Use of maternal plasma for non-invasive prenatal diagnosis of fetal ABO genotypes.

BACKGROUND: Measurement of free fetal DNA in maternal plasma opened a door for non-invasive prenatal diagnosis. Prenatal diagnosis of fetal ABO genotypes can provide a basis for the prevention and therapy of maternal-fetal incompatibility. We identified fetal ABO genotypes using fetal DNA in plasma from pregnant women with blood group O. The aim of the study was to investigate the accuracy and feasibility of this method. METHODS: A total of 105 blood group O women in middle or late pregnancy were enrolled. Fetal DNA in maternal plasma and genomic DNA in umbilical vein blood from newborns were extracted using a QIAamp DNA Blood Kit. DNA was amplified to identify ABO genotypes by PCR with sequence-specific primers (PCR-SSP). The genotype results were evaluated using serologic tests for ABO phenotyping. RESULTS: Using DNA from umbilical vein blood, ABO genotypes of 105 newborns were successfully identified by PCR-SSP. Using fetal DNA from maternal plasma, 88.6% (93/105) fetal ABO genotypes was correct; 12 false results were from 66 pregnant women with fetuses of type non-O. The accuracy in middle pregnancy was lower than that in late pregnancy, although the difference was not significant (0.05

[Screening of differentially expressed genes in placentas with hepatitis B virus infection by suppression subtractive hybridization technique].

OBJECTIVE: To screen differentially expressed genes in placentas with hepatitis B virus (HBV) infection and to discuss the molecular mechanism of HBV intrauterine infection. METHODS: Thirty placenta tissue specimens from HBsAg and HBV DNA positive pregnant women were used as the study group and 30 placenta tissue specimens from normal pregnant women with HBsAg and HBV DNA negativity were served as the control group. The suppression subtractive hybridization (SSH) technique was used. Total RNAs of placenta tissue of the study group were mixed as the tester, and total RNAs of placenta tissue of the control group were mixed as the driver. A subtractive cDNA library was constructed by PCR-selective cDNA subtraction systems. Amplifications of the library were carried out with E. coli strain DH5alpha by reverse spot hybridization. RT-PCR confirmed that phosphatidylinositol 3-kinase (PI3K) was up-regulated in placenta tissue with HBV infection. RESULTS: Colony PCR showed that the clones contained 200 - 1000 bp inserts. Thirty five clones were confirmed by reverse spot hybridization and analyzed by sequencing and bioinformatics. Thirty three known genes and 2 genes with unknown function were obtained. RT-PCR preliminarily confirmed that PI3K gene was up-regulated in HBV infected placenta. CONCLUSIONS: The differentially expressed genes in placentas with hepatitis B virus (HBV) infection using SSH technique has been screened out successfully. These differentially expressed genes encoding proteins participating in cell vital metabolism and malformation, and signal conduction-antiapoptosis pathway. This finding brings some new clues for studying the mechanisms of HBV intrauterine infection.

[The role of peripheral blood mononuclear cells (PBMC) of HBV-infected mothers in the intrauterine infection of their fetuses].

OBJECTIVE: To study the role of the HBV-infected mothers' PBMC in intrauterine transmission of HBV to their fetuses. METHODS: Thirty pregnant women with serum HBV DNA negative and PBMC HBV DNA positive and their newborns were used as the study group. Ten pregnant women with serum HBV negative and their infants served as the control group. HBV DNA in serum and in PBMC was detected using nested polymerase chain reaction (n-PCR). The mothers' PBMC in newborns' peripheral blood was examined using heminested-PCR. RESULTS: Four newborns were serum HBV DNA positive and 8 newborns were HBV DNA positive in PBMC in the study group. Among them, 2 newborns were HBV DNA positive in both serum and PBMC, 6 cases were positive in PBMC only, and 2 cases were positive in serum only. Five mothers had the GSTM1 gene; and it was not detected in 3 newborns. Among the 8 newborns with HBV DNA positive in PBMC, 3 did not have the GSTM1 gene, at the same time their mothers possessed the GSTM1 gene. Mothers' PBMC were detected in all of these three newborns' peripheral blood. HBV DNA in serum and in PBMC of the control group infants were all negative. CONCLUSION: HBV-infected PBMC of the mother may serve as a vector in HBV intrauterine infection.

[Expression of human annexin V in different fetal tissues].

OBJECTIVE: To investigate the expression of human annexin-V (HA-V) in relation to HBV infection in different fetal tissues. METHODS: Immunohistochemistry was employed to detect the expression and distribution of HA-V in the liver, kidney, ovary, heart, fallopian tube, spleen, and thymus gland of human fetus. RESULTS: HA-V expression was detected in different tissues including the ovary, liver, intrahepatic bile duct, heart, kidney, lymphocytic cells in the thymus gland, epithelial cells of the fallopian, and cortical and medullary cells of the spleen. HA-V was distributed mainly in the cytoplasm of the cells. The liver tissues exhibited greater gray scale for HA-V expression than in the other tissues (P<0.05) and no significant difference was observed in the other tissues than the liver (P>0.05) in image analysis with Photoshop 7.0. CONCLUSION: HA-V is an inherent protein in fetal tissues with possible relation to HBV infection of different tissues as a HBV receptor. Greater amount of HA-V in the liver may account for the vulnerability of the liver to HBV infection.

[Peripheral blood mononuclear cell of neonates infected with hepatitis B virus].

OBJECTIVE: To study the mechanism and significance of peripheral blood mononuclear cell (PBMC) of neonates infected with hepatitis B virus (HBV). METHODS: Eighty-four HBsAg-positive and HBeAg-negative mothers and their newborns were recruited in this study. Sixteen hepatitis B virus markers (HBVM)-negative mothers and their neonates were served as control. All these cases had no symptoms of hepatitis, serious pregnancy complications and preexisting disease. Age, gestational age and the method of delivery were matched in two groups (P > 0.05). Five ml blood samples were taken from the peripheral vein of the pregnant women before delivery and from neonates within 24 hours after birth, before inoculation of HBV vaccine (HBVac). Serum and PBMC were isolated from 2 ml and 3 ml samples respectively. The sera, PBMC and the last supernatant of PBMC washing were stored at -80 degrees C. HBVM of neonates were detected by using enzyme linked immunosorbent assay (ELISA). HBV DNA in serum, PBMC and the last supernatant of PBMC washing of mothers and neonates were detected by using a nested-polymerase chain reaction (n-PCR). Two pairs of oligonucleotide primers, the outer primer pair for first PCR and inner primer pair for second PCR, designed according to region S of HBV genome were synthesized at Shanghai Cell Biology Institute of Chinese Academy of Sciences. The neonates who were HBV DNA positive in PBMC but HBsAg and HBV DNA negative in serum were followed up for one year, HBsAb in serum and HBV DNA in PBMC were observed in the neonates. RESULTS: (1) The positive rate of HBV DNA in 84 serum and PBMC of mothers were 53.57% and 40.48%, respectively (chi(2) = 2.891, P > 0.05). All the results were weakly positive. (2) Twenty-four (28.57%) newborns in the study group were infected, including 7 who were only HBV DNA positive in serum, 11 only HBV DNA positive in PBMC and 6 in both, all the results were weakly positive. HBsAg was negative in all the newborns. None of the neonates in control group was infected with HBV. There was significant difference between the two groups (chi(2) = 4.55, P < 0.05). (3) Of all the study cases, 11 (13.10%) neonates were HBV DNA weakly positive in PBMC but HBsAg and HBV DNA negative in serum. Of their mothers, 5 were only HBV DNA positive in serum, 2 only positive in PBMC and 4 positive in both serum and PBMC. Seven of the 11 neonates were followed up for one year and at the end of follow-up, 4 were HBsAb positive and HBV DNA negative in PBMC; 3 were HBsAb negative, and among the 3 cases HBV DNA in 2 was still positive in PBMC, HBsAg and HBV DNA in serum were negative in all the 7 neonates. CONCLUSION: (1) HBV DNA positivity either in serum or in PBMC in mothers can result in infection of PBMC with HBV in their neonates. (2) PBMC infection with HBV can exist for a long time in neonates while HBsAg and HBV DNA are negative in serum, and may result in vaccination failure in neonates.

Expression of HBsAg and HBcAg in the ovaries and ova of patients with chronic hepatitis B.

AIM: To investigate the expression and distribution of HBV in the ovaries and ova. METHODS: The immunohistochemistry method was used to detect the HBsAg and HBcAg in the ovaries of patients with chronic hepatitis B. RESULTS: Expression of HBsAg in the ova, granular and interstitial cells of the ovaries was located in the cytomembrane and cytoplasm. Expression of HBcAg in the ova, granular, interstitial and endothelial cells of interstitial blood vessels of the ovaries was found in the cytomembrane, cytoplasm, and nuclei. CONCLUSION: HBV can infect the ova at different stages of development and replicate in it.

[Clinical significance of detecting neonatal peripheral blood mononuclear cells infected by HBV].

OBJECTIVE: To understand the HBV infection rate of peripheral blood mononuclear cells (PBMCs) from fetuses of HBsAg positive mothers, associated risk factors and to explore the clinical significance of detecting HBV infected PBMCs. METHODS: Sixty eight pregnant women who were delivered at the First Hospital of Xi'an Jiaotong University, China from August 1995 to February 1997, and their newborns were studied. They were divided into two groups according to their status of HBV serological markers. The study group included 50 cases who were serum HBsAg positive and 18 cases without any HBV serum markers served as control group. All these cases had no symptoms of hepatitis, high risk premature labor, premature delivery and hypertensive disorder complicating pregnancy. Age and gestational age were matched in two groups. Blood samples (5 mL) were taken from the peripheral vein of pregnant women before delivery and from newborns within 24 h after birth, before inoculation of HBV vaccine (HBVac) and injection of hepatitis B immunoglobulin (HBIG). PBMCs were isolated. The sera and PBMCs were stored at -80 degrees C. HBV-DNA in serum and PBMCs were detected with nested polymerase chain reaction (n-PCR). Two pairs of oligonucleotide primers, the outer primer pair for first PCR and inner primer pair for second PCR, designed according to region S of HBV genome were synthesized by Shanghai Cell Biology Institute of Chinese Academy of Science. RESULTS: The detection rate of HBV-DNA in serum and PBMCs from HBsAg positive pregnant women was 60.0% (30/50) and 40.0% (20/50), respectively. The detection rate of HBV-DNA in serum and PBMCs from newborns of HBsAg positive pregnant women was 46.0% (23/50) and 30.0% (15/50), respectively. Ten newborns were HBV-DNA positive in serum only, 2 were positive in PBMCs only and 13 were positive in both serum and PBMCs. In the control group, HBV-DNA was not detected in PBMC nor in serum. The positive rate of HBV-DNA in PBMCs of newborns was significantly higher in the group of mothers who were HBV-DNA or HBeAg positive in serum (P < 0.05, P < 0.01); the positive rate was significantly higher in the group of mothers who were HBV-DNA positive in both serum and PBMC than that in the group of mothers who were serum HBV-DNA positive only (P < 0.01); and it was markedly higher in the group of mothers who were PBMC HBV-DNA positive than that in group of mothers who were HBV-DNA negative in PBMCs (P < 0.01). The positive rate of HBV-DNA in PBMCs of newborns was significantly higher in the group of newborns who were HBV-DNA positive in serum than that in the group of newborns who were HBV-DNA negative in serum (P < 0.05). CONCLUSIONS: The positive rate of HBV-DNA in PBMCs from newborns of HBsAg positive pregnant women was 30.0% (15/30). It was related to HBV viremia level and HBV-DNA status in PBMCs of mothers and newborns. Detection of HBV-DNA in PBMCs may be an important supplementary method to determine intrauterine HBV infection, and can predict the response to HBV vaccine.

Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique.

AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection. METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BamH I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ. After 48 h, cells were collected and detected for the expression of beta-galactosidase (beta-gal). Suppression subtractive hybridization and bioinformatics techniques were used. The mRNA of HepG2 cells transfected with pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector was isolated, and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR) method, and cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification. RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of beta-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete S was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively. Sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques, one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877). CONCLUSION: The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.

[Study on intrauterine infection of hepatitis B virus in pregnant women with hepatitis B surface antigen and hepatitis B e antigen negative].

OBJECTIVE: To explore the significance of intrauterine infection of hepatitis B virus in pregnant women with hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) negative by nested polymerase chain reaction (PCR). METHODS: Twenty-four pregnant women with HBsAg and HBeAg negative but other HBV markers positive together with their infants were included as study group. Sixteen pregnant women with HBV marker negative and their infants were in the control group. HBV DNA in sera and peripheral blood mononuclear cells (PBMCs) of two groups was detected by nested PCR respectively. RESULTS: (1) In the study group, the positive rates of HBV DNA of pregnant women were 33% (8/24) in the sera and 42% (10/24) in PBMCs. Three women were detected HBV DNA in both serum and PBMCs. The rate of HBV infection was 63% (15/24); (2) also in the study group, the positive rates of HBV DNA of the infants were 13% (3/24) in the sera and 25% (6/24) in PBMCs. One newborn was detected HBV DNA in both serum and PBMCs, the rate of intrauterine infection of HBV was 33% (8/24); (3) HBV DNA was detected in sera and/or in PBMCs from four newborns of pregnant women with HBV DNA positive only in PBMCs, the positive ratio was 4/7. CONCLUSIONS: HBV intrauterine infection is possible in pregnant women with HBsAg and HBeAg negative. Detecting HBV-DNA in sera and PBMCs of pregnant women and their newborns by PCR is important clinical significance.

[Study on relationship between serum cholylglycine and placental apoptosis in patients with intrahepatic cholestasis of pregnancy].

OBJECTIVE: To explore the possible mechanism of placental dysfunction in patients with intrahepatic cholestasis of pregnancy (ICP). METHODS: Serum level of cholylglycine (CG) in 30 cases with ICP (ICP group) and 27 normal pregnant women (control group) was examined by radio-immunoassay before delivery. bax and bcl-2 level in placenta was detected by immunohistochemistry method. RESULTS: (1) Serum CG level in ICP group was (51.8 +/- 5.9) micro mol/L, and in control group it was (9.4 +/- 5.6) micro mol/L. The difference between the two groups was significant (P < 0.05). (2) bax expression level in syncytiotrophoblast of ICP group was: 2(-), 9(+), 11 (++), 8(+++), and bcl-2 expression level was: 12(+), 12(++), 6(+++); bax expression level in control group was: 9 (-), 13(+), 4(++), 1(+++) and bcl-2 expression level was: 3(+), 4(++), 20(+++). bax expression level in syncytiotrophoblast of ICP group was higher than that of the control (P < 0.0005), and bcl-2 expression level was lower than in the control (P < 0.0005), both significantly. A significant positive correlation between CG level and bax expression level and a negative correlation between CG and bcl-2 were found in ICP group (P < 0.005, P < 0.005). CONCLUSION: One of the possible mechanisms involved in placental dysfunction of ICP is overexpression of bax in syncytiotrophoblast caused by high concentrations of bile acid, leading to increased apoptosis.

[Study on the mechanism of intrauterine infection of hepatitis B virus].

OBJECTIVE: To study the possible mechanism of intrauterine infection of hepatitis B virus (HBV). METHODS: HBV DNA was examined in amniotic fluid, and vaginal secretion of 59 HBsAg positive mothers and in cord blood of their neonates by PCR. Ten negative hepatitis B virus marker (HBVM) mothers and their neonates were served as control. HBsAg and HBcAg in placenta were examined by avidin biotin complex (ABC) method. RESULTS: The detection rate of HBV DNA in amniotic fluid, vaginal secretion and neonatal cord blood of the study group were 47.5% (28/59), 52.5% (31/59) and 45.8% (27/59) respectively. HBsAg and HBcAg in placenta was distributed in the following descending order: maternal decidual cells, trophoblastic cells, villous mesenchymal cells and villous capillary endothelial cells. But the distribution was in reverse order in 4 placentas. HBsAg and HBcAg were detected in amniotic epithelial cells in 32 mothers. CONCLUSION: The main route of HBV transmission from mother to fetus is transplacental, from maternal side of placenta to fetal side. However, HBV intrauterine infection may take place through other routes.

Mechanism of intrauterine infection of hepatitis B virus.

AIM: To explore the possible mechanism of intrauterine infection of hepatitis B virus (HBV). METHODS: HBV DNA was detected in vaginal secretion and amniotic fluid from 59 HBsAg-positive mothers and in venous blood of their newborns by PCR. HBsAg and HBcAg in placenta were determined by ABC immunohistochemistry. RESULTS: The rate of HBV intrauterine infection was 40.1% (24/59). HBV DNA was detected in 47.5% of amniotic fluid samples and 52.5% of vaginal secretion samples respectively. HBsAg and HBcAg were detected in placentas from HBsAg-positive mothers. The concentration of the two antigens decreased from the mother's side to the fetus's side, in the following order: maternal decidual cells > trophoblastic cells > villous mesenchymal cells > villous capillary endothelial cells. However, in 4 placentas the distribution was in the reverse order. HBsAg and HBcAg were detected in amniotic epithelial cells from 32 mothers. CONCLUSION: The main route of HBV transmission from mother to fetus is transplacental, from the mother side of placenta to the fetus side. However, HBV intrauterine infection may take place through other routes.

Relationship between HBV viremia level of pregnant women and intrauterine infection:neated PCR for detection of HBV DNA.

AIM:To determine the incidence of hepatitis B virus (HBV) in trauterine infection and to explore the relationship between HBV viremia level of pregnant women and HBV intrauterine infection.METHODS: Sixty-nine pregnant women were divided into three groups. Group A, 41 HBsAg positive patients, 14 of them were HBeAg positive (group A1), and 27 HBeAg negative (group A2); Group B, 12 HBsAg negative patients, but positive for anti-HBs and/or anti-HBe and/or anti-HBc; and Group C, 16 patients negative for all HBV markers. Blood samples of mothers were taken at delivery, samples of their infants were collected within 24 hours after birth (before injection of HBIG and HBV vaccine). All the serum samples were stored at -20°. HBV serum markers were tested by radioimmunoassay and HBV NDA were detected by nested polymerase chain reaction.RESULTS: In group C, all of 16 newborns were negative for HBsAg and HBV DNA. In group A, 7 infants were HBsAg positive (17.1%), and 17 (41.5%) were HBV DNA positive (P < 0.05). The incidence of intrauterine HBV infection was much higher in group A1 than that in group A2 (HBsAg 42.9% vs 3.7%, HBV DNA 92.9% vs 14.8%, P < 0.05). The incidence of HBV intrauterine infection was significantly different between high and low HBV viremia of mothers (93.3% vs 42.9%,P < 0.05).CONCLUSION: The incidence of HBV intrauterine infection is high when HBV DNA in newborns detected with nested PCR is used as a marker of HBV infection. It is related to HBV viremia level of mothers.