| OBJECTIVE:To investigate the relationship between PBMC maternal-fetal transfer and HBV intrauterine infection and understand the biological basis of its role. To study the influence of placental apoptosis on placental permeability and understand the relationship between it and PBMC maternal-fetal transfer and neonatal HBV infection. To research the role mechanism of PBMC that is as HBV carrier and transport HBV from pregnant women into fetus and induce HBV intrauterine infection.METHODS:1population studies:HBsAg positive pregnant women and their newborns were consecutively collected from January (Jan.),2005to February (Feb.),2009in Taiyuan infectious hospital(third people's hospital of Taiyuan city). The above HBsAg positive pregnant women were selected by means of prenatal screening for HBsAg in provincial and municipal level hospitals of Taiyuan city and prenatal care and delivery in Obstetrics and Gynecology of Taiyuan infectious hospital. HBsAg positive pregnant women were continuously observed from prenatal examination to delivery. After pregnant women who had other infectious diseases or immune therapy were ruled out,450pairs HBsAg positive pregnant women and their newborns were collected as the research object. The epidemiology base line data involving gestation and postpartum were also collected, maternal elbow vein blood and femoral vein blood from newborns not only within24hours but also before infecting hepatitis B immuno-globulin (HBIG) were collected. At the same time, mature placenta were also collected under sterile conditions. Following studies were performed:(1) GSTM1and ACE gene polymorphism in PBMC were detected by allele-specific polymerase chain reaction (As-PCR) from HBsAg-positive pregnant women and their newborn. Mother-baby pairs informative cases for GSTM1would have the mother possessing the GSTM1gene and the baby possessing the null allele. And mother-baby pairs informative cases for ACE would have the mother possessing heterozygous ID gene type and the baby possessing homozygous insert Ⅱ genotype or homozygous deletion of DD genotype. In a word, informative cases for either GSTM1or ACE polymorphisms was subjected to As-PCR analysis for an allele which the mother possessed but which the baby did not. For the detection of the transfer of PBMC from the mother to the fetus,GSTM1or ACE allele were used as a maternal marker. PBMC maternal-fetal transfer was determined by detecting maternal marker in newborns' PBMC from informative cases.(2) HBeAg,HBe-Ab,HBc-Ab in peripheral blood of HBsAg positive pregnant women who came from informative cases and HBsAg in peripheral blood of newborns who came from informative cases were determined by ELISA.(3) HBV DNA level in serum and PBMC and HBV cccDNA level in PBMC in mothe-baby pairs informative cases were detected by fluorescence quantitative polymerase chain reaction (FQ-PCR).(4) Maternal marker was detected by FQ-PCR in PBMC of newborn from informative cases. ACt value(Cttarget gene/Ctβ-globin value) was used as showing the level of PBMC maternal-fetal transfer.(5) HBsAg in placentas were detected by immunohistochemistry ABC method.(6) TdT-mediated dUTP nick end labeling(TUNEL) method was used for informative cases placental samples to detect apoptosis index(AI). The expression of Caspase3in placenta was detected by a SP combination of immunohistochemistry techniques. Real time RT-PCR was used to detect the expression of Caspase3mRNA in placenta tissue.(7) Risk factors of maternal-fetal cell transfer,HBV infection and duplication in PBMC and HBV intrauterine infection were analyzed by nested case-control studies.(8) The expression of HBsAg and GST in PBMCs'glass slide of informative newborns were detected by the double immunofluorescent labeling method combined with confocal laser scanning microscope.2. Experimental studies in vitro:(1)100μl HBV DNA positive serum (the content of HBV DNA is5.0x106copies/ml) was added into PBMC in vitro for coculture. Then Cell Counting Kit-8was used to detect cell growth status at12h,24h,48h,72h. After coculture cells were washed four times by PBS,HBV DNA content in PBMC and lotion were detected by FQ-PCR.(2) The fusion phenomenon between PBMC infected HBV and Bewo cells in the transwell upper chamber (24-mm-diameter and1μm pore size) was observed by fluorescence activated cells sorting(FACs). Then PBMC transfer was studied in the transwell chamber (24-mm-diameter and8μm pore size). PBMCs labeled with green fluorescent dye in the below chamber were detected by fluorescence activated cells sorting(FACs).(3) This study had three groups:Bewo group(control group), HBV and Bewo coculture group, PBMC infected HBV and Bewo coculture group. They were respectively cultured for Oh,12h,24h,48h. FACs were used to detect apoptosis rate of Bewo cells at different time point of every group.(4) Caspase3mRNA expression of Bewo cells was detected by Real-time RT-PCR at different time point (0h,12h,24h,48h) in PBMC infected HBV and Bewo coculture group.(5) In PBMC infected HBV and Bewo coculture group FQ-PCR was used to detect HBV DNA and HBV cccDNA content of PBMCs in the below chamber. 3. Statistical analysis:Check and input the data, all of the data were analyzed by SPSS16.0for windows.χ2-test, t-test, ANOVAL and correlation analysis were used in the studies. RESULTS:1. The relationship between PBMC maternal-fetal transfer of HBsAg positive pregnant women and maternal HBV infection and neonatal HBV infection.(1) GSTM1and ACE gene polymorphism in PBMC were detected by allele-specific polymerase chain reaction (As-PCR) from450pairs ofHBsAg-positive pregnant women and their newborn. GSTM1genotyping revealed86informaitive cases in which the mother possessed an allele not present in the baby. ACE genotyping was then performed on the cases and revealed84informative cases. Thus, a total of155informative cases for the detection of maternal cells in fetal circulation were found uising these two systems.(2) The level of PBMC maternal-fetal transfer was detected by FQ-PCR. There was no statistical differences between HBsAg, HBeAg both positive mothers and negative mothers. The same result emerged respectively in the comparison between Big Sanyang or Small Sanyang mothers and negative mothers. There was no statistical differences of the level of PBMC maternal-fetal transfer among four groups of serum HBV DNA content in mothers.(3)ΔCt value (PBMC transportion Ct/β-globin Ct values) of PBMC HBV DNA positive group was lower than the negative group in HBsAg positive pregnant women, and the difference was statistically significant (t=6.42,P=0.00). ΔCt value (PBMC transportion Ct/β-globin Ct values) of PBMC HBV cccDNA positive was more than the negative group in HBsAg positive pregnant women, and the difference was also statistically significant (t=2.13,P=0.04).(4) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of ΔCt value (PBMC transportion Ct/β-globin Ct values) between HBV intrauterine infection group and negative group0=0.34, P=0.73). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of ΔCt value (PBMC transportion Ct/β-globin Ct values) between HBV intrauterine infection group and negative group (t'=6.25,P=0.00)2. The relationship between HBV infection in placenta and PBMC maternal-fetal transfer and neonatal HBV infection.(1) There was no statistical differences of ΔCt value (PBMC transportion Ct/β-globin Ct values) between HBV infection group and negative group in placenta (t=0.38, P=0.70). The same result was respectively showed among HBV infection group in placenta (t=0.38, P=0.70; t=0.49, P=0.63;t=0.17,P=0.87;t=1.72,P=0.09)(2) HBV infection in placenta was associated with HBsAg in newborns'serum (χ2=4.88, P=0.03), but it was independent of HBV DNA in newborns'serum and PBMC HBV infection in placenta was respectively independent of HBV intrauterine infection and PBMC HBV cccDNA in newborns.3. The relationship between cell apoptosis in placenta and PBMC maternal-fetal transfer and neonatal HBV infection.(1) The apoptosis index(AI) of155placental samples were detected by TdT-mediated dUTP nick end labeling(TUNEL) method. Placental cell nucleus appeared brown particle for expression of apoptosis positive signal. The Al was respectively4.53±0.07,5.56±0.06,4.66±0.05and4.05±0.06from the maternal side to the fetal side in the placental cell layers. The total of AI was4.75±0.05. Apoptotic index in the overall distribution of placental layers of cells of HBsAg-positive pregnant women were significantly different (F=102.60, P=0.00). The trophoblast cell apoptosis was higher than others, and villous capillary endothelial cell apoptosis was rare. Placental apoptosis index of each layer was compared with each other by using the analysis method of SNK. The results were that there were statistical significance between decidual cells and trophoblastic cells and villous capillary endothelial cells(P values less than0.05). But there were not statistical significance between decidual cells and villous mesenchymal cells.(2) Placental apoptosis index of PBMC maternal-fetal transfer positive group was more than the negative group, and the difference was also statistically significant (t=3.44, P=0.00). The same result was showed in the placental cell layers(t=2.86, P=0.01;t'=4.86, P=0.00; t=2.53, P=0.01; t=2.23, P=0.03).(3) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of placental apoptosis index between HBV intrauterine infection group and negative group (t=1.97, P=0.05). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of placental apoptosis index between HBV intrauterine infection group and negative group (t=3.22, P=0.00)(4) Placental apoptosis index of PBMC HBV DNA positive group in newborns was more than the negative group in decidual cells and trophoblastic cells, and the difference was also statistically significant (t=2.08, P=0.04;t=3.47, P=0.00). But placental apoptosis was respectively independent of PBMC HBV DNA in newborns in villous mesenchymal cells and villous capillary endothelial cells.(5) The expression of Caspase3in placentas were detected by immunohistochemistry SP method. Placental cell plasma and/or cell membrane appeared brown particle for expression of Caspase3positive signal. The gray value of Caspase3was respectively98.40±1.86,82.05±1.39,86.41±1.57and89.10±1.70from the maternal side to the fetal side in the placental cell layers. The total of gray value of Caspase3was88.99±1.47. The optical density of Caspase3was respectively15.58±0.33,18.59±0.29,17.67±0.31and17.31±0.35from the maternal side to the fetal side in the placental cell layers. The total of optical density of Caspase3was17.29±0.29. The expression of Caspase3in the overall distribution of placental layers of cells of HBsAg-positive pregnant women were significantly different (F=17.81,15.54, P value less than0.05). The expression of Caspase3of each layer was compared with each other by using the analysis method of SNK.The trophoblast cell apoptosis was higher than others, and decidual cells apoptosis was rare.(6) The gray value of Caspase3of PBMC maternal-fetal transfer positive group was lower than the negative group, and the difference was also statistically significant (t=2.80, P=0.01), and the optical density of Caspase3of PBMC maternal-fetal transfer positive group was more than the negative group, and the difference was also statistically significant (t=3.11, P=0.00). The same result was showed in the placental cell layers.(7) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of the expression of Caspase3between HBV intrauterine infection group and negative group(P>0.05). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of the optical density of Caspase3between HBV intrauterine infection group and negative group (t=2.20, P=0.03), but there was no statistical differences between both groups if the expression of Caspase3was showed by the gray value of Caspase3.(8) The expression of Caspase3of PBMC HBV DNA positive group in newborns was more than the negative group in trophoblastic cells, and the difference was also statistically significant (t'=2.71, P=0.01; t=2.33, P=0.02). But the expression of Caspase3was respectively independent of PBMC HBV DNA in newborns in other placental cell layers.(9) There was significantly negative correlation among Caspase3mRNA ΔCt value (CtCaspase3/CtGAPDH value) and the Al, optical density of Caspase3in placenta(r=-0.66, P=0.00; r=-0.18, P=0.03),and there was significantly positive correlation between Caspase3mRNA ΔCt value and gray value of Caspase3in placenta(r=0.23,P=0.00).(10) Caspase3mRNA ΔCt value (CtCaspase3/CtGAPDH value) of PBMC maternal-fetal transfer positive group was lower than the negative group, and the difference was also statistically significant (t=3.86, P=0.00).(11) When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no statistical differences of the mRNA of Caspase3between HBV intrauterine infection group and negative group (t=0.45, P=0.66). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was statistical differences of the mRNA of Caspase3between HBV intrauterine infection group and negative group (t=2.46, P=0.02)4. Risk factors of PBMC maternal-fetal transfer and HBV infection in neonatal were studied.(1) The risk factors of PBMC maternal-fetal transfer were analysis by logistic regression model. PBMC HBV DNA in pregnant women, the protein levels of Caspase3in placental decidual cells and apoptosis index of placental trophoblast cells were all introduced to the regression equation. OR values and95%CI were respectively14.60(5.61-38.00),1.13(1.01-1.27) and4.09(1.89-8.85), and there were interaction among of them (P<0.05, OR95%Cl did not include one).(2) The risk factors of PBMC HBV DNA positive in newborns were analysis by logistic regression model. PBMC HBV DNA in pregnant women and PBMC maternal-fetal transfer were introduced to the regression equation. OR values and95%CI were respectively41.21(11.13-152.64) and17.09(4.91-59.56), and there was an synergistic interaction between the two factors (P<0.05, OR95%CI did not include one).(3) The risk factors of PBMC HBV cccDNA positive in newborns were analysis by logistic regression model. PBMC HBV cccDNA in pregnant women and apoptosis index of placenta were introduced to the regression equation. OR values and95%CI were respectively40.87(9.63-173.41) and0.21(0.07-0.62), and there was an interaction both of them (P<0.05, OR95%CI did not include one).(4) The risk factors of HBV intrauterine infection in newborns were analysis by logistic regression model. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, HBeAg positive in pregnant women, mode of delivery, apoptosis index of placental trophoblast cells and age of pregnant women were introduced to the regression equation. OR values and95%CI were respectively3.42(1.28-9.12),0.33(0.12-0.95),2.14(1.14-4.04) and0.87(0.77-0.99). Interaction analysis showed that among apoptosis index of placental trophoblast cells and mode of delivery, age of pregnant women had no interaction, and among of other factors had interaction (P<0.05, OR95%CI did not include1). When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, PBMC HBV DNA in pregnant women, PBMC maternal-fetal transfer and apoptosis index of placental trophoblast cells were introduced to the regression equation. OR values and95%CI were respectively6.95(2.71-17.82),5.82(1.95-17.36) and2.56(1.33-4.91), and there were interaction among of them (P<0.05, OR95%CI did not include one). 5. Role of placental apoptosis on maternal-fetal transfer of PBMC infected with HBV was studied.(1) HBsAg positive rate was23.3%(20/86) in86cases of newborns'PBMC, that is23.3%of PBMC HBV infection in neonatal. The expression positive rate of GST was36.0%(31/86), that is36.0%of the mother-baby pairs occurring PBMC maternal-fetal transfer. There were13cases which showed coexistence of HBsAg and GST in the same PBMC of newborns, and the positive rate was15.12%(13/86), or15.12%of the mother-baby pairs occurring maternal-fetal transfer of PBMC infected with HBV. There were41.94%(13/31) of maternal-fetal transfer of PBMC infected with HBV in newborns who occurred PBMC maternal-fetal transfer.(2) The risk of HBsAg positive in PBMC of newborns who occurred PBMC transportion was more than that of non transportion, and the risk multiple was4.952(χ2=9.477,P=0.002).(3) Placental apoptosis index of PBMC maternal-fetal transfer positive group was more than the negative group, and the difference was statistically significant (t'=2.38, P=0.02). The higher rate of apoptosis in the trophoblast cells was closely related to PBMC maternal-fetal transfer(t=2.75, P=0.01). PBMC transfer from mother to baby was independent of the expression of Caspase3in placenta, and transfer of PBMC infected with HBV was also independent of the expression of Caspase3in placenta.(4) There were no correlation among HBsAg positive in newborns'PBMC and placental apoptosis index, the expression of Caspase3in placenta(P>0.05).6. Role of placental apoptosis on maternal-fetal transfer mechanism of PBMC was studied in vitro.(1) The cell counts cultured at12h,24h and48h with positive serum were increasing,but declining at72h.HBV DNA can be detected in PBMCs co-cultured with HBV DNA positive serum for48hours and can't be detected in washing liquid after be washed four times by PBS(2) A redistribution of the fluorescent dye was observed from the apical HBV-infected PBMCs to Bewo cells,indicating a fusion between HBV-infected PBMCs and Bewo cells in the Transwell model (1-mm porosity). In the Transwell model (8-mm porosity),PBMCs loaded with a fluorescent dye were detected in the basolateral chamber,which demonstrated that co-culture model in vitro to simulate transcytosis of the placental barrier was successfully established.(3) When HBV and Bewo cells group, HBV-infected PBMCs and Bewo group and control Bewo group were cultured for0h,12h,24h,48h. The difference of early apoptotic rates between HBV and Bewo cells group, HBV-infected PBMCs and Bewo group and control Bewo group was not statistically significant with all the value of P more than0.05. However, while cocultured time was24h or48h, total apoptotic rates had a statistically significant difference in three groups(P<0.05). early apoptotic rates and total apoptotic rates were all statistically significant(P<0.05) compared to respectively control group at the time0h,12h,24h or48h.when the cocultured time was48h, total apoptotic rates were higher in the HBV and Bewo cells group.(4) Compare caspase3mRNA of Bewo cells at different times in HBV+PBMCand Bewo cell groups. There was statistically significant difference (F=38.114,P=0.002). The relative expression of caspase3mRNA in48h group was higher than Oh,12h and24h group.(P<0.05),but there was also not significant difference between12h group and Oh group(P>0.05)(5) HBV DNA and HBV cccDNA expression of PBMCs in the basolateral chamber were detected.HBV DNA content was (2.565±0.361)×103copies/ml and HBV cccDNA content was (1.3550±2.473)X103copies/ml.These results demonstrated that HBV can infected PBMCs and reproducted in them.CONCLUSIONS:1. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no correlation between HBV intrauterine infection and PBMC maternal-fetal transfer. PBMC maternal-fetal transfer is correlated with PBMC HBV DNA in newborns. When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, it is correlated with PBMC maternal-fetal transfer.2. HBsAg positive in placenta is associated with HBsAg in newborns'serum. HBsAg positive in placenta is independent of HBV infection in newborns.3. Placental layers of cells show apoptosis. The trophoblast cell apoptosis is higher than others. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was no relation between placental apoptosis and HBV intrauterine infection. When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, there was correlation between HBV intrauterine infection and placental apoptosis. PBMC HBV DNA positive in newborns is related with placental apoptosis in trophoblastic cells.4. PBMC HBV DNA in pregnant women, the protein levels of Caspase3in placental decidual cells and apoptosis index of placental trophoblast cells are risk factors for PBMC maternal-fetal transfer. PBMC HBV DNA in pregnant women and PBMC maternal-fetal transfer are risk factors for PBMC HBV DNA positive in newborns. PBMC HBV DNA in pregnant women is a risk factor for PBMC HBV cccDNA positive in newborns, but apoptosis index of placenta is a protective factor. When any of HBsAg, HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, HBeAg positive in pregnant women and apoptosis index of placental trophoblast cells are risk factors for HBV intrauterine infection, but cesarean section and age of pregnant women are protective factors for HBV intrauterine infection. When any of HBsAg, HBV DNA and PBMC HBV DNA positive in newborns were used to judge neonatal HBV intrauterine infection index, PBMC HBV DNA in pregnant women, PBMC maternal-fetal transfer and apoptosis index of placental trophoblast cells are risk factors for HBV intrauterine infection.5. HBsAg in PBMC is as indicator of HBV infection in PBMC. Transfer of PBMC infected with HBV is independent of the placental apoptosis. PBMC HBsAg positive in newborns is related with PBMC transfer. There is no correlation among HBsAg positive in newborns'PBMC and placental apoptosis.6. PBMC HBV DNA positive in newborns can be used as one of diagnostic criteria for HBV intrauterine infection. PBMC can be used as the carrier of the HBV which transport HBV from the mothers to their fetal blood circulation, and can cause fetal infection. PBMC maternal-fetal transfer may be another infection route of HBV intrauterine infection.7. The relationship between placental apoptosis and transportion of PBMC infected with HBV was studied by imitating placental barrier in vitro. A positive correlation is found between the rate of placental apoptosis and the rate of PBMC transfer. After PBMCs infected with HBV come to below chamber of transwell through placental barrier, PBMCs below chamber can be infected. Thus, an inference come into being, that is PBMCs infected with HBV of mothers' transport to the fetal blood circulation and result in PBMC HBV infection in fetus.
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