Arch Gynecol Obstet
DOI 10.1007/s00404-013-2893-x
MATERNAL-FETAL MEDICINE
Role of peripheral blood mononuclear cell transportation
from mother to baby in HBV intrauterine infection
Qingliang Shao • Xiaxia Zhao • M. D. Yao Li
Received: 28 January 2013 / Accepted: 11 May 2013
! Springer-Verlag Berlin Heidelberg 2013
Abstract
Purpose We aimed to investigate the role of peripheral
blood mononuclear cell transportation from mother to baby
in hepatitis B virus (HBV) intrauterine infection.
Methods Thirty HBsAg-positive pregnant women in the
second trimester and their aborted fetuses were included in
this study. Enzyme-linked-immunosorbent-assay was uti-
lized to detect HBsAg in the peripheral blood of pregnant
women and the femoral vein blood of their aborted fetuses.
HBV-DNA in serum and peripheral blood mononuclear cells
(PBMC) and GSTM1 alleles of pregnant women and their
aborted fetuses were detected by nested polymerase chain
reaction (PCR) and seminested PCR, respectively. We also
examined the location of placenta HBsAg and HBcAb using
immunohistochemical staining. The expression of placenta
HBV-DNA was detected by in situ hybridization.
Results For the 30 aborted fetuses, the HBV intrauterine
infection rate was 43.33 %. The HBV-positive rates of
HBsAg in peripheral blood, serum, and PBMC were 10 %
(3/30), 23.33 % (7/30), and 33.33 % (10/30), respectively.
Maternal–fetal PBMC transport was significantly positively
correlated with fetal PBMC HBV-DNA (P = 0.004).
Meanwhile, the rates of HBV infection gradually decreased
from the maternal side to the fetus side of placenta (decidual
cells [ trophoblastic cells [ villous mesenchymal cells [
villous capillary endothelial cells). However, no significant
correlation between placenta HBV infection and HBV
intrauterine infection was observed (P = 0.410).
Q. Shao ! X. Zhao ! M. D. Yao Li (&)
Department of Pediatrics, The Second Affiliated Hospital
of Harbin Medical University, 148 Bao Jian Street,
Nan Gang District, Harbin, Heilongjiang 150081,
People’s Republic of China
e-mail: yaoli201305@163.com
Conclusions HBV intrauterine infection was primarily
due to peripheral blood mononuclear cell maternal–fetal
transportation in the second trimester in pregnant women.
Keywords HBV intrauterine infection ! PBMC transport !
HBV placenta infection
Introduction
Hepatitis B is an infectious disease that poses a serious
threat to human health [1, 2]. It has been suggested that
routine immunization of infants and young children could
control hepatitis B virus (HBV) infection effectively [3].
However, if there is intrauterine HBV infection and hep-
atitis B virus e antigen (HBeAg)-positive blood transmitted
from the pregnant women to their fetuses, the fetuses will
be infected with HBV and therefore postnatal immuniza-
tion will not be successful [4, 5].
China exhibits a high prevalence of hepatitis B, with
approximately 690 million infected persons and 120 mil-
lion carriers, as well as 20 million people with chronic
hepatitis [6]. The intrauterine infection rate of HBsAg-
positive pregnant women is 5–40.1 % [7]. Thus, intrauterine
infection is an important route of HBV transmission.
Elucidation of the mechanism of HBV maternal–fetal
transmission is crucial to develop methods to control and
block intrauterine HBV infection. To date, there have been
few reports focusing on this subject; moreover, the results
are conflicting [8–12].
Previously, some researchers considered that intrauterine
HBV infection occurred only during the last trimester of
pregnancy. They gave HBsAg-positive pregnant women
hepatitis B immunoglobulin (HBIG) to interrupt HBV
intrauterine infection during late pregnancy [13, 14]. In
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addition, most of the previous studies that focused on
maternal–fetal hepatitis B virus transport and placenta HBV
infection were conducted during the last trimester of preg-
nancy. To further understand the potential role of PBMC
transportation from mother to baby in HBV intrauterine
infection, we conducted a cross-sectional study in pregnant
women and their aborted fetuses at the second trimester.
Methods
Subjects and specimens
All subjects were recruited from the pregnant women who
accepted prenatal care in the Department of Gynaecology
and Obstetrics, the Second Affiliated Hospital of Harbin
Medical University, from January 2007 to January 2009.
The HBV markers (HBsAg, anti-HBs, HBeAg, anti-HBe,
anti-HBc) in the serum of pregnant women were detected.
Thirty HBsAg-positive women who had fetuses aborted in
second trimester and their aborted fetuses were recruited.
Informed consent was obtained from all pregnant women
or their family members prior to sample collection. Our
study was conducted in accordance with the Code of
Practice Harbin Medicine University Ethics Committees.
In total, 3 ml venous blood specimens from each preg-
nant woman before delivery and 3 ml venous blood spec-
imens from the femoral vein of the aborted fetuses within
24 h of birth were collected. For serum, 1 ml blood sample
was separated; 2 ml sample was utilized for PBMC using
2 % EDTA anticoagulants.
Placenta tissue samples (approximately 1 cm 9 1 cm 9
1 cm) from women upon delivery were stripped and fixed
in 4 % (w/v) paraformaldehyde/0.02 M PBS (pH 7.2) within
30 min. Next, they were dehydrated by gradient alcohol and
transparentized in xylene following embedded in paraffin.
Each one was sectioned to slices of 4–5 lm in thickness.
Laboratory analysis
Determination of serum HBsAg
HBsAg determination of peripheral blood from the preg-
nant women was performed by ELISA (Diagnostic Auto-
mation/Cortez Diagnostics, Inc. USA).
Quantification of HBV-DNA in serum and PBMC
HBV-DNA levels in serum and PBMC were screened and
sequenced by nested-PCR. The primers of polymerase chain
reaction (PCR) were as follows: P1 50 -AGGTCTTGCCCA
AGGTCTTAC-30 (nt 1633–1653), P2 50 -CAAGGCACAGC
TTGGAGGCTT-30 (nt 1888–1868), P3 50 -CCGACCACGG
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Arch Gynecol Obstet
GGCGCACCTCT-30 (nt 1515–1535), P4 50 -CTCAGA
GGCCTTGTAACAAGT-30 (nt 2046–2026).
The PCR conditions were as follows: initial denaturation
at 94 "C for 5 min, followed by 30 cycles of 94 "C for 60 s,
55 "C for 60 s, 72 "C for 60 s, and followed by a final
extension of 5 min at 72 "C. In addition, PCR products were
subjected to direct 2 % agarose gel electrophoresis staining.
If the result (first run) was negative, the other products were
taken as templates for the second run. Each experiment was
assayed to positive, negative and blank control.
Detection of maternal cells in the peripheral blood
of aborted fetus and maternal–fetal PBMC transport
Genomic DNA samples were extracted using kit [Tiangen
Biotech (Beijing) Co., Ltd.]. Based on the polymorphisms
of glutathione S transferase M1 (GSTM1) gene, maternal
GSTM1 (?/-, ?/?) and infant GSTM1 (-/-) genes were
chosen using nested-PCR. The primers were as follows: 50 -G
AACTCCCTGAAAAGCTAAAGC-30 , 50 -GTTGGGCTC
0
AAATATACGGTGG-3 . If GSTM1 (?/-, ?/?) was
detected from GSTM1 (-/-) infants, it indicated that
maternal–fetal PBMC transport exists. In this way, GST
products of PCR were amplified again. Each experiment was
allocated to positive, negative and blank control.
Detection of placenta HBV infection
Placenta HBsAg and HBcAb were examined by strepta-
vidin–biotin complex (SABC) method. HBV-DNA was
detected using in situ hybridization. We detected the
HBsAg, HBcAb, and HBV-DNA for the tissues from
decidual cells (DC), trophoblastic cells (TC), villous mes-
enchymal cells (VMC), and villous capillary endothelial
cells (VCEC) of placenta, respectively.
Statistical analysis
Statistical analysis was performed by SPSS version 16.0
software package. Statistical significance was tested by
Chi-square test. Pearson’s R and spearman correlation were
also generated by Chi-square test. The P value of \0.05
was considered to indicate a statistically significant result.
Results and discussion
Results
Intrauterine HBV infection rate
Among the 30 aborted fetuses, 3, 7, and 10 were HBsAg
positive in peripheral blood, HBV-positive DNA in serum,
Arch Gynecol Obstet
Fig. 1 The result of detecting maternal GSTM1 in fetal peripheral
blood by nested-PCR
and PBMC, respectively, with a rate of 10 % (3/30),
23.33 % (7/30), and 33.33 % (10/30), respectively. The
results showed that the positive rate of HBV-DNA in serum
or PBMC was higher compared to HBsAg-positive
peripheral blood. In this study, we defined the occurrence
of HBV intrauterine infection when one of the three indi-
cators (including HBsAg in peripheral blood, HBV-DNA
in serum, or PBMC) was positive. The rate of HBV
intrauterine infection was 43.33 % (13/30).
PBMC maternal–infant transport and HBV intrauterine
infection
The agarose gel electrophoresis results for the detection of
maternal GSTM1 in fetal peripheral blood are shown in
Fig. 1. Maternal GSTM1 (?/-, ?/?) gene was detected in
16 cases of fetuses with GSTM1 (-/-) gene. The inci-
dence rate of maternal to fetal cell transport was 53.33 %
(16/30).
As shown in Table 1, there was a significant correlation
between fetal PBMC HBV-positive DNA and maternal
PBMC transport (P value 0.004). Both the Pearson’s R and
Spearman’s R were 0.520; this indicated that there was a
significant correlation between maternal–fetal PBMC
transport and HBV intrauterine infection based on PBMC
HBV-DNA. However, no significant correlation was found
between maternal PBMC transport and the status of HBsAg
in fetal peripheral blood (P = 0.156). Similarly, there was
no significant correlation between maternal PBMC trans-
port and serum HBV-DNA (P = 0.346).
Placenta HBV infection and HBV intrauterine infection
As shown in Fig. 2, HBsAg and HBcAb in placenta
appeared as inhomogeneous dark brown yellow granules in
all cell types. The HBV-DNA was blue and was present in
the nuclei of positive cells that were distributed from
centralization to decentralization, including 16.67 % (5/30)
of maternal DC, 13.33 % (4/30) of TC, 10 % (3/30) of
VMC, and 3.33 % (1/30) of VCEC. The same rates of
HBV-positive DNA and HBsAg-positive were observed in
the different cells of placenta. Moreover, the number of
HBsAg, HBcAg, and HBV-DNA positive cells gradually
decreased from maternal DC to VCEC (Tables 2, 3).
However, there was no significant correlation between
placenta HBV infection and HBV intrauterine infection
(P = 0.410).
Discussion
Mother-to-child transmission (MTCT) is one of the prin-
cipal HBV infection pathways [15, 16]. In China, one in
three HBsAg-positive carriers is caused by MTCT and the
infection rate of newborns with HBsAg-positive mothers is
approximately 50 % [17]. Currently, there is no clinical
method to diagnose the HBV intrauterine infection accu-
rately in the second trimester. Extraction of the umbilical
cord blood through the mother’s womb by centesis is a
method that is invasive and not risk free. Moreover,
although detection results may be positive for HBsAg,
HBV intrauterine infection cannot be confirmed. A few
previous studies regarding the transmission mechanism of
HBV intrauterine infection in last trimester of pregnant
women have been published; however, the results were
conflicting. To elucidate the mechanism underlying HBV
intrauterine infection, we analyzed the correlation of HBV
intrauterine infection with PBMC transport and HBV pla-
centa infection in 30 HBsAg-positive pregnant women and
their aborted fetuses.
Because umbilical cord blood collection is feasible and
harmless to fetuses, intrauterine infection was determined
based on HBV-DNA detection results of umbilical cord
blood in some previously published studies. However, it
has been suggested that the false positive rate is difficult to
avoid because umbilical cord blood is easily contaminated
with maternal blood [18]. Therefore, in this study, 3 ml
femoral venous blood of aborted fetuses was collected
within 24 h of birth.
In the study by Zhang et al. [7], 24 full-term newborns
were HBV-DNA positive in venous blood samples of 59
newborns with HBsAg-positive mothers; the rate of HBV
intrauterine infection was 40.1 %. In this study, the rate of
positive HBV-DNA in venous blood from aborted fetuses
was 23.33 % (7/30); this indicated that the HBV intra-
uterine infection occurred at the second trimester. In
addition, the intrauterine HBV infection rate increased
rapidly during the last trimester of pregnancy.
Several studies have indicated that HBV-DNA is
potentially replicated in extra-hepatic tissues, such as
spleen, kidney, and pancreas, as well as in PBMC
[19–21]. Shi et al. [22] found that both maternal serum
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 Arch Gynecol Obstet

Table 1 Correlations between fetal peripheral blood HBsAg, serum HBV-DNA, PBMC HBV-DNA and maternal PBMC transportation
Fetal peripheral blood Maternal PBMC transport Total v2 P Pearson’s R Spearman correlation
? –

HBsAg
? 1 2 3 2.016 0.156 0.259 0.259
– 2 25 27
Serum HBV-DNA
? 2 5 7 0.887 0.346 0.157 0.157
– 4 19 23
PBMC HBV-DNA
? 9 1 10 8.103 0.004 0.520 0.520
– 7 13 20
Bold data indicated the significant correlations
PBMC peripheral blood monouclear cells, HBV hepatitis B virus

Fig. 2 HBsAg, HBcAb and

HBV-DNA expressed in
placenta cells. a,
b Streptavidin–biotin complex
(SABC) staining showed brown
was HBsAg and HBcAb
location in cytoplasm or
nucleus, respectively. c In situ
hybridization (ISH) revealed
that the blue HBV-DNA was in
nuclei

Table 2 Rate of HBsAg-positive, HBcAb-positve, and HBV-positive In the study by Zhang et al. [7], the concentration of
DNA in placenta cells HBsAg and HBcAg decreased from VCEC to maternal
Placenta HBsAg-positive HBcAb-positive HBV-positive decidual cells in the placentas of four cases. Based on the
cells (%) (%) DNA (%) detection results of HBsAg and HBcAg in amniotic epi-
dermic cells and HBV-DNA in amniotic fluid and vaginal
DC 16.67 10.00 16.67
secretion, they hypothesized that HBV in vaginal secretion
TC 13.33 6.67 13.33
potentially infected fetal membrane, amniotic fluid, the
VMC 10.00 3.33 10
fetus, and cells of different layers in placenta. In contrast,
VCEC 3.33 0 3.33 in this study, the rates of HBV infection gradually
DC decidual cells, TC trophoblastic cells, VMC villous mesenchymal decreased from the maternal side to the fetus side of pla-
cells, VCEC villous capillary endothelial cells, HBV hepatitis B virus centa (DC [ TC [ VMC [ VCEC) based on the detection
results of HBsAg, HBcAg, and HBV-DNA; this indicated
HBV-DNA and PBMC HBV-DNA were associated with that the HBV-infected cells potentially transfer from
fetal PBMC HBV infection; moreover, the long-term maternal decidua to villous capillary endothelia, then to
presence of PBMC HBV-positive DNA in newborns VMC and VCEC, finally resulting in infection of the fetus.
influenced the production of HBsAb after immunization. However, we did not find a significant correlation between
Similarly, according to our results, fetal PBMC HBV- placenta infection and intrauterine infection (P = 0.410).
positive DNA and maternal PBMC transport were sig- Therefore, it remains unclear whether the mechanism of
nificantly positively associated (P = 0.004). A significant intrauterine infection is caused by ‘‘transplacental leakage’’
correlation between HBV intrauterine infection and in the second trimester.
maternal–fetal PBMC transport was observed; the cor- Compared with the collection of HBsAg-positive preg-
relation indicated that HBV was given access to fetal nant women in the last trimester and the umbilical cord
blood circulation by PBMCs and replicated in lymphoid blood samples from live birth infants, it was much more
organs in the second trimester. difficult to study and collect HBsAg-positive women in the

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Table 3 Correlation between placenta and intrauterine HBV infection in second trimester
Placenta infection Intrauterine infection Total
? -
? 3 2 5
– 10 15 25
HBV hepatitis B virus
second trimester and their aborted fetuses. Although the
sample size was relatively small in this study, this is the
first study to report correlation analysis between HBV
intrauterine infection with maternal–fetal PBMC transport
and HBV placenta infection in the second trimester.
Conclusions
In this study, a significant correlation between fetal PBMC
HBV-positive DNA and maternal–fetal PBMC transport
was observed. It indicated that the HBV intrauterine
infection was primarily caused by maternal–fetal PBMC
transport in the second trimester of pregnancy. Further
research to investigate potential block of the maternal–fetal
PBMC HBV transportation is necessary in HBsAg-positive
women in the second trimester.
Conflict of interest None.
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