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Original article
Analysis on the outcomes of hepatitis B virus perinatal vertical transmission: nested case–control study Nan Lv, Xiao-Dan Chu, Yu-Hong Sun, Shu-Yu Zhao, Pei-Ling Li and Xuan Chen Objective Hepatitis B virus (HBV) infection is a public health problem worldwide, with vertical transmission as the leading transmission route. Therefore, it is very important to explore the risk factors associating with HBV perinatal transmission, providing valuable information for preventive and curative means for HBV perinatal infections. In this study, we systematically investigated the impact of adverse outcomes of HBV maternal infection on fetal intrauterine infection. Patients and methods This is a nested case–control study that included 42 hepatitis B surface antigen (HBsAg)positive pregnant women. Gestational weeks, delivery modes, alanine aminotransferase levels, platelet counts, liver ultrasonography results as well as adverse pregnancy outcomes for the pregnant mothers and Apgar scores at both 1 and 5 min, birth weight, and height for the newborn infants were recorded. Vein blood from pregnant mothers and cord blood immediately after delivery were collected for the detection of HBsAg, antibodies to hepatitis B surface antigen, hepatitis B e antigen (HBeAg), antibody to hepatitis B e antigen, hepatitis B core antigen, and HBV DNA. Placental tissues were collected for detection of HBV DNA. Results Positive HBeAg as well as HBV DNA in the mother’s serum were correlated closely with HBV intrauterine infection. Mother’s age, delivery mode, alanine
Introduction Hepatitis B virus (HBV) infection is a major health problem worldwide, with a chronically infected population of around 350–400 million worldwide [1]. In China, which has a high prevalence of HBV, the infected population accounts for one-third of the infected population worldwide [2,3]. HBV is predominantly transmitted through mother-to-child vertical transmission, with perinatal transmission being the main route [4,5]. Perinatal transmission can be subcategorized into three categories: intrauterine transmission, labor transmission, and postnatal transmission. Among about 120 million HBV carriers in China, 30–50% are infected with HBV through vertical transmission [2,6]. Since the introduction of HBV vaccination in the entire population including newborn infants in China in 1992, the hepatitis B surface antigen (HBsAg)-positive rate has reduced markedly. However, because of the underdeveloped immune system in the neonatal infants, the combined neonatal HBV vaccine and hepatitis B immunoglobulin have led to 20–30% of immunization failure in infants [7,8]. HBV perinatal transmission through the 0954-691X © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
aminotransferase, blood platelet count, clinical HBV infection features, premature labor, gestational diabetes mellitus, pre-eclampsia, fetal growth retardation, fetal distress, Apgar scores of the infant as well as the HBV infection status of the placenta all failed to show a statistically significant correlation with intrauterine infection. Conclusion High level of HBV in maternal blood was one of the risk factors accounting for intrauterine infection. Eur J Gastroenterol Hepatol 26:1286–1291 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. European Journal of Gastroenterology & Hepatology 2014, 26:1286–1291 Keywords: hepatitis B virus, perinatal outcome, placenta, vertical transmission Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Harbin Medical University, Harbin, China Correspondence to Pei-Ling Li, PhD, Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin 150086, China Tel: + 86 451 866 05141; fax: + 86 451 86297003; e-mail: [email protected] and Co-correspondence to Xuan Chen, MM, Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin 150086, China Tel: + 86 451 86605050; fax: + 86 451 86605343; e-mail: [email protected] Received 1 June 2014 Accepted 21 July 2014
mother to child can result in hepatitis fetuses. Without treatment, newborn infants with HBV infection have a considerably high possibility of becoming chronic carriers [9]. Therefore, it is very important to explore the risk factors associated with HBV perinatal transmission, providing valuable information for the preventive and curative means for HBV perinatal infections. As previous studies on HBV perinatal transmission are limited, in the current study, we have investigated the adverse outcomes of HBV maternal and fetal intrauterine infection by determining the relationship of possible adverse outcomes for pregnancy, the growth and development of fetuses, and the infection of placental cells in each layer with HBV intrauterine infection.
Methods Patients
Forty-two HBsAg-positive pregnant women, 22–43 years of age, without an antepartum hemorrhage history, who had undergone a prenatal examination and delivered in the Second Affiliated Hospital of Harbin Medical DOI: 10.1097/MEG.0000000000000189
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HBV perinatal vertical transmission Lv et al. 1287
University from January, 2011 to June, 2012 were recruited into the study. Patient clinical baseline characteristics
Among the 42 HBsAg-positive pregnant women, 10 were both HBsAg and hepatitis B e antigen (HBeAg) positive whereas 18 were HBV DNA positive. Gestational weeks, delivery modes, alanine aminotransferase (ALT) levels, platelet counts, liver ultrasonography results as well as adverse pregnancy outcomes (gestational diabetes mellitus (GDM), premature labor, pre-eclampsia, fetal distress, fetal growth retardation, etc.) for the pregnant mothers and Apgar scores [10] at both 1 and 5 min, birth weight, and height of the newborn infants were recorded. Criterion of intrauterine infection
Nested case controls were applied in the study. Cord blood HBV DNA more than 5 × 102 copies was considered the criterion of intrauterine infection. The selected babies were subsequently divided into an intrauterine infection group and a control group. Detection of HBV markers and HBV DNA in blood
Vein blood samples of the pregnant mothers 28 weeks before child birth as well as cord blood samples immediately after birth delivery were collected for the detection of markers of HBsAg, antibodies to hepatitis B surface antigen (anti-HBs), HBeAg, antibody to hepatitis B e antigen (anti-HBe), hepatitis B core antigen (antiHBc), and HBV DNA. Twelve months after birth, vein blood samples of the chosen newborns were collected for the detection of markers of HBV, HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, and HBV DNA. Serologic HBV markers and HBV DNA in the blood samples were measured by ELISA (Shanghai Kehua Bio-engineering Co., Ltd, Shanghai, China) and PCR, respectively, following the manufacturer’s instructions. HBV DNA in-situ hybridization
To analyze the relationship between placental cells in each layer of HBV DNA infection and intrauterine infection, HBV DNA in-situ hybridization on placental tissues was used. Placental specimens were collected and cut into a size of about 1 cm × 1 cm × 1.5 cm, fixed in 10% neutral-buffered formalin for 12–20 h, and subsequently embedded and cut into 3 μm sections. HBV DNA in the tissue sections were detected using in-situ hybridization with a digoxigenin system (Fujian Triplex International Biosciences Co., Ltd, Fujian, China) and brown-colored spots were HBV DNA-positive. The HBV-negative placental tissues were used as negative controls whereas HBsAg-positive liver tissues were used as positive controls. Ethical statement
All protocols in this study were reviewed and approved by the ethical board of Harbin Medical University.
Informed written consents were obtained from the pregnant women or the family of the newborn infants. Statistical analysis
Multivariate logistic regression analysis was used to analyze the adverse outcomes of pregnancy, clinical features in HBV-infected pregnant women, and the relationship between the Apgar score [10] of the newborns after birth and intrauterine infection. The odds ratios and 95% confidence intervals were calculated. For statistical comparisons between two groups, a t-test or Fisher’s exact test was used for enumeration data, and a P value less than 0.05 was considered statistically significant. All analyses were carried out using SPSS 11.0 (SPSS Inc., Chicago, Illinois, USA).
Results HBeAg positivity in maternal blood was correlated with intrauterine HBV infection of infants
Among 42 newborn infants, three had intrauterine infection, with HBV DNA in cord blood more than 5 × 102 copies (5 × 107, 5.215 × 103 and 3.879 × 105 copies, respectively). The rate of intrauterine infection was 7.14%. Of the three infants with intrauterine infection, one of their mothers was both HBsAg and HBeAg positive. Further analysis showed that positive HBeAg in mothers was correlated with intrauterine HBV infection in infants (P = 0.0105) (Tables 1 and 2). Maternal serum HBV DNA level was related positively to that in the fetal cord blood
Of 42 HBsAg-positive pregnant women, 18 were HBV DNA positive, with seven of these having more than 107 copies. Further analysis was carried out to examine the relationship between maternal serum HBV DNA levels and fetal cord blood HBV DNA levels. The results showed that maternal serum HBV DNA levels were related positively to that in the fetal cord blood (P = 0.0429) (Table 2), suggesting that high copies of HBV DNA in maternal blood was one of the risk factors for intrauterine infection. Mother’s age, delivery mode, adverse pregnancy outcome, clinical HBV infection feature as well as the Relationship between the peripheral blood of HBeAgpositive pregnant women and fetal intrauterine infection
Table 1
Intrauterine infection
Control
n (%)
3 0 3
7 32 39
10 (30.00) 32 (0) 42 (7.14)
HBeAg (+ ) HBeAg (− ) n HBeAg, hepatitis B e antigen. P = 0.0105. P < 0.05.
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Table 2
European Journal of Gastroenterology & Hepatology 2014, Vol 26 No 11
Maternal serum HBV DNA copies and newborn infants with HBV intrauterine infection (n < 40)
Maternal serum HBV DNA ≥ 107 copies (n = 7) Maternal serum HBV DNA < 107 copies (n = 11)
Intrauterine infection (n = 3)
Control (n = 15)
3 0
4 11
HBV, hepatitis B virus. Fisher’s exact probabilities, P = 0.0429, P < 0.05.
Clinical characteristics and adverse pregnancy outcomes of HBV-infected mothers
Table 3
Age at delivery (years) < 25 25–34 ≥ 35 Adverse pregnancy outcomes Spontaneous delivery Cesarean delivery GDM (no) GDM (yes) Term birth Preterm birth Pre-eclampsia (no) Pre-eclampsia (yes) Fetal distress (no) Fetal distress (yes) FGR (no) FGR (yes) 1 min Apgar score 8–10 4–7 5 min Apgar score 8–10 4–7 Clinical characteristics of hepatitis B Thrombocytopenia (no) Thrombocytopenia (yes) Transaminase normal Transaminase abnormal Liver ultrasonography normal Liver ultrasonography abnormal
Cases of HBV intrauterine infection (n = 3) [n (%)]
Controls (n = 39) [n (%)]
0 2 (67) 1 (33)
5 (13) 26 (67) 8 (20)
0 3 3 0 3 0 3 0 1 (33) 2 (66) 3 0
7 (18) 32 (82) 31 (79) 8 (21) 33 (85) 6 (15) 31 (79) 8 (21) 31 (79) 8 (21) 36 3
3 0
31 (79) 8 (21)
3 0
35 (90) 4 (10)
(66) (33) (66) (33) (66) (33)
35 4 36 3 37 2
2 1 2 1 2 1
(90) (10) (92) (8) (95) (5)
FGR, fetal growth restriction; GDM, gestational diabetes mellitus; HBV, hepatitis B virus.
infant’s Apgar score were not statistically related to intrauterine infection
To further explore the impact of mother’s age, delivery mode, adverse pregnancy outcome, clinical HBV infection feature as well as the infant’s Apgar score on intrauterine infection, logistic regression analysis was used (Table 3). Our results showed that all the tested factors, including mother’s age, delivery mode, ALT, blood platelet count, clinical HBV infection features, premature labor, GDM, pre-eclampsia, fetal growth retardation, fetal distress, and Apgar score of the infant, failed to show a statistically significant correlation with HBV intrauterine infection (P > 0.05) (Tables 4 and 5). HBV infection of placental cells was not correlated with intrauterine infection
HBV can infect placenta; we consequently tested whether HBV DNA levels in the placenta are related to
Table 4 Multivariate logistic regression analyses for HBV intrauterine infection
Age at delivery (years) < 25 25–34 ≥ 35 Adverse pregnancy outcomes Cesarean delivery GDM Preterm birth Pre-eclampsia Fetal distress FGR 1 min Apgar score 4–7 5 min Apgar score 4–7 Clinical characteristics of hepatitis B Thrombocytopenia Transaminase abnormal Liver ultrasonography abnormal
OR (95% CI)
P
– –
0.9975 0.9974
– – – – – –
0.9970 0.9968 0.9972 0.9968 0.9970 0.9970
–
0.9968
–
0.9965
0.229 (0.017–3.120) 0.167 (0.011–2.416) 0.108 (0.007–1.761)
0.2684 0.1891 0.1182
CI, confidence intervals; FGR, fetal growth restriction; GDM, gestational diabetes mellitus; HBV, hepatitis B virus; OR, odds ratio. P > 0.05.
intrauterine infection. Our results of HBV DNA in-situ hybridization showed that HBV DNA was detected in every cell layer of the placenta (Fig. 1). The infection rates of decidual cells (DC), trophoblast cells (VTC), villous stroma cells (VMC), and capillaries endothelial cells (VCEC) were 40.48% (17/42), 90.48% (38/42), 38.10% (16/42), and 83.33% (35/42), respectively. In placental tissues, HBV DNA in VTC and in VCEC was higher than its expression in DC and VMC. However, HBV DNA infection in every placental cell layer was not statistically significantly correlated with HBV intrauterine infection (P > 0.05) (Table 6).
Discussion HBV infection is a public health problem worldwide. In China, 6.70–8.25% of pregnant women are HBsAgpositive [11–13]. Perinatal transmission is the most frequent route of transmission in China. Consequently, it is very important to explore the risk factors associated with HBV perinatal transmission, providing valuable information for preventive and curative measures for HBV perinatal infections. In this study, we systematically investigated the impact of adverse outcomes of HBV maternal infection on fetal intrauterine infection. Our results indicated that positive HBeAg as well as HBV DNA in the mother’s serum were correlated closely with HBV intrauterine infection. We further analyzed the
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HBV perinatal vertical transmission Lv et al. 1289
Table 5
Effects of maternal HBV infection and HBV intrauterine infection on fetal development
HBeAg (+ ) (n = 10) HBeAg (− ) (n = 32) HBV DNA (+ ) (n = 18) HBV DNA (− ) (n = 24) ALT/AST abnormal (n = 3) ALT/AST normal (n = 39) Cord blood HBV DNA (+ ) (n = 3) Cord blood HBV DNA (− ) (n = 39)
Birth weight
Body length
1 min Apgar score
5 min Apgar score
3.25 ± 0.50 3.47 ± 0.51 3.54 ± 0.38 3.36 ± 0.58 3.35 ± 0.91 3.44 ± 0.49 3.93 ± 0.16 3.40 ± 0.51
49.00 ± 2.00 50.06 ± 2.05 50.00 ± 2.08 49.71 ± 2.07 50.00 ± 3.00 49.82 ± 2.02 51.66 ± 1.52 49.69 ± 2.04
7.70 ± 1.15 8.18 ± 0.69 8.11 ± 0.75 8.16 ± 0.81 7.66 ± 0.57 8.17 ± 0.79 8.33 ± 0.57 8.12 ± 0.80
8.20 ± 1.13 8.46 ± 0.71 8.27 ± 1.01 8.58 ± 0.58 8.33 ± 0.57 8.46 ± 0.82 8.66 ± 0.57 8.94 ± 3.39
FGR (%) 10.0 6.2 10.0 12.5 33.33 5.13 0 7.69
(1/10) (2/32) (1/10) (3/24) (1/3) (2/39) (0/3) (3/39)
ALT, alanine aminotransferase; AST, aspartate aminotransferase; FGR, fetal growth restriction; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus. P > 0.05.
Fig. 1
In-situ hybridization detection of hepatitis B virus (HBV) DNA in placental tissues (a) decidual cells show positive expression of HBV DNA (×40); (b) trophoblast cells (VTC) and villous stroma cells (VMC) show positive expression of HBV DNA (×40); (c) capillaries endothelial cells (VCEC) show positive expression of HBV DNA (×20); (d) negative control: vascular branch (×10).
Relationship between placental HBV DNA infection and fetal intrauterine infection
Table 6
Cases of HBV intrauterine infection (n = 3)
DC VTC VMC VCEC
Controls (n = 39)
Positive
Negative
Positive
Negative
2 2 1 2
1 1 2 1
15 36 15 33
24 3 24 6
OR
95% CI
P
3.2 0.27–38.44 0.5557 0.17 0.01–2.42 0.2652 0.8 0.07–9.16 1.0000 0.36 0.03–4.62 1.0000
CI, confidence intervals; DC, decidual cells; HBV, hepatitis B virus; OR, odds ratio; VCEC, capillaries endothelial cells; VMC, villous stroma cells; VTC, trophoblast cells. P > 0.05.
relationship of a series of factors with intrauterine infection and found that mother’s age, mode of delivery, ALT, blood platelet count, clinical HBV infection features, premature labor, GDM, pre-eclampsia, fetal growth retardation, fetal distress, Apgar score of the infant as well as the HBV infection status of the placenta all failed to a statistically significant correlation with intrauterine infection. The results in this study suggested that a high level of HBV in maternal blood was one of the risk factors accounting for intrauterine infection. HBsAg, HBeAg, and their antibodies could enter the fetus through the placenta; however, a definite
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conclusion on intrauterine infection cannot be drawn according to the results of HBsAg and HBeAg test in cord blood as the presence of HBeAg and HBsAg in the cord blood does not necessarily indicate an HBV infection of the fetus. Unlike HBsAg and HBeAg, the HBV DNA in the cord blood is a good indicator for screening HBV intrauterine infection [14,15]. In this study, our results showed that HBeAg as well as HBV DNA in the mother’s blood during pregnancy were correlated with HBV intrauterine infection, suggesting that these two markers might have the potential to predict intrauterine infection. We further analyzed the relationship of a series of factors with intrauterine infection. Surprisingly, our results showed that mother’s age, delivery mode, ALT, blood platelet count, clinical HBV infection features, premature labor, GDM, pre-eclampsia, fetal growth retardation, fetal distress, Apgar score of the infant as well as the HBV infection status of the placenta all failed to a statistically significant correlation with intrauterine infection. Our results were consistent with previous studies, which reported that ALT and fetal distress were not associated with HBV intrauterine infection [16,17]. However, some factors not included in the current study, such as maternal first-degree family history of HBV, intrahepatic cholestasis, and premature rupture of membranes, have been described as risk factors for perinatal transmission of HBV [16]. Therefore, to better understand the possible risk factors for HBV intrauterine infection, studies with a more comprehensive inclusion of potential factors, although beyond the scope of the current study, should be carried out. Our study also found that HBV can infect every layer of the placenta, with the HBV DNA levels in VTC and VCEC significantly higher than those in DC and VMC. However, no statistically significant correlation was observed between the HBV DNA levels in the placenta and HBV intrauterine infection. Previous studies by others have also shown that HBV DNA could be detected in VTC in the placenta of pregnant women with hepatitis B [18]. The status of placenta HBV infection in HBsAg-positive pregnant women varies considerably. The rates of placental HBV infection, on the basis of HBsAg-positive placental tissue, were 16.6% [19] and 20% [20], and 67% [21] and 81.5% [22], on the basis of HBsAg and/or HBcAg positive, respectively. Mingkai Cao et al. also reported that the detection rate of HBV DNA in placenta was 100%, but the intrauterine infection was low [23], providing more evidence that, consistent with our results in the current study, placental infection did not indicate intrauterine infection. The reason why placental cells were infected with HBV DNA, but fetal intrauterine infection did not occur, was probably because the placenta protected the fetus against the viruses. It has been reported that placental trophoblast cells, when invaded by a pathogen, can release chemokines, which consequently recruit immunocytes including T lymphocytes and monocytes to the site of
infection [24]. However, detailed underlying mechanisms should be further defined. Conclusion
In summary, our results showed that positive HBeAg as well as HBV DNA in the mother’s serum were correlated closely with HBV intrauterine infection. We further analyzed the relationship of a series of factors with intrauterine infection and found that mother’s age, delivery mode, ALT, blood platelet count, clinical HBV infection features, premature labor, GDM, pre-eclampsia, fetal growth retardation, fetal distress, Apgar score of the infant as well as the HBV infection status of the placenta all failed to show a statistically significant correlation with intrauterine infection. The results in this study suggested that a high level of HBV in maternal blood was one of the risk factors accounting for intrauterine infection.
Acknowledgements The authors thank Fenghe (Shanghai) Information Technology Co., Ltd. Their ideas and help provided a valuable added dimension to our research. This study was supported by the Scientific research subject of Heilongjiang province health department (No.2010-062), the Science and technology research project of Heilongjiang province department of education (No.12521347), the Overseas scholars research project of Heilongjiang province department of education (No. 1153h14), and the Hospital youth fund (No. QN2010-14). Conflicts of interest
There are no conflicts of interest.
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Original article
Analysis on the outcomes of hepatitis B virus perinatal vertical transmission: nested case–control study Nan Lv, Xiao-Dan Chu, Yu-Hong Sun, Shu-Yu Zhao, Pei-Ling Li and Xuan Chen Objective Hepatitis B virus (HBV) infection is a public health problem worldwide, with vertical transmission as the leading transmission route. Therefore, it is very important to explore the risk factors associating with HBV perinatal transmission, providing valuable information for preventive and curative means for HBV perinatal infections. In this study, we systematically investigated the impact of adverse outcomes of HBV maternal infection on fetal intrauterine infection. Patients and methods This is a nested case–control study that included 42 hepatitis B surface antigen (HBsAg)positive pregnant women. Gestational weeks, delivery modes, alanine aminotransferase levels, platelet counts, liver ultrasonography results as well as adverse pregnancy outcomes for the pregnant mothers and Apgar scores at both 1 and 5 min, birth weight, and height for the newborn infants were recorded. Vein blood from pregnant mothers and cord blood immediately after delivery were collected for the detection of HBsAg, antibodies to hepatitis B surface antigen, hepatitis B e antigen (HBeAg), antibody to hepatitis B e antigen, hepatitis B core antigen, and HBV DNA. Placental tissues were collected for detection of HBV DNA. Results Positive HBeAg as well as HBV DNA in the mother’s serum were correlated closely with HBV intrauterine infection. Mother’s age, delivery mode, alanine
Introduction Hepatitis B virus (HBV) infection is a major health problem worldwide, with a chronically infected population of around 350–400 million worldwide [1]. In China, which has a high prevalence of HBV, the infected population accounts for one-third of the infected population worldwide [2,3]. HBV is predominantly transmitted through mother-to-child vertical transmission, with perinatal transmission being the main route [4,5]. Perinatal transmission can be subcategorized into three categories: intrauterine transmission, labor transmission, and postnatal transmission. Among about 120 million HBV carriers in China, 30–50% are infected with HBV through vertical transmission [2,6]. Since the introduction of HBV vaccination in the entire population including newborn infants in China in 1992, the hepatitis B surface antigen (HBsAg)-positive rate has reduced markedly. However, because of the underdeveloped immune system in the neonatal infants, the combined neonatal HBV vaccine and hepatitis B immunoglobulin have led to 20–30% of immunization failure in infants [7,8]. HBV perinatal transmission through the 0954-691X © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
aminotransferase, blood platelet count, clinical HBV infection features, premature labor, gestational diabetes mellitus, pre-eclampsia, fetal growth retardation, fetal distress, Apgar scores of the infant as well as the HBV infection status of the placenta all failed to show a statistically significant correlation with intrauterine infection. Conclusion High level of HBV in maternal blood was one of the risk factors accounting for intrauterine infection. Eur J Gastroenterol Hepatol 26:1286–1291 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. European Journal of Gastroenterology & Hepatology 2014, 26:1286–1291 Keywords: hepatitis B virus, perinatal outcome, placenta, vertical transmission Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Harbin Medical University, Harbin, China Correspondence to Pei-Ling Li, PhD, Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin 150086, China Tel: + 86 451 866 05141; fax: + 86 451 86297003; e-mail: [email protected] and Co-correspondence to Xuan Chen, MM, Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin 150086, China Tel: + 86 451 86605050; fax: + 86 451 86605343; e-mail: [email protected] Received 1 June 2014 Accepted 21 July 2014
mother to child can result in hepatitis fetuses. Without treatment, newborn infants with HBV infection have a considerably high possibility of becoming chronic carriers [9]. Therefore, it is very important to explore the risk factors associated with HBV perinatal transmission, providing valuable information for the preventive and curative means for HBV perinatal infections. As previous studies on HBV perinatal transmission are limited, in the current study, we have investigated the adverse outcomes of HBV maternal and fetal intrauterine infection by determining the relationship of possible adverse outcomes for pregnancy, the growth and development of fetuses, and the infection of placental cells in each layer with HBV intrauterine infection.
Methods Patients
Forty-two HBsAg-positive pregnant women, 22–43 years of age, without an antepartum hemorrhage history, who had undergone a prenatal examination and delivered in the Second Affiliated Hospital of Harbin Medical DOI: 10.1097/MEG.0000000000000189
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HBV perinatal vertical transmission Lv et al. 1287
University from January, 2011 to June, 2012 were recruited into the study. Patient clinical baseline characteristics
Among the 42 HBsAg-positive pregnant women, 10 were both HBsAg and hepatitis B e antigen (HBeAg) positive whereas 18 were HBV DNA positive. Gestational weeks, delivery modes, alanine aminotransferase (ALT) levels, platelet counts, liver ultrasonography results as well as adverse pregnancy outcomes (gestational diabetes mellitus (GDM), premature labor, pre-eclampsia, fetal distress, fetal growth retardation, etc.) for the pregnant mothers and Apgar scores [10] at both 1 and 5 min, birth weight, and height of the newborn infants were recorded. Criterion of intrauterine infection
Nested case controls were applied in the study. Cord blood HBV DNA more than 5 × 102 copies was considered the criterion of intrauterine infection. The selected babies were subsequently divided into an intrauterine infection group and a control group. Detection of HBV markers and HBV DNA in blood
Vein blood samples of the pregnant mothers 28 weeks before child birth as well as cord blood samples immediately after birth delivery were collected for the detection of markers of HBsAg, antibodies to hepatitis B surface antigen (anti-HBs), HBeAg, antibody to hepatitis B e antigen (anti-HBe), hepatitis B core antigen (antiHBc), and HBV DNA. Twelve months after birth, vein blood samples of the chosen newborns were collected for the detection of markers of HBV, HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, and HBV DNA. Serologic HBV markers and HBV DNA in the blood samples were measured by ELISA (Shanghai Kehua Bio-engineering Co., Ltd, Shanghai, China) and PCR, respectively, following the manufacturer’s instructions. HBV DNA in-situ hybridization
To analyze the relationship between placental cells in each layer of HBV DNA infection and intrauterine infection, HBV DNA in-situ hybridization on placental tissues was used. Placental specimens were collected and cut into a size of about 1 cm × 1 cm × 1.5 cm, fixed in 10% neutral-buffered formalin for 12–20 h, and subsequently embedded and cut into 3 μm sections. HBV DNA in the tissue sections were detected using in-situ hybridization with a digoxigenin system (Fujian Triplex International Biosciences Co., Ltd, Fujian, China) and brown-colored spots were HBV DNA-positive. The HBV-negative placental tissues were used as negative controls whereas HBsAg-positive liver tissues were used as positive controls. Ethical statement
All protocols in this study were reviewed and approved by the ethical board of Harbin Medical University.
Informed written consents were obtained from the pregnant women or the family of the newborn infants. Statistical analysis
Multivariate logistic regression analysis was used to analyze the adverse outcomes of pregnancy, clinical features in HBV-infected pregnant women, and the relationship between the Apgar score [10] of the newborns after birth and intrauterine infection. The odds ratios and 95% confidence intervals were calculated. For statistical comparisons between two groups, a t-test or Fisher’s exact test was used for enumeration data, and a P value less than 0.05 was considered statistically significant. All analyses were carried out using SPSS 11.0 (SPSS Inc., Chicago, Illinois, USA).
Results HBeAg positivity in maternal blood was correlated with intrauterine HBV infection of infants
Among 42 newborn infants, three had intrauterine infection, with HBV DNA in cord blood more than 5 × 102 copies (5 × 107, 5.215 × 103 and 3.879 × 105 copies, respectively). The rate of intrauterine infection was 7.14%. Of the three infants with intrauterine infection, one of their mothers was both HBsAg and HBeAg positive. Further analysis showed that positive HBeAg in mothers was correlated with intrauterine HBV infection in infants (P = 0.0105) (Tables 1 and 2). Maternal serum HBV DNA level was related positively to that in the fetal cord blood
Of 42 HBsAg-positive pregnant women, 18 were HBV DNA positive, with seven of these having more than 107 copies. Further analysis was carried out to examine the relationship between maternal serum HBV DNA levels and fetal cord blood HBV DNA levels. The results showed that maternal serum HBV DNA levels were related positively to that in the fetal cord blood (P = 0.0429) (Table 2), suggesting that high copies of HBV DNA in maternal blood was one of the risk factors for intrauterine infection. Mother’s age, delivery mode, adverse pregnancy outcome, clinical HBV infection feature as well as the Relationship between the peripheral blood of HBeAgpositive pregnant women and fetal intrauterine infection
Table 1
Intrauterine infection
Control
n (%)
3 0 3
7 32 39
10 (30.00) 32 (0) 42 (7.14)
HBeAg (+ ) HBeAg (− ) n HBeAg, hepatitis B e antigen. P = 0.0105. P < 0.05.
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Table 2
European Journal of Gastroenterology & Hepatology 2014, Vol 26 No 11
Maternal serum HBV DNA copies and newborn infants with HBV intrauterine infection (n < 40)
Maternal serum HBV DNA ≥ 107 copies (n = 7) Maternal serum HBV DNA < 107 copies (n = 11)
Intrauterine infection (n = 3)
Control (n = 15)
3 0
4 11
HBV, hepatitis B virus. Fisher’s exact probabilities, P = 0.0429, P < 0.05.
Clinical characteristics and adverse pregnancy outcomes of HBV-infected mothers
Table 3
Age at delivery (years) < 25 25–34 ≥ 35 Adverse pregnancy outcomes Spontaneous delivery Cesarean delivery GDM (no) GDM (yes) Term birth Preterm birth Pre-eclampsia (no) Pre-eclampsia (yes) Fetal distress (no) Fetal distress (yes) FGR (no) FGR (yes) 1 min Apgar score 8–10 4–7 5 min Apgar score 8–10 4–7 Clinical characteristics of hepatitis B Thrombocytopenia (no) Thrombocytopenia (yes) Transaminase normal Transaminase abnormal Liver ultrasonography normal Liver ultrasonography abnormal
Cases of HBV intrauterine infection (n = 3) [n (%)]
Controls (n = 39) [n (%)]
0 2 (67) 1 (33)
5 (13) 26 (67) 8 (20)
0 3 3 0 3 0 3 0 1 (33) 2 (66) 3 0
7 (18) 32 (82) 31 (79) 8 (21) 33 (85) 6 (15) 31 (79) 8 (21) 31 (79) 8 (21) 36 3
3 0
31 (79) 8 (21)
3 0
35 (90) 4 (10)
(66) (33) (66) (33) (66) (33)
35 4 36 3 37 2
2 1 2 1 2 1
(90) (10) (92) (8) (95) (5)
FGR, fetal growth restriction; GDM, gestational diabetes mellitus; HBV, hepatitis B virus.
infant’s Apgar score were not statistically related to intrauterine infection
To further explore the impact of mother’s age, delivery mode, adverse pregnancy outcome, clinical HBV infection feature as well as the infant’s Apgar score on intrauterine infection, logistic regression analysis was used (Table 3). Our results showed that all the tested factors, including mother’s age, delivery mode, ALT, blood platelet count, clinical HBV infection features, premature labor, GDM, pre-eclampsia, fetal growth retardation, fetal distress, and Apgar score of the infant, failed to show a statistically significant correlation with HBV intrauterine infection (P > 0.05) (Tables 4 and 5). HBV infection of placental cells was not correlated with intrauterine infection
HBV can infect placenta; we consequently tested whether HBV DNA levels in the placenta are related to
Table 4 Multivariate logistic regression analyses for HBV intrauterine infection
Age at delivery (years) < 25 25–34 ≥ 35 Adverse pregnancy outcomes Cesarean delivery GDM Preterm birth Pre-eclampsia Fetal distress FGR 1 min Apgar score 4–7 5 min Apgar score 4–7 Clinical characteristics of hepatitis B Thrombocytopenia Transaminase abnormal Liver ultrasonography abnormal
OR (95% CI)
P
– –
0.9975 0.9974
– – – – – –
0.9970 0.9968 0.9972 0.9968 0.9970 0.9970
–
0.9968
–
0.9965
0.229 (0.017–3.120) 0.167 (0.011–2.416) 0.108 (0.007–1.761)
0.2684 0.1891 0.1182
CI, confidence intervals; FGR, fetal growth restriction; GDM, gestational diabetes mellitus; HBV, hepatitis B virus; OR, odds ratio. P > 0.05.
intrauterine infection. Our results of HBV DNA in-situ hybridization showed that HBV DNA was detected in every cell layer of the placenta (Fig. 1). The infection rates of decidual cells (DC), trophoblast cells (VTC), villous stroma cells (VMC), and capillaries endothelial cells (VCEC) were 40.48% (17/42), 90.48% (38/42), 38.10% (16/42), and 83.33% (35/42), respectively. In placental tissues, HBV DNA in VTC and in VCEC was higher than its expression in DC and VMC. However, HBV DNA infection in every placental cell layer was not statistically significantly correlated with HBV intrauterine infection (P > 0.05) (Table 6).
Discussion HBV infection is a public health problem worldwide. In China, 6.70–8.25% of pregnant women are HBsAgpositive [11–13]. Perinatal transmission is the most frequent route of transmission in China. Consequently, it is very important to explore the risk factors associated with HBV perinatal transmission, providing valuable information for preventive and curative measures for HBV perinatal infections. In this study, we systematically investigated the impact of adverse outcomes of HBV maternal infection on fetal intrauterine infection. Our results indicated that positive HBeAg as well as HBV DNA in the mother’s serum were correlated closely with HBV intrauterine infection. We further analyzed the
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HBV perinatal vertical transmission Lv et al. 1289
Table 5
Effects of maternal HBV infection and HBV intrauterine infection on fetal development
HBeAg (+ ) (n = 10) HBeAg (− ) (n = 32) HBV DNA (+ ) (n = 18) HBV DNA (− ) (n = 24) ALT/AST abnormal (n = 3) ALT/AST normal (n = 39) Cord blood HBV DNA (+ ) (n = 3) Cord blood HBV DNA (− ) (n = 39)
Birth weight
Body length
1 min Apgar score
5 min Apgar score
3.25 ± 0.50 3.47 ± 0.51 3.54 ± 0.38 3.36 ± 0.58 3.35 ± 0.91 3.44 ± 0.49 3.93 ± 0.16 3.40 ± 0.51
49.00 ± 2.00 50.06 ± 2.05 50.00 ± 2.08 49.71 ± 2.07 50.00 ± 3.00 49.82 ± 2.02 51.66 ± 1.52 49.69 ± 2.04
7.70 ± 1.15 8.18 ± 0.69 8.11 ± 0.75 8.16 ± 0.81 7.66 ± 0.57 8.17 ± 0.79 8.33 ± 0.57 8.12 ± 0.80
8.20 ± 1.13 8.46 ± 0.71 8.27 ± 1.01 8.58 ± 0.58 8.33 ± 0.57 8.46 ± 0.82 8.66 ± 0.57 8.94 ± 3.39
FGR (%) 10.0 6.2 10.0 12.5 33.33 5.13 0 7.69
(1/10) (2/32) (1/10) (3/24) (1/3) (2/39) (0/3) (3/39)
ALT, alanine aminotransferase; AST, aspartate aminotransferase; FGR, fetal growth restriction; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus. P > 0.05.
Fig. 1
In-situ hybridization detection of hepatitis B virus (HBV) DNA in placental tissues (a) decidual cells show positive expression of HBV DNA (×40); (b) trophoblast cells (VTC) and villous stroma cells (VMC) show positive expression of HBV DNA (×40); (c) capillaries endothelial cells (VCEC) show positive expression of HBV DNA (×20); (d) negative control: vascular branch (×10).
Relationship between placental HBV DNA infection and fetal intrauterine infection
Table 6
Cases of HBV intrauterine infection (n = 3)
DC VTC VMC VCEC
Controls (n = 39)
Positive
Negative
Positive
Negative
2 2 1 2
1 1 2 1
15 36 15 33
24 3 24 6
OR
95% CI
P
3.2 0.27–38.44 0.5557 0.17 0.01–2.42 0.2652 0.8 0.07–9.16 1.0000 0.36 0.03–4.62 1.0000
CI, confidence intervals; DC, decidual cells; HBV, hepatitis B virus; OR, odds ratio; VCEC, capillaries endothelial cells; VMC, villous stroma cells; VTC, trophoblast cells. P > 0.05.
relationship of a series of factors with intrauterine infection and found that mother’s age, mode of delivery, ALT, blood platelet count, clinical HBV infection features, premature labor, GDM, pre-eclampsia, fetal growth retardation, fetal distress, Apgar score of the infant as well as the HBV infection status of the placenta all failed to a statistically significant correlation with intrauterine infection. The results in this study suggested that a high level of HBV in maternal blood was one of the risk factors accounting for intrauterine infection. HBsAg, HBeAg, and their antibodies could enter the fetus through the placenta; however, a definite
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conclusion on intrauterine infection cannot be drawn according to the results of HBsAg and HBeAg test in cord blood as the presence of HBeAg and HBsAg in the cord blood does not necessarily indicate an HBV infection of the fetus. Unlike HBsAg and HBeAg, the HBV DNA in the cord blood is a good indicator for screening HBV intrauterine infection [14,15]. In this study, our results showed that HBeAg as well as HBV DNA in the mother’s blood during pregnancy were correlated with HBV intrauterine infection, suggesting that these two markers might have the potential to predict intrauterine infection. We further analyzed the relationship of a series of factors with intrauterine infection. Surprisingly, our results showed that mother’s age, delivery mode, ALT, blood platelet count, clinical HBV infection features, premature labor, GDM, pre-eclampsia, fetal growth retardation, fetal distress, Apgar score of the infant as well as the HBV infection status of the placenta all failed to a statistically significant correlation with intrauterine infection. Our results were consistent with previous studies, which reported that ALT and fetal distress were not associated with HBV intrauterine infection [16,17]. However, some factors not included in the current study, such as maternal first-degree family history of HBV, intrahepatic cholestasis, and premature rupture of membranes, have been described as risk factors for perinatal transmission of HBV [16]. Therefore, to better understand the possible risk factors for HBV intrauterine infection, studies with a more comprehensive inclusion of potential factors, although beyond the scope of the current study, should be carried out. Our study also found that HBV can infect every layer of the placenta, with the HBV DNA levels in VTC and VCEC significantly higher than those in DC and VMC. However, no statistically significant correlation was observed between the HBV DNA levels in the placenta and HBV intrauterine infection. Previous studies by others have also shown that HBV DNA could be detected in VTC in the placenta of pregnant women with hepatitis B [18]. The status of placenta HBV infection in HBsAg-positive pregnant women varies considerably. The rates of placental HBV infection, on the basis of HBsAg-positive placental tissue, were 16.6% [19] and 20% [20], and 67% [21] and 81.5% [22], on the basis of HBsAg and/or HBcAg positive, respectively. Mingkai Cao et al. also reported that the detection rate of HBV DNA in placenta was 100%, but the intrauterine infection was low [23], providing more evidence that, consistent with our results in the current study, placental infection did not indicate intrauterine infection. The reason why placental cells were infected with HBV DNA, but fetal intrauterine infection did not occur, was probably because the placenta protected the fetus against the viruses. It has been reported that placental trophoblast cells, when invaded by a pathogen, can release chemokines, which consequently recruit immunocytes including T lymphocytes and monocytes to the site of
infection [24]. However, detailed underlying mechanisms should be further defined. Conclusion
In summary, our results showed that positive HBeAg as well as HBV DNA in the mother’s serum were correlated closely with HBV intrauterine infection. We further analyzed the relationship of a series of factors with intrauterine infection and found that mother’s age, delivery mode, ALT, blood platelet count, clinical HBV infection features, premature labor, GDM, pre-eclampsia, fetal growth retardation, fetal distress, Apgar score of the infant as well as the HBV infection status of the placenta all failed to show a statistically significant correlation with intrauterine infection. The results in this study suggested that a high level of HBV in maternal blood was one of the risk factors accounting for intrauterine infection.
Acknowledgements The authors thank Fenghe (Shanghai) Information Technology Co., Ltd. Their ideas and help provided a valuable added dimension to our research. This study was supported by the Scientific research subject of Heilongjiang province health department (No.2010-062), the Science and technology research project of Heilongjiang province department of education (No.12521347), the Overseas scholars research project of Heilongjiang province department of education (No. 1153h14), and the Hospital youth fund (No. QN2010-14). Conflicts of interest
There are no conflicts of interest.
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