CN113791224B - Early warning method for recurrent abortion caused by unknown reasons based on follicular fluid protein expression - Google Patents
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The invention belongs to the field of reproductive medicine. The technical proposal is as follows: the technical scheme provided by the invention is as follows: an early warning method based on follicular fluid HRG and C4B protein expression level as recurrent abortion caused by unknown reasons, wherein the method is not used for disease diagnosis and treatment; the method comprises the following steps: 1) Collecting and preserving follicular fluid: picking up the collected follicular fluid at constant temperature, collecting the residual follicular fluid, centrifuging at 1500rpm to obtain supernatant, packaging, and freezing at-80deg.C; 2) C4B, HRG follicular fluid protein concentration ELISA assay: adopting ELISA detection kit of corresponding protein to detect the concentration of corresponding protein in follicular fluid; 3) And (5) carrying out result statistics and analysis. The method should have a certain predictive value for pregnancy outcome in the patients with URSA.
Description
Technical Field
The invention belongs to the field of reproductive medicine, and particularly relates to a method for early warning of patients with recurrent abortion caused by unknown reasons.
Background
In women of childbearing age, the chance of miscarriage occurs at a single pregnancy is about 15-25% and most miscarriages occur at early gestation. About 2-5% of women in childbearing age experience 2 or more abortions. Fetal loss of 3 or more times before 28 weeks of gestation is commonly referred to as recurrent abortion, and the recurrent abortion has complex etiology, and common etiology of recurrent abortion in early gestation period includes genetic factors, anatomical abnormality, infective factors, autoimmune diseases, endocrine abnormality, pre-thrombotic state and environmental factors, etc. However, more than about half of cases of RSA cannot be diagnosed with an exact etiology, recurrent abortion (unexplained recurrent spontaneous abortion, urs a), known as unknown causes, and the personalized treatment of such patients and the prevention of recurrence of abortion are currently a significant problem in clinical work of gynaecology and obstetrics science due to the unknown etiology.
Study statistics show that the risk of recurrent abortion increases with the number of past abortions: pregnant women with primary pregnancy have a miscarriage rate of 11% and women with a history of 3 and more miscarriages have a recurrent pregnancy rate increased to 40%. Therefore, the specialist in China recommends that 2 abortions occur consecutively, and the risk of the abortions occurring again is similar to that of 3 abortions, namely, the abortions should be emphasized and evaluated. However, the research on the pathogenesis of recurrent abortion caused by unknown reasons is not clear, a specific treatment means is lacked, and effective molecular markers and an evaluation system are not available for the prognosis of recurrent abortion recurrence risk in clinical work, so that the research on establishing an early warning mechanism of abortion occurrence is important for the treatment of RSA patients.
With the development of various high-throughput histology technologies in recent years, technologies such as proteomics and metabonomics play an important role in the exploration of the pathogenesis of URSA and the search of molecular markers. The use of proteomics, RNA-seq and bioinformatics analysis techniques to detect proteomic changes in aborted villi, decidua and endometrial tissues of recurrent aborted patients has been reported in the literature; however, the villus, decidua and endometrial tissue components are complex, and the large sample is difficult to collect and verify, and is difficult to be used for clinical assessment of the risk of re-pregnancy. On the other hand, a body fluid such as serum is a common sample for detection of a molecular marker, and recently, research has been reported on proteomics and metabonomics of serum samples of patients with URSA by using iTRAQ proteomics and PRM techniques and (1) H NMR techniques, and a series of potential molecular markers have been found. However, since key factors such as hormone and protein related to female reproduction are greatly affected by menstrual cycles, the sampling time of serum samples is difficult to be unified well, and the sampling time is greatly affected by other systems of the whole body, so that the verification of subsequent large samples is difficult.
Disclosure of Invention
The invention aims to overcome the defects of the background technology, and provides an early warning method for recurrent abortion based on the expression level of HRG and C4B proteins of follicular fluid, which is used as unknown reasons, based on follicular fluid proteomics research of IVF performed on recurrent abortion patients with unknown reasons in the early stage and later stage enlarged sample verification, and the method can have a certain predictive value for pregnancy ending of URSA patients.
The technical scheme provided by the invention is as follows:
an early warning method based on follicular fluid HRG and C4B protein expression level as recurrent abortion caused by unknown reasons, wherein the method is not used for disease diagnosis and treatment; the method comprises the following steps:
1) Collecting and preserving follicular fluid
Picking up the collected follicular fluid at constant temperature, collecting the residual follicular fluid, centrifuging at 1500rpm to obtain supernatant, packaging, and freezing at-80deg.C;
2) ELISA determination of C4B, HRG follicular fluid protein concentration
Adopting ELISA detection kit of corresponding protein to detect the concentration of corresponding protein in follicular fluid;
3) Result statistics and analysis
ELISA protein concentration data of follicular fluid obtained by detection; analyzing and comparing with ROC (receiver operator characteristic curve) curve of follicular fluid molecular marker of URSA patient;
if the HRG concentration of the follicular fluid protein obtained by detection is less than 35.80mg/dl, the probability that the patient can obtain live birth in the current egg taking period is lower; or/and, if the C4B concentration of the follicular fluid protein obtained by detection is higher than 2739.7ng/ml, the probability that the patient can obtain a live birth in the current egg taking period is lower;
or, taking a predicted probability value (predicted probability) obtained by carrying out logistic regression calculation on the HRG concentration and the C4B concentration of follicular fluid protein of the same patient as a test variable, and drawing a ROC curve by taking whether the current egg taking period of the patient obtains live birth or not as a state variable; when the predicted probability value of the patient is larger than 0.5816, the probability that the patient can obtain a live birth in the current egg taking period is lower.
In step 3), the HRG concentration of the follicular fluid protein is less than 35.80mg/dl, and the C4B concentration of the follicular fluid protein is more than 2739.7ng/ml, which are respectively derived from the Cut-off value of the ROC curve of the HRG concentration and the Cut-off value of the ROC curve of the C4B concentration;
and the ROC curve of HRG concentration and the ROC curve of C4B concentration are respectively drawn by taking the HRG concentration and the C4B concentration of follicular fluid protein of a plurality of normal control and URSA patients as test variables and taking whether the current egg taking period of the corresponding patients obtains live birth or not as state variables.
In step 3), the predicted probability value of the patient is >0.5816 (corresponding protein concentration is HRG <39.84mg/dl, C4B >2374.47 ng/ml), derived from the Cut-off value of the ROC curve; the prediction probability ROC curve is formed by drawing a prediction probability value obtained by performing logistic regression calculation on HRG concentration and C4B concentration of follicular fluid protein of a plurality of normal control samples and URSA patient as a test variable and whether a live birth is obtained in the current egg taking period of the patient or not as a state variable.
In step 3), the patient has a predicted probability value of >0.5816, corresponding protein concentration of HRG <39.84mg/dl, C4B >2374.47ng/ml.
The analysis was compared to a mapping and statistical analysis using GraphPad Prism 7 software and a ROC curve was plotted and statistical analysis using MedCalc (MedCalc Software Ltd, belgium) software.
The follicular fluid collection in step 1) is performed by collecting the follicular fluid with a HCG trigger, namely 36 hours after LH peak, and obtaining the follicular fluid through vaginal ovum extraction.
The beneficial effects of the invention are as follows: according to the invention, follicular fluid of a patient suffering from recurrent abortion caused by unknown reasons for assisted reproduction is used as a detection sample, and a system of HRG and C4B serving as molecular markers for predicting the risk of recurrent abortion is established through TMT proteomics and ELISA verification of an enlarged sample. Mainly comprises the following steps:
1. the invention combines the past medical history of the patient with the recurrent abortion caused by unknown reasons and the outcome of the assisted fertility treatment of the current row in the sample collection process, and carries out proteomic comparison analysis on the change of the follicular fluid protein level of the patient which does not obtain normal clinical pregnancy for recurrent abortion caused by unknown reasons by using the TMT technology for the first time; differential proteins with predictive value for the end of regeneration are screened pertinently.
2. The method is characterized by firstly finding that the HRG expression in follicular fluid of a patient with the prior URSA and the IVF/ICSI of the current line still does not obtain normal pregnancy is reduced, and the C4B expression is increased; and when the enlarged sample is verified, the ROC curve analysis is utilized to further prove that the protein expression levels of HRG and C4B in follicular fluid have a certain predictive value on the pregnancy ending of a URSA patient.
3. The ROC curve is utilized for the first time, and the cut-off value of the auxiliary reproductive pregnancy ending predicted by the corresponding protein expression is calculated, so that the method has practical application value.
Drawings
FIG. 1 shows the results of ELISA detection of the HRG concentration of follicular fluid protein according to the example of the present invention.
FIG. 2 is a ROC curve of whether an egg taking period patient obtains a live birth as a state variable with HRG follicular fluid concentration as a test variable.
FIG. 3 shows results of ELISA detection of follicular fluid protein C4B concentration according to an embodiment of the present invention.
FIG. 4 is a ROC curve of whether the patient acquired live birth as a state variable for this time of aspiration with C4B follicular fluid concentration as a test variable.
Fig. 5 is a graph of ROC curve drawn with predicted probability value (predicted probability) as a test variable and whether the patient in the egg taking period has a live birth as a state variable (the predicted probability value is obtained by performing logistic regression calculation on the same samples HRG and C4B).
Detailed Description
The inventors collected recurrent abortion (unexplained recurrent spontaneous abortion, URSA) of unknown origin and did not obtain normal clinical pregnancy in this egg taking cycle, and patients with infertility due to tubular factors and live birth in this egg taking cycle were used as normal controls (health control), and the determination of proteomic level of differentially expressed proteins was performed on follicular fluid by TMT (Tandem mass tag) technique. HRG was found to be expressed at low levels in URSA patients and C4B was found to be expressed at high levels by preliminary screening of differentially expressed proteins and ELISA validation of further expanded samples. Further ROC (receiver operator characteristic Curve) Curve analysis shows that, by taking HRG follicular fluid concentration as a test variable and taking whether the patient in the current egg taking period obtains a live birth as a state variable, an ROC Curve is drawn, the Area Under the Curve (AUC) is 0.785 (P < 0.01), and by taking C4B follicular fluid concentration as a test variable and taking whether the patient in the current egg taking period obtains a live birth as a state variable, the ROC Curve is drawn, and the AUC is 0.710 (P < 0.01). The above results further define the predictive value of the corresponding proteins for clinical pregnancy outcome. The two proteins can be used as molecular markers for early warning of the assisted reproductive outcome of a URSA patient.
The invention will be further described with reference to examples of embodiments shown in the drawings.
1. Collecting follicular fluid:
the samples collected by the invention are mature follicular fluid of a patient to be subjected to In Vitro Fertilization (IVF) treatment, the patient is treated by controlled ovarian hyperstimulation (Controlled Ovarian Hyperstimulation), the ovarian response is detected by vaginal B-ultrasound monitoring of the follicular size, serum E2, P, LH, FSH values and other indexes during the period, and the dosage of the medicament is appropriately adjusted according to the individual response of the patient. When at least 2 dominant follicles in the dominant follicular group were >18mm in diameter and reference to patient estrogen levels, a single injection of HCG 5000-10000 units or HCG + dabigard induced ovulation as appropriate, followed by negative ultrasound guided ova aspiration after 36 hours. The follicles were aspirated with a single lumen tube during the operation, and the residual follicular fluid was collected after picking up the cumulus complexes at a constant temperature, centrifuged at 1500rpm, sub-packaged, and frozen at-80 ℃. In the invention, follicular fluid of 44 HC and 43 URSA patients is collected, and the concentration of HRG and C4B proteins is subjected to expanded sample detection.
2. C4B, HRG follicular fluid protein concentration ELISA assay.
ELISA detection kit for corresponding protein is purchased, and detection of corresponding protein concentration is carried out according to the operation steps suggested by manufacturers. The kit mainly comprises: HRG (Cloud-Clone Corp, SEC534 Hu), C4B (Cloud-Clone Corp, SEB305 Hu).
3. And (5) carrying out result statistics and analysis.
Follicular fluid ELISA protein concentration results were plotted and statistically analyzed using GraphPad Prism 7 software (GraphPad Software, san Diego, california, USA).
4. Data processing analysis
(1) Results of the follicular fluid protein HRG concentration ELISA assay (see fig. 1).
(2) Drawing an ROC curve by taking the HRG follicular fluid concentration as a test variable and taking whether a patient in an egg taking period obtains a live birth as a state variable, wherein the ordinate is sensitivity, the abscissa is 1-specificity, the AUC is the area under the curve, and the Cut-off value is less than 35.80mg/dl; that is, when the HRG concentration in follicular fluid is less than 35.80mg/dl, the probability that the patient can obtain live birth at this time in the egg taking period is low, the detection sensitivity is 56.1%, and the accuracy is 88.6% (see FIG. 2).
(3) Results of the follicular fluid protein C4B concentration ELISA assay (see fig. 3).
(4) Drawing an ROC curve by taking the concentration of C4B follicular fluid as a test variable and taking whether a patient obtains live birth or not in the current egg taking period as a state variable, wherein the ordinate is sensitivity, the abscissa is 1-specificity, the AUC is the area under the curve, and the Cut-off value is >2739.7ng/ml; that is, when the C4B concentration in follicular fluid is >2739.7ng/ml, the probability that the patient can obtain viable birth at this time in the egg taking period is low, the detection sensitivity is 56.4%, and the accuracy is 78.4% (see FIG. 4).
(5) And (3) carrying out logistic regression calculation on HRG concentration and C4B concentration of a plurality of normal control samples and URSA follicular fluid protein to obtain a predicted probability value (predicted probability), and drawing an ROC curve by taking the predicted probability value as a test variable and taking whether a patient obtains live birth as a state variable in the current egg taking period. * P <0.001, P <0.05. The Cut-off value of the predicted probability value is >0.5816, the corresponding HRG is <39.84mg/dl, and C4B is >2374.47ng/ml; that is, when the predicted probability value of the patient is >0.5816, the probability that the patient can obtain a live birth in the current egg taking period is low, the detection sensitivity is 77.8%, and the accuracy is 81.8% (see fig. 5).
The invention is characterized in that:
1. the follicular fluid of utilizing mature follicular as detection sample, its sampling time unifies to be the HCG trigger, and 36 hours after the LH peak promptly gets in the ovum art through the vagina again, and sample homogeneity is better, to the patient who carries out supplementary fertility, can obtain sufficient sample when getting the ovum art, and the sample is convenient, and follicular fluid is getting the ovum postoperative and belongs to discarding the sample, can not cause the damage to the patient. The components of the follicular fluid are necessary for important reproductive physiological processes such as ovum development, fertilization and the like, and are more stable compared with serum; the follicular fluid is used for detection and analysis, so that the influences of the sampling time, menstrual cycle and other systems of the organism of samples such as serum, urine and the like are overcome, and the state of the follicular growth microenvironment can be more intuitively reflected.
2. The invention combines the past medical history of the patient with recurrent abortion caused by unknown reasons and the outcome of the assisted reproductive treatment of this row in the sample collection process, and specifically screens the differential protein with predictive value for the outcome of the recurrent abortion.
3. The protein concentration of HRG and C4B in follicular fluid is further proved to have a certain prediction value on fertility outcome by ROC curve analysis while the sample is expanded for verification, and the cut-off values of the two in separate prediction and combined prediction of fertility outcome are calculated, and when the HRG and C4B protein expression level is used for combined prediction, the accuracy and the sensitivity are both greatly improved.
The invention discloses a differential expression protein spectrum of follicular fluid of a URSA patient by means of preliminary analysis of TMT proteomics of follicular fluid of the URSA patient and utilizes a big data set method, and the C4B and the HRG are screened out as candidate molecular markers of the URSA by biological information analysis methods such as GO function enrichment analysis, KEGG pathway enrichment analysis, protein interaction network analysis (PPI) and the like and previous literature research, so that the invention has better innovation and pertinence, and further confirms the prediction value of corresponding proteins on clinical pregnancy ending through expanded sample ELISA verification and ROC (receiver operator characteristic curve) curve analysis, and has stronger theory and experimental basis.
Claims (4)
1. Use of a reagent for detecting the expression levels of HRG protein and C4B protein in follicular fluid for the preparation of a detection reagent for use in a method for pre-warning recurrent abortion of unknown origin, said pre-warning method comprising the steps of:
1) Picking up the collected follicular fluid at constant temperature, collecting the residual follicular fluid, centrifuging at 1500rpm to obtain supernatant, packaging, and freezing at-80deg.C;
2) ELISA determination of C4B and HRG follicular fluid protein concentration
Adopting ELISA detection kit of corresponding protein to detect the concentration of corresponding protein in follicular fluid;
3) Result statistics and analysis
Taking a predicted probability value obtained by carrying out logistic regression calculation on the HRG concentration and the C4B concentration of follicular fluid protein of the same patient as a test variable, and drawing an ROC curve by taking whether the current egg taking period of the patient obtains live birth or not as a state variable; when the predicted probability value of the patient is larger than 0.5816, the probability that the patient can obtain a live birth in the current egg taking period is lower.
2. The use of a reagent for detecting the expression levels of HRG protein and C4B protein in follicular fluid according to claim 1 for the preparation of a detection reagent for a method of early warning recurrent abortion of unknown origin, characterized in that: in step 3), the predicted probability value of the patient is >0.5816, derived from the Cut-off value of the predicted probability value ROC curve; and a predicted probability value ROC curve, wherein a predicted probability value obtained by performing logistic regression calculation on the HRG concentration and the C4B concentration of the plurality of normal control samples and the URSA follicular fluid protein is used as a test variable, and whether a live birth is obtained in the current egg taking period of the patient is used as a state variable is drawn.
3. The use of a reagent for detecting the expression levels of HRG protein and C4B protein in follicular fluid according to claim 2 for the preparation of a detection reagent for a method of early warning recurrent abortion of unknown origin, characterized in that: in step 3), the patient has a predicted probability value of >0.5816, corresponding protein concentration of HRG <39.84mg/dl, C4B >2374.47ng/ml.
4. The use of the reagent for detecting the expression levels of the HRG protein and the C4B protein in follicular fluid according to claim 3, for preparing a detection reagent for an early warning method for recurrent abortion of unknown origin, characterized in that: the result statistics and analysis are plotted and statistically analyzed by using GraphPad Prism 7 software, and ROC curves are plotted and statistically analyzed by using MedCalc software.
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