lymphocytes and B lymphocytes; and the cells involved in the immune response are selected as one or more of plasma cells, natural killer cells, granulocytes, antigen-presenting cells, and cells of the mononuclear phagocyte system.

[0022] Optionally, in the above-described method for assessing the immune microenvironment of the endometrium, the endometrial health status being assessed is selected from one or more of the following: endometritis, endometriosis, endometrial polyps, uterine fibroids, uterine cysts, endometrial cancer, and embryonic pregnancy.

[0023] The second inventive point of this application is to provide a multicolor immunofluorescence detection method for evaluating the immune microenvironment of the endometrium, wherein the evaluation method for the immune microenvironment of the endometrium is as described above; the multicolor immunofluorescence detection method includes the following steps:

[0024] T1. Collect endometrial samples;

[0025] T2. Select the appropriate antibody fluorescent dye based on the at least two primary antibodies and at least one secondary antibody used;

[0026] T3. The sample is treated sequentially with primary antibody, secondary antibody, antibody fluorescent dye, and blocking agent to obtain stained sample;

[0027] T4. Statistical analysis of data on immune cell aggregation foci in stained samples and data on the samples themselves.

[0028] Optionally, in the above-described multicolor immunofluorescence detection method, the primary antibody is selected from one or more of CD20 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD3 monoclonal antibody, CD45 monoclonal antibody, and Ki-67 monoclonal antibody; the secondary antibody is selected from antibodies capable of binding to the primary antibody, preferably HRP-labeled secondary antibody; and the antibody fluorescent dye is selected from fluorescent dyes capable of labeling antibodies, preferably one or more of TSA fluorescent dyes and quantum dot fluorescent dyes.

[0029] The third inventive aspect of this application is to provide a kit specifically for the above-mentioned multicolor immunofluorescence detection method. The kit contains at least two primary antibodies, at least one secondary antibody, corresponding fluorescent dyes for the antibodies, and a blocking agent. The primary antibody is selected from one or more of CD20 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD3 monoclonal antibody, CD45 monoclonal antibody, and Ki-67 monoclonal antibody. The secondary antibody is selected from antibodies capable of binding to the primary antibody, preferably HRP-labeled secondary antibody. The antibody fluorescent dye is selected from fluorescent dyes capable of labeling the antibody, preferably one or more of TSA fluorescent dyes and quantum dot fluorescent dyes.

[0030] Multicolor immunofluorescence detection methods can be categorized into four-color, three-color, two-color, and single-color primary antibody schemes, based on the type of primary antibody, and each corresponds to a specific reagent kit.

[0031] In the four-color primary antibody scheme, the primary antibody selections are CD20 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody and Ki-67 monoclonal antibody;

[0032] In the three-color primary antibody protocol, the primary antibody selection is CD20 monoclonal antibody, CD4 monoclonal antibody and CD8 monoclonal antibody;

[0033] In the primary antibody two-color scheme, the primary antibodies selected are CD20 monoclonal antibody and CD3 monoclonal antibody;

[0034] In the primary antibody monochromatic scheme, the primary antibody selected is a CD45 monoclonal antibody;

[0035] The above are some of the primary antibody options that the applicant has recommended based on extensive experiments, but the applicant is not limited to these few antibodies.

[0036] Meanwhile, any type of secondary antibody and fluorescent dye can be selected, as long as they can bind to the primary antibody and the antibody is labeled.

[0037] The preferred secondary antibody is an HRP-labeled secondary antibody (mixed type, mouse and rabbit);

[0038] The fluorescent dye is preferably a fluorescent dye capable of labeling antibodies, such as one or more of TSA fluorescent dyes and quantum dot fluorescent dyes;

[0039] Specifically, these could be: TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD20 monoclonal antibodies, TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD4 monoclonal antibodies, TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD8 monoclonal antibodies, TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD3 monoclonal antibodies, TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD45 monoclonal antibodies, and TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to Ki-67 monoclonal antibodies.

[0040] Compared with the prior art, this application has the following advantages:

[0041] This application provides a method for assessing the endometrial immune microenvironment and its application. By statistically analyzing the proportion of immune cell aggregation foci on the endometrium, the endometrial microenvironment can be assessed efficiently and accurately, thereby effectively improving the quality of early diagnosis of endometrial-related diseases such as chronic endometrial inflammation and pregnancy outcomes. Furthermore, by assessing the endometrial immune microenvironment, the predictive quality of embryo implantation and live birth outcomes can be further improved, providing a new direction for clinical intervention and treatment of infertile patients. Attached Figure Description

[0042] Figure 1 shows the results of negative staining of lung adenocarcinoma tissue sections according to an embodiment of this application.

[0043] Figure 2 is a staining result of lymphocyte aggregation foci in a lung adenocarcinoma tissue section according to an embodiment of this application;

[0044] Figure 3 shows the results of vaginal staining of a section of endometrial tissue to be analyzed in one embodiment of this application.

[0045] Figure 4 shows the staining effect of lymphocyte aggregation foci in endometrial tissue section 3-1 to be analyzed in one embodiment of this application.

[0046] Figure 5 shows the staining effect of lymphocyte aggregation foci in endometrial tissue section 4-1 to be analyzed in one embodiment of this application.

[0047] Figure 6 shows the staining effect of lymphocyte aggregation foci in endometrial tissue section 5-1 to be analyzed in one embodiment of this application.

[0048] Figure 7 shows the staining effect of lymphocyte aggregation foci in endometrial tissue section 5-2 to be analyzed in one embodiment of this application.

[0049] Figure 8 is a diagram showing the effect of the first group of negative samples in high-throughput staining of endometrial tissue in one embodiment of this application.

[0050] Figure 9 is a diagram showing the positive seroconversion effect of the first group of high-throughput staining samples applied to endometrial tissue in one embodiment of this application.

[0051] Figure 10 is a diagram showing the effect of high-throughput staining of the second group of samples in endometrial tissue in one embodiment of this application.

[0052] Figure 11 is a diagram showing the positive seroconversion effect of the second group of high-throughput staining samples applied to endometrial tissue in one embodiment of this application.

[0053] Figure 12 shows the effect of sample 15 in the first group of high-throughput staining samples applied to endometrial tissue in one embodiment of this application.

[0054] Figure 13 shows the effect of sample No. 31 in the second group of high-throughput staining samples applied to endometrial tissue in one embodiment of this application.

[0055] Figure 14 is a schematic diagram of the sample acquisition and information tracking process in one embodiment of this application;

[0056] Figure 15 is a statistical graph showing the difference in the abundance of lymphocyte aggregation foci in the endometrium under different embryo implantation outcomes / different live birth outcomes in one embodiment of this application (statistics are performed using the number / area ratio and area / area ratio, respectively).

[0057] Figure 16 shows the predictive value of ROC curve analysis of lymphocyte aggregation foci abundance on embryo implantation outcome / live birth in one embodiment of this application (using number / area ratio and area / area ratio statistics, respectively).

[0058] Figure 17 shows the detection results of the four-color immunofluorescence detection method and the three-color immunofluorescence detection method in one embodiment of this application.

[0059] Figure 18 shows the detection results of the three-color immunofluorescence detection method and the two-color immunofluorescence detection method in one embodiment of this application.

[0060] Figure 19 shows the correlation analysis results of four-color, three-color, two-color, and single-color immunofluorescence detection methods in one embodiment of this application.

[0061] Figure 20 shows different degrees of inflammation of the endometrium in one embodiment of this application. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0063] CD20 is a surface marker of B cells, primarily used to identify B cells and their subsets. In immunological research, CD20 is frequently used as a target for B cells, for example, in some antibody therapies.

[0064] CD4: This is a surface marker of helper T cells (Th cells). CD4+ T cells play a crucial role in the immune response; for example, they can assist B cells in producing antibodies and activate other immune cells.

[0065] CD8: Primarily found on the surface of cytotoxic T cells (Tc cells). CD8+ T cells play a crucial role in cell-mediated immunity, directly killing infected or tumor cells.

[0066] Ki-67 is a nuclear protein associated with cell proliferation and is often used as a marker to assess the proliferative activity of tumor cells. High Ki-67 expression usually indicates strong cell proliferative activity.

[0067] B cells are part of the immune system and are primarily responsible for producing antibodies to fight pathogens.

[0068] CD4+ T cells: also known as helper T cells, which are crucial for initiating and maintaining immune responses.

[0069] CD8+ T cells: also known as cytotoxic T cells, whose main function is to directly kill cells infected by pathogens or tumor cells.

[0070] HRP-labeled secondary antibodies: In immunohistochemical staining, HRP (horseradish peroxidase) is often used as an enzyme marker to bind to secondary antibodies (i.e., antibodies against primary antibodies). Once the primary antibody binds to the target antigen in the sample, the HRP-labeled secondary antibody can bind to it, allowing visualization of the antigen's location through a colorimetric reaction with the enzyme substrate.

[0071] TSA fluorescent dye: It is a fluorescent dye used for tyramide signal amplification (TSA). It is commonly used in biomedical research techniques such as immunohistochemistry (IHC), immunocytochemistry (ICC), and fluorescence in situ hybridization (FISH) to improve the sensitivity and specificity of the signal.

[0072] H&E examination report: This refers to the pathological report obtained from histological examination using the H&E staining method. H&E refers to two dyes, hematoxylin and eosin, which are often used together to stain tissue samples. Hematoxylin dye primarily makes the chromatin in the cell nucleus appear blue-purple, highlighting the structure of the cell nucleus. Eosin dye primarily makes the cytoplasm and extracellular matrix appear red, thus highlighting the cell outline and extracellular structures.

[0073] Rabbit IgG, Mouse IgG: IgG is an abbreviation for immunoglobulin G, the most abundant immunoglobulin in blood. Rabbit IgG refers to IgG derived from rabbits, while mouse IgG is immunoglobulin G derived from mice. They are commonly used in antibody research or as control reagents.

[0074] DAPI staining: DAPI is a fluorescent dye that binds to DNA. DAPI staining is commonly used in cell biology and genetics research to visualize the location and morphology of the cell nucleus. DAPI staining can clearly show the nuclear morphology of cells, thus helping researchers understand the state and function of cells.

[0075] ROC curve: ROC curve is an abbreviation for "Receiver Operating Characteristic Curve". It is a tool used to evaluate the performance of binary classifiers by plotting the relationship between the True Positive Rate and the False Positive Rate to show the classifier's performance. ROC curves can help researchers select the optimal classification threshold and evaluate the overall performance of the classifier.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0077] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0078] Example 1

[0079] The assessment method for the immune microenvironment of the endometrium is to evaluate the health status of the endometrium by assessing the aggregation status of immune cells.

[0080] Aggregation refers to the proportion of immune cell aggregation foci per unit area of ​​the endometrium.

[0081] Immune cell aggregation foci are defined as immune cell clusters with a number of ≥20 cells and / or an immune cell density of ≥20 cells / 3000 μm. 2 An aggregate of immune cells;

[0082] Preferably, the number of immune cells is greater than or equal to 30 and the immune cell density is greater than or equal to 30 cells / 3000μm. 2 An aggregate of immune cells.

[0083] The specific evaluation method is as follows:

[0084] S1. Take samples from at least one point on the endometrium; sampling is usually done using a negative pressure tube, taking samples from the functional layer of the endometrial surface tissue, avoiding the basal tissue and blood (samples are cylindrical or strip-shaped).

[0085] S2. Detect and statistically analyze the immune cell aggregation foci in the samples obtained in step S1;

[0086] S3. Calculate the proportion of immune cell aggregation foci in the unit area of ​​the endometrial sample in step S2;

[0087] S4. Assess the endometrial immune microenvironment using the percentage values ​​obtained in step S3.

[0088] In step S1, at least three sampling points are selected; the sample volume is selected to be greater than or equal to 8 mm³. 3 .

[0089] In step S2, the detection method for immune cell aggregation foci is selected as one or more of multicolor immunofluorescence detection or H&E staining; preferably, multicolor immunofluorescence detection is used.

[0090] In step S3, the ratio of immune cell aggregation foci per unit area of ​​the endometrial sample is selected as either the ratio of aggregation foci area or the ratio of aggregation foci number.

[0091] In step S4, the percentage value is compared with the standard value to assess the immune microenvironment of the endometrium.

[0092] The immune cells are selected from one or more of lymphocytes and cells involved in the immune response; the lymphocytes are selected from one or more of T lymphocytes and B lymphocytes; the cells involved in the immune response are selected from one or more of plasma cells, natural killer cells, granulocytes, antigen-presenting cells and cells of the mononuclear phagocyte system.

[0093] The endometrial health status to be assessed is selected from one or more of the following: endometritis, endometriosis, endometrial polyps, uterine fibroids, uterine cysts, endometrial cancer, and embryonic pregnancy.

[0094] A multicolor immunofluorescence assay method for assessing the endometrial immune microenvironment is also provided, the assessment method for the endometrial immune microenvironment being as described above; the multicolor immunofluorescence assay method includes the following steps:

[0095] T1. Collect endometrial samples;

[0096] T2. Select the appropriate antibody fluorescent dye based on the at least two primary antibodies and at least one secondary antibody used;

[0097] T3. The sample is treated sequentially with primary antibody, secondary antibody, antibody fluorescent dye, and blocking agent to obtain stained sample;

[0098] T4. Statistical analysis of data on immune cell aggregation foci in stained samples and data on the samples themselves.

[0099] The primary antibody is selected from one or more of the following: CD20 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD3 monoclonal antibody, CD45 monoclonal antibody, and Ki-67 monoclonal antibody.

[0100] The secondary antibody is selected as an antibody that can bind to the primary antibody, preferably an HRP-labeled secondary antibody;

[0101] The antibody fluorescent dye is selected as a fluorescent dye capable of labeling antibodies, preferably one or more of TSA fluorescent dyes and quantum dot fluorescent dyes.

[0102] Multicolor immunofluorescence detection methods can be categorized into four-color, three-color, two-color, and single-color primary antibody schemes, based on the type of primary antibody, and each corresponds to a specific reagent kit.

[0103] In the four-color primary antibody scheme, the primary antibody selections are CD20 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody and Ki-67 monoclonal antibody;

[0104] In the three-color primary antibody protocol, the primary antibody selection is CD20 monoclonal antibody, CD4 monoclonal antibody and CD8 monoclonal antibody;

[0105] In the primary antibody two-color scheme, the primary antibodies selected are CD20 monoclonal antibody and CD3 monoclonal antibody;

[0106] In the primary antibody monochromatic scheme, the primary antibody selected is a CD45 monoclonal antibody;

[0107] The above are some of the primary antibody options that the applicant has recommended based on extensive experiments, but the applicant is not limited to these few antibodies.

[0108] Meanwhile, any type of secondary antibody and fluorescent dye can be selected, as long as they can bind to the primary antibody and the antibody is labeled.

[0109] The preferred secondary antibody is an HRP-labeled secondary antibody (mixed type, mouse and rabbit);

[0110] The fluorescent dye is preferably a fluorescent dye capable of labeling antibodies, such as one or more of TSA fluorescent dyes and quantum dot fluorescent dyes;

[0111] Specifically, these could be: TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD20 monoclonal antibodies, TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD4 monoclonal antibodies, TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD8 monoclonal antibodies, TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD3 monoclonal antibodies, TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to CD45 monoclonal antibodies, and TSA fluorescent dyes or quantum dot fluorescent dyes corresponding to Ki-67 monoclonal antibodies.

[0112] Example 2:

[0113] The staining reagent kit includes:

[0114] Primary antibody, secondary antibody, and fluorescent dye corresponding to the primary antibody;

[0115] Primary antibodies include mouse anti-human CD20 monoclonal antibody, mouse anti-human CD4 monoclonal antibody, rabbit anti-human CD8 monoclonal antibody and mouse anti-human Ki-67 monoclonal antibody.

[0116] The combination of staining reagents used to predict embryonic pregnancy outcomes has the following advantages, purposes, and effects:

[0117] (1) Simultaneous detection of multiple antibodies: The combination of staining reagents can simultaneously characterize multiple indicators, covering specific marker antibodies for different cell types and functions, and obtain the expression results of multiple antibodies in one experiment.

[0118] (2) Comprehensive assessment of inflammatory status: Endometrial inflammation often involves abnormal expression of various immune cells and cytokines. A combination of multiple antibodies can comprehensively assess inflammation-related cell types (such as CD4+). + T cells, CD8 + T cells, B cells, etc.), cell proliferation activity, and inflammatory status.

[0119] (3) Provide more comprehensive information: Through the combined characterization of multiple antibodies, more detailed information on the endometrial immune microenvironment can be obtained, providing a more comprehensive basis for the next step of clinical intervention.

[0120] (4) Increase the accuracy and reliability of results: The simultaneous application of multiple antibodies can mutually verify the results, thereby improving the accuracy and reliability of the evaluation results.

[0121] (5) Personalized treatment guidance: Based on the expression of different antibodies, individualized treatment guidance and strategies can be provided to patients, which helps to develop more precise treatment plans.

[0122] By integrating the expression results of multiple antibodies, the staining reagent combination can provide comprehensive and accurate information on the endometrial immune microenvironment for scientific research and clinical practice, helping doctors make more targeted treatment decisions.

[0123] The secondary antibody is a mixed HRP-labeled secondary antibody from mice and rabbits.

[0124] Secondary antibodies are used to bind with primary antibodies to form complexes, and are labeled with different probes or enzymes to detect and enhance staining signals.

[0125] Secondary antibody plays an important role in the combination of staining reagents for predicting embryonic pregnancy outcomes:

[0126] (1) Enhanced staining signal: HRP-labeled secondary antibody can bind to primary antibody and amplify staining signal through enzyme reaction, making the target substance easier to detect.

[0127] (2) Multiple antibody binding: Due to its mixed-type characteristics, this secondary antibody can simultaneously recognize and bind to primary antibodies from multiple sources.

[0128] Its advantages include:

[0129] (1) Universality of multiple primary antibodies: Since mixed HRP-labeled secondary antibodies can bind to primary antibodies from different sources, multiple primary antibodies can be used in the same staining reagent combination.

[0130] (2) Simplified operation: Using mixed secondary antibody can simplify experimental steps and reduce the number of reagents and operation time.

[0131] (3) Enhanced signal: HRP-labeled secondary antibody can amplify the staining signal through enzyme reaction, thereby improving the sensitivity of detection.

[0132] The fluorescent dyes include: a first monochromatic TSA fluorescent dye corresponding to mouse anti-human CD20 monoclonal antibody, a second monochromatic TSA fluorescent dye corresponding to mouse anti-human CD4 monoclonal antibody, a third monochromatic TSA fluorescent dye corresponding to rabbit anti-human CD8 monoclonal antibody, and a fourth monochromatic TSA fluorescent dye corresponding to mouse anti-human Ki-67 monoclonal antibody.

[0133] In the staining reagent kit, the fluorescent dye is a single-color TSA (Tyramide Signal Amplification) fluorescent dye corresponding to the primary antibody. Details are as follows:

[0134] (1) The first monochromatic TSA fluorescent dye, such as PPD520, corresponds to the mouse anti-human CD20 monoclonal antibody and emits a green fluorescent signal;

[0135] (2) The second monochromatic TSA fluorescent dye, for example, can be PPD570, which corresponds to the mouse anti-human CD4 monoclonal antibody and emits a yellow fluorescent signal;

[0136] (3) The third single-color TSA fluorescent dye, such as PPD480, can be used, which corresponds to the rabbit anti-human CD8 monoclonal antibody and emits a cyan fluorescent signal;

[0137] (4) The fourth single-color TSA fluorescent dye, such as PPD650, corresponds to the mouse anti-human ki-67 monoclonal antibody and emits a red fluorescent signal.

[0138] In addition, the above-mentioned dyes can all be other dyes with the same or corresponding functions.

[0139] These fluorescent dyes correspond to primary antibodies and can be used to enhance and visualize the fluorescence signal of the target, offering the following advantages:

[0140] (1) Signal amplification: Monochromatic TSA fluorescent dyes introduce fluorescent labels into the sample through an enzyme-catalyzed reaction, thereby amplifying the target analyte. This signal amplification technique can increase signal intensity and improve staining sensitivity.

[0141] (2) Multicolor labeling: Different wavelengths of fluorescent dyes are used to correspond to different primary antibodies, which can simultaneously characterize multiple targets in the same sample and achieve multiple fluorescent labeling.

[0142] (3) High resolution: Fluorescence microscopes and other equipment can detect and distinguish fluorescence signals of different wavelengths, thereby enabling simultaneous observation and analysis of multiple targets.

[0143] In summary, using fluorescent dyes corresponding to primary antibodies can amplify and multi-fluorescently label the target, thereby improving the accuracy and reliability of staining and meeting the needs of multicolor signal statistics and analysis.

[0144] In addition, the staining reagent combination may also include other reagents. The specific reagents and their corresponding specifications included in the staining reagent combination are shown in Table 1.

[0145] Table 1

[0146] This staining reagent combination can simultaneously present the expression of four antigen targets on a single sample slice, facilitating the observation of target protein interactions and co-localization, and significantly reducing the amount of tissue sample required. The staining reagent combination can easily, accurately, and efficiently indicate lymphocyte aggregation foci and thus predict embryonic pregnancy outcomes, overcoming the bottlenecks of lack of predictive indicators and standardized technical procedures for embryonic pregnancy outcomes in clinical practice.

[0147] It also provides multicolor immunofluorescence detection methods, including:

[0148] S1, collect biochemical samples and prepare dried sections corresponding to the biochemical samples;

[0149] S2, based on the primary antibody, secondary antibody and fluorescent dye in the staining reagent combination, the primary antibody reagent, secondary antibody reagent and dye working solution are prepared respectively, and the blocking solution is prepared.

[0150] S3, using primary antibody reagent, secondary antibody reagent, dye and blocking solution to perform fluorescent staining on dried sections to obtain stained samples;

[0151] S4. Scan the stained sample to obtain a multicolor immunofluorescence microscopic panoramic image corresponding to the stained sample, and use the multicolor immunofluorescence microscopic panoramic image as the multicolor immunofluorescence result.

[0152] In step S4, an imaging scanning instrument (such as the Olympus VS200 research-grade whole slide scanning system) can be used to set the program to scan the entire slide and obtain the corresponding multicolor immunofluorescence microscopic panoramic image.

[0153] The multicolor immunofluorescence detection method employs fully automated staining, enabling high-throughput and standardized operation of clinical tissue samples. Up to 30 pathological slides can be tested simultaneously in a single run, while avoiding the drawbacks of long operation time and large errors of manual operation. It provides clinical laboratory personnel with a simple and efficient experimental operation process and analysis method.

[0154] Step S1: Collect biochemical samples and prepare dried sections corresponding to the biochemical samples, including:

[0155] S11, collect endometrial tissue from the subject as a biochemical sample;

[0156] S12, after the biochemical sample is washed, fixed, dehydrated, embedded and sectioned in sequence, dried sections are prepared.

[0157] In this step, endometrial tissue (volume not less than 8 mm) is collected according to the standard sampling procedure. 3 Place it in a culture dish containing physiological saline to remove obvious blood clots and mucus from the tissue.

[0158] The obtained endometrial tissue was cleaned, fixed, dehydrated, embedded, and sectioned to prepare dried sections with a tissue thickness of 4 μm for subsequent staining of lymphocyte aggregation foci.

[0159] Step S2 includes:

[0160] The preparation method of the primary antibody reagent is as follows:

[0161] Take primary antibody, rabbit IgG and mouse IgG, and dilute them respectively with antibody diluent to obtain primary antibody reagents corresponding to each primary antibody, as well as working solutions corresponding to rabbit IgG and mouse IgG;

[0162] The formula for preparing the amount of primary antibody reagent is: Primary antibody reagent amount = A + B × n;

[0163] In the formula, all values ​​are in μL; n is the total number of slides to be stained in each batch of experiments; A is the dead volume of the open container; and B is the amount used per slide. For example, A = 300, B = 150.

[0164] Specifically, take out the antibody stock solution corresponding to the primary antibody, briefly centrifuge it in a centrifuge, and prepare the working solution in a burette (Titration Kit) using antibody diluent according to the dilution ratio in the table below. Label the outside of the burette with the dye name and preparation time, and store it in a refrigerator at 4°C for later use. The antibody preparation parameters are shown in Table 2.

[0165] Table 2

[0166] The preparation method for the dye working solution is as follows:

[0167] Take the fluorescent dye and dilute it with a dye diluent to obtain a dye working solution corresponding to each fluorescent dye;

[0168] The formula for preparing the amount of dye working solution is: Dye working solution amount = A + B × n;

[0169] In the formula, all values ​​are in μL; n is the total number of slides to be stained in each batch of experiments; A is the dead volume of the open container; and B is the amount used per slide. For example, A = 300, B = 150.

[0170] The specific steps are as follows: In a burette (Titration Kit), prepare the working solution with diluent according to the dilution ratio in the table below, wrap the burette with aluminum foil, mark the dye name and preparation time on the outside of the burette, and store it in a refrigerator at 4°C for later use. The preparation parameters of the fluorescent dye are shown in Table 3.

[0171] Table 3

[0172] The formula for preparing the secondary antibody reagent and blocking solution is: Total amount = A + B × n;

[0173] In the formula, all values ​​are in μL; n is the total number of slides to be stained in each batch of experiments; A is the dead volume of the open container; and B is the amount used per slide. For example, A = 300, B = 150.

[0174] When preparing the secondary antibody reagent, use a pipette to transfer the HRP-labeled secondary antibody-rabbit mixture into a burette (the specific amount is calculated according to the formula above).

[0175] When preparing the 2% BSA blocking solution, weigh out BSA and prepare a 2% BSA (mg / mL) solution with 1×PBS. Mix well by inverting and transfer to a burette (the amount of blocking solution is calculated according to the above formula).

[0176] Step S3: The dried sections are fluorescently stained using primary antibody reagent, secondary antibody reagent, dye, and blocking solution to obtain stained samples, including:

[0177] S31, after dewaxing and rehydration of the dried sections, they are blocked with a blocking solution;

[0178] S32, After blocking, the sections are sequentially stained with antibodies based on primary antibody reagent, secondary antibody reagent and dye working solution;

[0179] S33, DAPI staining is performed on the antibody-labeled slides to obtain the stained sample.

[0180] Step S32: After blocking, the slides are sequentially stained with antibodies based on primary antibody reagent, secondary antibody reagent, and dye working solution, including:

[0181] S321, after blocking, the slides are subjected to the following treatments in sequence: primary antibody incubation with the primary antibody reagent corresponding to antibody CD20, secondary antibody incubation, and staining with the working solution of TSA fluorescent dye-PPD520; primary antibody incubation with the primary antibody reagent corresponding to antibody CD4, secondary antibody incubation, and staining with the working solution of TSA fluorescent dye-PPD570; primary antibody incubation with the primary antibody reagent corresponding to antibody CD8, secondary antibody incubation, and staining with the working solution of TSA fluorescent dye-PPD480; primary antibody incubation with the primary antibody reagent corresponding to antibody Ki-67, secondary antibody incubation, and staining with the working solution of TSA fluorescent dye-PPD650, thus obtaining the positive control sample.

[0182] S322, after blocking, the slides were incubated with mouse IgG and rabbit IgG, then with secondary antibody, and finally with the corresponding dye working solution to obtain negative control samples.

[0183] The prepared tissue sections were stained using the prepared reagents according to the specific procedures in Tables 4 and 5 (fully automated multiplex immunostaining instrument).

[0184] The fully automated multicolor immunofluorescence staining procedure (applicable to positive control samples and samples to be tested) is shown in Table 4.

[0185] The fully automated multicolor immunofluorescence staining procedure (for negative control samples) is shown in Table 5.

[0186] Specifically, it can be divided into procedures applicable to positive control samples and samples to be tested, and procedures applicable to negative control samples.

[0187] Table 4

[0188] Table 5

[0189] In addition, after staining, the slides need to be mounted and preserved.

[0190] After the tissue sections have been stained, aspirate all the liquid.

[0191] Add 1-2 drops of enhanced anti-fluorescence quenching mounting medium to each tissue section, cover with a coverslip, and ensure that the enhanced anti-fluorescence quenching mounting medium completely covers the tissue without air bubbles;

[0192] Seal the area around the coverslip with clear nail polish. After the nail polish has hardened, store it in a 4°C refrigerator away from light until scanning is complete.

[0193] In addition, an application of a staining reagent combination in the preparation of a product for predicting embryonic pregnancy outcomes is provided, wherein the product is stained by the method described above and a multicolor immunofluorescence microscopic panoramic image corresponding to each biochemical sample is obtained; and the ratio of the target lymphocyte aggregation foci to the endometrial tissue area is calculated based on the multicolor immunofluorescence microscopic panoramic image.

[0194] Multicolor immunofluorescence microscopic panoramic images are panoramic images corresponding to each biochemical sample obtained after staining using the above method.

[0195] After staining, multicolor immunofluorescence microscopic panoramas can be used to further obtain the area of ​​the target lymphocyte aggregation foci corresponding to each multicolor immunofluorescence microscopic panoramas using an image recognition system (such as the HALO pathological image analysis system from Indica Labs).

[0196] Methods for obtaining the area of ​​target lymphocyte aggregation foci include:

[0197] For each biochemical sample, the multicolor immunofluorescence microscopic panorama is defined as follows: green signal represents CD20 positive signal, yellow signal represents CD4 positive signal, cyan signal represents CD8 positive signal, and red signal represents Ki-67 positive signal. If the number of lymphocytes in the four positive signal clusters is greater than or equal to 30 and the lymphocyte density is greater than or equal to 30 cells / 3000 μm, then the positive signal is considered positive. 2 If so, it is considered a focus of lymphocyte aggregation;

[0198] The sum of the areas of all positive signal aggregation foci in the multicolor immunofluorescence microscopy panoramic image was used as the area of ​​the target lymphocyte aggregation foci.

[0199] When the ratio of the area of ​​the target lymphocyte aggregation foci to the area of ​​the endometrial tissue is <0.186, the embryo implantation outcome is predicted to be successful in infertile patients.

[0200] When the ratio of the area of ​​the target lymphocyte aggregation foci to the area of ​​the endometrial tissue is ≥0.186, the embryo implantation outcome is predicted to be failure in infertile patients.

[0201] When the ratio of the area of ​​the target lymphocyte aggregation foci to the area of ​​the endometrial tissue is <0.139, the predicted live birth outcome for infertile patients is successful.

[0202] When the ratio of the area of ​​the target lymphocyte aggregation foci to the area of ​​the endometrial tissue is ≥0.139, the predicted live birth outcome for infertile patients is failure.

[0203] Another method of determination is to count the sum of the number of all positive signal aggregation foci in the multicolor immunofluorescence microscopy panoramic image as the number of target lymphocyte aggregation foci;

[0204] When the ratio of the number of target lymphocyte aggregation foci to the area of ​​endometrial tissue is <0.314, the embryo implantation outcome is predicted to be successful in infertile patients.

[0205] When the ratio of the number of target lymphocyte aggregation foci to the area of ​​endometrial tissue is ≥0.314, the embryo implantation outcome is predicted to be failure in infertile patients.

[0206] When the ratio of the number of target lymphocyte aggregation foci to the area of ​​endometrial tissue is <0.106, the predicted live birth outcome for infertile patients is considered successful.

[0207] When the ratio of the number of target lymphocyte aggregation foci to the endometrial tissue area is ≥0.106, the predicted live birth outcome for infertile patients is failure.

[0208] The ratios listed above are all clinical decision values ​​(i.e., cut-off values), not absolute values. They are derived from the combination of available data volume, sensitivity, and specificity. These values ​​will also change by ±10% when the amount of data changes.

[0209] The product can be any one or more of the following: reagent kits, testing equipment, and data analysis systems.

[0210] It should be noted that embryo implantation outcome and live birth outcome for infertile patients refer to the implantation and development of the embryo in the uterus during assisted reproductive technology (such as in vitro fertilization), and ultimately, whether a live birth is successfully delivered. Specifically:

[0211] (1) Embryo implantation outcome: In the in vitro fertilization (IVF) process, the embryo is implanted into the mother's uterus. If the embryo successfully implants and develops, it is called successful embryo implantation. If the embryo fails to implant, it is called failed embryo implantation. Therefore, the embryo implantation outcome is evaluated as success or failure.

[0212] (2) Live birth outcome: Live birth outcome refers to the result of successful embryo implantation, in which the mother is able to carry the pregnancy to term and deliver a live birth. If complications occur during the pregnancy or during delivery, the live birth may fail. Therefore, the live birth outcome is also evaluated as success or failure.

[0213] Using success or failure to evaluate embryo implantation and live birth outcomes is because these outcomes are clearly binary—either successful or unsuccessful. This evaluation method facilitates research and statistical data analysis, directly reflecting the treatment effectiveness and success rate of infertile patients during the IVF process. It helps doctors and patients make further treatment decisions, influencing adjustments to assisted reproductive protocols and improving patient pregnancy success rates.

[0214] Through extensive experiments, the inventors discovered a correlation between the ratio of target lymphocyte aggregation foci to endometrial tissue area and embryo implantation and live birth outcomes in infertile patients. Specifically, there was a significant difference in the ratio between the successful embryo implantation group and the failed embryo implantation group. Similarly, there was a significant difference between the successful live birth group and the non-live birth group. Large-scale ROC curve analysis of the samples determined optimal cut-off values ​​of 0.186 and 0.139, respectively. The provided staining reagent combination can be used to prepare products predicting embryonic pregnancy outcomes, enabling quantitative analysis of lymphocyte aggregation foci. The abundance of lymphocyte aggregation foci was found to be strongly correlated with pregnancy outcome, and a suitable cut-off value was ultimately determined to successfully predict pregnancy outcomes.

[0215] Example 3:

[0216] Differences between the four-color immunofluorescence detection method and the three-color immunofluorescence detection method (Figure 17):

[0217] In the four-color primary antibody scheme, the primary antibody selections are CD20 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody and Ki-67 monoclonal antibody;

[0218] In the three-color primary antibody protocol, the primary antibody selection is CD20 monoclonal antibody, CD4 monoclonal antibody and CD8 monoclonal antibody;

[0219] The main difference between the two lies in whether or not Ki67 monoclonal antibody is used. Ki67 primarily indicates lymphocyte proliferation; the absence of Ki67 does not affect the quantification of lymphocyte aggregates. Therefore, the results of four-color immunofluorescence assay and three-color immunofluorescence assay are not significantly different. In fact, as described in Example 5, Ki67 plays an important role in the identification of severe endometrial inflammation.

[0220] Example 4:

[0221] CD3-mediated cell function mainly includes CD4-mediated cells. + and CD8 + For cells, four-color or three-color fluorescence detection can also be replaced by two-color immunofluorescence detection of CD20 and CD3, as shown in Figure 18;

[0222] The inventors also compared the lymphocyte aggregates indicated by two-color fluorescence detection (i.e., CD3 monoclonal antibody + CD20 monoclonal antibody) and single-color fluorescence detection (i.e., CD45 monoclonal antibody). The comparison results showed that the single-color fluorescence detection method using CD45 monoclonal antibody can also indicate lymphocyte aggregates to some extent. However, because CD45 monoclonal antibody can include more immune cells, the boundaries of lymphocyte aggregates in this single-color fluorescence detection method are more blurred than those of other detection methods (four-color, three-color, and two-color fluorescence detection).

[0223] In the primary antibody two-color scheme, the primary antibodies selected are CD20 monoclonal antibody and CD3 monoclonal antibody;

[0224] In the primary antibody monochromatic scheme, the primary antibody selected is CD45 monoclonal antibody.

[0225] The correlation analysis results of four-color, three-color, two-color and single-color immunofluorescence detection methods are shown in Figure 19.

[0226] Example 5:

[0227] Different degrees of endometrial inflammation (Figure 20 shows mild, moderate, and severe inflammation from left to right):

[0228] Mild (the endometrium shows a smaller number and area of ​​lymphocyte aggregation, with both T cells and B cells involved);

[0229] (The endometrium shows a large number and large area of ​​lymphocyte aggregation, with both T cells and B cells involved, but B cells are the main aggregates).

[0230] The endometrium showed a large number and extensive aggregation of lymphocytes, with both T cells and B cells involved. B cell aggregation was significant, and they exhibited marked proliferation (Ki67). + )

[0231] Example 6:

[0232] In this embodiment, a verification experiment was conducted on the staining effect and repeatability applied to endometrial tissue, and the verification results were obtained.

[0233] 1. Experimental Methods:

[0234] Two slides of lung adenocarcinoma tissue with a clear lymphocyte aggregation were taken as a positive control.

[0235] Four more endometrial tissue samples were randomly selected, and one slice was taken from each sample. Sample No. 5 was sliced ​​twice consecutively.

[0236] After the sample sections were prepared, lymphocyte aggregation foci were stained according to the aforementioned technical solution provided in this invention. The sample grouping is shown in Table 6 below.

[0237] Table 6

[0238] 2. Experimental Results:

[0239] After the staining and smearing steps were completed, the results of multiplex immunofluorescence scanning were analyzed using the HALO pathological image analysis system. The staining effect is shown in Figures 1-7:

[0240] Figure 1 shows the results of negative staining of lung adenocarcinoma tissue sections, Figure 2 shows the staining results of lymphocyte aggregation foci in lung adenocarcinoma tissue sections, Figure 3 shows the results of negative staining of endometrial tissue sections, and Figures 4-7 show the staining effects of lymphocyte aggregation foci in three endometrial tissue sections to be tested.

[0241] (1) As shown in Figure 1, the negative control of lung adenocarcinoma tissue did not show obvious positive signals for CD4, CD8, CD20, and Ki-67; as shown in Figure 2, the positive control sample of lung adenocarcinoma tissue section showed significant positive signals (CD4, CD8, and CD20 were located in the cell membrane, and Ki-67 was located in the cell nucleus. The marker localization was as expected, there was no obvious cross-coloring between different markers, and the staining was uniform), and obvious positive signal aggregation areas were visible (outlined by white dashed lines). The results in Figures 1 and 2 show that the provided technical solution has low staining background, high staining specificity of each marker, and correct subcellular localization. HALO system analysis shows that it is feasible to identify and recognize lymphocyte aggregation foci.

[0242] (2) As shown in Figure 3, the negative reference endometrial tissue did not show obvious positive signals for CD4, CD8, CD20, and Ki67, and there was no nonspecific staining. As shown in Figures 4-7, the staining results of the endometrial tissue sections showed significant positive signals, the staining of each marker was correctly located, there was no obvious cross-coloring between different markers, and the staining was specific and uniform. In the staining results of sample 5 (Figures 6-7), obvious lymphocyte aggregation foci were visible, outlined by white dashed lines. The results of Figures 3-7 demonstrate that the technical solution provided in this application is feasible for identifying and recognizing lymphocyte aggregation foci in endometrial tissue samples.

[0243] (3) Meanwhile, two consecutive sections of sample 5 were repeated. The staining results of section 5-1 (Figure 6) were basically consistent with those of section 5-2 (Figure 7), indicating that the technical solution provided in this application has stability and reproducibility.

[0244] Conclusion: The fully automated multicolor immunofluorescence staining kit used in the examples is prepared based on the staining reagent combination in this application. Using the fully automated multicolor immunofluorescence staining kit and standardized procedures, the staining results for endometrial tissue showed that the subcellular localization of each marker was correct, there was no non-specific staining, and lymphocyte aggregation foci could be accurately identified. The results passed acceptance testing, the experimental conditions were stable and reliable, and it can be used for the identification and statistical analysis of lymphocyte aggregation foci in endometrial samples.

[0245] Example 7:

[0246] In this embodiment, to test whether the present application can achieve high-throughput detection of endometrial tissue, 50 paraffin-embedded endometrial samples from women of reproductive age in the proliferative phase were selected from the sample bank of Hospital A for sectioning and staining of lymphocyte aggregation foci (all 50 samples underwent high-quality embryo transfer within 6 months after endometrial biopsy).

[0247] In this embodiment, following the staining reagent combination of this application and the method for obtaining multicolor immunofluorescence results based on the staining reagent combination, a fully automated multiplex immunostaining instrument was used to divide the above-mentioned slides into two groups according to serial numbers 1-28 (first group) and 29-50 (second group) for staining (Note: For each experiment, two slides of endometrial tissue 20231999 were used for the control experiment, one for the positive control experiment and one for the IgG isotype control). After staining, the slides were mounted, scanned, image analyzed, and data processed.

[0248] The staining results of multiplex immunofluorescence scanning were analyzed using the HALO pathological image analysis system. The specific staining effects are shown in Figures 8-13. Figures 8 and 9 show the staining results of the negative control and positive control of the first group of samples, respectively. Figures 10 and 11 show the staining results of the negative control and positive control of the second group of samples, respectively. Figures 12 and 13 show the staining results of randomly selected samples from the first group (sample 15 from No. 1-28 in the first group) and the second group (sample 31 from No. 29-50 in the second group), respectively.

[0249] The results showed that the antibody staining of the endometrial samples was normal, with no cross-staining or non-specific staining. The staining effect was stable, and obvious lymphocyte aggregation foci were visible in the endometrial samples. This technique, combined with a fully automated multiplex immunostaining instrument, enables automated staining of 30 slides at a time. The staining results passed acceptance, the experimental conditions were stable and reliable, and high-throughput analysis of lymphocyte aggregation foci in endometrial samples was successfully achieved.

[0250] Example 8:

[0251] In this embodiment, to explore the predictive effect of quantitative analysis of lymphocyte aggregation foci on pregnancy outcomes, a total of 198 paraffin-embedded endometrial samples from women of childbearing age in the proliferative phase were selected from sample banks of four hospitals. The samples were then stained and statistically analyzed for lymphocyte aggregation foci. At the same time, their clinical pregnancy outcomes and live birth outcomes were tracked and collected (all 198 samples underwent high-quality embryo transfer within 6 months after endometrial biopsy, Figure 14).

[0252] Experimental results:

[0253] (1) Based on the outcome of the detection node "B-ultrasound detection of gestational sac", infertile patients were divided into two groups: successful embryo implantation (gestational sac visible on B-ultrasound) and failed embryo implantation (gestational sac not visible on B-ultrasound). The abundance difference of lymphocyte aggregation foci in the endometrium of the two groups was statistically analyzed.

[0254] As shown in Figure 15, a significant difference was found between the ratio of target lymphocyte aggregation foci to endometrial tissue area in the embryo implantation failure group (Mean±SEM=13.2±2.2828, N=20) and the ratio in the embryo implantation successful group (Mean±SEM=5.03±0.7284, N=30) (p<0.0001).

[0255] The ratio of target lymphocyte aggregation foci to endometrial tissue area in the embryo implantation failure group (Mean±SEM=13.2±2.2828, N=20) was significantly different from that in the embryo implantation success group (Mean±SEM=5.03±0.7284, N=30) (p<0.0001).

[0256] (2) Regarding the outcome of the detection node "whether a live birth occurred", infertile patients were divided into two groups: those who had a successful live birth and those who had not. The abundance difference of lymphocyte aggregation foci in the endometrium of the two groups was statistically analyzed.

[0257] As shown in Figure 15, a significant difference was found between the ratio of target lymphocyte aggregation foci to endometrial tissue area in the group with successful live birth (Mean±SEM=4.75±0.8113, N=24) and the ratio in the group with unsuccessful live birth (Mean±SEM=11.58±2.5266, N=26) (p<0.0001).

[0258] (3) ROC curve analysis showed that the ratio of target lymphocyte aggregation foci to endometrial tissue area had a predictive value for embryo implantation outcome. As shown in Figure 16, the area under the ROC curve for predicting embryo implantation outcome was 0.76, the optimal cut-off value was 0.186, the sensitivity (SE) was 0.71, and the specificity (SP) was 0.70.

[0259] (4) ROC curve analysis showed that the ratio of target lymphocyte aggregation foci to endometrial tissue area was 0.79, the optimal cut-off value was 0.139, the sensitivity (SE) was 0.69, and the specificity (SP) was 0.79.

[0260] The technical solution and the calculation and statistics of target lymphocyte aggregation foci provided in this application can help predict the embryonic pregnancy outcome in infertile patients in clinical practice. Specifically, the judgment and prediction methods can be as follows:

[0261] When the ratio of the target lymphocyte aggregation foci to the area of ​​endometrial tissue is <0.186, the embryo implantation outcome in infertile patients is predicted to be successful.

[0262] When the ratio of the target lymphocyte aggregation foci to the area of ​​endometrial tissue is ≥0.186, the predicted embryo implantation outcome for infertile patients is failure.

[0263] When the ratio of the target lymphocyte aggregation foci to the area of ​​endometrial tissue is <0.139, the predicted live birth outcome for infertile patients is successful.

[0264] When the ratio of the target lymphocyte aggregation foci to the area of ​​endometrial tissue is ≥0.139, the predicted live birth outcome for infertile patients is failure.

[0265] Conclusion: The results of the above multi-group comparative experiments and clinical relevance tests show that the staining reagent combination and technical solution provided in this application can detect the abundance of lymphocyte aggregation foci in the endometrium with high throughput, stability and accuracy, and thus predict the embryonic pregnancy outcome, providing a new direction for clinical intervention and treatment of infertile patients.

[0266] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for assessing the immune microenvironment of the endometrium, characterized in that, The assessment method evaluates the immune microenvironment of the endometrium by assessing the aggregation status of immune cells on the endometrium, thereby differentiating and determining the health status of the endometrium.

2. The method for assessing the endometrial immune microenvironment according to claim 1, characterized in that, The aggregation state of immune cells on the endometrium refers to the proportion of immune cell aggregation foci per unit area of ​​the endometrium.

3. The method for assessing the endometrial immune microenvironment according to claim 2, characterized in that, The term "foci of immune cell aggregation" refers to a concentration of 20 or more immune cells and / or an immune cell density of 20 or more cells per 3000 μm. 2 An aggregate of immune cells; preferably 30 or more and with an immune cell density of 30 or more per 3000 μm. 2 An aggregate of immune cells.

4. The method for assessing the endometrial immune microenvironment according to claim 3, characterized in that, The specific evaluation method is as follows: S1. Take samples from at least one location of the endometrium; S2. Detect and statistically analyze the immune cell aggregation foci in the endometrial samples obtained in step S1; S3. Calculate the proportion of immune cell aggregation foci in the unit area of ​​the endometrial sample in step S2; S4. Assess the endometrial immune microenvironment using the proportions obtained in step S3.

5. The method for assessing the endometrial immune microenvironment according to claim 4, characterized in that, In step S1, at least three sampling points are selected; the sample volume is selected to be greater than or equal to 8 mm³. 3 .

6. The method for assessing the endometrial immune microenvironment according to claim 4, characterized in that, In step S2, the detection method for immune cell aggregation foci is selected from one or more of multicolor immunofluorescence detection or H&E staining; preferably, multicolor immunofluorescence detection is used.

7. The method for assessing the endometrial immune microenvironment according to claim 4, characterized in that, In S3, the proportion of immune cell aggregation foci in a unit area of ​​the endometrial sample is selected as any one or more of the ratio of immune cell aggregation foci area and the ratio of immune cell aggregation foci number.

8. The method for assessing the endometrial immune microenvironment according to claim 4, characterized in that, In S4, the percentage value is compared with the standard value to assess the endometrial immune microenvironment.

9. The method for assessing the endometrial immune microenvironment according to any one of claims 1-8, characterized in that, The immune cells are selected from one or more of lymphocytes and cells involved in the immune response; the lymphocytes are selected from one or more of T lymphocytes and B lymphocytes; the cells involved in the immune response are selected from one or more of plasma cells, natural killer cells, granulocytes, antigen-presenting cells, and cells of the mononuclear phagocyte system.

10. The method for assessing the endometrial immune microenvironment according to any one of claims 1-8, characterized in that, The endometrial health status to be assessed is selected from one or more of the following: endometritis, endometriosis, endometrial polyps, uterine fibroids, uterine cysts, endometrial cancer, and embryonic pregnancy.

11. A multicolor immunofluorescence assay for evaluating the immune microenvironment of the endometrium, characterized in that, The method for assessing the endometrial immune microenvironment is as described in any one of claims 1-8; the multicolor immunofluorescence detection method includes the following steps: T1. Collect endometrial samples; T2. Select the appropriate antibody fluorescent dye based on the at least one primary antibody and at least one secondary antibody used; T3. The sample is treated sequentially with primary antibody, secondary antibody, corresponding antibody fluorescent dye, and blocking agent to obtain stained sample; T4. Statistical analysis was performed on the data of immune cell aggregation foci in the stained samples and the data of the samples themselves.

12. The multicolor immunofluorescence detection method for evaluating the endometrial immune microenvironment according to claim 11, characterized in that, The primary antibody is selected from one or more of CD20 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD3 monoclonal antibody, CD45 monoclonal antibody, and Ki-67 monoclonal antibody; the secondary antibody is selected from antibodies capable of binding to the primary antibody, preferably HRP-labeled secondary antibody; the antibody fluorescent dye is selected from fluorescent dyes capable of labeling the antibody, preferably one or more of TSA fluorescent dyes and quantum dot fluorescent dyes.

13. The kit for a multicolor immunofluorescence assay for evaluating the endometrial immune microenvironment as described in claim 11 or 12, characterized in that, The kit contains at least one primary antibody, at least one secondary antibody, a corresponding fluorescent dye for the antibody, and a blocking agent; the primary antibody is selected from one or more of CD20 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD3 monoclonal antibody, CD45 monoclonal antibody, and Ki-67 monoclonal antibody; the secondary antibody is selected as an antibody capable of binding to the primary antibody, preferably an HRP-labeled secondary antibody; the antibody fluorescent dye is selected as a fluorescent dye capable of labeling the antibody, preferably one or more of TSA fluorescent dye and quantum dot fluorescent dye.

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