Levels Of Th1 And Th2 Cells And Th1/Th2 Balance In Peripheral Blood Of Patients With Recurrent Spontaneous Abortion

Objective:In recent years,the number of patients with recurrent spontaneous abortion?RSA?due to abnormalities in the immune system has continued to increase.Helper T cells?Th cells?are an important part of the immune system.According to different secreted cytokines,Th cells can be divided into helper type T cells?Helper T1 cell,Th1 cell?,helper type T cells?Helper T2 cell,Th2 cell?,and helper type 3 T cells?Helper?.T3 cell,Th3cell),helper type T cells?Th9 cell?and helper type 17 T cells?Helper T17 cell,Th17 cell?and other subtypes.Thl cells mainly secrete cytokines such as IL-2,IL-21,IFN-?,TNF-?,TNF-?and IL-10.Th2 cells mainly secrete cytokines such as anti-inflammatory factors IL-4,IL-5,IL-6,IL-9,IL-10,IL-13.The cellular immune response mediated by Th1 cells can produce an immune rejection effect,and an increase in the content of Th1 cytokines adversely affects recurrent spontaneous abortion mediated by the same immune factor.Th2 cells mainly secrete anti-inflammatory factors,which can maintain the immune tolerance of the maternal-fetal interface and maintain the smooth progress of pregnancy.The Th1/Th2 balance between the mother and the embryo is an important condition for ensuring normal pregnancy.If the Th1/Th2 balance is destroyed,it may cause placental damage and miscarriage.It is currently clinically found that approximately 60%of recurrent abortions are associated with Th1 cytokine and Th2 cytokine levels and Th1/Th2 balance state ratio.At present,there is no normal reference interval and clinical index threshold for the ratio of Th1 cells,Th2 cells and Th1/Th2 equilibrium in recurrent abortion in China.Clinicians can only rely on clinical experience and rheumatology to obtain rheumatoid arthritis.The reference interval for the ratio of Th1 cells,Th2 cells,and Th1/Th2 balance states associated with immune diseases was treated with oral prednisone acetate.Based on this,this study intends to use the rheumatology and immunology department of Peking University People's Hospital to study the normal reference interval of Th1cells,Th2 cells and Th1/Th2 equilibrium state ratios(the reference interval of Th1 cells is 5.53%-19.82%,the reference interval of Th2 cells was1.25%-2.88%,and the reference interval of Th1/Th2 equilibrium ratio was4.16%-13.47%.The reference standard was used to determine the female patients with a history of recurrent abortion by flow cytometry.The levels of Th cells,Th1 and Th2 cells in peripheral blood of normal women of childbearing age without recurrent abortion,calculated the ratio of Th1 cells,Th2 cells and Th1/Th2 balance,compared with patients with recurrent abortion history and no recurrence The ratio of Th1 cells,Th2 cells and Th1/Th2 balance in peripheral blood of patients with a history of abortion;and whether the ratio of Th1 cells and Th2 cells and Th1/Th2 balance status have potential value for the diagnosis and treatment of recurrent spontaneous abortion.Methods:1.Subjects:The experimental group was 477 female patients with a history of recurrent miscarriage who had a history of recurrent abortion at the outpatient clinic of the Sixth Medical Center of the Sixth Medical Center of the People's Liberation Army General Hospital from October 2016 to October2018.The control group was 60 normal women of childbearing age who were in the period of pregnancy during the pregnancy period from October 2016 to October 2018 at the outpatient clinic of the Sixth Medical Center of the People's Liberation Army General Hospital.2.Inclusion criteria:A preliminary diagnosis is made on the patient by collecting medical history,and the patient is assisted according to the initial diagnosis.The specific auxiliary examination includes chromosome examination,gynecological examination and vaginal B-ultrasound exam-ination,blood routine examination,and seven sex hormone tests.On the third day after ovulation and on the fifth day after ovulation,three tests for sex hormones,thyroid function test,hepatitis B,syphilis and hepatitis C test,platelet aggregation test,D-Diner test,anti-?2 antibody test,natural killer cell test The anticardiolipin antibody?ACA?test,antinuclear antibody?ANA?test,and auxiliary test results were all determined to be enrolled.Inclusion criteria:normal chromosomes in both husband and wife;normal genital tract structure;no menstrual disorders,early ovarian insufficiency?POI?,premature ovarian failure?POF?,luteal insufficiency;no hepatitis B Infectious diseases such as hepatitis C and syphilis;no coagulopathy,anti-cardiolipin syndrome,allergic diseases;patients who have not received immunosuppressive therapy for the past three months.3.Reference standard:The reference interval of the Th1cells,Th2 cells and Th1/Th2 equilibrium state ratios related to rheumatic immune diseases studied by the Department of Rheumatology and Immunology,Peking University People's Hospital.The reference interval of Th1 cells is 5.53%-19.82%,the reference interval of Th2 cells was 1.25%-2.88%,and the reference interval of Th1/Th2 equilibrium state ratio was 4.16%-13.475%.4.The study has obtained informed consent from the patient and his or her family.5.Experimental detection index:Peripheral blood of the experimental group and the control group were collected,and the levels of Th cells,Th1cells and Th2 cells were obtained by peripheral blood incubation,flow sample preparation and flow cytometry detection and analysis.Among them,the detection indexes of Th cells are CD₃ PerCP and CD₈ APC,and the Th cell population is represented by the regions where CD₃^?and CD₈^-are located.The detection indicators of Th1 cells are CD₃ PerCP and IFN-?FITC,and the Th1cell population is represented by the region where CD₃^?and IFN-?^?are located.The detection indicators of Th2 cells are CD₃ PerCP and IL-4 PE,and the Th2cell population is represented by the regions where CD₃^?and IL-4^?are located,and the results are expressed as percentages.The Th1/Th2equilibrium state ratio was calculated from Th1 cells/Th2 cells.6.Statistical methods:statistical analysis of data using SPSS21.0.All data were first analyzed by k-s test for normal distribution.The general data of the patients were consistent with the normal distribution,and the results were expressed as mean±standard deviation.The ratios of Th1 cells,Th2 cells and Th1/Th2 balance in the peripheral blood of the experimental group and the control group did not conform to the normal distribution,and the results were based on the median?Median?and interquartile range?IQR?25-75??.The mean,standard deviation,median,and IQR values were calculated using SPSS 21.0.Flow cytometry and scatter plots were used to analyze the distribution of Th1 and Th2 cell levels and the ratio of Th1/Th2 equilibrium.Non-normal data were analyzed by rank sum test to compare the ratio of Th1cells,Th2 cells and Th1/Th2 balance in peripheral blood of experimental group and control group:P<0.05 indicated that the difference was statistically significant.The underlying area of the ROC curve?AUC?and the Youden Index?YI?were used to evaluate the diagnostic value of the ratio of Th1 cells,Th2 cells and Th1/Th2 balance to recurrent spontaneous abortion:when the area under the ROC curve is close to 0.5 When there is no diagnostic value;the area under the ROC curve is less than 0.7,indicating that the diagnostic accuracy is low;the area under the ROC curve is between 0.7 and 0.9,indicating that the diagnostic accuracy is medium;the area under the ROC curve is above 0.9,indicating that the diagnostic accuracy is higher.The value of the Youden index is between 0-1,and the larger the value,the better the diagnostic authenticity.Results:1.Levels of Th cells,Th1 cells and Th2 cells in peripheral blood of different subjects.Healthy control group:Flow cytometry results of one of 60 healthy control women showed that the levels of Th cells?CD₃⁺,CD₈^-?in peripheral blood were 32.4%,and Th1 cells?CD₃⁺,IFN-?⁺?The level was 15.0%and the level of Th2 cells?CD₃⁺,IL-4⁺?was 1.51%.Recurrent abortion group:Flow cytometry results of 477 women with recurrent abortion group,the level of Th cells?CD₃⁺,CD₈^-?in peripheral blood was 41.7%,and Th1 cells?CD₃⁺,IFN-?⁺?level was 12.7%,and the level of Th2 cells?CD₃⁺,IL-4⁺?was 0.66%.2.Distribution of Th1 cell and Th2 cell levels and Th1/Th2 equilibrium state ratio in peripheral blood of different subjects.The results were obtained by summarizing and statistically analyzing the data from the results of flow cytometry in 60 healthy controls and 477female patients with a history of recurrent abortion:Healthy control group:The median level of Th1 cells in peripheral blood?IQR25-75?was 11.37%?IQR9.04%-14.84%?Recurrent abortion group:The median level of Th1 cells in peripheral blood?IQR25-75?was 10.24%?IQR7.90%-13.65%?.The level of Th1 cells in peripheral blood of patients with a history of recurrent spontaneous abortion was lower than that of healthy controls Group?P<0.05?.Healthy control group:The median?IQR25-75?level of Th2 cells in peripheral blood was 1.62%?IQR1.50%-1.88%?.Recurrent abortion group:The median level of Th2 cells in peripheral blood?IQR25-75?was 1.76%?IQR1.46%-2.11%?.Patients with a history of recurrent spontaneous abortion did not have Th1 cells in peripheral blood and healthy controls.Difference?P>0.05?.Healthy control group:The median?IQR25-75?ratio of Th1/Th2balance in peripheral blood was 6.97%?IQR5.36%-8.52%?.Recurrent abortion group:The median?IQR25-75?ratio of Th1/Th2balance in peripheral blood was 5.97%?IQR4.69%-7.41%?,and the Th1/Th2balance in peripheral blood of patients with recurrent abortion history The ratio was lower than the healthy control group?P<0.05?.3.The value of Th1 cell and Th2 cell levels and Th1/Th2 equilibrium status in peripheral blood for the diagnosis of recurrent spontaneous abortion.The ROC curve of Th1 cell and Th2 cell level and Th1/Th2 equilibrium state ratio was established.The area under the ROC curve represents the difference between recurrent abortion and normal control.The ROC was evaluated at the Th1 cell level,the AUC was 0.42?95%CI:0.35-0.49?,and when the Th1 cell level was 17.39%,the Yoden index was0.015.ROC evaluation was performed at Th2 cell level with an AUC of 0.57,?95%CI:0.51-0.63?.When the Th2 cell level was 2.05%,the Yoden index was 0.204.The ROC evaluation was performed with a Th1/Th2 equilibrium state ratio,and the AUC was 0.37?95%CI:0.30-0.44?.When the Th1/Th2equilibrium state ratio is 16.92%,the Yoden index is 0.002.Conclusion:Patients with a history of recurrent abortion have lower levels of Th1 cells in peripheral blood than those in normal women of childbearing age.There was no difference in the level of Th2 cells in peripheral blood between patients with a history of recurrent abortion and those in normal women of childbearing age.The ratio of Th1/Th2 balance in peripheral blood of patients with a history of recurrent abortion was lower than that of women of normal childbearing age.