CN112708653B

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

Info

Publication number: CN112708653B
Application number: CN202110108676.2A
Authority: CN
Inventors: 魏海明; 傅斌清; 童先宏; 杜祥慧; 高敏; 孙汭; 田志刚
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2020-11-26
Filing date: 2021-01-26
Publication date: 2023-06-16
Anticipated expiration: 2041-01-26
Also published as: CN112708653A

Abstract

The present invention relates to a method for predicting recurrent abortion and/or diagnosing the cause of recurrent abortion by using menstrual blood. The proportion and phenotype of the NK cell subsets of menstrual blood are analyzed by using a flow cytometer, so that the proportion change of the NK cell subsets of the menstrual blood is presumed, the proportion change of the NK cell subsets of the menstrual blood is positively related to the occurrence of recurrent abortion, and whether pregnancy diseases such as recurrent abortion caused by unknown reasons are related to immune factors, especially the proportion and dysfunction of the NK cell of the endometrium can be clinically judged.

Description

Detection method for predicting recurrent abortion and/or diagnosing cause of recurrent abortion by menstrual blood

Technical Field

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

Background

Recurrent abortion (Recurrent miscarriage) refers to abortion after multiple (usually 3 or more) embryo implantation, and its etiology is quite large, and there may be nuclear abnormality of couple chromosome, female genital tract deformity, endocrine disturbance, and systemic infection; abnormal semen, poor sperm quality, etc. in men. However, it is counted that about 50% -80% of patients with recurrent abortion have unknown etiology detected by the existing technical means, and may be closely related to immune disorder. Wherein recurrent abortion (recurrent spontaneous abortion, RSA) refers to abortion after 3 or more consecutive natural pregnancies with the same partner. Recurrent Implantation Failure (RIF) refers to failure of a woman to try In Vitro Fertilization (IVF) three times with good embryo quality, some of which are implantation failure and some of which are normal but post-implantation abortions. Many patients with RSA and RIF have multiple abortions of unknown cause, and many scholars currently consider the cause to be closely related to the immunological effect between maternal and fetal.

During pregnancy, fertilized eggs invade the endometrium of a mother through trophoblasts, and serve as a type of allograft of the mother, and the mother generates protective immune response by means of immunoregulation of maternal-fetal interface, and the fertilized eggs are received and tolerized, and meanwhile, rely on normal development of placenta and decidua to ensure survival of the fertilized eggs. Thus, defining the immune mechanism and actively correcting immune imbalance may reduce the incidence of RSA and RIF.

In maternal-fetal interface decidua tissue three months before gestation, natural killer cells (Natural killer cells, NK) account for 70% of all lymphocytes, playing a central role. Current studies show that NK cells in the decidua uteri support the invasion of trophoblasts into uterine arteries and remodelling spiral arterioles by secreting cytokines during pregnancy, to ensure adequate placental blood perfusion during pregnancy, to inhibit inflammatory cells to maintain normal maternal immune tolerance, and to secrete multipotent growth factors to maintain and promote fetal growth. NK cells in the decidua uteri have different sub-populations and perform different functions. Therefore, understanding NK cell subsets in the uterus is of great importance for diagnosis and treatment of RSA and RIF for unknown reasons.

The prior art means: 1. the results were very inaccurate by detecting the status of peripheral blood NK cells in response to the endometrial status and only detecting the proportion and number of NK cells without distinguishing subpopulations. 2. NK cells are obtained from the pre-pregnancy endometrium for detection by minimally invasive surgery, but are difficult to manipulate and cause trauma to the endometrium due to the need to obtain more endometrium tissue.

Summary of The Invention

The technical scheme of the invention is as follows:

1. use of an agent capable of quantifying at least one of the following NK cell subsets for the preparation of a kit for predicting a subject's risk of recurrent abortion by menstrual blood and/or for diagnosing whether the cause of recurrent abortion in a subject is associated with endometrial NK cell abnormalities:

CD56 ⁺ CD16 ⁺ NK cell subpopulations, CD56 ⁺ CD49a ⁺ NK cell subset, CD49a ⁺ CD16 ^- NK cell subset and/or CD49a ^- CD16 ⁺ A subpopulation of NK cells.

2. The use according to claim 1, 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.

3. The use of any one of the preceding claims, wherein the recurrent abortion includes recurrent abortion and recurrent implantation failure.

4. The use as in item 3 above, wherein CD56 when menstrual blood ⁺ CD3-NK cell subgroup is less than or equal to 19.4% and CD56 ⁺ CD16 ^- NK cell subset is more than or equal to 39.3%, CD56 ⁺ CD49a ⁺ NK cell subset is less than or equal to 56.8%, CD49a ⁺ CD16 ^- NK cell subset less than or equal to 48.2% and/or CD49a ^- CD16 ⁺ NK cell subset ≡ 33.1%, preferably CD56 ⁺ CD49a ⁺ NK cell subset less than or equal to 56.8% and/or CD49a ⁺ CD16 ^- When NK cell subset is less than or equal to 48.2%, then diagnosing the subject as having the risk of recurrent abortion or diagnosing the cause of recurrent abortion in the subject as being related to endometrial NK cell abnormality.

5. The use as in item 3 above, wherein CD49a in menstrual blood ^- CD16 ⁺ NK≥31.7％、CD56 ⁺ CD16 ⁺ NK cell subgroup is more than or equal to 28.6%, CD56 ⁺ CD49a ⁺ NK cell subset less than or equal to 65.1% and/or CD49a ⁺ CD16 ^- NK cell subsets 60% or less, preferably CD49a ^- CD16 ⁺ NK.gtoreq.31.7%, diagnosing the subject as having a risk of suffering recurrent implantation failure, or diagnosing the cause of recurrent implantation failure in the subject as being related to endometrial NK cell abnormality.

6. The use of any one of the preceding claims, wherein the agent is an antibody.

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

8. The use of any one of the preceding claims, wherein the proportion of menstrual blood NK cell subpopulations and/or the proportion of menstrual blood NK cell subpopulations is detected by flow cytometry.

9. A repeated abortion diagnostic kit comprising reagents and/or menstrual blood collectors, preferably menstrual cups, capable of quantifying at least one of the following NK cell subsets: CD56 ⁺ CD16 ⁺ NK cell subpopulations, CD56 ⁺ CD49a ⁺ NK cell subset, CD49a ⁺ CD16 ^- NK cell subset and/or CD49a ^- CD16 ⁺ A subpopulation of NK cells.

10. The kit of item 9, wherein the reagent is an antibody, preferably the antibody is conjugated with fluorescein.

11. The kit of claim 9 or 10, wherein the reagent is used to quantify the proportion of NK cell subpopulations in menstrual blood to total NK cells in menstrual blood in a subject.

12. The kit of any one of the preceding claims, wherein the recurrent abortion includes recurrent abortion and recurrent implantation failure.

13. The kit of claim 12, wherein the subject is diagnosed as having a risk of recurrent abortion or as having a cause of recurrent abortion in the subject associated with endometrial NK cell abnormalities when CD56+CD3-NK.ltoreq.19.4%, CD56+CD16-NK.ltoreq.39.3%, CD56+CD49 a+NK.ltoreq.56.8%, CD49 a+CD16-NK.ltoreq.48.2% and/or CD49 a-CD16+NK.gtoreq.33.1%, preferably CD56+CD49 a+CD16-NK.ltoreq.56.8% in menstrual blood.

14. The kit of claim 12, wherein CD49a in menstrual blood ^- CD16 ⁺ NK≥31.7％、CD56 ⁺ CD16 ⁺ NK cell subgroup is more than or equal to 28.6%, CD56 ⁺ CD49a ⁺ NK cell subset less than or equal to 65.1% and/or CD49a ⁺ CD16 ^- NK cell subsets 60% or less, preferably CD49a ^- CD16 ⁺ NK.gtoreq.31.7%, diagnosing the subject as having a risk of suffering recurrent implantation failure, or diagnosing the cause of recurrent implantation failure in the subject as being related to endometrial NK cell abnormality.

15. The kit of any one of the preceding claims, wherein the proportion of menstrual blood NK cell subpopulations and/or the proportion of menstrual blood NK cell subpopulations is detected by flow cytometry.

Detailed Description

Women of childbearing age can proliferate from about 0.5mm to about 5-7mm endometrium in each menstrual cycle under the stimulation of hormones, and with periodic withdrawal of progesterone levels, endometrium falls off to form menstrual blood containing a large number of NK cells. Compared with the method of obtaining endometrial tissues through surgery and the like, the method has extremely low invasion of endometrial NK cells separated from menstrual blood, no ethical disputes exist, and the separation and identification means are mature at present. The present disclosure shows that menstrual NK cells (i.e., menstrual NK cells containing a part of endometrial components more similar to endometrium than peripheral blood) can reflect the state of endometrial NK cells very well, and that the proportion of menstrual NK cell subpopulations and abnormal state are positively correlated with the occurrence of recurrent abortion disease.

The invention aims to solve the technical problems that: a method which is easy to operate and can accurately predict NK cell subsets in endometrium is established. Solves the problems of traumatism and potential iatrogenic infection existing in the peripheral blood NK cell detection that can not respond to the state of endometrium or directly obtain the tissue from the endometrium.

The scheme for solving the problems is as follows: according to the invention, menstrual blood formed by periodic shedding of endometrium of women of childbearing age is utilized, menstrual blood of women of childbearing age on the second day of menstruation is collected by a menstrual cup, and the proportion and phenotype of NK cell subsets of the menstrual blood are analyzed by a flow cytometry, so that the state of NK cells of the endometrium is estimated, and whether pregnancy diseases such as repeated abortion caused by unknown reasons are related to immune factors, especially the proportion and dysfunction of the NK cells of the endometrium can be clinically judged.

The specific method of the invention is to use monoclonal antibodies such as CD56, CD3, CD16, CD49a and the like to respectively or jointly label menstrual blood NK cells of normal breeding people and people with recurrent abortion caused by unknown reasons. Analysis of NK cell subpopulations in menstrual blood for proportion and phenotypic changes by flow cytometry, found CD56 in menstrual blood from persons repeatedly aborted due to unknown cause compared to normal fertility population ⁺ CD16 ⁺ NK cell subpopulation ratio is increased, CD56 ⁺ CD49a ⁺ NK、CD49a ⁺ CD16 ^- The reduced proportion of NK cell subsets has obvious correlation with repeated abortion caused by unknown reasons and has diagnostic significance.

The invention collects a large amount of menstrual blood of normal childhood population and people with repeated abortion caused by unknown reasons and further analyzes NK cell CD56 ⁺ CD16 ⁺ NK cell subpopulations, CD56 ⁺ CD49a ⁺ NK cell subset, CD49a ⁺ CD16 ^- NK cell subset and/or CD49a ^- CD16 ⁺ NK cell subset proportion, obtained by statistical treatment: the Cut-Off value of the recurrent abortion caused by unknown reasons predicted by the proportion of NK cell subsets of menstrual blood is as follows: CD56 ⁺ CD3-NK≤19.4％、CD56 ⁺ CD16 ^- NK≥39.3％，CD56 ⁺ CD49a ⁺ NK≤56.8％，CD49a ⁺ CD16 ^- NK.ltoreq.48.2% and/or CD49a ^- CD16 ⁺ NK is larger than or equal to 33.1%, wherein the NK cell subgroup with highest accuracy is CD56 ⁺ CD49a ⁺ And CD49a ⁺ CD16 ^- A sub-population; the Cut-Off value of RIF, which predicts the unknown cause of the proportion of NK cells in menstrual blood, is: CD49a ^- CD16 ⁺ NK cell subgroup not less than 31.7% and CD56 ⁺ CD16 ⁺ NK cell subgroup is more than or equal to 28.6%, CD56 ⁺ CD49a ⁺ NK cell subset less than or equal to 65.1% and/or CD49a ⁺ CD16 ^- NK cell subsets 60% or less, preferably CD49a ^- CD16 ⁺ NK≥31.7％。

Advantages and positive effects

The detection method has simple operation and can solve the problems of traumata and potential iatrogenic infection existing in the case that the detection of peripheral blood NK cells can not respond to the state of endometrium or the tissue can be directly obtained from the endometrium. The detection result of menstrual blood NK cells is highly similar to the state of endometrial NK cells, can be used for accurately predicting the state of NK cell subsets in the endometrium, can be further used for diagnosing repeated abortion caused by unknown reasons, and can provide important indexes for judging the state of the endometrium, determining the implantation time phase of an embryo, carrying out related immunotherapy aiming at the abnormal endometrial NK cells and other clinical related problems.

Terminology:

as used herein, "mononuclear cells" refers to cells having a single nucleus in tissue and peripheral blood, including lymphocytes, which in turn include NK cells, T cells, B cells, and the like.

As used herein, "NK cells in decidua tissue" refers to NK cells enriched in what is called decidua tissue after decidua of the endometrium during gestation.

As used herein, "NK cells in menstrual blood" refers to NK cells in menstrual blood that contain a portion of the endometrial components and are thus more similar to endometrium.

Drawings

FIG. 1 shows CD3 in decidua tissue, menstrual blood and peripheral blood of an RSA patient of unknown origin ⁺ T、CD56 ⁺ NK and CD3 ⁺ CD56 ⁺ Changes in the number of NKT cells, FIG. 1A is CD3 ⁺ T、CD56 ⁺ NK and CD3 ⁺ CD56 ⁺ FIG. 1B is a flow cytometry results of NKT cells showing CD3 ⁺ T、CD56 ⁺ NK and CD3 ⁺ CD56 ⁺ A graph of the statistical results of the number of NKT cells as a percentage of the total mononuclear cell number.

FIG. 2 shows CD56 in decidua tissue, menstrual blood and peripheral blood of an RSA patient of unknown origin ⁺ CD16 ⁺ NK subpopulations and CD56 ⁺ CD49a ⁺ Changes in subpopulations, wherein FIG. 2A is CD56 ⁺ CD16 ⁺ NK cell subsets and CD56 ⁺ CD49a ⁺ NK cellsFlow cytometry results for subpopulations, FIG. 2B is a CD56 ⁺ CD16 ⁺ NK subpopulations and CD56 ⁺ CD49a ⁺ Statistical results plot of cell number of subpopulations as a percentage of total NK cell number.

FIG. 3 shows CD49 in decidua tissue, menstrual blood and peripheral blood of an RSA patient of unknown origin ⁺ CD16 ^- NK subpopulations, CD49-CD16 ⁺ NK subpopulations and CD49a ⁺ EMOES ⁺ Changes in subpopulations, wherein FIG. 3A is CD49 ⁺ CD16 ^- NK subpopulations, CD49 ^- CD16 ⁺ NK subpopulations and CD49a ⁺ EMOES ⁺ Flow cytometry results for subpopulations, FIG. 3B is a CD49 ⁺ CD16 ^- NK subpopulations, CD49-CD16 ⁺ NK subpopulations and CD49a ⁺ EMOES ⁺ Statistical results plot of cell number of subpopulations as a percentage of total NK cell number.

FIG. 4 shows menstrual NK cell CD56 ⁺ (i.e., CD 56) ⁺ CD3 ^- ) NK cells (FIG. 4A), CD16 ⁺ (i.e., CD 56) ⁺ CD16 ⁺ ) NK cell subset (FIG. 4B), CD56 ⁺ CD49a ⁺ NK cell subset (FIG. 4C) and CD49a ⁺ CD16 ^- NK cell subset (FIG. 4D), and CD49a ^- CD16 ⁺ The statistical working curve (roc curve) of NK cell subpopulations (fig. 4E), combined with the prediction of RSA occurrence, fig. 4F is the predicted efficacy statistics of menstrual NK cells and subpopulations thereof on recurrent abortion.

FIG. 5 shows NK cell CD56 in menstrual blood of RIF patients ⁺ CD16 ⁺ NK cell ratio, CD49a ^- CD16 ⁺ The cell proportion is elevated compared to control normal human menstrual blood. FIG. 5A is a CD56 ⁺ CD16 ⁺ NK cell subset, CD56 ⁺ CD49 ⁺ NK cell subset, CD49a ⁺ CD16 ^- 、CD49a ^- CD16 ⁺ Flow cytometry results for cell subsets; FIG. 5B is a CD56 ⁺ CD16 ⁺ NK cell subset, CD56 ⁺ CD49 ⁺ NK cell subset, CD49a ⁺ CD16 ^- 、CD49a ^- CD16 ⁺ Flow cytometry statistics of cell subsets.

FIG. 6 shows menstrual blood CD49a of RIF patient ^- CD16 ⁺ NKCell subpopulations (fig. 6A), CD56 ⁺ CD49a ⁺ NK cell subset (FIG. 6B), CD49a ⁺ CD16 ^- NK cell subset (FIG. 6C), CD56 ⁺ CD16 ⁺ The statistical working curve (roc curve) of the cell subpopulations (fig. 6D) jointly predicts the occurrence of RIF. FIG. 6E is a table showing CD49a ^- CD16 ⁺ NK cell subset, CD49a ⁺ CD16 ^- NK cell subset, CD56 ⁺ CD16 ⁺ Cell subpopulations, CD56 ⁺ CD49a ⁺ Accuracy, sensitivity and threshold (cut-off) of prediction of NK cell subpopulations statistics of predicted potency of menstrual NK cell subpopulations against RIF.

Detailed Description

The following description of embodiments further illustrates the principles of operation of specific embodiments of the present invention, such as menstrual blood collection, cell enrichment, labeling, and detection methods.

Example 1:

subjects were 48 women of childbearing age ranging from 30.5±5.2, all from the first affiliated hospital of the university of chinese science and technology, of which 26 had RSA of unknown origin (with a history of two or more natural miscarriages and normal embryonic chromosomes), 11 had RIF of unknown origin (normal embryo implantation but miscarriages occurred after implantation and over 2 occurrences), and the rest were healthy subjects. The study is approved by ethical committee of the university of science and technology, national academy of science and technology.

Example 2:

and (3) collecting menstrual blood samples: on menstrual period day 2, all women of child bearing age in example 1 were placed outside the cervical os using menstrual cups for 3-4 hours. Carefully remove menstrual cup from vagina, collect menstrual blood volume of about 4-5ml, pour into 15ml centrifuge tube with RPMI1640 medium or normal saline, immediately process or store in fridge at 4deg.C.

Separation of menstrual blood mononuclear cells: the collected menstrual blood sample is filtered through a 50-mesh nylon sieve, collected into a 50ml centrifuge tube, washed with 1 Xphosphate buffer (PBS) to remove large blood clots and mucus, the volume of the collected menstrual blood is about 30ml, after being fully and reversely mixed, the menstrual blood is slowly added to 15ml Ficoll liquid by using a Pasteur pipe or a 50ml pipette, and mononuclear cells are separated by a gradient density centrifugation method under the conditions of 20 ℃ and 600g centrifugal force and 1-liter 0 centrifugation for 20 min. The obtained mononuclear cell suspension was washed once with 1 XPBS of the same volume, centrifuged at 650g at 4℃for 10min with 9L and 9L, the supernatant was discarded, and the cell pellet was resuspended in 2-3ml of 1 XPBS and counted by a cell counter.

Example 3:

flow cytometry detects NK cell subtypes.

1. Menstrual blood NK cell markers: for each subject, mononuclear cells in menstrual blood of each subject obtained in example 2 were added to an external standard tube and an internal standard tube (1.5 ml EP tube), respectively, to ensure that the cell concentration in each external standard tube was not less than 5X 10 ⁵ The cell concentration in each internal standard tube is not less than 1X 10 per ml ⁶ And each ml. The cell pellet was resuspended in 100ul of 1 XPBS buffer, washed once with 1 XPBS, and the T cells, NKT cells and NK cell surface molecules stained with the following monoclonal antibodies conjugated with fluorescein: CD3-BV605 (Biolegend, labeled T cell marker), CD56-BV421 (labeled NK cell marker), CD45-BV510 (Biolegend, labeled lymphocyte marker), CD16-PP5.5 (Biolegend, labeled NK subgroup), CD49a-647 (Biolegend, labeled NK subgroup) were incubated at 4℃for 30 minutes in the absence of light. The intracellular staining sample tube was fixed with a fixing solution for 30min after the completion of the labeling of the external standard, the corresponding internal standard antibody was added, labeling was performed at 4℃for 30min, the antibody coupled to EOMES-PE (eBioscience, labeled NK cell transcription factor) was used for intracellular staining, the supernatant was discarded after washing with 1 XPBS, the cell pellet was resuspended with 200ul of 1 XPBS, and if the sample in the external standard tube could not be detected in time, the fixed cells were resuspended with 200ul of 1% paraformaldehyde and detected by flow cytometry, and the obtained results were analyzed using Flowjo software.

2. Negative control, positive control settings: the negative control is set in two ways, namely, voltage regulation and compensation are respectively carried out on a blank control and a isotype control, and the position of a negative ball is determined, wherein the blank control, namely, the detected cells are not marked by fluorescent antibodies, the treatment process is consistent with that of a marking tube, but no fluorescent antibodies are added in the whole process, the isotype control adopts immunoglobulins with the same species source, the same subtype and the same dosage to replace the target fluorescent antibodies, and the isotype control is (monoclonal antibodies of Biolengd company) CD16-PP5.5-Mouse IgG1, kappa respectively; CD49a-APC-Mouse IgG1, kappa; CD45-BV510-Mouse IgG1, kappa; CD56-BV421-Mouse IgG1, kappa; CD3-BV605-Mouse IgG1, kappa; EOMES-PE-Mouse IgG1, single label tube was used for kappa positive control, and each channel was labeled as follows: CD3-FITC; CD45-PE; CD3-5.5; CD8-PCY7; CD56-APC; CD56-BV421; CD45-BV510; CD3-BV605 was used to adjust the voltage and offset and determine the detected target cell location.

3. The door setting method comprises the following steps: by regulating SSC and FSC voltages, the lymphocyte group is distinguished, and CD45 is circled after target lymphocyte group is circled ⁺ White blood cells, further in CD45 ⁺ NK and T cells are circled by CD56, CD3 molecules in the cell population, CD56 ⁺ CD3 ^- The NK cell related subgroup is further circled in the NK cell population, and the expression condition of each molecule is determined according to the negative control and the positive control.

4. Statistical methods: the experiment is mainly to compare the normal group with the experimental group in pairs, and adopts a two-independent sample t test method to carry out statistical analysis, and P is smaller than 0.05 to consider that the difference has statistical significance.

Example 4:

menstrual blood NK cells can mimic endometrial NK cell status.

To verify whether menstrual blood represents the status of endometrial NK cells better than a peripheral blood sample, we compared the peripheral blood of normal control (i.e., healthy subjects) and patients repeatedly aborted for unknown reasons, menstrual blood and endometrial (i.e., decidua) samples at the early stages of pregnancy and examined for changes in various immune cells.

The experimental method comprises the following steps:

1. peripheral blood specimen collection: the collection of peripheral blood samples is performed simultaneously with the collection of menstrual blood samples. The subject of example 1 was left with menstrual blood samples the next day of the menstrual cycle, 5ml of the corresponding peripheral blood samples were collected by negative pressure elbow vein blood sampling using heparin sodium anticoagulation tube, and after mixing for 8 times upside down, immediately processed or put in a 4 ℃ refrigerator for temporary storage.

Peripheral blood NK cell mononuclear cell isolation: the collected 5ml peripheral blood is diluted by adding 1 XPBS with equal volume, and slowly added to 5ml Ficoll liquid after fully and uniformly mixing, and the subsequent mononuclear cell separation and detection methods are consistent with those of the embodiment 2.

2. Decidua tissue collection: normal decidua is derived from healthy subjects who voluntarily choose to terminate pregnancy.

Decidua tissue mononuclear cell isolation: decidua tissue collected from healthy subjects and RSA patients of unknown origin in example 1 was placed in a cell culture dish, washed clean with 1×pbs, specimens with blood clots removed, liquid discarded, and the tissue was minced to 1mm using scissors ³ Size, add to 50ml centrifuge tube, add 10ml collagenase IV at a concentration of 2mg/ml, digest 40-50min in shaker at 37℃and 200rpm until no tissue mass is evident to the naked eye. The digested tissue suspension was filtered using a 50 mesh nylon sieve, collected into a new 50ml centrifuge tube, the screen was washed with 1 XPBS, the incompletely digested tissue was filtered off, 1 XPBS was added to 50ml, after thoroughly reversing and mixing, the supernatant was discarded by centrifugation at 650g up to 9 g down for 10min, after re-suspending the cell pellet with 20ml of 1 XPBS, the cell suspension was slowly added to 15ml Ficoll solution using a pasteur or 50ml pipette, and the subsequent decidua tissue single nucleus isolation and detection procedure was consistent with that of example 2.

Experimental results: as shown in FIG. 1, CD3 in mononuclear cells of menstrual blood from RSA patients of unknown origin ⁺ T ratio increases, CD56 ⁺ Reduced NK ratio, CD3 ⁺ CD56 ⁺ The ratio of NKT was not significantly different, and was consistent with that in decidua abortion.

It is shown that the proportion of decidua NK cells of RSA patients is significantly reduced for unknown reasons, and the proportion of menstrual blood NK cells is also reduced, which is consistent with the condition of decidua tissue, but the peripheral blood does not appear in the condition, unlike the decidua tissue. Conclusion: compared with peripheral blood detection, the detection of menstrual blood can reflect the state of decidua NK cells.

Example 5:

menstrual blood CD56 of patient with RSA caused by unknown reasons ⁺ CD16 ⁺ NK subpopulations and CD56 ⁺ CD49a ⁺ The subgroup changes were consistent with those in decidua.

NK cells are multiple sub-populations of immune cells with apparent heterogeneity, and different sub-populations perform different functions. For example CD16 ⁺ NK cells have a high ratio in peripheral blood NK, mainly perform a killing function, and CD16 expression in decidua NK is very low; CD49a is an important index for our laboratory findings that can characterize the resident characteristics of human decidua NK cell tissue, CD49a ⁺ NK cells are almost absent in peripheral blood, whereas more than 85% of NK cells express CD49a in decidua tissue. CD49a was found in our earlier study ⁺ NK cells have an important role in promoting embryonic development. The conventional clinical detection generally detects NK cells in a general way, but the subgroup of the NK cells is not refined, and the real state of the NK cells cannot be accurately reflected.

Experimental results: as shown in FIG. 2, menstrual blood CD56 of RSA patients of unknown cause ⁺ CD16 ⁺ NK is significantly elevated, CD56 ⁺ CD49a ⁺ NK was significantly reduced and consistent with the duty cycle in decidua abortus. While peripheral blood detection did not show such changes. Further proves that the detection of menstrual blood can reflect whether NK cells of decidua are abnormal or not more than peripheral blood, and provides important support for clinical diagnosis and treatment.

Example 6:

menstrual blood CD49a of patient with RSA caused by unknown reasons ⁺ CD16 ^- NK subpopulations, CD49a ^- CD16 ⁺ NK subpopulations, CD49a ⁺ EOMES ⁺ NK cell industry group changes were consistent with those in decidua.

In order to better find the best ways to characterize the NK subgroup changes of the abnormal state of patients with recurrent abortion due to unknown reasons, we further tried CD49a ⁺ CD16 ^- NK subpopulations and CD49a ⁺ EOMES ⁺ Both subgroups were highly dominant (more than 80%) in normal pregnancy decidua, but were significantly reduced in RSA patients of unknown origin. We compared the menstrual blood of RSA patients with unknown reasons with the menstrual blood of normal control, and found that the same subpopulations were alsoA significant decrease occurs. Whereas peripheral blood was unchanged (fig. 3).

Example 7:

menstrual NK cell subpopulation analysis predicts endometrial NK cell subpopulation status threshold (cut-off) value settings.

The difference index on NK cells measured by the flow of a normal group and an experimental group mainly relates to CD56 ⁺ CD3 ^- ，CD56 ⁺ CD16 ⁺ NK，CD56 ⁺ CD49a ⁺ NK，CD49a ⁺ CD16 ^- NK，CD49a ^- CD16 ⁺ NK cell percentages ROC curve analysis was performed. By calculating the about step index (sensitivity ⁺ Specificity-1), the percentage of NK cells corresponding to the maximum about dengue index is found to be the critical value. Sensitivity, namely specificity in theoretical sense is given in an ROC curve generated according to Graphpad statistical software, sensitivity and specificity given by the software are used, and Cut-off value corresponding to the maximum about dengue index is found by calculating about dengue index= (sensitivity + specificity-1). As can be seen from fig. 4F, the accuracy of predicting recurrent abortion by the above five cells is 80%, 76.7%, 85.7%, 82.1% and 76% in order, and the corresponding threshold values are 19.4%, 39.3%, 56.8%, 48.32% and 33.1%, respectively, and then the threshold value (cut-off) for predicting recurrent abortion by analyzing menstrual NK cell subpopulations can be set according to the previous embodiment: CD56 ⁺ CD3 ^- NK≤19.4％、CD56 ⁺ CD16 ^- NK≥39.3％，CD56 ⁺ CD49a ⁺ NK≤56.8％，CD49a ⁺ CD16 ^- NK.ltoreq.48.2% and/or CD49a ^- CD16 ⁺ NK is more than or equal to 33.1 percent. Wherein the NK cell subset with highest accuracy is CD56 ⁺ CD49a ⁺ And CD49a ⁺ CD16 ^- A sub-population. Thus, the critical value (cut-off) for predicting recurrent abortion by menstrual blood NK cell subpopulation analysis can be preferably set as: CD56 ⁺ CD49a ⁺ Less than or equal to 56.8 percent; and/or CD49a ⁺ CD16 ^- 48.2 (FIG. 4).

Example 8:

the changes in individual NK cell subsets in menstrual blood of RIF patients relative to RIF patients in healthy subjects of the control group were compared by flow cytometry also for RIF patients of unknown origin in example 1, as per example 2.

As shown in FIG. 5, the RIF patient's menstrual blood CD56 is of unknown origin, similar to that of the RSA patient ⁺ CD16 ⁺ NK subpopulations, CD49a ^- CD16 ⁺ The proportion of cell subsets to total NK cells is obviously increased compared with menstrual blood of healthy subjects in a control group, and CD56 ⁺ CD49a ⁺ NK subpopulations, CD49a ⁺ CD16 ^- The proportion of NK subpopulations to total NK cells was significantly reduced compared to menstrual blood of healthy subjects in the control group, showing that recurrent abortion of RIF patients after implantation was also associated with endometrial microenvironment NK cell abnormalities.

Example 9:

the difference index on NK cells measured by the normal group and the unidentified RIF patient experimental group mainly relates to CD56 ⁺ CD49a ⁺ NK cell subset, CD56 ⁺ CD16 ⁺ NK cell subset, CD49a ^- CD16 ⁺ NK cell subset, CD49a ⁺ CD16 ^- The percentage of NK cell subpopulations was analyzed by ROC curve. The same procedure as used in example 7 was used to calculate the predicted efficacy of NK cells and sub-populations thereof for RIF of unknown origin.

As can be seen from fig. 6E, the above four kinds of cell indicators of the unknown cause RIF are 84.0%, 94.7% and 74.0% in order, and the corresponding threshold values are 65.1%,28.6%,31.7% and 60.0%, respectively, so that the threshold value (cut-off) of the analysis and prediction of the NK cell subset of menstrual blood for the unknown cause RIF can be set according to the previous embodiment: CD56 ⁺ CD49a ⁺ NK cell subgroup is less than or equal to 65.1%, CD56 ⁺ CD16 ⁺ NK cell subset not less than 28.6%, and/or CD49a ⁺ CD16 ^- NK.ltoreq.60% and/or CD49a ^- CD16 ⁺ NK cell subsets were not less than 31.7%. Wherein the NK cell subset with highest accuracy is CD49a ^- CD16 ⁺ A subpopulation of NK cells. Thus, the critical value (cut-off) value of the analysis and prediction of the cause of unknown RIF of the menstrual blood NK cell subpopulation can be preferably set as: CD49a ^- CD16 ⁺ NK cell subsets were not less than 31.7%.

Claims

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;

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.