Study On Hepatitis B Virus Infection Of Placenta And Intrauterine Transmission

Objective1. To explore the influence of pregnant women hepatitis B virus (HBV) infection ongrowth of fetus and placenta.2. To study the changes of HBV infection on placental morphology and functionfrom histological experiments.3. To study the infection status of HBV in trophoblast cells, preliminary discussthe possibility of intracellular replication in infected trophoblasts in vitroexperiments.Methods1. The first part:Retrospectively analysised the clinical data of194hepatitis B surface antigen(HBsAg)positive pregnant women and their newborns,while296HBsAg negative women andtheir newborns were selected randomly as control group.Compared the pregnantcomplications, perinatal outcome and the fetal-placental growth of the two groups,anddiscussed the relationship between these indexes with HBV intrauterine infection.2. The second part:Collected HBsAg positive women term placental tissues25cases as case group,andHBsAg negative women placental tissues10cases as control group, the expression ofHBsAg were detected by PV-6002two steps of immunohstochemical (IHC) stainingin case group; HBV covalently closed circular DNA (cccDNA) was detected byfluorescence quantitative PCR(FQ-PCR) in case group; The villous capillaries werestained with anti-CD34and the changes of microvascular length density and volume density were evaluated stereologically; TUNEL method for the detection of placentalcell apoptosis index(AI).3. The third part:Collected HBV carrying pregnant women term placenta12cases,the chorion tissueswere separated and purified by the combination of modified gradient gravitycentrifugation with repeated adherent method,then to culture; Flow cytometric(FCM)detection of trophoblast cell cytokeratin-7(CK-7) expression,for the cell purityidentification;Immune double-labeling combined with laser scanning confocalmicroscopy (LSCM)techniques for the detection of intracellular CK-7and Vimentin,to identify the morphology; The culture supernatants were collected at24h,48h,72hand subjected to HBsAg/HBeAg detection by Microparticle Enzyme Immunoassay(MEIA) as well as to HBV DNA measurement by FQ-PCR; more than98%puritytrophoblasts were selected by FCM,and FQ-PCR detected the HBV cccDNAexpression in trophoblast cells.Results1. The first part:1) Compared with the HBsAg negative women, HBsAg positive pregnant womenpregnant frequency increased, placental volume were smaller, and their neonateshad small birth weight (P<0.05); The placental volume of HBV DNA positivepregnant women were smaller than the ones of HBVDNA negative,but theplacenta volume of glutamic transaminase(ALT) abnomal group were largerthan the nomal group(P<0.05);2) HBsAg positive newborns had smaller birth weights,heights and placentalvolume,but higher incidence of fetal growth restriction(FGR),compared with theHBsAg negative newborns(P<0.05); Hepatitis B e antigen(HBeAg) positivenewborns had higher incidence of FGR,and smaller placental volume than theHBeAg negative newborns(P<0.05);The placental volume of hepatitis B surface antibody (HBsAb) positive newborns were larger than the HBsAb negativenewborns(P<0.05);3) Placental volume was the protective factor of intrauterine HBV(OR=0.996，p=0.005); while maternal serum HBV DNA as risk factor(OR=1.396，p=0.004).2. The second part:1) There were13HBsAg positive placentas in the25cases (the positive rate was52.0%); The positive signals of HBsAg was mostly located in trophoblasticcells(TC),villous meseachymal cells(VMC) and villous capillary endothelialcells(VCEC);2) There were7HBV cccDNA positive placentas in the25cases (the positive ratewas35.0%),and their quantities as follows:6.04×10~5、3.82×10~3、8.94×10~3、2.91×10~4、7.02×10~3、5.28×10~2、1.12×10~4copies/ml; Compared with the thenegative group,the abnormal rate of ALT in HBV cccDNA positive placenta washigher, and pregnant frequency increased(P<0.05); There were significant andpositive correlation between the titers of maternal serum HBV DNA and placentalcccDNA(r=0.994，P=0.000); Pregnant frequency and HBsAg positive in placentawere risk factors of HBV cccDNA positive in placenta (OR=4.85;OR=16.02);3) Placental villus vascular stereology showed that, CD34almost marked all thevillus vascular,both HBV placenta and normal placentas were detected the brownpositive signals; There is no difference in the number of microvascular lengthdensity and volume density between HBV placenta and normalplacentas(P>0.05); Microvascular volume density were significantly andpositively related to the titers of placental HBV cccDNA(r=0.944，P=0.001);4) Apoptosis cells were present in HBV placenta and normal placenta,and thepositive signals are expressed in different types cell of placenta; Apoptosis indexof trophoblast cells and mesenchymal cells in HBV infection placenta were bothhigher than the nomal group(P<0.05); Maternal serum HBV DNA titers andapoptosis index showed a moderate positive correlation(r=0.422，p=0.045)； Microvascular length density and apoptosis index of placental cells showed amoderate negative correlation(r=-0.464，p=0.022).3. The third part:1) The supernatants of trohpoblast cell from12HBV carrying pregnant women,which cultured in vitro for24,48,72h,could be detected the positive expression ofHBV DNA, HBsAg and HBeAg, and all the expression were hightest at24h;Withthe passage of time, HBV DNA titer decreased gradually, while HBsAg andHBeAg titer were first decline and then rising, but the trend were not statisticallydifferent at three time points;The expression of HBsAg and HBeAg at threepoints showed a positive correlation(P<0.05).2) There were4HBV cccDNA positive placentas in the12trohpoblast samples (thepositive rate was33.3%),and their quantities as follows:5.67×10~3、2.12×10~4、1.72×10~4、4.90×10~4copies/ml; Maternal serum HBV DNA titers were positiverelated to the HBV cccDNA titers of trophoblasts(r=0.997，p=0.023).Conclusion1. HBV infection can affect fetal and placental growth,which results in smallplacental size and low birth weight.2. Placental volume is the protective factor of intrauterine HBV infection,Theplacental volume of HBsAb positive newborns are larger than the HBsAbnegative newborns; Maternal serum high viral load and elevated transaminasesare risk factors of intrauterine HBV infection.3. Pregnant frequency and HBsAg positive in placenta are risk factors of HBVcccDNA positive in placenta. With the rising of maternal blood HBV DNA titerrises, placental HBV cccDNA titer rises, and microvascular volume densityincreases.4. In HBV infection placentas, apoptosis index of trophoblast cells andmesenchymal cells increase. 5. HBV could infect placental trophoblast cells and replicate in trophoblasts.