| BackgroundAt present,50%of the 93 million cases of hepatitis B virus carriers in China are transmitted from mother to child.The incidence of HBV intrauterine infection reported in previous studies was up to 9.4%-44.4%.At present,a series of studies have expanded and revised the traditional HBV intrauterine infection,and a new HBV intrauterine transmission was formed.Meanwhile,recent studies reveal that the immune response rate of hepatitis B vaccine of the offspring of HBs Ag positive mothers was 73.26%only.Hence,such population was more liable to generate HBV mother to child transmission.How to primary screen and warn the high risk group of HBV mother-to-infant transmission in early stage is a blank field of current research.Since HBV infection can induce unbalanced immune response of Th1/Th2 cells,it becomes main cause of chronic HBV infection,TLR is a hot spot in virus immunity.Therefore,based on the new connotation of HBV intrauterine transmission,adopted nested case-control and cohort study methods.By describing the levels of Th1/Th2 cytokines and TLR 9 in the peripheral blood of HBs Ag positive pregnant women in the third trimester of pregnancy.As a rusult,the study to screen the early-warning indicators of HBV intrauterine transmission and hepatitis B vaccine no response of the offspring,so as to establish a theoretical foundation for the prevention and control of HBV intrauterine transmission.Objectives1.To obtain the expression characteristics of Th1/Th2 cytokines in the peripheral blood of pregnant women with HBs Ag positive in the third trimester of pregnancy,and screen Th1/Th2 cytokines relating to HBV intrauterine transmission of the offspring.2.To obtain the expression characteristics of TLR 9 in the peripheral blood of pregnant women with HBs Ag positive in the third trimester of pregnancy,and understand the relationship with HBV intrauterine transmission of the offspring.3.Based on the follow-up visit on HBV infection status and hepatitis B vaccine immune response against infants,who born from pregnant and lying-in women in HBs Ag positive and completed the planned hepatitis B vaccine immunization throughout the course,to obtain the correlation of maternal cytokine and TLR 9 levels in the third trimester of pregnancy with HBV infection and immune response of hepatitis B vaccine in infants.Subject and Methods1 Subject1.1 A nested case-control study of cytokines:Peripheral blood of pregnant women with HBs Ag positive in the third trimester of pregnancy 341 HBs Ag positive pregnant women and 344 newborns(3 twin)and 74 healthy pregnant women and newborns and their epidemiological investigation information was collected.All of the subjects were admitted in Northwest Women and Children Hospital,Shaanxi Province for delivery.HBV intrauterine dominant infection was found in the peripheral blood of the offspring within24 hours(without HBV high titer immunoglobulin injection,HBV vaccine and BCG vaccine)with HBs Ag positive,and HBV intrauterine occult infection was found in the blood with HBs Ag negative and HBV-DNA≥200IU/ml.Both are referred to as HBV intrauterine transmission.1.2 A nested case-control study of TLR 9:290 HBs Ag positive pregnant women and 291newborns(1 twin)and 45 healthy pregnant women and newborns in hospital delivery in Northwest women and children Hospital of Shaanxi Province were collected.1.3 A cohort study:HBs Ag positive pregnant women delivered in Northwest Women and Children Hospital in Shaanxi Province and their young children who have completed the entire hepatitis B vaccine immunization plan and 91 cases signed the informed consent,followed up by questionnaire and successfully collected their peripheral blood,and 83cases were finally included in the follow-up cohort.2 Epidemiological investigation(1)Epidemiological baseline survey:including general information of pregnant women and newborn babies.(2)Epidemiological follow-up survey:including neonatal feeding and vaccination.3 Laboratory testing(1)Peripheral blood of pregnant women and newborn babies was collected and ELISA method was used to test 5 serum hepatitis B indexes.(2)Real-time fluorescence quantitative PCR was used to detect serum HBV-DNA levels in pregnant women and newborns.(3)Suspension array technology was used to detect the level of Th1/Th2 cytokines in maternal serum,Th1 cytokines include IL-2 IL-12,IL-18,INF-γ,Th2 cytokines include IL-4,IL-6 and IL-10.(4)ELISA method was used to detect the level of TLR 9 in maternal serum.4 Data statistical analysis methodEpi Data was used to establish the database,and SPSS19.0 software was used for statistical analysis of data.The measurement data was tested by normality test.Through two-group comparison,t test was used for those with the homogenous variance and Mann-Whitney U test was used for those with non-homogenous variance.Through multi-variable analysis,ANOVA was used for those with homogenous variance and Kruskai-Wallis test was used for those with non-homogenous variance.χ~2 test was used for statistical data.Logistic regression was used for single factor and multi factor analysis.Area under ROC curve was used for diagnosis efficiency.The significance criteria were set at 0.05 for all tests.Results1 Epidemiological study on Th1 cytokines in the third trimester of pregnancy and HBV intrauterine transmission of the offspring1.1 The incidence of HBV intrauterine dominant infection(DBI),occult HBV intrauterine infection(OBI),HBV intrauterine transmission(BIT)and HBV intrauterine non-transmission(NBIT)were11.34%(39/344),36.63%(126/344),47.97%(165/344),52.03%(179/344),respectively.1.2 Study on the level of Th1 cytokines in peripheral blood in HBs Ag positive pregnant women and intrauterine transmission groups.1.2.1 For HBs Ag positive pregnant women and BIT,NBIT,DBI and OBI groups,IL-2level in peripheral blood was no significant difference between HBs Ag negative control group(All P>0.05).1.2.2 For HBs Ag positive pregnant women and BIT,NBIT,DBI and OBI groups,IFN-γlevel in peripheral blood was significantly higher than that in HBs Ag negative control group(P<0.0001;P<0.0001;P<0.0001;P=0.021;P<0.0001).1.2.3 For HBs Ag positive pregnant women and BIT,NBIT,DBI and OBI groups,IL-12level in peripheral blood was significantly lower than that in HBs Ag negative control group(P<0.0001;P<0.0001;P=0.002;P<0.0001).IL-12 level in NBIT group was significantly higher than that in OBI group(P=0.047).1.2.4 For HBs Ag positive pregnant women and BIT and OBI groups,IL-18 level in peripheral blood was significantly higher than that in HBs Ag negative control group(P=0.022;P=0.014;P=0.007).1.3 Level of Th1 cytokines of HBs Ag positive pregnant women with different HBV infection states1.3.1 IFN-γand IL-18 levels in HBe Ag positive group were significantly higher than those in HBs Ag negative control group(P<0.0001;P<0.0001).However,IL-12 level of pregnant women in HBe Ag positive group was significantly lower than that in HBs Ag negative control group(P=0.015).1.3.2 Taking 10~3IU/ml and 10~6IU/ml as the cut-off points,it was divided into three layers.The level of IL-18 in the 10~3-10~6IU/ml HBV DNA load group increased,and the risk of DBI occurrence of the offspring also increased(P=0.045).1.4 Correlation between pregnancy intervention of HBs Ag positive pregnant women and Th1 cytokine level1.4.1 In the group with HBIG injection,the level of IFN-γin the DBI group was significantly lower than that in the NBIT group(P=0.008).The level of IL-12 in the OBI group was significantly lower than that in the NBIT group(P=0.008).The level of IFN-γin the NBIT group with HBIG injection was significantly higher than that in the group without HBIG injection(P=0.006).The level of IL-18 in the group with HBIG injection was significantly higher than that in the group without HBIG injection(P=0.040).1.4.2 For antiviral treatment group and hepatitis B vaccine injection group,the level of IFN-γshowed intragroup differentiation.The level of IFN-γin OBI group was significantly lower than that in NBIT group(P=0.027,P=0.029).For the group without hepatitis B vaccine injection and the group with HBIG injection,the level of IL-18 in OBI group was significantly higher than that in NBIT group(P=0.047,P=0.036).1.5 Single factor analysis of Th1 cytokines and HBV intrauterine transmission showed no significant correlation.1.6 Logistic regression analysis of Th1 cytokines and multiple variable factors with HBV intrauterine transmission was conducted,and no significant correlation factors were screened out.2 Epidemiological study on Th2 cytokines in the third trimester of pregnancy and HBV intrauterine transmission of the offspring2.1 For HBs Ag positive pregnant women and NBIT,DBI and OBI groups,IL-4,IL-6 and IL-10 levels of Th2 cytokines in peripheral blood were significantly higher than those in HBs Ag negative control group(P<0.05).2.2 Level of Th2 cytokine in HBs Ag positive pregnant women with different HBV infection states2.2.1 For pregnant women in the HBe Ag negative group and the HBe Ag positive group,the levels of IL-4,IL-6 and IL-10 were significantly higher than those in HBs Ag positive control group(P<0.05).2.2.2 In the group with HBV-DNA content<10~3IU/ml,the level of IL-4 in DBI group was significantly higher than that in OBI group(P=0.011)and NBIT group(P=0.007).In the group with HBV-DNA>10~6IU/ml,the level of IL-10 in OBI group was significantly lower than that in NBIT group(P=0.031).In the group with HBV-DNA>10~6IU/ml,the level of IL-4 in DBI group was significantly lower than that in the group with HBV-DNA<10~6IU/ml group(P=0.016).2.3 For the group without antiviral treatment and the group with hepatitis B vaccine injection,the level of IL-6 in DBI group was significantly lower than that in OBI group(P=0.008;P=0.012).2.4 Single factor analysis showed that IL-4 was significantly correlated with HBV intrauterine transmission.The probability of BIT of offspring with high level of IL-4 was1.003 times higher than that of NBIT(OR=1.003,95%CI:1.000-1.006,P=0.046).2.5 Multi factor analysis showed that with the increase of maternal peripheral blood IL-4 level,the risk of DBI increased.The probability of DBI of offspring with high level of maternal IL-4was 1.005 times higher than that of NBIT(OR=1.005,95%CI:1.001-1.010,P=0.025).3 Study on the effect of TLR 9 in the third trimester of pregnancy on HBV intrauterine transmission of the offspring3.1 Rates of DBI,OBI and BIT occurrence in the subject were 9.28%(27/291),40.21%(117/291)and 49.48%(144/291),respectively.3.2 For HBs Ag positive pregnant women group and NBIT and OBI groups,the level of TLR 9 in peripheral blood was significantly lower than that in HBs Ag negative control group(P=0.001;P<0.0001;P<0.0001).Amongst others,the level of TLR 9 in NBIT group was significantly lower than that in BIT group(P=0.015),and the level of TLR9 in DBI group was significantly higher than that in NBIT group and OBI group(P<0.0001;P<0.0001).3.3 Level of TLR 9 in HBs Ag positive pregnant women with different HBV infection states3.3.1 The level of TLR 9 in HBe Ag negative pregnant women group was significantly lower than that in HBs Ag negative control group(P<0.0001).According to the BIT severity,it was divided into DBI group,OBI group and NBIT group.With the increase of BIT severity,the level of TLR 9 in DBI group was significantly higher than that in OBI group and NBIT group(P<0.01).3.3.2 Taking 10~3IU/ml and 10~6IU/ml as the cut-off points,it was divided into three layers.With the increase of BIT severity,the level of TLR 9 in each layer increased(P<0.05).The level of TLR 9 in each layer in DBI group was significantly higher than that in OBI group and NBIT group(P<0.05).3.4 Whether antiviral treatment or not,HBIG injection or not and hepatitis B vaccine injection group,the level of TLR 9 in maternal peripheral blood increased with the increase of BIT severity.The level of TLR 9 in maternal peripheral blood in DBI group was significantly higher than that in OBI group and NBIT group(P<0.0001;P<0.0001).3.5 Single factor analysis showed that the risk of DBI increased with the increase of TLR9level in maternal peripheral blood.The probability of DBI in offspring of pregnant women with high TLR 9 expression is 1.39 times higher than that of NBIT(OR=1.392,95%CI:1.224-1.582,P<0.0001).The probability of DBI is 1.36 times that of OBI(OR=1.360,95%CI:1.195-1.549,P<0.0001).3.6 Multi factor analysis found that:(1)The probability of DBI in offspring of pregnant women with high TLR 9 expression is 1.44 times higher than that of NBIT(OR=1.442,95%CI:1.247-1.667,P<0.0001).The probability of DBI is 0.14 times higher than that of NBIT when HBV-DNA load is 10~3-10~6 IU/ml(OR=0.135,95%CI:0.023-0.781,P=0.025).(2)The probability of DBI in offspring with high TLR 9 expression is 1.43 times that of OBI(OR=1.434,95%CI:1.237-1.663).In the group without HBIG injection,the probability of DBI of the offspring was 0.13 times higher than that of OBI(OR=0.131,95%CI:0.043-0.399).4 A cohort study on the relationship between the expression of cytokines and TLR 9in late pregnancy and the prognosis of infants4.1 Eighty-three infants and their mothers were followed up in this study.Among them,27(32.53%)had occult HBV infection and 56(67.47%)had no HBV infection.The overall hepatitis B immune response rate of infants was 72.29%.The hepatitis B vaccine response rates in DBI group,OBI group and NBIT group were 81.82%(9/11),80.00%(28/35)and62.16%(23/37),respectively.The hepatitis B immune response rates of infants as followed up in the group with occult infection and the group without infection were88.89%(24/27)and 64.29%(36/56),respectively.4.2 For the outcome of infants with high level of IL-2 in maternal peripheral blood in the third trimester of pregnancy,the risk of HBV infection decreased(P=0.014).4.3 The level of IL-2 in the group of infants as followed up without hepatitis B vaccine immune response was significantly higher than that in the group of antibody positive infants(P=0.013).4.4 Th1/Th2 cytokines and TLR 9 tested in the study were included in HBV intrauterine transmission for ROC curve analysis.Results showed that the sensitivity,specificity and Youden index of TLR 9 was 43.90%,76.20%,and 0.201,respectively.Area under curve was 0.582(95%CI:0.516-0.649).The threshold of TLR 9 was 1.690pg/ml.Therefore,when TLR 9 was higher than this threshold,it indicates that HBV intrauterine transmission may have occurred.4.5 Th1/Th2 cytokines and TLR 9 tested in the study were included in HBV infection of infants for ROC curve analysis.Results showed that the sensitivity,specificity and Youden index of IL-2 was 92.30%,52.40%,and 0.447,respectively.Area under curve was 0.755(95%CI:0.592-0.917).The threshold of IL-2 was 2.275pg/ml.Therefore,when IL-2 was lower than this threshold,the risk of HBV infection of infants as followed up would increase.4.6 Th1/Th2 cytokines and TLR 9 level were included in the HBV immune response of infants for ROC curve analysis.Results showed that the sensitivity,specificity and Youden index of IL-2 was 80.90%,61.10%,and 0.420,respectively.Area under curve was 0.677(95%CI:0.521-0.833).The threshold of IL-2 was 2.490pg/ml.Therefore,when IL-2 was lower than this threshold,the ability of infants as followed up to generate immune response would decrease.ConclusionBased on the new intension of HBV intrauterine transmission,Through case-control study and cohort study to select IL-12,IL-18,IL-4 and TLR 9 in the peripheral blood of HBs Ag positive pregnant in late pregnancy as the monitoring indexes for early predicting of the occurrence of HBV intrauterine transmission.IL-2 in the peripheral blood of late pregnancy of HBs Ag positive pregnant women could be used as the reference index for predicting the occurrence of HBV infection of the offspring and the immune response of hepatitis B vaccine.1.The levels of IL-12 and IL-18 in HBs Ag positive pregnant and lying-in women decreased.The possibility of occurrence of HBV intrauterine transmission increased due to the high expression level of IL-4 and TLR 9,which can be used as a cytokine to some extent for an early-warning indicator of HBV intrauterine transmission.To some extent,due to the decrease of IL-10 level,HBV intrauterine transmission may occur easily,which means IL-10 level may be used as an effective reference index for predicting HBV intrauterine transmission.2.Injection of HBIG may stimulate the increase of maternal IFN-γ,IL-12 and IL-18 levels,therefore it is necessary to promptly correct the immune disorder caused by HBV and reduce the probability of HBV intrauterine transmission,thereby providing an effective intervention index for blocking HBV intrauterine transmission.3.HBV can inhibit the expression of TLR 9 in pregnant women.However,with the increase of intrauterine transmission of HBV,TLR 9 shows a compensatory increase and intragroup differentiation.Conclusively,TLR 9 may provide a reference for the monitoring and management of HBs Ag positive pregnant women.4.The level of IL-2 in the third trimester of pregnancy may predict the risk of HBV infection in the course of children’s outcome.Meanwhile,the secretion of IL-2 in pregnant women may be involved in the expression of hepatitis B antibody in infants.5.When TLR 9 level is higher than 1.690pg/ml in the third trimester of pregnancy,it indicates that HBV intrauterine transmission may have occurred.When the level of IL-2 in late pregnancy is lower than 2.275pg/ml,the risk of HBV infection may increase in the child as followed up.When the level of IL-2 is lower than 2.490pg/ml,the capability of the child to produce immune response of HBV vaccine may decrease. |
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