CN115323050A - Marker and method for predicting pregnancy placenta dysfunction of recurrent abortion population - Google Patents
Marker and method for predicting pregnancy placenta dysfunction of recurrent abortion population Download PDFInfo
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Abstract
The invention discloses a marker and a method for predicting gestational placental dysfunction of recurrent abortion people, wherein the level of PLGF in plasma is used as a sensitive biomarker for predicting gestational placental dysfunction of recurrent abortion people; firstly, separating platelets in plasma of a URSA patient by using a differential centrifugation method, then performing treatment digestion of nuclear genome DNA interference on platelet precipitates by using DNA restriction enzymes, directly performing bisulfite treatment on digestion products, and detecting the methylation level of the respiratory chain related gene locus of platelet mtDNA by combining a pyrosequencing method; the invention has the advantages that: methylation level detection is carried out by selecting mitochondrial loci related to URSA, and the diagnostic efficacy of the biomarker is evaluated by using AUC, so that the prediction capability of the risk of placental dysfunction is improved.
Description
Technical Field
The invention relates to a biomarker, in particular to a marker and a method for predicting placental dysfunction in pregnancy of recurrent abortion people, and belongs to the field of biomarkers.
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. The cause of abortion in 40% -50% of patients is unknown, and is called recurrent abortion (URSA).
Many studies have shown that placental dysfunction during pregnancy is also one of the important factors leading to URSA. The placenta is an important channel connecting the pregnant woman and the fetus, and is a main site for gas exchange, nutrient absorption, and metabolite excretion between the mother and the fetus. Placental growth factor (PLGF) is one of the important angiogenic factors secreted by the placenta, and has the effects of helping the placenta maintain a normal blood supply, inducing proliferation of fetal vascular endothelial cells, and promoting angiogenesis. When the placenta function is abnormal and the PLGF level is lower than the normal physiological range, serious pregnancy complications such as pregnancy hypertension, preeclampsia, fetal growth restriction, premature delivery, abortion and the like are easily caused. Therefore, the PLGF level in the plasma is probably a sensitive biomarker of RSA caused by the placenta dysfunction in pregnancy and a new target point and target for future intervention treatment. In recent years, the probability of RSA is increased year by year under the influence of factors of placental dysfunction during pregnancy, and the increase of the RSA probability has adverse effect on female pregnancy. Therefore, it is an urgent need in the present society to diagnose the placental dysfunction caused by RSA in an effective manner as soon as possible, so as to implement effective intervention, improve pregnancy rate, improve pregnancy outcome, and avoid RSA.
Many URSA caused by placenta dysfunction have difficulty in defining the specific pathogenesis in clinic, 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 pathological state of pregnancy is often accompanied by the increase of oxidative stress response, and mitochondria are also the target of oxidative stress damage as the main place for the generation of active oxygen in cells. When the placenta function is abnormal in the pregnancy, the placenta of the pregnant woman generates oxidative stress, so that mitochondria of the pregnant woman are easy to damage.
Therefore, analyzing the correlation between the methylation level of the key sites of the platelet mitochondrial DNA (mtDNA) and the fetal disc 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 placental dysfunction, and taking the sensitive CpG sites as a potential biomarker, the method is beneficial to the occurrence risk prediction, diagnosis and individualized treatment of the placental dysfunction events during pregnancy. However, until now, no relevant population research and exploration is found, and corresponding population research data is lacked. There is therefore a great need to determine CpG sites and methylation levels of placental dysfunction in the URSA population.
The identification of high risk group with placental dysfunction in pregnancy at early stage and the adoption of targeted treatment measures are the key to reduce URSA and obtain 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 comprehensive evaluation indexes for evaluating the accuracy of the prediction effect, and has more accurate prediction efficiency. Prediction of the methylation level of a mitochondrial site 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 placental dysfunction during pregnancy. Epigenetic techniques are combined with epidemiological approaches to assess an individual's risk of developing a disease and, based thereon, to take earlier or stronger intervention to benefit the patient. The capability of disease risk prediction can be effectively improved.
Disclosure of Invention
The invention aims to design a marker and a method for predicting placental dysfunction of recurrent abortion population in pregnancy, and the diagnosis efficiency of the biomarker is evaluated by using AUC (AUC), so that the prediction capability of the risk of placental dysfunction is improved.
The technical scheme of the invention is as follows:
a marker for predicting placental dysfunction in pregnancy in a population with recurrent abortion, wherein the biomarker is sensitive biomarker for predicting placental dysfunction in pregnancy in a population with recurrent abortion, and the level of PLGF in plasma is used as the sensitive biomarker.
A method for predicting placental dysfunction in pregnancy of recurrent abortion population comprises collecting 5ml of fasting venous blood of pregnant woman by vacuum blood taking needle venipuncture, centrifuging at 3000r/min for 10min, collecting upper layer serum, and determining PLGF level by double antibody enzyme-linked immunosorbent assay. The method simultaneously utilizes an epigenetic method, firstly, the differential centrifugation method is used for separating the blood platelets in the blood plasma of a URSA patient, then DNA restriction enzyme is used for carrying out treatment and digestion of nuclear genome DNA interference on the blood platelet sediment, then the digestion product is directly treated by bisulfite, and the methylation level of the respiratory chain related gene sites MT-ATP8, MT-COX1, MT-COX2, MT-COX3, MT-ND5 and MT-TRNA of the blood platelets is detected by combining a pyrosequencing method; pyrosequencing can rapidly detect methylation levels so as to accurately qualitatively and quantitatively detect methylation sites related to the PLGF level in plasma.
Compared with the prior art for diagnosing recurrent abortion, the diagnosis method is more efficient and quicker. By detecting the level of PLGF in plasma, recurrent abortion caused by abnormal placenta function in pregnancy can be predicted more timely, so that pregnancy outcome is improved. Meanwhile, a new idea and a new target point are provided for future clinical intervention and treatment.
The invention has the beneficial effects that: methylation level detection is carried out by selecting mitochondrial loci 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 placenta dysfunction is improved.
The invention is further illustrated by the following figures and examples.
Drawings
FIG. 1 is a schematic structural diagram of the prediction of placental dysfunction during pregnancy by using conventional risk factors according to an embodiment of the present invention;
FIG. 2 is a graph of placental dysfunction during pregnancy predicted by the combined sites of COX1p1+ COX1p2+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p 2;
FIG. 3 is a graph of the prediction of placental dysfunction during pregnancy at the combined sites COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ TRNAp1+ TRNAp2 according to the example of the invention;
FIG. 4 is a graph showing the prediction of placental dysfunction during pregnancy at the combined site of ATP8p1+ ATP8p2+ COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 according to an embodiment of the invention;
FIG. 5 is a graph of the COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 combination sites of the example of the invention predicting pregnancy; stage placental dysfunction;
FIG. 6 shows the prediction of placental dysfunction during pregnancy at the COX2p1+ COX2p2+ ND5p1+ ND5p2 combined site for example of the invention;
FIG. 7 shows the prediction of placental dysfunction during pregnancy by the combination of conventional risk factors with COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p2 combined sites according to an embodiment of the invention;
FIG. 8 shows that the placenta dysfunction during pregnancy is predicted by combining traditional risk factors with COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COXp2+ TRNAp1+ TRNAp2 combined sites according to an embodiment of the invention;
FIG. 9 shows that the conventional risk factor of the embodiment of the present invention can predict placental dysfunction during pregnancy by combining the site of ATP8p1+ ATP8p2+ COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COXp 2;
FIG. 10 is a graph showing the prediction of placental dysfunction during pregnancy by the combination of the conventional risk factors and the combined sites COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COXp2 in an embodiment of the invention;
FIG. 11 is a graph showing the prediction of placental dysfunction during pregnancy for a combination of conventional risk factors and COX2p1+ COX2p2+ ND5p1+ ND5p2 combined sites in an embodiment of the invention.
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.
Example 1
As shown in figures 1-11, a marker and method for predicting placental dysfunction in pregnancy of recurrent abortion population.
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 platelets in plasma by using a differential centrifugation method, then treating and digesting a platelet sediment 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 genes (MT-ATP 8, MT-COX1, MT-COX2, MT-COX3, MT-ND5 and MT-TRNA) sites in platelet mtDNA by combining a Pyrosequencing method (Pyrosequencing).
1. Study population and inclusion criteria
The study object is nest type case control population of recurrent abortion (URSA) of unknown cause, and is from the natural population cohort of ELEFANT in Tianjin City, which is established by cooperation of Tianjin medical university, total Hospital of Tianjin medical university and Tianjin City family planning research institute. The study protocol was approved by the local research institutes ethical committee and all participants 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 stains 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 normal, ovulation normal, thyroid gland function normal, diabetes mellitus, polycystic ovary syndrome. (5) Phospholipid antibody spectrum, antinuclear antibody spectrum, lupus anticoagulant detection are normal, and thyroid antibody is negative. (6) The detection of the prothrombotic state (blood coagulation function, homocysteine and protein C/S) has no abnormality. (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 criteria of risk groups, 141 participants with complete data on both follow-up data and the methylation level of the platelet mtDNA locus were selected in the analysis. After baseline placental dysfunction was excluded, the PLGF indices of 72 subjects were changed by follow-up, resulting in placental dysfunction during pregnancy.
2. Method for measuring placenta function abnormality index
Collecting fasting venous blood by vacuum blood taking needle venipuncture, centrifuging at 3000r/min for 10min, collecting upper layer serum, and determining PLGF level by double antibody enzyme-linked immunosorbent assay (ELISA). Blood sample determinations were performed at the clinical laboratory center of the department of obstetrics and gynecology genetics, general Hospital, tianjin medical university. The genetic chamber is established in 1970, is one of laboratories for carrying out cytogenetic detection in China at the earliest, and researches and clinical work related to RSA placenta dysfunction factors are carried out from 2000.
The diagnosis criteria for placental dysfunction were: 5-15 weeks (32.00 pg/mL), 16-20 weeks (60.00 pg/mL), and 20 weeks later (100.00 pg/mL). The standard of diagnosis refers to "teaching materials of gynaecology and obstetrics 8 th edition".
3. Platelet mtDNA site selection and prediction model grouping
Platelet mtDNA sites related to placental dysfunction (PLGF levels) during pregnancy, namely ATP8, COX1, COX2, COX3, ND5, TRNA sites I, II and mean methylation levels of the sites thereof, are selected, and methylation levels of the corresponding sites are detected by a pyrosequencing method. And then, obtaining the prediction effect of the single site or the plurality of combined sites of the platelet mtDNA on the placental dysfunction in the pregnancy by utilizing the Area (AUC) under the ROC curve, and establishing a placental dysfunction prediction model group by combining the traditional risk factors according to the accuracy of the prediction efficiency and considering the economic cost.
4. Statistical method
And (3) evaluating the relevance of the single sites of the platelet mtDNA and the occurrence of the pregnancy placental dysfunction event by using logistic regression analysis, and establishing a prediction model group by utilizing the area under the ROC curve (AUC) and combining traditional risk factors so as to evaluate the accuracy of different platelet mtDNA sites on the placental dysfunction prediction.
5. Results of the study
1. Basic characteristics of the study population
The baseline profile of 141 URSA subjects selected in this study is shown in Table 1, where the mean level of PLGF was 68.78. + -. 44.17 at 5-15 weeks gestational time, all within the normal diagnostic range, and no placental dysfunction occurred during pregnancy.
2. Relationship between single platelet mtDNA locus and pregnancy placental dysfunction
In the following population, a total of 18 sites (ATP 8p1, ATP8p2, ATP8, COX1p1, COX1p2, COX1, COX2p2, COX2p1, COX2p2, COX3p1, COX3, ND5p1, ND5p2, ND5, TRNAp1, TRNAp2, TRNA) were involved, as indicated in table 2 for individual platelet mtDNA sites associated with placental dysfunction. The result shows that the placental abnormality in pregnancy has no significant statistical significance (p is more than 0.05) compared with the single platelet mtDNA locus.
3. Prediction of placental dysfunction during pregnancy by combination of traditional risk factors and multiple platelet mtDNA loci
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 placental dysfunction, so that the prediction ability can be significantly increased by combining a plurality of platelet mtDNA sites and combining the area under the ROC curve (AUC). AUC is one of comprehensive evaluation indexes of prediction effect accuracy, generally AUC is used to represent general grading standard, lower prediction efficiency is represented as 0.5-straw AUC less than or equal to 0.7, medium prediction efficiency is represented as 0.7-straw AUC less than or equal to 0.9, and higher prediction efficiency is represented as AUC > 0.9.
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 placenta abnormality in 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 conventional risk factors predict placental abnormality AUC during pregnancy to be 0.5549 (0.5-straw AUC ≦ 0.7), indicating that the prediction efficiency is low.
2) Multiple platelet mtDNA locus association
As shown in tables 3.2-3.6 below, placental dysfunction during pregnancy was not statistically significant (p > 0.05) with the following 5 groups of associated platelet mtDNA sites.
Group 1:
COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ ND5p1+ ND5p2 combination sites predicted placental dysfunction AUC to be 0.8357 (0.7 and AUC ≦ 0.9), indicating that it was predicted to be moderately potent.
Group 2:
the combined sites of COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2+ TRNAp1+ TRNA2 predicted placental dysfunction AUC to be 0.7286 (0.7 Ap AUC ≦ 0.9), indicating moderate predicted potency.
Group 3:
the combined site of ATP8p1+ ATP8p2+ + COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 predicts a placental dysfunction AUC of 0.7000 (0.5 Ap AUC ≦ 0.7), indicating that it is less predictive of potency.
Group 4:
the COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 combined site predicted placental dysfunction AUC to be 0.7000 (0.5 straw AUC ≦ 0.7), indicating that it was predicted to be less potent.
Group 5:
the combined site of COX2p1+ COX2p2+ ND5p1+ ND5p2 predicted placental dysfunction AUC was 0.7286 (0.7-Ap ≦ 0.9), indicating moderate prediction potency.
3) Traditional risk factor binding platelet mtDNA combined site
The above results indicate that the efficacy of predicting placental dysfunction during pregnancy by only relying on a single traditional risk factor or platelet mtDNA locus is moderate or even low. Therefore, the traditional risk factors are combined with the platelet mtDNA combined site to jointly predict the risk of occurrence of placental dysfunction in pregnancy.
As shown in the following tables 3.7-3.11, the pregnancy placental dysfunction and the traditional risk factor combined with the platelet mtDNA combined site have no significant statistical significance (p is more than 0.05).
Group 1:
traditional risk factors combine COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2
The AUC of the + ND5p1+ ND5p2 combined locus prediction pregnancy placental dysfunction is 0.9214 (AUC > 0.9), which indicates that the prediction efficiency is higher.
Group 2:
traditional risk factors combine COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2
The AUC of the + TRNAp1+ TRNAp2 combined site prediction pregnancy placental dysfunction is 0.8571 (0.7-straw AUC is less than or equal to 0.9), which indicates that the prediction potency is moderate.
Group 3:
traditional risk factors binding ATP8p1+ ATP8p2+ COX1p1+ COX1p2+ COX2p1+ COX2p2
The AUC of + COX3p1+ COX3p2 combined site prediction pregnancy placental dysfunction is 0.8500 (0.7-Ap AUC ≦ 0.9), indicating that the prediction potency is moderate.
Group 4:
the conventional risk factors are combined with COX1p1+ COX1p2+ COX2p1+ COX2p2+ COX3p1+ COX3p2 combined sites to predict placenta dysfunction AUC during pregnancy to be 0.8357 (0.7 straw AUC is less than or equal to 0.9), which indicates that the predicted efficacy is moderate.
Group 5:
the combination of the traditional risk factors and COX2p1+ COX2p2+ ND5p1+ ND5p2 combined sites predicts the placenta dysfunction AUC of 0.9143 (AUC > 0.9) during pregnancy, indicating that the prediction efficiency is higher.
4. Pregnancy placental dysfunction 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 suggested that the conventional factors are combined with the COX1+ COX2+ COX3+ ND5 combined site to predict the placental dysfunction during pregnancy, such as the COX1+ COX2+ COX3+ ND5 combined site to predict the placental dysfunction during pregnancy in consideration of the predicted potency and economic cost.
Claims (3)
1. The marker for predicting the placental dysfunction in pregnancy of recurrent abortion population is characterized in that: the biomarker is a sensitive biomarker for predicting placental dysfunction in pregnancy of a recurrent abortion population by taking the level of PLGF in plasma as a marker.
2. A method for predicting placental dysfunction during pregnancy in a population with recurrent pregnancy loss, comprising: the method adopts an epigenetic method, firstly, the differential centrifugation method is used for separating the blood platelets in the plasma of a URSA patient, then DNA restriction enzyme is used for carrying out nuclear genome DNA interference treatment and digestion on the blood platelet sediment, then the digestion product is directly treated by bisulfite, and the methylation levels of the respiratory chain related gene sites MT-ATP8, MT-COX1, MT-COX2, MT-COX3, MT-ND5 and MT-TRNA of the blood platelet are detected by combining the pyrosequencing method.
3. The method of claim 2 for predicting placental dysfunction during pregnancy in a population with recurrent pregnancy loss, wherein: the method comprises the steps of collecting 5ml of fasting venous blood of a pregnant woman by utilizing vacuum blood taking needle venipuncture, centrifuging at a speed of 3000r/min for 10min, taking upper-layer serum after centrifugation, and determining the PLGF level by adopting a double-antibody enzyme-linked immunosorbent assay method.
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