International Journal of Gynecology and Obstetrics 125 (2014) 158–161

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CLINICAL ARTICLE

Risk factors for intrauterine infection with hepatitis B virus Zhao Zhang a,1, Aizhen Li a,b,1, Xiaomin Xiao a,⁎ a b

Department of Obstetrics and Gynecology, First Affiliated Hospital of Jinan University, Guangzhou, China Department of Obstetrics, Obstetrics and Gynecology Affiliated Hospital of Fudan University, Shanghai, China

a r t i c l e

i n f o

Article history: Received 1 August 2013 Received in revised form 31 October 2013 Accepted 29 January 2014 Keywords: Hepatitis B virus Intrauterine infection Risk factors

a b s t r a c t Objective: To investigate risk factors for hepatitis B virus (HBV) intrauterine infection. Methods: Peripheral blood samples and clinical data were collected from 174 pregnant women who were positive for hepatitis B surface antigen (HBsAg). Their 176 neonates received an active–passive immunization schedule at 0, 1, and 6 months. Blood samples from the infants, collected before immune prophylaxis administration, were tested for HBV markers and HBV DNA. Results: The intrauterine infection rate at 6 months after birth was 5.1%. Maternal HBV DNA positivity (OR 11.362; 95% CI, 1.389–92.931), hepatitis B e antigen (HBeAg) positivity (OR 7.278; 95% CI, 1.734–30.538), and thalassemia minor (OR 15.619; 95% CI, 2.239–108.964) were associated with intrauterine infection. The intrauterine infection rate for mothers with 105 copies/mL of serum HBV DNA or more was 18.2%, compared with 0.8% for mothers with less than 105 copies/mL. Conclusion: A positive HBsAg test at 24 hours and/or 1 month of age followed by a positive test at 6 months is an objective and comprehensive criterion for the diagnosis of HBV intrauterine infection. Maternal HBV DNA positivity (especially 105 copies/mL of HBV DNA or more), HBeAg positivity, and thalassemia minor are risk factors for HBV intrauterine infection. © 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction

2. Materials and methods

Hepatitis B virus (HBV) infection is a major global health problem, and more than 240 million people develop chronic liver infection as a result [1]. Vertical transmission accounts for 40–50% of all chronic HBV infections, making it an important mode of HBV infection and an important reason why there are so many HBV carriers in the population [2]. The Advisory Committee on Immunization Practices recommends that all infants born to mothers who are positive for hepatitis B surface antigen (HBsAg) should undergo postexposure immunoprophylaxis with both the hepatitis B vaccine and hepatitis B immunoglobulin (HBIG) [3–6]. However, such a remedial measure is not sufficient to completely prevent the vertical transmission of HBV. Additionally, the standard passive–active immunoprophylaxis with HBIG and hepatitis B vaccine in neonates after birth may have a failure rate as high as 10–15% [7]. Previous work [7–9] indicates that one of the underlying causes of vaccine failure may be intrauterine infection. The present study investigated the risk factors of HBV intrauterine infection among HBsAgpositive pregnant women, and aimed to identify new possible ways for the prevention of intrauterine infection.

The present study included pregnant women who gave birth at the obstetric department of the First Affiliated Hospital of Jinan University, Guangzhou, China, between August 1, 2004, and August 31, 2005. The study protocol was approved by the Ethics Committee of the National Natural Science Foundation of China. The following criteria needed to be met for eligibility: (1) HBsAg seropositivity; (2) normal liver and kidney function; (3) no history of infection with toxoplasmosis, other (syphilis, hepatitis, zoster), rubella, cytomegalovirus, or herpes simplex (TORCH infection); (4) no receipt of any research drugs, antivirals, immunomodulating or cytotoxic medications, or steroid hormones during pregnancy; (5) husband was no HBV carrier or hepatitis B patient; (6) ability to give written informed consent. A total of 174 pregnant women and their 176 neonates (including 2 twins) were eligible for the study (Fig. 1). Clinical data including gravidity, parity, history of abortion, mode of delivery, maternal complications, and placental data were obtained from the participants’ medical records. Peripheral blood samples were collected from the median cubital vein of all participants before delivery. The newborns received 100 IU of HBIG intramuscularly in the anterolateral thigh and 5 μg of yeast-derived recombinant hepatitis B vaccine in the contralateral anterolateral thigh within 24 hours of birth, and were vaccinated again at 1 and 6 months without the simultaneous administration of HBIG (standard 0-, 1-, and 6-month vaccination schedule). Before each administration of immune prophylaxis, blood samples were drawn from the infants’ femoral vein.

⁎ Corresponding author at: No. 613 West of Huangpu Avenue, Guangzhou, 510632 China. Tel.: +86 20 38088660; fax: +86 20 38688606. E-mail address: [email protected] (X. Xiao). 1 Authors contributed equally.

0020-7292/$ – see front matter © 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijgo.2013.10.028

Z. Zhang et al. / International Journal of Gynecology and Obstetrics 125 (2014) 158–161

Table 2 Univariate analysis of associations between maternal and pregnancy characteristics and HBV intrauterine infection (n = 176).a

Expectant mothers in the hospital (n=1237)

HBsAg-positive

HBsAg-negative

(n=212)

(n=1025)

Collection of clinical data (n=212)

Pregnant women with regular prenatal care (n=189)

Infants followed-up for 6 months (n=174)

Exclusion (n=1025)

Pregnant women without regular prenatal care (n=23)

Infants not followed-up for 6 months (n=15)

Enrollment (n=174)

159

Exclusion (n=23)

Exclusion (n=15)

Fig. 1. Flow chart of the patient selection process. Abbreviations: HBsAg, hepatitis B surface antigen.

The serum levels of HBsAg and hepatitis B e antigen (HBeAg) were measured by enzyme-linked immunosorbent assay (Sino-American Biotechnology Company, Henan, China). The levels of HBV DNA were determined by fluorogenic quantitative polymerase chain reaction (Shanghai Kehua Bio-engineering Company, Shanghai, China). All procedures were performed according to the manufacturers’ instructions. If the level of HBV DNA was below 103 copies/mL (the fluorescence detection threshold given in the user manual), the sample was considered to be negative; otherwise it was considered to be positive. Intrauterine transmission was defined as a positive HBsAg test at 24 hours and/or 1 month of age, followed by a positive test at 6 months of age [10]. In the univariate analysis, χ2 tests were used for categorical data and t tests were used for continuous variables. The Fisher exact test was used for categorical analyses. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to measure the association between maternal characteristics and intrauterine transmission. All analyses were performed with SPSS version 13.0 (IBM, Armonk, NY, USA). P b 0.05 was considered statistically significant.

3. Results Of the 176 newborns in the study, 9 tested positive for HBsAg at 24 hours and/or 1 month of age and at 6 months, giving an intrauterine infection rate of 5.1% (Table 1). In the univariate analysis (Table 2), maternal HBV DNA positivity (P = 0.011), HBeAg positivity (P = 0.006), and thalassemia minor

t value

P value

Characteristic

No intrauterine infection (n = 167)

Intrauterine infection (n = 9)

Maternal age, y Gravidity Parity Number of abortions Duration of pregnancy, wk Umbilical cord length, cm Placental volume, cm3 Maternal ALT, U/L Maternal HBV DNA positivity Maternal HBeAg positivity Han ethnicity Mode of delivery (V/C) PROM Threatened premature labor Threatened abortion Preterm birth Placenta previa Hyperemesis gravidarum Polyhydramnios Oligohydramnios MSAF Cord entanglement Hysteromyoma GDM Mild anemia Thalassemia minor Placental calcification Fetal distress Breech presentation History of FGR Increase of AFP level

28.53 ± 3.82 1.80 ± 1.02 1.13 ± 0.37 0.67 ± 0.98 38.60 ± 1.45 52.43 ± 8.14 762.06 ± 174.92 21.68 ± 13.67 69 (41.3) 36 (21.6) 164 (98.2) 85/82 (50.9/49.1) 48 (28.7) 4 (2.4) 9 (5.4) 12 (7.2) 1 (0.6) 1 (0.6) 3 (1.8) 12 (7.2) 38 (22.8) 51 (30.5) 7 (4.2) 5 (3.0) 4 (2.4) 3 (1.8) 7 (4.2) 29 (17.4) 5 (3.0) 9 (5.4) 26 (15.6)

27.56 ± 4.03 0.745 0.457 1.89 ± 1.27 −0.245 0.807 1.11 ± 0.33 0.162 0.871 0.78 ± 0.97 −0.318 0.751 38.78 ± 1.20 −0.363 0.717 51.11 ± 6.01 0.477 0.643 657.00 ± 178.69 1.753 0.081 20.78 ± 12.20 0.193 0.847 8 (88.9) 0.011 6 (66.7) 0.006 9 (100.0) N0.99 8/1 (88.9/11.1) 0.060 2 (22.2) N0.99 0 (0.0) N0.99 1 (11.1) 0.417 0 (0.0) N0.99 0 (0.0) N0.99 0 (0.0) N0.99 0 (0.0) N0.99 0 (0.0) N0.99 3 (33.3) 0.437 3 (33.3) N0.99 0 (0.0) N0.99 0 (0.0) N0.99 0 (0.0) N0.99 2 (22.2) 0.022 0 (0.0) N0.99 2 (22.2) 0.660 0 (0.0) N0.99 1 (11.1) 0.417 3 (33.3) 0.169

Abbreviations: AFP, α-fetoprotein; ALT, alanine aminotransferase; FGR, fetal growth restriction; GDM, gestational diabetes mellitus; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; MSAF, meconium-stained amniotic fluid; PROM, premature rupture of membranes; V/C, vaginal delivery/cesarean delivery. a Values are given as mean ± SD or number (percentage).

(P = 0.022) were significantly associated with HBV intrauterine transmission, and the risk for HBV intrauterine infection was considerably increased among women with these characteristics (Table 3). An analysis stratified by maternal HBV DNA level showed that the intrauterine infection rate was considerably higher among women with higher HBV DNA levels (Table 4). Because some frequencies were too small for R × C χ2 analysis, maternal HBV DNA levels with similar intrauterine infection rates were grouped together. There was a significant difference between the intrauterine infection rates of women with less than 105 copies/mL of HBV DNA and those with 105 copies/mL or more (P b 0.001, Fisher exact test) (Table 5). 4. Discussion The absence of unified standards for the diagnosis of HBV intrauterine infection has led to an inconsistency in reported intrauterine infection rates [2,10–13]. The ideal method to measure the intrauterine infection rate is to detect HBV DNA levels from neonatal liver tissue,

Table 1 Frequency of HBV serum markers among the newborns in the study (n = 176).a Age, mo

HBsAg

HBsAb

HBeAg

HBeAg

HBV DNA

0 1 6

16 (9.1) 11 (6.3) 9 (5.1)

50 (28.4) 92 (52.3) 131 (74.4)

33 (18.8) 9 (5.1) 6 (3.4)

20 (11.4) 0 (0.0) 0 (0.0)

7 (4.0) 12 (6.8) 4 (2.3)

Abbreviations: HBeAg, HBeAg-positive but HBsAg-negative; HBeAg, hepatitis B e antigen; HBsAb, hepatitis B surface antibody; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus. a Values are given as number (percentage).

Table 3 Maternal risk factors for intrauterine HBV infection (n = 176). Risk factor

OR

95% CI

P value

Positive HBV DNA test Positive HBeAg test Thalassemia minor

11.362 7.278 15.619

1.389–92.931 1.734–30.538 2.239–108.964

0.011 0.006 0.022

Abbreviations: CI, confidence interval; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; OR, odds ratio.

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Table 4 Maternal HBV DNA level and intrauterine infection rate (n = 176). Maternal HBV DNA level

Intrauterine infection (n = 9)a

No intrauterine infection (n = 167)a

Intrauterine infection rate, %

b103 copies/mL 103–104 copies/mL 104–105 copies/mL 105–106 copies/mL 106–107 copies/mL 107–108 copies/mL N108 copies/mL

1 (11.1) 0 (0.0) 0 (0.0) 1 (11.1) 1 (11.1) 2 (22.2) 4 (44.4)

98 (58.7) 22 (13.2) 12 (7.2) 7 (4.2) 8 (4.8) 10 (6.0) 10 (6.0)

0.01 0.00 0.00 12.50 11.10 16.70 28.60

a

Values are given as number (percentage).

but this is not feasible in practice. At present, the most widely used diagnostic approach is to determine whether the serum of newborns is HBsAg-positive and/or HBeAg-positive, or whether their peripheral blood is HBV-DNA-positive during the first 24 hours after birth, before the administration of active or passive immune prophylaxis [14]. However, it is not sufficient to simply determine whether intrauterine infection has occurred based on the detection of HBV markers in the newborn’s peripheral blood during the 24 hours after birth. The neonate requires follow-up care to avoid false-positives and to exclude the possibility of missing “delayed expression” [10,15]. Thus, in the present study, intrauterine transmission was defined as HBsAg seropositivity at 24 hours and/or at 1 month of age, followed by another positive test at 6 months of age. We considered this diagnostic criterion to be more objective and comprehensive, and of greater practicality in the clinical setting. Based on this criterion, the intrauterine infection rate was 5.1% (9/176) in the present study, which is close to the rate reported by Li et al. [10]. Intrauterine infection with HBV is a fundamental reason why there are so many chronic HBV carriers in China [16]. To date, there are no consistent and reliable measures that could be used to prevent and defend against HBV intrauterine transmission. Therefore, to prevent and block intrauterine infection effectively it is necessary to study the risk factors associated with it. However, the risk factors of HBV intrauterine transmission reported in the current literature are both inconsistent and controversial. In the present study, based on univariate analysis, maternal HBV DNA positivity and HBeAg positivity increased the risk of intrauterine transmission, which is consistent with the majority of earlier studies [17,18]. Serum levels of HBeAg can be used to indirectly measure HBV virus replication in vivo and to determine the infectiousness of the host. Furthermore, HBV DNA is the most direct and reliable measure of viral replication in vivo. Serum positivity for HBeAg and HBV DNA indicates that there is a lot of virus in the blood stream and that it is highly infectious. Importantly, active viral replication is a strong risk factor for intrauterine infection [2,7,19]. Furthermore, the stratified analysis in the present study revealed that the intrauterine infection rate for mothers with HBV DNA levels of 105 copies/mL or more was significantly higher than that for mothers with HBV DNA levels below 105 copies/mL (18.2% versus 0.8%). This result further confirms the link between high levels of HBV DNA and high risk of HBV intrauterine infection.

Table 5 Comparison of intrauterine infection rates by maternal HBV DNA level (n = 176).a Maternal HBV DNA level

Intrauterine infection (n = 9)

No intrauterine infection (n = 167)

Intrauterine infection rate, %

b105 copies/mL ≥105 copies/mL

1 (11.1) 8 (88.9)

132 (79.0) 44 (26.4)

0.8b 18.2b

a b

Values are given as number (percentage). P b 0.001 for the difference between the infection rates.

In the univariate analysis, thalassemia minor was a strong risk factor for HBV intrauterine infection (OR 15.619; 95% CI, 2.239–108.964). However, no relationship was observed between mild anemia and HBV intrauterine infection (P b 0.99). Previous studies have not reported the link between thalassemia minor and HBV intrauterine infection. Thalassemia is a hemoglobinopathy characterized by the impaired production of normal globin peptide chains. Sheiner et al. [20] collected clinical details from 261 pregnant women with β-thalassemia minor and found that thalassemia minor was associated with intrauterine growth restriction and oligohydramnios. This work indicates that the hypoxemic state caused by thalassemia minor might contribute to hypoxia of the placenta, and might ultimately cause intrauterine growth restriction and oligohydramnios. Roozbeh et al. [21] showed that minor β-thalassemia related to anemia and hypoxemia during pregnancy might lead to damage of the placenta, which could in turn result in pre-eclampsia. Accordingly, we hypothesize that the hypoxemia caused by impaired production of normal globin peptide chains and hemolysis in thalassemia minor is more serious than the hypoxemia caused by mild anemia. Therefore, the pathologic changes (e.g. edema) of the placenta caused by the hypoxemia in thalassemia might contribute to the placental leakage of HBV-infected blood that infects the fetus. It is also possible that patients with thalassemia have certain genes that increase their susceptibility of HBV intrauterine infection. However, the present results might be attributable to a sampling error. For this reason, further extensive sampling and in-depth studies are needed to confirm the present findings. Another issue of common concern is the influence of the mode of delivery on intrauterine infection. Some studies [18,22] indicate that cesarean delivery could limit direct contact of the fetus with infected secretions or blood in the maternal genital tract and may thus be protective of HBV intrauterine transmission. However, a systematic review [22] pointed out that although elective cesarean delivery appears to be effective in preventing the mother-to-child transmission of HBV, this finding must be interpreted with great caution because of a high risk of bias in each included study (grade C evidence). In the present study, we found no relationship between the mode of delivery and HBV intrauterine infection (P = 0.060). This is consistent with the findings of Wang et al. [23]. These results indicate that cesarean delivery is not a more advantageous mode of delivery for HBsAg-positive pregnant women, and that the mode of delivery should be chosen based on the actual obstetric condition of the pregnant woman. Additionally, the present study found that maternal age, gravidity, parity, pregnancy duration, number of abortions, umbilical cord length, placental volume, maternal alanine aminotransferase, Han ethnicity, premature rupture of membranes, threatened premature labor, threatened abortion, preterm birth, placenta previa, hyperemesis gravidarum, polyhydramnios, oligohydramnios, meconium-stained amniotic fluid, cord entanglement, hysteromyoma, gestational diabetes mellitus, placental calcification, fetal distress, breech presentation, history of fetal growth restriction, and increase of α-fetoprotein level were not associated with HBV intrauterine infection, which is consistent with previous studies [18,24,25]. In conclusion, the present results show that HBV DNA positivity (especially for mothers with serum levels of 105 copies/mL or more) and HBeAg positivity are risk factors for HBV intrauterine transmission. Furthermore, thalassemia minor in pregnant women might increase the risk of HBV intrauterine transmission. Further studies are warranted to determine whether active–passive HBV immunization should be administered in cases of maternal thalassemia.

Acknowledgments The present work was supported by a grant from the Ministry of Education of China (20070559006) and the National Natural Science Foundation of China (C30973214).

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Conflict of interest The authors have no conflicts of interest.

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