CN114574572A

CN114574572A - Method for predicting immune dysfunction of recurrent abortion population in pregnancy

Info

Publication number: CN114574572A
Application number: CN202210234646.0A
Authority: CN
Inventors: 郭丽琼; 张颖; 刘子泉; 刘琪思婧; 于媛媛
Original assignee: Wenzhou Safety Emergency Research Institute Of Tianjin University
Current assignee: Wenzhou Safety Emergency Research Institute Of Tianjin University
Priority date: 2022-03-10
Filing date: 2022-03-10
Publication date: 2022-06-03

Abstract

The invention discloses a method for predicting the immune dysfunction of recurrent abortion people in pregnancy, which comprises the steps of separating blood platelets in plasma of a URSA patient by using a differential centrifugation method, then carrying out nuclear genome DNA interference treatment digestion on the blood platelet sediment by using DNA restriction endonuclease, then directly carrying out bisulfite treatment on a digestion product, and detecting the methylation levels of respiratory chain related gene loci including MT-ATP8, MT-COX1, MT-COX2, MT-3, MT-ND5 and MT-TRNA in platelet mtDNA by combining a pyrosequencing method; the invention has the advantages that: the methylation level detection is carried out by selecting the mitochondrial site related to URSA, and the diagnosis efficiency of the biomarker is evaluated by using AUC, so that the prediction capability of the risk of the abnormal coagulation function is improved.

Description

Method for predicting immune dysfunction of recurrent abortion population in pregnancy

Technical Field

The invention relates to a method for predicting immune dysfunction, in particular to a method for predicting immune dysfunction in pregnancy of recurrent abortion people, and belongs to the field of genetics.

Background

Spontaneous abortion accounts for 15% -40% of the incidence rate of all pregnancies, and spontaneous abortion for 2 or more consecutive times is called Recurrent Spontaneous Abortion (RSA), and causes RSA in complex and various forms, mainly including genetic factors, infectious factors, endocrine factors, anatomical factors, immune factors, blood coagulation factors, and the like. 40% -50% of patients have unknown abortion reasons, and are called recurrent abortion (URSA). Blood coagulation dysfunction is thought to be the leading cause of RSA. Studies have shown that nearly 55% to 62% of patients with poor pregnancy outcome exhibit abnormal clotting function, usually in a hypercoagulable state of blood, an increased risk of thrombosis, and the like. The blood coagulation function can affect the whole body and placenta blood circulation state, when the blood coagulation function index exceeds the normal physiological range, the placenta ischemia and hypoxia can be caused, the occurrence rate of abortion, fetal distress and the like can be improved, and the pregnancy safety and pregnancy outcome can be affected. The levels of D-Dimer (D-Dimer), protein s (ps), protein c (pc) and blood Homocysteine (HCY) in plasma are RSA-independent risk factors caused by coagulation dysfunction and targets for therapeutic intervention. In recent years, the probability of RSA has been increasing year by year under the mutual influence of various blood coagulation factors, and the increase in RSA probability has an adverse effect on female pregnancy. Therefore, it is an urgent need in the present society to diagnose the blood coagulation factors causing RSA as early as possible, monitor the relevant index of blood coagulation function closely, and intervene if necessary, to maintain the normal blood coagulation function and avoid the occurrence of RSA.

Many URSA caused by immune factors have difficulty in defining the specific pathogenesis clinically, and are usually the result of long-term interaction among multiple genes or between multiple genes and environmental factors. Mitochondria play an important role in maintaining intracellular energy homeostasis and participate in a variety of important biological processes such as oxidative phosphorylation, Reactive Oxygen Species (ROS) regulation, calcium ion homeostasis, signal transduction, and the like. The immune function of the body is closely related to the mitochondria. Therefore, analyzing the correlation between the methylation level of the key sites of the platelet mitochondrial DNA (mtDNA) and the immune dysfunction in the URSA population, defining the sensitive CpG sites in the platelet mtDNA which have the prediction effect on the occurrence risk of the immune dysfunction, and taking the sensitive CpG sites as a potential biomarker, the method is beneficial to the disease risk prediction, diagnosis and individualized treatment of the immune dysfunction event during pregnancy. However, no relevant population research and exploration is found so far, and corresponding population research data is lacked. There is therefore a great need to determine the CpG sites and the methylation levels of immune dysfunction in the population of URSA.

Disclosure of Invention

The invention aims to design a method for predicting immune dysfunction of recurrent abortion people in pregnancy, wherein methylation level detection is carried out by selecting mitochondrial loci related to URSA, and AUC is used for evaluating the diagnostic efficiency of biomarkers, so that the capability of predicting the risk of immune dysfunction is improved.

The technical scheme of the invention is as follows:

a method for predicting immune dysfunction in pregnancy in a population with recurrent abortion, comprising the steps of:

step (1): separating platelets in plasma of a URSA patient by using a differential centrifugation method, then treating and digesting the platelet precipitate by using DNA restriction enzyme through nuclear genome DNA interference, then directly carrying out bisulfite treatment on a digestion product, and detecting the methylation level of respiratory chain related gene loci including MT-ATP8, MT-COX1, MT-COX2, MT-COX3, MT-ND5 and MT-TRNA in platelet mtDNA by combining a pyrosequencing method;

the pyrosequencing method is a new sequence analysis method, and can be used for rapidly detecting the methylation frequency and accurately qualitatively and quantitatively detecting methylation sites in a sample.

The present invention by accurately quantifying methylation frequency at a single, continuous CpG site, pyrosequencing itself can detect and quantify subtle changes in methylation levels. During the sequence extension process, the C-T ratio of a single site is quantitatively determined according to the incorporation amount of C and T. Thus, changes in the methylation rate of different CpG sites can be accurately detected. Methylation status can be presented in sequence form as pyrosequencing provides authentic sequence data. The standard procedure is used for operation and detection, and the average value is obtained by repeating twice on all sample detection values. And (3) evaluating the relation between the single sites of the platelet mtDNA and the onset risk of the immune dysfunction by using logistic regression analysis, calculating the relative risk degree OR of the dangerous methylation sites, and predicting the relation between the methylation level of the platelet mtDNA sites and the immune dysfunction by utilizing the area AUC under the ROC curve. Combining traditional factors, establishing prediction model groups according to the results of single sites or a plurality of combined sites, and screening the platelet mtDNA sites capable of accurately predicting the immune dysfunction in pregnancy.

In addition, the inclusion criteria for the risk population of URSA are: (1) history of fetal loss (identity concomitance) prior to 28 weeks of 2/more pregnancies; (2) the karyotype of the peripheral blood chromosome of both couples is normal; (3) the anatomical deformity of the genital duct is confirmed by gynecology, B-ultrasonic, hysteroscope, hysterosalpingography and the like; (4) no abnormal endocrine function including menstrual cycle normality, ovulation normality, thyroid gland function normality, non-diabetes, polycystic ovary syndrome; (5) the phospholipid antibody spectrum, the antinuclear antibody spectrum and the lupus anticoagulant detection are normal, and the thyroid antibody is negative; (6) the detection of the prothrombotic state comprises that the blood coagulation function, the homocysteine and the protein C/S are not abnormal; (7) the pathogenic microorganism related to genital tract infection is detected as negative, and genital tract lesion and infection are absent; (8) has no acute and chronic diseases of heart, liver and kidney, and no infectious diseases.

Further, the measurement method of the immunological abnormality index comprises the following steps: venous blood was collected by venipuncture with a vacuum blood collection needle, and the LA and ANA levels were measured.

Further, LA in blood plasma is detected by adopting a fully automatic blood coagulation analyzer and matching reagents thereof to dilute Vipera venom time and silica coagulation time, and ANA is detected by adopting an indirect immunofluorescence method. The diagnosis index of the immune dysfunction is as follows: LA positive and/or ANA positive.

Step (2) platelet mtDNA site selection and prediction model grouping: selecting platelet mtDNA sites related to immune dysfunction in pregnancy, including LA and ANA, including ATP8, COX1, COX2, COX3, ND5, TRNA, site one, site two and average methylation level of sites. The methylation level of the corresponding site was first detected by pyrosequencing. Then, obtaining the prediction effect of a single site or a plurality of combined sites of the platelet mtDNA on the immune dysfunction in the pregnancy by utilizing the area AUC under the ROC curve, and establishing immune dysfunction prediction model groups by combining traditional factors according to the accuracy of prediction efficiency and considering the economic cost;

step (3), counting: and (3) evaluating the relevance of the single site of the platelet mtDNA and the occurrence of the pregnancy clotting dysfunction event by using logistic regression analysis, and establishing a prediction model group by using the area AUC under the ROC curve and combining the traditional risk factors so as to evaluate the accuracy of different platelet mtDNA sites on the clotting dysfunction prediction.

Early identification of high risk population with immune dysfunction during pregnancy and adoption of targeted treatment measures are key to reducing URSA occurrence and obtaining healthy pregnancy outcome. Risk prediction can provide referenceable information for disease prevention, government decision making, and evaluation of the efficacy of health intervention programs, as well as clinical treatment. The area under the ROC curve (AUC) is one of the comprehensive evaluation indexes for evaluating the accuracy of the prediction effect, and has more accurate prediction efficiency. Prediction of the methylation level of mitochondrial sites using AUC was used to determine whether the methylation level of the relevant site in platelet mtDNA could be used as a marker for predicting immune dysfunction during pregnancy. The epigenetic technology is combined with an epidemiological method to evaluate the individual risk of disease, and earlier or stronger intervention measures are taken according to the individual risk of disease, so that patients benefit from the individual risk of disease, and the disease risk prediction capability is effectively improved.

The invention has the beneficial effects that: the methylation level detection is carried out by selecting the mitochondrial loci related to URSA, and the diagnosis efficiency of the biomarker is evaluated by using AUC, so that the prediction capability of the incidence risk of the immune dysfunction is improved.

The invention is further illustrated by the following figures and examples.

FIG. 1 is a graph of the prediction of pregnancy clotting dysfunction for conventional risk factors;

FIG. 2 is a graph of immune dysfunction at the combined site of ATP8p1+ ATP8p2+ + COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p2+ TRNAp1+ TRNAp2 predicted for pregnancy;

FIG. 3 is a graph of the immune dysfunction at the joint site prediction of COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1ND5p2 during pregnancy;

FIG. 4 is a graph of immune dysfunction at COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ TRNAp1+ TRNAp2 combined with site prediction during pregnancy;

FIG. 5 is a graph of the immune dysfunction at the combined site of ATP8p1+ ATP8p2+ COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 predicted during pregnancy;

FIG. 6 is a graph of the predicted immune dysfunction at the combined site COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 during pregnancy;

FIG. 7 is a graph of COX2p1+ COX2p2+ ND5p1+ ND5p2 combined site predicted immune dysfunction during pregnancy;

FIG. 8 is a graph of the prediction of immune dysfunction during pregnancy for the combination of conventional risk factors binding ATP8p1+ ATP8p2+ COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p2+ TRNAp1+ TRNAp2 at the site;

FIG. 9 is a graph of the prediction of immune dysfunction during pregnancy for the combination of a traditional risk factor with COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p2 combined sites;

FIG. 10 is a graph of the prediction of immune dysfunction during pregnancy for the combination of a traditional risk factor with COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ TRNAp1+ TRNAp2 at the combined site;

FIG. 11 is a graph of the combined site predictive pregnancy immunological abnormality of a traditional risk factor binding ATP8p1+ ATP8p2+ COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p 2;

FIG. 12 is a graph of the prediction of immune dysfunction during pregnancy at the combined COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 sites;

FIG. 13 is a graph of the prediction of immune dysfunction during pregnancy for the combination of a traditional risk factor with COX2p1+ COX2p2+ ND5p1+ ND5p2 combined sites.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.

Examples

A method for predicting immune dysfunction in pregnancy of recurrent abortion population comprises the following steps:

first, research experiment scheme

141 URSA patients were selected as study subjects and followed for pregnancy, pregnancy outcome and neonatal status. Subject blood samples were collected and data were collected in questionnaires for past disease history, family history, birth history, socioeconomic status, occupational/environmental exposure, psychological stress, anxiety, smoking/passive smoking/drinking, drug use, lifestyle (including exercise), URSA-related drug use (glucocorticoids, folic acid, progestational hormones, aspirin, low molecular weight heparin, etc.), sleep, and physical examination/pregnancy.

Separating blood platelets in plasma by using a differential centrifugation method, then treating and digesting the blood platelet sediment by using DNA restriction endonuclease through nuclear genome DNA interference, then directly carrying out bisulfite treatment on a digestion product, and detecting the methylation level of respiratory chain related genes (MT-ATP 8, MT-COX1, MT-COX2, MT-COX3, MT-ND5 and MT-TRNA) sites in blood platelet mtDNA by combining a Pyrosequencing method (Pyrosequencing).

Pyrosequencing technology is used as a new sequence analysis technology, and can rapidly detect the frequency of methylation and accurately, qualitatively and quantitatively detect methylation sites in a sample. By accurately quantifying the frequency of methylation at a single contiguous CpG site, pyrosequencing by itself can detect and quantify subtle changes in the level of methylation. During the sequence extension process, the C-T ratio of a single site is quantitatively determined according to the incorporation amount of C and T. Thus, changes in the methylation rate of different CpG sites can be accurately detected. Methylation status can be presented in sequence form as pyrosequencing provides authentic sequence data. The standard procedure is used for operation and detection, and the average value is obtained by repeating twice on all sample detection values. And (3) evaluating the relation between the single site of the platelet mtDNA and the onset risk of the immune dysfunction by using logistic regression analysis, calculating the relative risk (OR) of dangerous methylation sites, and predicting the relation between the methylation level of the platelet mtDNA site and the immune dysfunction by using the area under the ROC curve (AUC). Combining traditional factors, establishing prediction model groups according to the results of single sites or a plurality of combined sites, and screening the platelet mtDNA sites capable of accurately predicting the immune dysfunction in pregnancy.

Second, research population and inclusion criteria

The study object is nested case control population of unexplained recurrent abortion (URSA), and is from Tianjin medical university, Total Hospital of Tianjin medical university and ELEFANT natural population cohort in Tianjin City co-established with the planned parentage research institute. The study protocol was approved by the ethical committee of the local research institute and the participants all signed written informed consent.

Inclusion criteria for the risk population of URSA were: (1) the past 2/more than 2 pregnancies had a history of fetal loss (concomitance) before 28 weeks of gestation. (2) The karyotype of the peripheral blood chromosome of both couples is normal. (3) The anatomical deformity of the genital duct is confirmed by gynecology, B-ultrasonic, hysteroscope, hysterosalpingography and the like. (4) Endocrine function is not abnormal, including menstrual cycle, ovulation, thyroid gland function, diabetes, polycystic ovary syndrome. (5) The phospholipid antibody spectrum, the antinuclear antibody spectrum and the lupus anticoagulant detection are normal, and the thyroid antibody is negative. (6) The detection of the prothrombotic state (blood coagulation function, homocysteine and protein C/S) is not abnormal. (7) The pathogenic microorganism related to genital tract infection is detected as negative, and genital tract lesion and infection are absent. (8) Has no acute and chronic diseases of heart, liver and kidney, and no infectious diseases.

According to the inclusion standard of risk population, 141 participants with complete data of follow-up data and the methylation level of the platelet mtDNA locus are analyzed and selected. After baseline immune dysfunction is eliminated, the indexes of LA and ANA of 36 study subjects are changed respectively through follow-up, and further immune dysfunction in pregnancy is caused.

Third, measuring method for immunity abnormity index

Venous blood was collected by venipuncture with a vacuum blood collection needle, and the LA and ANA levels were measured. Blood sample determination was performed at the clinical laboratory center of the department of obstetrics and gynecology genetics at the general hospital of Tianjin medical university. The genetic room was established in 1970, is one of the earliest laboratories for carrying out cytogenetic detection in China, and researches and clinical work related to RSA immune factors are carried out from 2000.

Adopts a full-automatic blood coagulation analyzer (ACL-TOP 700) and matched reagents thereof to dilute viper venom time and silica coagulation time to detect LA in blood plasma, and ANA is determined by an indirect immunofluorescence method. The diagnosis index of the immune dysfunction is as follows: LA positive and/or ANA positive. The diagnostic standard refers to the Chinese guidelines for the diagnosis and treatment of antiphospholipid syndrome and the International Classification Standard of Sapporo.

Platelet mtDNA site selection and prediction model grouping

Platelet mtDNA sites associated with immune dysfunction (LA, ANA) during pregnancy were selected, as ATP8, COX1, COX2, COX3, ND5, site one, site two of TRNA, and the mean methylation levels of their sites, respectively. The methylation level of the corresponding site was first detected by pyrosequencing. And then, obtaining the prediction effect of the single site or the plurality of combined sites of the platelet mtDNA on the immune dysfunction in the pregnancy by utilizing the Area (AUC) under the ROC curve, and establishing immune dysfunction prediction model groups by combining the traditional factors according to the accuracy of the prediction efficiency and considering the economic cost.

Fourth, statistical method

And (3) evaluating the relevance of the single sites of the platelet mtDNA and the occurrence of the immune dysfunction event in the pregnancy by using logistic regression analysis, and establishing a prediction model group by utilizing the area under the ROC curve (AUC) and combining traditional factors so as to evaluate the accuracy of different platelet mtDNA sites on the immune dysfunction prediction.

Examples of the experiments

1 basic characteristics of the study population

2 relationship between single platelet mtDNA locus and immune dysfunction in pregnancy

In the following population, 18 sites (ATP 8p1, ATP8p2, ATP8, COX1p1, COX1p2, COX1, COX2p1, COX2p2, COX2, COX3p1, COX3p2, COX3, ND5p1, ND5p2, ND5, TRNAp1, TRNAp2, TRNA) were involved in a single platelet mtDNA site associated with immune dysfunction, as shown in table 2. The result shows that the immune abnormality in pregnancy has no significant statistical significance (p is more than 0.05) with the single platelet mtDNA locus.

3 traditional risk factors and a plurality of platelet mtDNA loci jointly predict immune dysfunction in pregnancy

From the above results, it is seen that the methylation level change of a single platelet mtDNA site has relatively weak effect on predicting pregnancy immune dysfunction, so that the prediction capability can be remarkably increased by combining a plurality of platelet mtDNA sites and combining the area under the ROC curve (AUC). AUC is one of the comprehensive evaluation indexes of the accuracy of the prediction effect, the general grading standard is usually expressed by AUC, the lower prediction efficiency is expressed as 0.5< AUC < 0.7, the medium prediction efficiency is expressed as 0.7< AUC < 0.9, and the higher prediction efficiency is expressed as AUC > 0.9.

3.1 traditional Risk factors

The common traditional risk factors mainly comprise age, pre-pregnancy BMI, pregnancy and other factors, and the result shows that the immune abnormality in the pregnancy has no significant statistical significance (p is more than 0.05) with the age, the pre-pregnancy BMI, the pregnancy and other factors. As shown in figure 1, the AUC of the immune abnormality in pregnancy predicted by the traditional risk factors is 0.6185 (0.5 < AUC ≦ 0.7), which indicates that the prediction efficiency is low.

3.2 multiple platelet mtDNA site Association

As shown in tables 3.2-3.7 below, immunological abnormalities during pregnancy had no significant statistical significance with the 6 following groups in combination with the platelet mtDNA site (p > 0.05).

Group 1:

the combined site of ATP8p1+ ATP8p2+ + COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p2+ TRNAp1+ TRNAp2 predicted that the AUC of immune dysfunction was 0.7880, indicating that the predicted potency was moderate (0.7 < AUC ≦ 0.9).

Group 2:

the COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1 combined site predicted immune dysfunction AUC of 0.7427, indicating that the predicted potency was moderate (0.7 < AUC ≦ 0.9).

Group 3:

COX1p1+COX1p2+COX2p1+COX2p2+COX3p1+COX3p2+TRNAp1+

TRNAp2 combined with site predicted immune dysfunction AUC was 0.6550, indicating that its predicted potency was lower (0.7 < AUC ≦ 0.9).

Group 4:

ATP8p1+ATP8p2+COX1p1+COX1p2+COX2p1+COX2p2+COX3p1+COX3p2

the AUC of the combined site prediction immune dysfunction is 0.7120, which shows that the prediction efficacy is moderate (0.7 < AUC ≦ 0.9).

Group 5:

the COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 combined site predicted immune dysfunction AUC of 0.6433, indicating that it predicted less potent (0.5 < AUC ≦ 0.7).

Group 6:

the COX2p1+ COX2p2+ ND5p1+ ND5p2 combined site predicted immune dysfunction AUC of 0.7061, indicating that the predicted potency is moderate (0.5 < AUC ≦ 0.7).

3.3 traditional Risk factors binding to platelet mtDNA combination sites

The above results indicate that the efficacy of predicting pregnancy immune dysfunction is moderate or even low by only relying on a single traditional risk factor or platelet mtDNA locus. Therefore, the traditional risk factors are combined with the platelet mtDNA combined site to jointly predict the risk of the immunological abnormality in pregnancy. As shown in the following tables 3.8-3.13, the conventional risk factors combined with the methylation level changes of ATP8p1+ ATP8p2+ COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p2+ TRNAp1+ TRNAp2 site, and the methylation level changes of ND5p1 site and the pregnancy and pregnancy immune function abnormality have significant statistical significance (p < 0.05), wherein the methylation level changes of ATP8p2 site (OR: 0.0195% CI: 0-0.736), ND5p1 site (OR: 0.1495% CI: 0.02-0.80) and the pregnancy (OR: 0.0795% CI: 0.01-0.53) are positively correlated with the pregnancy immune function abnormality, and the combination of the ATP8p 1p2+ COX1p1+ COX2p 3+ ND5p 3p 5p 3+ TRNAp 3p 5p 3p 5p 3p 5p 3p 5p 3p 5p 3p 5p 3p 5p 3p 5p 3p 0 p 3p 5p 0 p 3p 4 + ND5p 4 and ND5p 3p 5p 3p 5p 3.

Group 1:

the traditional risk factors combined with ATP8p1+ ATP8p2+ COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p2+ TRNAp1+ TRNAp2 combined site predicted immune dysfunction AUC at pregnancy to be 0.8787, indicating that it predicted moderate potency (0.7 < AUC ≦ 0.9).

Group 2:

the traditional risk factor combined with COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p2 combined site predicted immune dysfunction AUC during pregnancy to be 0.8319, indicating that the predicted potency was moderate (0.7 < AUC ≦ 0.9).

Group 3:

the traditional risk factors combined with COX1p1+ COX1p1+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ TRNAp1+ TRNAp2 combined sites predicted immune function AUC during pregnancy to be 0.7953, indicating that the predicted potency was moderate (0.7 < AUC ≦ 0.9).

Group 4:

the conventional risk factor node ATP8p1+ ATP8p2+ COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 combined site predicts an immune dysfunction AUC during pregnancy of 0.8216, indicating that the predicted potency is moderate (0.7 < AUC ≦ 0.9).

Group 5:

the traditional risk factors combined with COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 combined sites predicted immune dysfunction AUC during pregnancy to be 0.7924, indicating that the predicted potency was moderate (0.7 < AUC ≦ 0.9).

Group 6:

the conventional risk factor combined with COX2p1+ COX2p2+ ND5p1+ ND5p2 combined site predicts the immune dysfunction AUC during pregnancy to be 0.8085, which shows that the predicted efficacy is moderate (0.7 < AUC ≦ 0.9).

4 pregnancy immune anomaly risk prediction model grouping

From the above results, the classical risk factors (age, pre-pregnancy BMI, pregnancy) and the predicted potency of the platelet mtDNA site were grouped.

Therefore, it is preferable to predict the immunological abnormality during pregnancy by using a combination site of conventional factors combined with ATP8+ COX1+ COX2+ COX3+ ND5+ TRNA, and for example, a combination site of ATP8+ COX1+ COX2+ COX3+ ND5+ TRNA may be used to predict the immunological abnormality during pregnancy in consideration of the prediction of potency and economic cost.

Claims

1. The method for predicting the immune dysfunction in the pregnancy of the recurrent abortion population is characterized by comprising the following steps of:

step (2) platelet mtDNA site selection and prediction model grouping: selecting platelet mtDNA sites related to immune dysfunction in pregnancy, including LA and ANA, which are ATP8, COX1, COX2, COX3, ND5, first site and second site of TRNA and average methylation level of the sites; firstly, detecting the methylation level of a corresponding site by a pyrosequencing method; then, obtaining the prediction effect of a single site or a plurality of combined sites of the platelet mtDNA on the immune dysfunction in the pregnancy by utilizing the area AUC under the ROC curve, and establishing immune dysfunction prediction model groups by combining traditional factors according to the accuracy of prediction efficiency and considering the economic cost;

step (3), counting: and (3) evaluating the relevance of the single site of the platelet mtDNA and the occurrence of the coagulation dysfunction event in the pregnancy by using logistic regression analysis, and establishing a prediction model group by using the area AUC under the ROC curve and combining the traditional risk factors so as to evaluate the accuracy of different platelet mtDNA sites on the coagulation dysfunction prediction.

2. The method of claim 1, wherein the method comprises predicting immune dysfunction in the recurrent pregnancy in the miscarriage group: pyrosequencing is used as a new sequence analysis method, and can rapidly detect the frequency of methylation and accurately, qualitatively and quantitatively detect methylation sites in a sample.

3. The method of predicting immune dysfunction in pregnancy in a population with recurrent pregnancy loss according to claim 1 or 2, wherein: inclusion criteria for the risk population of URSA were: (1) the history of fetal loss before 28 weeks of more than 2/more than 2 pregnancies; (2) the karyotype of the peripheral blood chromosome of both couples is normal; (3) the anatomical deformity of the genital duct is confirmed by gynecology, B-ultrasonic, hysteroscope, hysterosalpingography and the like; (4) no abnormal endocrine function including menstrual cycle normality, ovulation normality, thyroid gland function normality, non-diabetes, polycystic ovary syndrome; (5) the phospholipid antibody spectrum, the antinuclear antibody spectrum and the lupus anticoagulant detection are normal, and the thyroid antibody is negative; (6) no abnormality is detected in the prothrombotic state; (7) the pathogenic microorganism related to genital tract infection is detected as negative, and genital tract lesion and infection are absent; (8) has no acute and chronic diseases of heart, liver and kidney, and no infectious diseases.

4. The method of predicting immune dysfunction in pregnancy in a population with recurrent pregnancy loss according to claim 1 or 2, wherein: the method for measuring the immunological abnormality indexes comprises the following steps: venous blood was collected by venipuncture with a vacuum blood collection needle, and the LA and ANA levels were measured.

5. The method of predicting immune dysfunction in pregnancy in a population with recurrent pregnancy loss according to claim 1 or 2, wherein: detecting LA in blood plasma by adopting viper venom time and silica coagulation time diluted by a complete automatic blood coagulation analyzer and a matched reagent thereof, wherein ANA is determined by adopting an indirect immunofluorescence method; the diagnosis index of the immune dysfunction is as follows: LA positive and/or ANA positive L.