CN112708653B - Detection method for predicting recurrent abortion and/or diagnosing cause of recurrent abortion by menstrual blood - Google Patents

Abstract

The present invention relates to a method for predicting recurrent miscarriage and/or diagnosing the cause of recurrent miscarriage using menstrual blood. It uses flow cytometry to analyze the proportion and phenotype of NK cell subsets in menstrual blood, so as to infer the change in the proportion of NK cell subsets in the endometrium, which is positively correlated with the occurrence of recurrent miscarriage, and can be clinically judged for unexplained recurrent miscarriage. Whether abortion and other pregnancy diseases are related to immune factors, especially the proportion and dysfunction of endometrial NK cells.

Description

A test for predicting recurrent miscarriage and/or diagnosing the cause of recurrent miscarriage using menstrual blood test method

technical field

The invention relates to the field of medical and biological detection, and mainly relates to a detection method for predicting recurrent miscarriage and/or diagnosing the cause of recurrent miscarriage by using menstrual blood.

Background technique

Recurrent miscarriage (Recurrent miscarriage) refers to multiple (usually 3 or more) miscarriages after embryo implantation. There are many causes, including abnormal karyotype of the husband and wife, deformity of the female reproductive tract, endocrine disorders, and systemic infection; Abnormal semen, poor sperm quality, etc. However, according to statistics, there are still about 50% to 80% of patients with recurrent miscarriage whose etiology is unknown by existing technical means, which may be closely related to immune disorders. Among them, recurrent spontaneous abortion (RSA) refers to abortion after 3 or more consecutive natural pregnancies with the same spouse. Recurrent implantation failure (RIF) refers to women who have failed three attempts at in vitro fertilization (IVF) with good embryo quality, some of which have failed implantation, and some have normal implantation but miscarriage after implantation. In these two diseases, RSA and RIF, there are a large number of unexplained multiple miscarriages in patients. At present, many scholars believe that the etiology is closely related to the immunological effect between mother and fetus.

During pregnancy, the fertilized egg invades the mother's endometrium through the trophoblast cells, and as a kind of allograft of the mother, by virtue of the immune regulation of the mother-fetal interface, the mother will have a protective immune response. At the same time, it depends on the normal development of placenta and decidua to ensure its survival. Therefore, clarifying the immune mechanism and actively correcting the immune imbalance may reduce the incidence of RSA and RIF.

Natural killer cells (Natural killer cells, NK) cells account for 70% of all lymphocytes in the decidua tissue at the maternal-fetal interface in the first trimester of pregnancy and play a central role. Current studies have shown that NK cells in the decidua during pregnancy support trophoblast cells to invade the uterine arteries and remodel the spiral arterioles by secreting cytokines to ensure sufficient placental blood perfusion during pregnancy and inhibit inflammatory cells to maintain normal maternal immune tolerance , and secrete pluripotent growth factors to maintain and promote fetal growth and development. There are different subsets of NK cells in decidua, which perform different functions. Therefore, understanding NK cell subsets in the uterus is of great significance for the diagnosis and treatment of unexplained RSA and RIF.

Existing technical means: 1. The state of endometrium is reflected by detecting the state of NK cells in peripheral blood, and only the proportion and quantity of NK cells are detected without distinguishing subgroups, and the results are very inaccurate. 2. Obtain NK cells from pre-pregnancy endometrium for detection by minimally invasive surgery, but the operation is difficult, and more endometrial tissue needs to be obtained, which will cause endometrial trauma.

Summary of the invention

Technical scheme of the present invention is:

1. A reagent capable of quantifying at least one of the following NK cell subsets is prepared for predicting the risk of recurrent miscarriage in a subject through menstrual blood and/or diagnosing whether the cause of recurrent miscarriage in a subject is related to endometrial NK Uses of kits related to cell abnormalities:

CD56 ⁺ CD16 ⁺ NK cell subsets, CD56 ⁺ CD49a ⁺ NK cell subsets, CD49a ⁺ CD16 ^- NK cell subsets and/or CD49a ^- CD16 ⁺ NK cell subsets.

2. The use according to item 1, wherein the reagent is used to quantify the proportion of NK cell subgroups in the menstrual blood of a subject to the total NK cells in menstrual blood.

3. The use according to any one of the preceding claims, wherein said recurrent miscarriage comprises recurrent miscarriage and recurrent implantation failure.

4. The use as described in item 3 above, wherein in menstrual blood, CD56 ⁺ CD3-NK cell subsets≤19.4%, CD56 ⁺ CD16 ^- NK cell subsets≥39.3%, CD56 ⁺ CD49a ⁺ NK cell subsets≤ 56.8%, CD49a ⁺ CD16 ^- NK cell subset ≤ 48.2% and/or CD49a ^- CD16 ⁺ NK cell subset ≥ 33.1%, preferably CD56 ⁺ CD49a ⁺ NK cell subset ≤ 56.8% and/or CD49a ⁺ CD16 ^- NK When the cell subgroup is ≤48.2%, the subject is diagnosed as having the risk of recurrent miscarriage, or the cause of recurrent miscarriage in the subject is diagnosed as being related to endometrial NK cell abnormalities.

5. The use as described in item 3 above, wherein in menstrual blood, CD49a ^- CD16 ⁺ NK ≥ 31.7%, CD56 ⁺ CD16 ⁺ NK cell subsets ≥ 28.6%, CD56 ⁺ CD49a ⁺ NK cell subsets ≤ 65.1% and / or CD49a ⁺ CD16 ^- NK cell subsets ≤ 60%, preferably CD49a ^- CD16 ⁺ NK ≥ 31.7%, the subject is diagnosed as having a risk of recurrent implantation failure, or the subject is diagnosed as having The cause of recurrent implantation failure was diagnosed as related to endometrial NK cell abnormalities.

6. Use according to any one of the preceding claims, wherein the agent is an antibody.

7. The use according to any one of the preceding claims, wherein the antibody is conjugated with fluorescein.

8. The use as described in any one of the foregoing, wherein the ratio of menstrual blood NK cell subsets and/or the ratio of menstrual blood NK cell subsets is detected by flow cytometry.

9. A recurrent miscarriage diagnostic kit comprising reagents and/or a menstrual blood collector, preferably a menstrual cup, capable of quantifying at least one of the following NK cell subsets: CD56 ⁺ CD16 ⁺ NK cell subsets, CD56 ⁺ CD49a ⁺ NK cell subsets population, CD49a ⁺ CD16 ^- NK cell subsets and/or CD49a ^- CD16 ⁺ NK cell subsets.

10. The kit according to item 9, wherein said reagent is an antibody, preferably said antibody is conjugated with fluorescein.

11. The kit according to item 9 or 10, wherein the reagent is used to quantify the ratio of subgroups of NK cells in the menstrual blood of a subject to the total NK cells in menstrual blood.

12. The kit of any one of the preceding claims, wherein said recurrent miscarriage comprises recurrent miscarriage and recurrent implantation failure.

13. The kit as described in item 12 above, wherein in menstrual blood, CD56+CD3-NK≤19.4%, CD56+CD16-NK≥39.3%, CD56+CD49a+NK≤56.8%, CD49a+CD16-NK ≤48.2% and/or CD49a-CD16+NK≥33.1%, preferably CD56+CD49a+NK≤56.8%, CD49a+CD16-NK≤48.2%, the subject is diagnosed as having the risk of recurrent miscarriage , or the cause of recurrent miscarriage in the subject was diagnosed as being related to endometrial NK cell abnormalities.

14. The kit as described in Item 12 above, wherein in menstrual blood, CD49a ^- CD16 ⁺ NK ≥ 31.7%, CD56 ⁺ CD16 ⁺ NK cell subsets ≥ 28.6%, CD56 ⁺ CD49a ⁺ NK cell subsets ≤ 65.1% and/or CD49a ⁺ CD16 ^- NK cell subsets ≤ 60%, preferably CD49a ^- CD16 ⁺ NK ≥ 31.7%, the subject is diagnosed as having a risk of recurrent implantation failure, or the subject is The cause of recurrent implantation failure was diagnosed as being related to endometrial NK cell abnormalities.

15. The kit as described in any one of the foregoing, wherein the ratio of menstrual blood NK cell subsets and/or the ratio of menstrual blood NK cell subsets is detected by flow cytometry.

Detailed description of the invention

Under the stimulation of hormones, in women of childbearing age, the endometrium can proliferate from about 0.5mm to about 5-7mm in each menstrual cycle. With the periodic retreat of progesterone levels, the endometrium will fall off and form menstrual blood, which contains a large amount of NK cells. Compared with obtaining endometrial tissue through surgery and other methods, the isolation of endometrial NK cells from menstrual blood is extremely low-invasive and has no ethical controversy. At present, the methods of isolation and identification are relatively mature. This disclosure shows that menstrual blood NK cells (that is, menstrual blood NK cells, including part of the endometrium, which are more similar to endometrium than peripheral blood) can very well reflect the state of endometrial NK cells, and the proportion of menstrual blood NK cell subsets Abnormal status and abnormalities were positively correlated with the occurrence of recurrent abortion disease.

The technical problem to be solved by the present invention is to establish a method for easily and accurately predicting NK cell subsets in the endometrium. Solve the traumatic and potential iatrogenic infection problems that the detection of peripheral blood NK cells cannot reflect the state of the endometrium or directly obtain tissue from the endometrium.

The solution to the above problems is: the present invention uses the menstrual blood formed by the periodic shedding of the endometrium of women of childbearing age every month, collects the menstrual blood of women of childbearing age on the second day of menstruation with a menstrual cup, and then analyzes the NK cells in the menstrual blood with a flow cytometer. Subgroup ratio and phenotype, so as to infer the status of endometrial NK cells, can be used to judge whether pregnancy diseases such as unexplained recurrent miscarriage are related to immune factors, especially the ratio and dysfunction of endometrial NK cells.

The specific method of the present invention is to use CD56, CD3, CD16, CD49a and other monoclonal antibodies respectively or in combination to mark the menstrual blood NK cells of normal fertile populations and unexplained recurrent abortion populations. Using flow cytometry to analyze the proportion and phenotype changes of NK cell subsets in menstrual blood, it was found that compared with normal reproductive population, the proportion of CD56 ⁺ CD16 ⁺ NK cell subsets in menstrual blood of unexplained recurrent abortion population increased, The proportion of CD56 ⁺ CD49a ⁺ NK and CD49a ⁺ CD16 ^- NK cell subsets decreased, which was significantly correlated with unexplained recurrent miscarriage, which has diagnostic significance.

The present invention collects the menstrual blood of a large number of normal fertile populations and unexplained repeated abortion populations, and further analyzes NK cell CD56 ⁺ CD16 ⁺ NK cell subsets, CD56 ⁺ CD49a ⁺ NK cell subsets, CD49a ⁺ CD16 ^- NK cell subsets and/or CD49a ^- The proportion of CD16 ⁺ NK cell subsets, after statistical processing, it is concluded that the cut-off value of the proportion of NK cell subsets in menstrual blood for predicting unexplained recurrent miscarriage is: CD56 ⁺ CD3-NK≤19.4%, CD56 ⁺ CD16 ^- NK ≥39.3%, CD56 ⁺ CD49a ⁺ NK ≤ 56.8%, CD49a ⁺ CD16 ^- NK ≤ 48.2% and/or CD49a ^- CD16 ⁺ NK ≥ 33.1%, where the NK cell subsets with the highest accuracy were CD56 ⁺ CD49a ⁺ and CD49a ⁺ CD16 ^- subsets; the cut-off values of menstrual blood NK cell subsets in predicting unexplained RIF: CD49a ^- CD16 ⁺ NK cell subsets ≥ 31.7%, CD56 ⁺ CD16 ⁺ NK cell subsets ≥ 28.6%, CD56 ⁺ CD49a ⁺ NK cell subset ≤ 65.1% and/or CD49a ⁺ CD16 ^- NK cell subset ≤ 60%, preferably CD49a ^- CD16 ⁺ NK ≥ 31.7%.

Advantages and Positive Effects

The detection method in the present invention has the advantages of simple operation, and can solve the problem of trauma and potential iatrogenic infection that the peripheral blood NK cell detection cannot reflect the state of the endometrium or directly obtain tissue from the endometrium. The test results of NK cells in menstrual blood are highly similar to the state of endometrial NK cells, which can be used to accurately predict the state of NK cell subsets in the endometrium, and then can be used to diagnose unexplained recurrent miscarriage, which can be used to judge the state of endometrium , to determine the phase of embryo implantation and to provide important indicators for clinically relevant issues such as immunotherapy for endometrial NK cell abnormalities.

the term:

As used herein, "mononuclear cells" refer to cells with a single nucleus in tissues and peripheral blood, including lymphocytes and monocytes, etc., and lymphocytes include NK cells, T cells, and B cells.

As used herein, "NK cells in decidual tissue" refers to NK cells enriched in so-called decidual tissue after decidualization of the endometrium during pregnancy.

As used herein, "NK cells in menstrual blood" refers to NK cells in menstrual blood, which contain part of the endometrium and thus are more similar to the endometrium.

Description of drawings

Figure 1 shows the changes in the number of CD3 ⁺ T, CD56 ⁺ NK and CD3 ⁺ CD56 ⁺ NKT cells in decidua tissue, menstrual blood and peripheral blood of patients with RSA of unknown cause, in which Figure 1A shows CD3 ⁺ T, CD56 ⁺ NK and CD3 ⁺ CD56 ⁺ The flow cytometry results of NKT cells, Figure 1B is the statistical results of the percentage of CD3 ⁺ T, CD56 ⁺ NK and CD3 ⁺ CD56 ⁺ NKT cells in the total number of mononuclear cells.

Figure 2 shows the changes of CD56 ⁺ CD16 ⁺ NK subpopulations and CD56 ⁺ CD49a ⁺ subpopulations in decidua tissue, menstrual blood and peripheral blood of RSA patients with unknown causes, in which Fig. 2A shows CD56 ⁺ CD16 ⁺ NK cell subpopulations and CD56 ⁺ CD49a ⁺ NK The flow cytometry results of cell subgroups, Figure 2B is the statistical results of the percentage of CD56 ⁺ CD16 ⁺ NK subgroups and CD56 ⁺ CD49a ⁺ subgroups in the total NK cell numbers.

Figure 3 shows the changes of CD49 ⁺ CD16 ^- NK subpopulation, CD49-CD16 ⁺ NK subpopulation and CD49a ⁺ EMOES ⁺ subpopulation in decidua tissue, menstrual blood and peripheral blood of RSA patients with unknown causes, and Fig. 3A shows the CD49 ⁺ CD16 ^- NK subpopulation population, CD49 ^- CD16 ⁺ NK subpopulation and CD49a ⁺ EMOES ⁺ subpopulation of flow cytometry results, Figure 3B is the CD49 ⁺ CD16 ^- NK subpopulation, CD49-CD16 ⁺ NK subpopulation and CD49a ⁺ EMOES ⁺ subpopulation Statistical graph of the percentage of cell number to total NK cell number.

Figure 4 shows menstrual blood NK cells CD56 ⁺ (ie CD56 ⁺ CD3 ^- ) NK cells (Figure 4A), CD16 ⁺ (ie CD56 ⁺ CD16 ⁺ ) NK cell subsets (Figure 4B), CD56 ⁺ CD49a ⁺ NK cell subsets (Figure 4B) 4C) and CD49a ⁺ CD16 ^- NK cell subsets (Figure 4D), and the statistical working curve (roc curve) of CD49a ^- CD16 ⁺ NK cell subsets (Figure 4E), jointly predicting the occurrence of RSA, Figure 4F is the menstrual blood NK Statistics on the predictive power of cells and their subsets for recurrent miscarriage.

Figure 5 shows that the proportion of NK cells CD56 ⁺ CD16 ⁺ NK cells and the proportion of CD49a ^- CD16 ⁺ cells in the menstrual blood of RIF patients are increased compared with those of normal controls. Figure 5A is the flow cytometry results of CD56 ⁺ CD16 ⁺ NK cell subsets, CD56 ⁺ CD49 ⁺ NK cell subsets, CD49a ⁺ CD16 ^- , CD49a ^- CD16 ⁺ cell subsets; Figure 5B is CD56 ⁺ CD16 ⁺ NK cells Subpopulations, CD56 ⁺ CD49 ⁺ NK cell subpopulations, CD49a ⁺ CD16 ^- , CD49a ^- CD16 ⁺ cell subpopulations flow cytometry statistical results.

Figure 6 shows the CD49a ^- CD16 ⁺ NK cell subsets (Figure 6A), CD56 ⁺ CD49a ⁺ NK cell subsets (Figure 6B), CD49a ⁺ CD16 ^- NK cell subsets (Figure 6C), CD56 ⁺ CD16 ⁺ cells in the menstrual blood of RIF patients Statistical working curve (roc curve) of the subgroups (Fig. 6D), jointly predicting the occurrence of RIF. Figure 6E table shows CD49a ^- CD16 ⁺ NK cell subsets, CD49a ⁺ CD16 ^- NK cell subsets, CD56 ⁺ CD16 ⁺ cell subsets, CD56 ⁺ CD49a ⁺ NK cell subsets prediction accuracy, sensitivity and cut-off value ( cut-off value) of menstrual blood NK cell subsets for the prediction performance statistics of RIF.

Detailed ways

In the following, referring to the accompanying drawings, the specific embodiments of the present invention, such as the working principles of menstrual blood collection, cell enrichment, labeling and detection methods, will be further explained through the description of the embodiments.

Example 1:

The subjects were 48 women of childbearing age with an age range of 30.5±5.2, all of whom were from the First Affiliated Hospital of the University of Science and Technology of China, and 26 of them suffered from unexplained RSA (with two or more spontaneous abortion histories, and the embryo chromosomes were normal ), 11 patients suffered from unexplained RIF (embryo implantation was normal, but miscarriage occurred after implantation, and this phenomenon occurred more than 2 times), and the rest were healthy subjects. This study was approved by the Ethics Committee of the School of Life Sciences, University of Science and Technology of China.

Example 2:

Collection of menstrual blood samples: On the second day of menstruation of all female subjects of childbearing age in Example 1, use a menstrual cup to cover the outside of the cervix and place it for 3-4 hours. Carefully take out the menstrual cup from the vagina, collect about 4-5ml of menstrual blood and pour it into a 15ml centrifuge tube added with RPMI1640 medium or saline, and process it immediately or temporarily store it in a refrigerator at 4°C.

Separation of mononuclear cells from menstrual blood: the collected menstrual blood samples are filtered through a 50-mesh nylon sieve, collected into a 50ml centrifuge tube, and the sieve is washed with 1× phosphate buffered saline (PBS) to filter out large blood clots and mucus The volume of collected menstrual blood is about 30ml. After fully inverting and mixing, use a Pasteur tube or a 50ml pipette to slowly add menstrual blood to 15ml Ficoll solution. Through gradient density centrifugation, at 20°C, 600g centrifugal force, liter Mononuclear cells were isolated by centrifugation at 1°C for 20 min. Add the same volume of 1×PBS to the obtained mononuclear cell suspension and wash it once, centrifuge at 650g at 4°C for 10 minutes at liters 9 and 9, discard the supernatant, resuspend the cell pellet with 2-3ml of 1×PBS and use A cell counter was used to count the cells.

Example 3:

NK cell subtypes were detected by flow cytometry.

1. Menstrual blood NK cell labeling: for each experimenter, the mononuclear cells in the menstrual blood of each experimenter obtained from Example 2 were added to the external standard tube and the internal standard tube (1.5mlEP tube) respectively In this method, ensure that the cell concentration in each external standard tube is not less than 5×10 ⁵ cells/ml, and the cell concentration in each internal standard tube is not less than 1×10 ⁶ cells/ml. Wash once with 1×PBS, discard the supernatant, resuspend the cell pellet in 100ul of 1×PBS buffer, stain the surface molecules of T cells, NKT cells and NK cells using the following monoclonal antibodies coupled with fluorescein : CD3-BV605 (Biolegend, marking T cell marker), CD56-BV421 (marking NK cell marker), CD45-BV510 (Biolegend, marking lymphocyte marker), CD16-PP5.5 (Biolegend, marking NK subgroup ), CD49a-647 (Biolegend Company, labeled NK subpopulation), incubated at 4°C for 30 minutes in the dark. The intracellular staining sample tube was fixed with fixative solution for 30 minutes after the external standard labeling was completed, and the corresponding internal standard antibody was added for labeling at 4°C for 30 minutes. Here, a tube coupled with EOMES-PE (eBioscience company, marking NK cell transcription factor) was used. For intracellular staining with antibodies, wash once with 1×PBS and discard the supernatant, resuspend the cell pellet with 200ul 1×PBS, if the samples in the external standard tube cannot be detected in time, you need to resuspend and fix the cells with 200ul 1% paraformaldehyde before use Flow cytometry detection, the obtained results were analyzed using Flowjo software.

2. Negative control and positive control settings: Negative controls are set in two ways, respectively, blank control and isotype control to adjust the voltage and compensation and determine the position of the negative ball. The blank control means that the detected cells are not labeled with fluorescent antibodies. The labeled tubes were the same, but no fluorescent antibody was added throughout the process. The isotype control was to use immunoglobulin from the same species, the same subtype, and the same dose to replace the target fluorescent antibody. The isotype controls were CD16-PP5 (monoclonal antibody from Biolengd Company). 5-Mouse IgG1, κ; CD49a-APC-Mouse IgG1, κ; CD45-BV510-Mouse IgG1, κ; CD56-BV421-Mouse IgG1, κ; CD3-BV605-Mouse IgG1, κ; EOMES-PE-Mouse IgG1, The κ positive control uses a single-labeled tube, and the labels of each channel are as follows: CD3-FITC; CD45-PE; CD3-5.5; CD8-PCY7; CD56-APC; CD56-BV421; CD45-BV510; Compensate and determine the location of the target cell for detection.

3. Gating method: By adjusting the voltage of SSC and FSC, distinguish the position of lymphocyte group, circle the target lymphocyte group, and then circle CD45 ⁺ leukocytes, and further circle CD56 and CD3 molecules in the CD45 ⁺ cell group For NK and T cells, in the CD56 ⁺ CD3 ^- NK cell population, NK cell-related subgroups were further circled, and the expression of each molecule was determined according to the negative control and positive control.

4. Statistical method: This experiment is mainly a pairwise comparison between the normal group and the experimental group, using the method of two independent samples t-test for statistical analysis, and P<0.05 is considered statistically significant.

Example 4:

Menstrual blood NK cells can mimic the state of endometrial NK cells.

In order to verify whether menstrual blood can better characterize the status of endometrial NK cells than peripheral blood samples, we compared the peripheral blood, menstrual blood and early pregnancy uterine A sample of the lining (i.e., the decidua) was tested for changes in various immune cells.

experimental method:

1. Collection of peripheral blood samples: the collection of peripheral blood samples is carried out at the same time as the menstrual blood samples. The subjects in Example 1 took menstrual blood samples on the second day of the menstrual cycle, and at the same time, collected 5ml of their corresponding peripheral blood samples through the cubital vein negative pressure blood collection method using a heparin sodium anticoagulant tube, and mixed them upside down 8 times. Process immediately or store in a 4°C refrigerator temporarily.

Separation of NK cells and mononuclear cells from peripheral blood: add an equal volume of 1×PBS to the collected 5ml of peripheral blood for dilution, mix well and then slowly add to 5ml of Ficoll solution, the subsequent mononuclear cell separation and detection methods are the same as in Example 2 The method is the same.

2. Collection of decidua tissue: Normal decidua were obtained from healthy subjects who voluntarily chose to terminate their pregnancy.

Isolation of mononuclear cells from decidual tissue: put the decidual tissue collected from healthy subjects and patients with RSA of unknown cause in Example 1 into a cell culture dish, wash it with 1×PBS, and remove blood clots Discard the liquid, use scissors to cut the tissue to a size of 1mm ³ , add it to a 50ml centrifuge tube, add 10ml of collagenase IV with a concentration of 2mg/ml, and digest it in a shaker at 37°C and 200rpm for 40-50min to No tissue clumps were apparent to the naked eye. Filter the digested tissue suspension through a 50-mesh nylon sieve, collect it into a new 50ml centrifuge tube, wash the sieve with 1×PBS, filter out incompletely digested tissue, add 1×PBS to 50ml, and mix thoroughly by inverting , 4°C, centrifuge at 650g up to 9 down to 9 for 10min, discard the supernatant, resuspend the cell pellet with 20ml of 1×PBS and mix well, then slowly add the cell suspension to 15ml Ficoll using a Pasteur tube or a 50ml pipette On the liquid, the method of isolation and detection of a single nucleus from the decidua tissue is consistent with the method in Example 2.

Experimental results: As shown in Figure 1, the proportion of CD3 ⁺ T in menstrual blood mononuclear cells of patients with unexplained RSA increased, the proportion of CD56 ⁺ NK decreased, and the proportion of CD3 ⁺ CD56 ⁺ NKT had no significant difference, which was similar to that in aborted decidua Be consistent.

It shows that compared with normal healthy people, the proportion of decidual NK cells in patients with unexplained RSA decreased significantly, and the proportion of NK cells in their menstrual blood also decreased, which was consistent with the situation in decidual tissue, but this situation did not occur in peripheral blood, which was consistent with the decidual tissue. different. Conclusion: Compared with peripheral blood detection, menstrual blood detection can better reflect the status of decidua NK cells.

Example 5:

The changes of CD56 ⁺ CD16 ⁺ NK subpopulation and CD56 ⁺ CD49a ⁺ subpopulation in menstrual blood of RSA patients with unknown cause were consistent with those in decidua.

NK cells are multi-subgroup immune cells with obvious heterogeneity, and different subgroups perform different functions. For example, CD16 ⁺ NK cells account for a high proportion in peripheral blood NK cells, mainly performing killing functions, while the expression of CD16 in decidual NK cells is very low; CD49a is an important indicator found in our laboratory that can characterize the tissue residence characteristics of human decidual NK cells, CD49a ⁺ NK cells were almost absent in peripheral blood, while more than 85% of NK cells expressed CD49a in decidua tissue. Our previous study found that CD49a ⁺ NK cells play an important role in promoting embryonic development. Previous clinical tests generally detect NK cells without refining their subgroups, which cannot accurately reflect the true state of NK cells.

Experimental results: As shown in Figure 2, CD56 ⁺ CD16 ⁺ NK in menstrual blood of patients with unexplained RSA was significantly increased, and CD56 ⁺ CD49a ⁺ NK was significantly decreased, which was consistent with the proportion in aborted decidua. However, there was no such change in the peripheral blood test. It is further proved that the detection of menstrual blood can better reflect the abnormality of decidua NK cells than peripheral blood, and provide important support for clinical diagnosis and treatment.

Embodiment 6:

The changes of CD49a ⁺ CD16 ^- NK subpopulation, CD49a ^- CD16 ⁺ NK subpopulation and CD49a ⁺ EOMES ⁺ NK subpopulation in menstrual blood of RSA patients with unknown cause were consistent with those in decidua.

In order to better find the NK subpopulation changes that best characterize the abnormal status of patients with unexplained recurrent miscarriage, we further tried the CD49a ⁺ CD16 ^- NK subpopulation and CD49a ⁺ EOMES ⁺ subpopulation, which were found in normal pregnant decidua These are very dominant subgroups (more than 80%), but this subgroup is significantly decreased in unexplained RSA patients. We compared the menstrual blood of patients with unexplained RSA and normal controls, and found that the same subgroup was also significantly reduced. There was no such change in peripheral blood (Figure 3).

Embodiment 7:

Analysis of NK cell subsets in menstrual blood to predict the cut-off value setting of endometrial NK cell subsets.

The difference indicators on NK cells measured by flow cytometry between the normal group and the experimental group mainly involve the percentage of CD56 ⁺ CD3 ^- , CD56 ⁺ CD16 ⁺ NK, CD56 ⁺ CD49a ⁺ NK, CD49a ⁺ CD16 ^- NK, CD49a ^- CD16 ⁺ NK cells Perform ROC curve analysis. By calculating the Youden index (sensitivity ⁺ specificity -1), find out the percentage of NK cells corresponding to the maximum Youden index, which is the critical value. According to the ROC curve generated by the Graphpad statistical software, the theoretical sensitivity or specificity will be given. Using the sensitivity and specificity given by the software, calculate the Youden index = (sensitivity + specificity - 1) and find The cut-off point corresponding to the maximum Oden index is the cut-off value. It can be seen from Figure 4F that the accuracies of the above five cell types in predicting recurrent miscarriage are 80%, 76.7%, 85.7%, 82.1% and 76%, and the corresponding critical values are 19.4%, 39.3%, 56.8%, 48.32% and 33.1%, according to the previous embodiment, the cut-off value of menstrual blood NK cell subsets analysis for predicting recurrent miscarriage can be set: CD56 ⁺ CD3 ^- NK ≤ 19.4%, CD56 ⁺ CD16 ^- NK ≥ 39.3%, CD56 ⁺ CD49a ⁺ NK ≤ 56.8%, CD49a ⁺ CD16 ^- NK ≤ 48.2% and/or CD49a ^- CD16 ⁺ NK ≥ 33.1%. The NK cell subsets with the highest accuracy were CD56 ⁺ CD49a ⁺ and CD49a ⁺ CD16 ^- subsets. Therefore, it may be preferable to set the cut-off value of menstrual blood NK cell subsets analysis to predict recurrent miscarriage as: CD56 ⁺ CD49a ⁺ ≤56.8%; and/or CD49a ⁺ CD16 ^- ≤48.2 (Fig. 4).

Embodiment 8:

For the unexplained RIF patient in Example 1, the changes of various NK cell subgroups in the menstrual blood of the RIF patient were compared with those of the healthy subjects RIF patients in the control group by flow cytometry according to the method of Example 2.

As shown in Figure 5, similar to the situation of RSA patients, the ratio of CD56 ⁺ CD16 ⁺ NK subpopulations and CD49a ^- CD16 ⁺ cell subpopulations to the total NK cells in menstrual blood of unexplained RIF patients was significantly higher than that of healthy subjects in the control group. High, CD56 ⁺ CD49a ⁺ NK subpopulation, the proportion of CD49a ⁺ CD16 ^- NK subpopulation in the total NK cells was significantly lower than that of healthy subjects in the control group, showing that the unexplained recurrent miscarriage after implantation in RIF patients is also associated with NK cell abnormalities associated with the endometrial microenvironment.

Embodiment 9:

The difference indicators on NK cells measured by flow cytometry between the normal group and the experimental group of RIF patients with unknown causes mainly involve CD56 ⁺ CD49a ⁺ NK cell subsets, CD56 ⁺ CD16 ⁺ NK cell subsets, CD49a ^- CD16 ⁺ NK cell subsets , CD49a ⁺ CD16 ^- NK cell subset percentages were analyzed by ROC curve. The same method as in Example 7 was also used to count the predictive performance of menstrual blood NK cells and their subpopulations on unexplained RIF.

It can be seen from Figure 6E that the accuracy of the above four types of cells in predicting unexplained RIF is 84.0%, 84.0%, 94.7% and 74.0%, and the corresponding critical values are 65.1%, 28.6%, 31.7% and 60.0%, respectively. In the previous embodiment, the cut-off value for the analysis of menstrual blood NK cell subsets to predict unexplained RIF can be set: CD56 ⁺ CD49a ⁺ NK cell subsets ≤ 65.1%, CD56 ⁺ CD16 ⁺ NK cell subsets ≥28.6%, and/or CD49a ^{+ CD16-} NK ≤60% and/or CD49a ^- CD16 ⁺ NK cell subsets ≥31.7%. Among them, the NK cell subset with the highest accuracy is the CD49a ^- CD16 ⁺ NK cell subset. Therefore, the cut-off value of menstrual blood NK cell subset analysis for predicting unexplained RIF can be preferably set as: CD49a ⁻ CD16 ⁺ NK cell subset ≥ 31.7%.

Claims (6)

1. Capable of quantifying CD56 ⁺ CD49a ⁺ Use of an agent of NK cell subpopulations in the preparation of a kit for predicting a subject's risk of recurrent abortion for unknown reasons by menstrual blood;

wherein the recurrent abortion of unknown origin is recurrent abortion of unknown origin and recurrent implantation failure of unknown origin.

2. Capable of quantifying CD56 ⁺ CD49a ⁺ NK cell subpopulation reagent and method for quantifying CD56 ⁺ CD16 ⁺ NK cell subset, CD49a ⁺ CD16-NK cell subset and/or CD49a ^- CD16 ⁺ Use of a combination of reagents of NK cell subpopulations in the preparation of a kit for predicting a subject's risk of recurrent abortion of unknown origin by menstrual blood;

wherein the recurrent abortion of unknown origin is recurrent abortion of unknown origin and recurrent implantation failure of unknown origin.

3. The use according to claim 1 or 2, wherein the agent is used to quantify the proportion of NK cell subsets in menstrual blood to total NK cells of menstrual blood in a subject.

4. The use of claim 1 or 2, wherein the agent is an antibody.

5. The use of claim 4, wherein the antibody is conjugated with fluorescein.

6. The use according to claim 3, wherein the proportion of menstrual blood NK cell subpopulations is detected by flow cytometry.

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