doi:10.1111/jog.14065 J. Obstet. Gynaecol. Res.

2019

Effect of viral load on pregnancy outcomes in chronic

hepatitis B infection

Canan Unal, Atakan Tanacan, Gunel Ziyadova, Erdem Fadiloglu and Mehmet S. Beksac
Division of Perinatology, Department of Obstetrics and Gynecology, Hacettepe University, Ankara, Turkey

Abstract
Aim: This study aimed to evaluate perinatal and neonatal outcomes in pregnant women with chronic hepa-
titis B virus infection based on infection status and to identify cut-off values based on hepatitis B virus DNA
viral load to predict composite adverse perinatal/neonatal outcomes.
Methods: Pregnant women with chronic hepatitis B virus who delivered at Hacettepe University between
2010 and 2018 were evaluated retrospectively. We included 95 patients. The patients were classified into two
groups based on laboratory findings and viral load: group 1 (n = 63), immune inactive; and group 2 (n = 32),
immune active. Maternal age, gravidity, parity, gestational week at birth, birth weight, 5th minute APGAR
scores and composite perinatal and neonatal outcomes were compared between groups.
Results: Gestational week at birth, birth weight and 5th minute APGAR score in group 2 were lower than
those in group 1 (P < 0.001, P < 0.005 and P < 0.001, respectively). The rates of composite adverse peri-
natal/neonatal outcome, preterm birth, fetal growth restriction, oligohydramnios, pre-eclampsia, admission
to the neonatal intensive care unit, small for gestational age and 5th minute APGAR score less than 7 were
significantly higher in group 2 (P < 0.001). Hepatitis B virus DNA viral load of 17 515 IU/mL (72.7% sensi-
tivity, 78.1% specificity) and 17 515 IU/mL (81.8% sensitivity, 80.8% specificity) were determined to be cut-
off values for composite adverse perinatal and neonatal outcomes, respectively.
Conclusion: Care should be taken in patients with a viral load of greater than 17 515 IU/mL, and pregnancy
should be postponed until the inactive phase of the disease for optimal results.
Key words: adverse neonatal outcome, adverse perinatal outcome, hepatitis B, pregnancy, viral load.

Introduction increased HBV replication due to the immunosup-

Infection with hepatitis B virus (HBV) is still an
important public health problem with its changing
epidemiology, mortality and morbidity. More than
240 million individuals are infected with HBV world-
wide.1 The prevalence of chronic hepatitis B virus
(CHB) infection in pregnancy is similar with the gen-
eral population. Approximately 5% of the mothers are
hepatitis B surface antigen (HbsAg) positive.2 More-
over, the interaction between pregnancy and CHB has
not been clearly defined yet.3–5 Higher estrogen and
progesterone levels during pregnancy may cause
pressive effects of these hormones.6 The immunologi-
cal changes that occur during pregnancy may affect
the clinical manifestations of CHB infection.7 There-
fore, hepatic flares and progression of the disease to
liver failure and HBV viremia may be encountered.6,7
Hepatic flares during pregnancy may be fatal in
20–30% of cases.8,9
To assess the severity of the disease in pregnant
women with CHB infection, hepatitis B e antigen
(HbeAg)/hepatitis B e antibody (anti-Hbe) positivity,
alanine aminotransferase (ALT) values and HBV viral
load, and coexisting viral diseases should be

Received: March 6 2019.

Accepted: June 22 2019.
Correspondence: Dr Canan Unal, Division of Perinatology, Department of Obstetrics and Gynecology, Hacettepe University, Tıp
Fakultesi Street, 06100 Ankara, Turkey, Email: unal_canann@hotmail.com

© 2019 Japan Society of Obstetrics and Gynecology 1

C. Unal et al.
evaluated.10 HBV DNA and ALT levels during preg-
nancy should be controlled at 3 months interval and
at the end of the third trimester.11
The perinatal and neonatal effects of CHB infection
in pregnancy are controversial. In many studies, preg-
nant women with CHB infection were compared with
HbsAg-negative pregnant women.5,12–15 Some studies
reported no difference in gestational week at birth,
birth weight or perinatal mortality, while some others
reported increased rates of gestational diabetes
mellitus, prematurity, lower birth weight and
antepartum hemorrhage.5,15,16 However, significantly
poor obstetric outcomes are reported for the patients
with severe chronic HBV infection resulting in com-
plications such as cirrhosis.17
This study aimed to evaluate the composite adverse
perinatal and neonatal outcomes in pregnant women
with CHB infection due to immune inactive and
immune active criteria and to identify the cut-off
values based on the HBV DNA viral load to predict
composite adverse perinatal/neonatal outcomes.
Methods
We retrospectively evaluated the data of pregnant
women with CHB who delivered at Hacettepe Uni-
versity Hospital Division of Perinatology between
2010 and 2018. Singleton pregnancies delivered alive
after the 28th gestational week were included in the
study. Pregnant women on anti-viral therapy before
or during pregnancy were excluded from the study.
HIV, hepatitis C virus (HCV), hepatitis D virus co-
infection cases were also excluded from the study.
The required data were extracted from the institu-
tional electronic patients’ database.
CHB infection was defined as the presence of
HBsAg for at least 6 months. Additional conditions
are; (i) variation of serum HBV DNA from
undetectable to several billion IU/mL; (ii) subdivision
of HBeAg as positive or negative. HBV DNA levels
are greater than 20 000 IU/mL in HbeAg-positive
CHB, and lower values (2000–20 000 IU/mL) are
often seen in HBeAg-negative CHB; (iii) normal or
elevated ALT and/or aspartate aminotransferase
(AST) levels; and (iv) liver biopsy results demonstrat-
ing chronic hepatitis with variable necroinflammation
and/or fibrosis.18
Pregnant women with CHB infection were classi-
fied into two groups: group 1, immune inactive and
group 2, immune active. The patients in the immune
2 © 2019 Japan Society of Obstetrics and Gynecology
inactive group were HBsAg positive, HBeAg nega-
tive, anti-HBe positive and had HBV DNA less than
2000 IU/mL and normal AST and ALT levels. The
patients in the immune active group were defined as;
HBsAg positive for greater than or equal to 6 months,
serum HBV DNA greater than 20 000 IU/mL in
HBeAg-positive CHB and greater than 2000 IU/mL in
HBeAg-negative CHB and intermittently or persis-
tently elevated ALT and/or AST levels.18
The upper limits of normal for ALT in healthy
adults are reported to be 19 to 25 U/L for females.
For purposes of guiding management of CHB, an
upper limit of normal for ALT of 25 U/L for females
is defined.19
HBV DNA was also studied by real time polymer-
ase chain reaction (PCR) technique during the study
period. The PCR primers and probe were prepared to
equally amplify all known HBV genotypes. Sequences
from GenBank were used for this purpose and a well
conserved region among all genotypes in the S gene
was selected. Thereafter, PCR primers and TaqMan
minor groove binder probe sequences were investi-
gated regarding base composition, melting tempera-
tures, guanine-cytosine content, internal folding by
using Primer Express software (Applied Biosystems).
The QIAamp DNA mini kit (QIAGEN) was used
according to manufacturer’s instructions in order to
isolate HBV DNA from the plasma. DNA was
extracted from 200 μL plasma with 1 μL of the inter-
nal control (1× IPC DNA; Applied Biosystems) and
eluted in 50 μL buffer. The Versant HBV DNA Assay
version 3.0 (bDNA – Siemens Medical Solutions Diag-
nostics) was applied in compliance with the manufac-
turer’s protocol. Amplification protocol was
performed by a 50 μL reaction solution containing: 1×
TaqMan Universal PCR Master Mix, 20 μM of each
primer and probe, and 5 μL of extracted DNA. Abso-
lute quantification of HBV DNA was performed with
ABI PRISM 7500 Real Time (Applied Biosystems).
Amplification protocol was started with an incubation
at 50
C for 2 min for uracil N0 -glycosylase inactivate
possible contaminating amplicons, followed by
10 min at 95
C that activates AmpliTaq Gold Poly-
merase and inactivates uracil N0 -glycosylase. PCR
cycling program consisted of 45 two-step cycles of
15 s at 95
C and 60 s at 60
C (universal conditions).20
Maternal age, gravidity, parity, gestational week at
birth, birth weight, 5th minute APGAR scores, and
composite perinatal and neonatal outcomes were
compared in these two groups. Composite adverse
perinatal outcomes involved preterm birth, fetal

growth restriction (FGR), oligohydramnios and pre-
eclampsia. Composite adverse neonatal outcomes
included small for gestational age, 5th minute
APGAR less than 7 score, respiratory distress syn-
drome (RDS), and admission to neonatal intensive
care unit (NICU).
The laboratory values of the patients before deliv-
ery were used for the comparison of the groups in this
study.
Statistical analyses were performed using the Statisti-
cal Package for the Social Sciences (SPSS 22; IBM SPSS
Statistics for Windows, Version 22.0., IBM Corp.). The
Kolmogorov–Smirnov test was used to evaluate the
normal data distribution. Because data were not nor-
mally distributed, the median values together with
interquartile range (IQR) values were used for continu-
ous variables. Chi-square or Fisher exact test was used
to compare categorical variables.
Receiver operating characteristic (ROC) curves were
used to assess the performance of viral load in
predicting composite adverse perinatal and neonatal
outcomes. ROC curves plot the true positive rate (sensi-
tivity) against the false-positive rate (1 − specificity) for
the possible cut-off values. The area under the curve
(AUC) corresponds to the probability that the criterion
will correctly classify a random observation. An AUC
greater than 0.5 indicates that the criterion is superior to
chance. The significance level was set at P < 0.05. Writ-
ten informed consent was obtained from all the patients,
and the study was approved by the institutional ethics
committee of Hacettepe University (GO 18/1006).
Results
We included 95 pregnant women with CHB infection
with a median age of 29 years (IQR: 7). Immune inac-
tive and immune active CHB infection were observed
in 63 (66%) and 32 (33%) pregnant women, respec-
tively. The median HBV DNA was 0 (IQR: 20) IU/mL
in the immune inactive group and 32 575 (IQR: 29587)
IU/mL in the immune active group.
Table 1 Demographic features and obstetric outcomes
Variables Group 1 (n = 63)
Maternal age 29 (IQR:7)
Gravidity 2 (IQR:2)
Parity 1 (IQR:1)
Gestational week at birth 38 (IQR:2)
Birth weight 3300 (IQR: 640)
APGAR Score (5th minute) 10(IQR:0)
IQR, interquartile range.
© 2019 Japan Society of Obstetrics and Gynecology
Pregnancy and hepatitis B
The demographic features and obstetric outcomes
were evaluated in groups 1 and 2. No statistically sig-
nificant differences were found between the groups for
maternal age, gravidity and parity (P = 0.679, 0.417
and 0.9, respectively). Gestational week at birth, birth
weight and 5th minute APGAR score in group 2 were
lower than those in group 1 (P < 0.001, P < 0.005 and
P < 0.001, respectively). The demographic features and
obstetric outcomes are summarized in Table 1.
Adverse perinatal and neonatal outcome percent-
ages were compared between groups 1 and 2. The
composite adverse perinatal outcome in groups 1 and
2 was of 6% and 56%, respectively (P < 0.001). Pre-
term birth, fetal growth restriction, oligohydramnios
and pre-eclampsia percentages were significantly
higher in group 2 (P < 0.001).
The percentage of composite adverse neonatal out-
come in groups 1 and 2 were 9.5% and 50%, respec-
tively (P < 0.001). The percentages of admission to the
NICU, SGA neonates and 5th minute APGAR score
less than 7 were significantly higher in group 2. Com-
parison of groups based on composite adverse perina-
tal and neonatal outcomes is summarized in Table 2.
Results of the ROC analysis for assessing the per-
formance of viral load in predicting composite
adverse perinatal and neonatal outcomes are shown
in Table 3, and ROC curves are shown in Figures 1
and 2. AUC values were 0.760 (95% confidence inter-
val (CI): 0.666–0.855) and 0.761 (95% CI: 0.637–0.884)
for composite adverse perinatal and neonatal out-
comes, respectively. As a result, HBV DNA viral load
of 17 515 IU/mL (72.7% sensitivity, 78.1% specificity)
and 17 515 IU/mL (81.8% sensitivity, 80.8% specific-
ity) was determined to be the cutoffs for composite
adverse perinatal and neonatal outcomes, respec-
tively, with highest sensitivity and specificity.
Discussion
CHB infection is a global public health problem, and
it is associated with further risk for hepatocellular
Group 2 (n = 32) P value
28,5 (IQR:10) P: 0.679
2(IQR:1) P: 0.417
1(IQR:1) P: 0.912
37(IQR:2) P < 0.001
2995 (IQR: 890) P: 0.005
9,5(IQR:3) P < 0.001
3
C. Unal et al.

Table 2 Composite adverse perinatal and neonatal outcomes

Variables Group 1 (n = 63) Group 2 (n = 32) P value
Composite adverse perinatal outcome 4 (%)6 18 (V) P < 0.001
Preterm 2 (%)3 6 (%)18.7
FGR 1 (%)1.5 4 (%)12.5
Oligohydroamniosis 1 (%)1.5 4 (%)12.5
Pre-eclampsia 0 4 (%)12.5
Composite adverse neonatal outcome 6 (%)9.5 16 (%)50 P < 0.001
SGA 3 (%)4.7 5 (%)15.6
APGAR < 7 1 (%)1.5 5 (%)15.6
RDS 2 (%)3 4 (%)12.5
Admission to NICU 0 2 (%)6
FGR, fetal growth restriction; NICU, neonatal intensive care unit; RDS, respiratory distress syndrome; SGA, small for gestational age.

Table 3 ROC curve analysis for assessing the performance of HBV viral load in predicting composite adverse perinatal
and neonatal outcomes
Composite adverse perinatal outcome, Cut-off value for Sensitivity Specifity P value
AUC: 0.828 (95% CI: 0.727–0.928)
17 515 72.7 78.1 <0.001
Composite adverse neonatal outcome,
AUC: 0.761
(95% CI: 0.637–0.884)
17 515 81.8 80.8 <0.001
AUC, area under the curve; CI, confidence interval; HBV, hepatitis B virus; ROC, receiver operating characteristic.

Figure 2 ROC curve analysis for composite adverse

Figure 1 ROC curve analysis for composite adverse neonatal outcomes. ROC, receiver operating
perinatal outcomes. ROC, receiver operating characteristic.
characteristic.

carcinoma.1 The World Health Organization states The course of CHB infection during pregnancy is
that HBV infection is the second most important often stable, but it is an important health problem that
cause of human carcinogens next to smoking.21 needs to be addressed. Pregnancy is generally well

4 © 2019 Japan Society of Obstetrics and Gynecology


tolerated in women with CHB infection without
advanced liver disease. Some studies claimed that
CHB infection did not affect perinatal and neonatal
outcomes.5,22 In a study comparing the perinatal out-
comes of 824 women with HBsAg to a control group
of 6281 women, the incidences of preterm birth, pre-
term prelabor rupture of membranes, prelabor rup-
ture of membranes, SGA, neonatal jaundice, fetal
distress, perinatal asphyxia, congenital abnormality,
gastrointestinal tract abnormality and perinatal mor-
tality were all similar between the groups.5
In contrast, composite adverse perinatal and neona-
tal outcomes were reported in various studies.6,7,16 In
a nationwide study from Singapore, pregnancy-
related complications including gestational diabetes
mellitus, preterm birth, FGR, pre-eclampsia,
antepartum hemorrhage and cholestasis were evalu-
ated for pregnant women with HBV and HCV. Logis-
tic regression analysis was used to examine the
association between HBV, HCV, HBV + HCV, and
pregnancy-related complications. Furthermore, 1446
had a coded diagnosis of HBV, HCV or both. Women
with HBV had an increased risk for preterm birth
(odds ratio (OR) 1.65, CI 1.3–2.0) but a decreased risk
for cesarean delivery (OR 0.686, CI 0.53–0.88). Patients
with both HBV and HCV co-infection had an
increased risk for antepartum hemorrhage (OR 2.82,
CI 1.1–7.2). However, viral hepatitis was not associ-
ated with FGR or pre-eclampsia.7 Furthermore, in a
retrospective study conducted in a German university
hospital, no significantly increased prevalence of
adverse pregnancy outcomes were found in patients
with HBsAg.16 In addition, based on a retrospective
cohort study that included 10 years of birth data from
Florida, singleton pregnancies with HBV and HCV
infection were evaluated. After using multivariable
modeling to adjust for important sociodemographic
variables and obstetric complications, women with
HBV infection were less likely to deliver SGA infants
(OR, 0.79; 95% CI, 0.66–0.95).6
All patients in this study had CHB infection differ-
ent from the previous studies in the literature.
Patients were grouped by considering viral load,
HBeAg and anti-HBe positivity or negativity, and
AST and ALT levels. In other words, we evaluated
our patients based on their immune active or inactive
phase for CHB infection. Adverse perinatal and neo-
natal outcomes in group 2 were significantly higher
than those in group 1.
Pregnancy is associated with significant
immune, metabolic, and hemodynamic changes.
© 2019 Japan Society of Obstetrics and Gynecology
Pregnancy and hepatitis B
When CHB infection in the immune active phase
is present during pregnancy, it may cause adverse
perinatal and neonatal outcomes. Pregnancy is a
physiologic immunosuppressant condition, and it
may alter the balance between humoral and cellu-
lar immunity.23,24
Chronic HBV infection is associated with increased
levels of pro-inflammatory cytokines, such as interleu-
kin (IL)-2, IL-6, IL-10, macrophage migration inhibi-
tory factor, and tumor necrosis factor-alpha (TNF-α),
and this effect is more, particularly in the active
phase.25,26 The active phase of CHB infection may
affect the fetoplacental unit through these cytokines
and mediators. Inflammation of the placenta may
cause abnormal remodeling of the spiral arteries,
defective trophoblast differentiation, and placental
hypoperfusion.27–29 An exaggerated systematic
inflammatory response is thought to be associated
with some obstetric complications, for example, pre-
eclampsia.30,31 In our study, pre-eclampsia was not
observed in group 1, whereas pre-eclampsia occurred
in approximately 12% of patients in group 2. The ratio
of SGA, FGR, and oligohydramnios was higher in
group 2 in relation to obstetric complications. A large
number of studies have shown that the increase of
pro-inflammatory cytokines, including IL-2 receptors,
IL-6, IL-8, TNF-α, and thrombin play an effective role
in premature birth and premature rupture of
membranes.32–34 In our study, the preterm delivery
rate was 18.7% and 2% in groups 2 and group
1, respectively, in accordance with this literature.
Therefore, the incidence of RDS, lower 5th minute
APGAR score and admission to NICU rates were
increased.
Therefore, normal functioning of the feto-placental
unit may be impaired, and composite adverse perina-
tal and neonatal outcomes may be observed.
The main strength of this study is the evaluation of
pregnant women with CHB in terms of viral load.
However, the limitations of the study were the rela-
tively small sample size, retrospective design and
single-center experience. Lack of possible changes in
viral load values during pregnancy may be consid-
ered as a limitation. Prospective, multicenter studies
involving large patient groups may be conducted to
confirm our results.
In conclusion, management of patients with CHB
infection within the framework of multidisciplinary
antenatal care programs seems to be the key factor for
achieving better perinatal/neonatal outcomes. Physi-
cians should be cautious regarding patients whose
5
C. Unal et al.
viral load is greater than 17 515 IU/mL, and preg-
nancy should be postponed until the inactive phase of
the disease for optimal outcomes.
Disclosure
Authors have not any conflict of interest.
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