The risk factors and mechanisms of hepatitis B virus intrauterine transmission

Outline

Elucidation of the mechanisms underlying the intrauterine transmission of hepatitis B virus (HBV) plays a key role in controlling the epidemic of viral hepatitis B in the world. Before 1990, although there were many reports about incidence rates of HBV intrauterine transmission, the related studies on its mechanisms still were very limited. Also the results were controversial not only in theory but also in practice. This chapter introduces our series of studies on the major risk factors of HBV intrauterine transmission, which includes the comprehensive analysis about the role of placental infection by using standard analytic and molecular epidemiological methods. The results showed that HBV intrauterine infection might be caused by two transmission modes, “hematogenous” and “transplacental cellular traffic”. We hope our studies offer a new model in combining macroscopic and microcosmic approaches together to investigate the occurrence, spreading and epidemic of a disease in human population.

Hepatitis B (HB) is still a major epidemic worldwide and threatens people’ s life and health tremendously. China is a prevalent area of HBV. The positive rate of HBsAg in normal population is up to 10 percent and over 120 million people are the carrier of HBV in China. Most of them have been caused by mother to infant transmission. It was presumed that HBV could be transmitted from pregnant woman to her fetus through the placenta, when HBV was first discovery [1]. However, presence of HBV intrauterine infection was not confirmed till mid-80s, when HBV DNA and HBsAg were detected in liver cells of aborted fetus from HBV positive pregnant women, by using immunophathologic and molecular biologic technology,

Intrauterine transmission refers to the transmission mode that HBV infects fetus through placenta. This infection is defined as intrauterine infection. HBV intrauterine transmission was not completely cut off by hepatitis B vaccine and hepatitis B immunoglubin. So the neonates suffering HBV infection by intrauterine transmission would become the chronic virus carriers throughout their life, and 25 to 50 percent of them would develop into liver cirrhosis or liver cancer. If female is infected, she could transmit HBV to her children by the same ways. In this cycle, intrauterine transmission will cause severe consequence. There are about 100 to 200 thousand neonates who develop into HBV carrier status by intrauterine infection every year in China. World Health Organization (WHO) has proclaimed to “Control the global epidemic of hepatitis B by 2010”. Therefore, understanding the mechanisms underlying HBV intrauterine transmission is one of the key requirements to control the epidemic of hepatitis B.

However, until the beginning of 1990′ s, the reports on the mechanism of HBV intrauterine transmission were still limited
in the world, although there were many studies about the incidence rate of HBV intrauterine transmission, including more than 20 reports in China. There were some reasons for that. First, it was still short history since population received regular inoculation of hepatitis B vaccine, and perinatal stage transmission and horizontal transmission were more predominant than intrauterine transmission with little attention to the prevention against intrauterine transmission. Secondly, it was difficult to facilitate researches about mechanism of HBV intrauterine transmission because of the involvement of fetus and/or neonates and/or placenta. In addition, advanced techniques, strict condition, long term follow-up visit, and large sample size etc are also required. Our understanding of mechanism of HBV intrauterine transmission, thus, has been a long and intricate process, which can be divided into three stages. At first, descriptive epidemiological studies demonstrated that the threatened abortion and threatened premature labor induced angiorrhexis of placenta capillary vessel could cause HBV intrauterine transmission. Then, HBV markers on the placenta were detected. It was considered that HBV intrauterine transmission was related to HBV replication in the placenta, instead of threatened abortion. At last, based on the combination of field research and laboratory technologies, molecular epidemiological studies were performed. The main risk factors in HBV intrauterine transmission and the distribution of HBV markers in the placenta and its localization pattern were elucidated. The results suggested that there existed two pathways for HBV intrauterine transmission, the hematogenous transfer and the celluar transfer.

1. Descriptive Study

Initially, the mechanism of HBV intrauterine transmission and its influencing factors were studied by some specialists from field of gynaecology and obstetrics, blood transfusion and pediatrics in Japan and Taiwan. In this cooperative study [2˜4], 22 pregnant women from Japan and 10 pregnant women from Taiwan whose HBsAg and HBeAg were both positive, and their neonates, were recruited. 32 neonates’ cord blood was detected for HBsAg by reverse passive hemagglutination (RPHA). If HBsAg were negative, the neonate would receive the anti-hepatis B immunoglobulin (HBIG) at once. In addition, HBeAg and anti-HBc IgM were detected by enzyme linked immunosorbent assay, HBV DNA by dot blot hybridization, and HBsAg in the placenta by peroxydase-antiperoxidase (PAP). As for the risk factors, the abnormal liver function, history of threatened abortion and/or threatened premature labor of the mothers were mainly taken into consideration.

The “intrauterine transmission/infestation” was defined as “the neonates are positive for HBsAg immediately after they were born or within one month after birth even receiving HBIG injection”.

The investigators conducted the studies to address the following three issues:

1) The relationship between HBV intrauterine transmission/infestation and threatened abortion and/or symptom of threatened premature labor that could cause leakage of the placenta.

The results indicated that HBV intrauterine transmission/infestation occurred in 5 neonates, of which, 3 neonates were positive for HBsAg immediately after they were born, the others became positive for HBsAg within 1 month after birth. 5 of
32 pregnant women had been exposed to threatened abortion and/or symptom of threatened premature labor. Of these 5 cases, 3 neonates (3/5) suffered intrauterine infection. While of the other 27 cases, only 2 neonates (2/27) suffered intrauterine infection (Table 15.1 ). The difference was statistically significant (P<0.05).

Table 15.1 The relationship between HBV intrauterine transmission/infestation and threatened abortion and/or symptom of threatened premature labor

HBV intrauterine transmission/infestation (n)	Threatened abortion and/or symptom of threatened premature labor^a(n)
	+	–
+ (5)	3	2
– (27)	2^b	25
^aThere is significant difference between the two groups (P<0.05). ^bThe two pregnant women gave birth at 30 and 31 gestational weeks respectively, less than one week after threatened premature labor (leakage of placenta)
(Date from Ohto, 1987)

The 5 parturients experiencing threatened abortion and/or threatened premature labor were divided into 2 types. ① In 3 cases, symptom of threatened premature labor (which could cause leakage of placenta) appeared 7 weeks before child delivery. They continued gestation after treatment. All of their neonates suffered HBV intrauterine infection. ② In the other 2 cases, the symptom of threatened premature labor appeared only one week before child delivery. They gave birth immediately after inefficient treatment (Table 15.1 ). Both of their neonates didn’t suffer HBV intrauterine infection. The anti-hepatitis B immunoglobulin (HBIG) administration after birth may prevent HBV infection.

Thus, the investigators presumed that there was close correlation between HBV intrauterine transmission and threatened abortion and/or symptom of threatened premature labor that could cause leakage of the placenta.

2) The relationship between HBV intrauterine transmission/infestation and HBsAg expression in the placenta tissues

The investigators tested the placenta tissues in 5 cases of HBV intrauterine transmission by using PAP. HBsAg was negative in all 5 cases. The investigators concluded that there wasn’t any evidence to indicate HBV replication in placenta tissues. Together with reviewing literatures, they assumed that trophoblastic cells in chorionic villus couldn’t bind HBV because they were lack of polyadenylic acid albumin receptor.

3) The relationship of HBV intrauterine transmission/ infestation with other HBV markers in the blood and hepatic function

The researchers compared the titre of HBeAg, anti-HBc IgM, HBV DNA, quantitation of IgM, IgA, IgD in the maternal blood and cord blood and hepatic function between the intrauterine infection group and non-intrauterine infection group. The results indicated that there was no significant difference between the two groups. As shown in Table 15.2, the titre of HBeAg in both maternal blood and cord blood were higher in intrauterine infection cases than in non-intrauterine infection cases, but the difference did not reach the significant level. Therefore, researchers considered that HBV intrauterine transmission had little relationship with the titre of HBeAg in the maternal blood and/or cord blood, and anti-HBc IgM, HBV DNA, quantitation of IgM, IgA, IgD, and hepatic function.

Collectively, it was concluded that HBV intrauterine transmission was correlated with threatened abortion and/or symptom of threatened premature labor that could cause leakage of the placenta, but wasn’ t correlated with HBV replication in
placenta, the titre of HBeAg in maternal blood and the level of HBV DNA.

Table 15.2 The titre of HBeAg in the maternal blood and cord blood

The titre of HBeAg	Intrauterine infection (n=5)	Non-intrauterine infection (n=27)
maternal blood	2.000˜8.000 (average 4.000)	1.0˜20.000 (average 2.000)	NS^a
cord blood	10˜200 (average 40)	1˜400 (average 20)	NS
maternal blood/cord blood (%)	0.5˜25 (average 2)	0.02˜80 (average 8)	NS
NS^a: no significance (Date from Ohto, 1987, revised slightly)

2. Laboratory detection of blood and placenta tissue samples

2.1 The researches in China

Tang’s group [5] repeated Ohto’s and Lin’ s investigation. But to come as a surprise, they had a completely opposite result to Ohto’s.

They collected the serum samples from pregnant women who were positive or negative for HBsAg as well as fetus samples by induction delivery, and did the research from the following three aspects.

1) The relationship between fetus infestation and the status of HBV infection in pregnant women

The subjects were classified into three groups according to the status of HBV markers: 11 cases in Group A, who were positive for HBeAg and/or HBV DNA; 24 cases in Group B, who were negative for both HBeAg and HBV DNA; 10 cases in Group C, who were positive for anti-HBe (Table 15.3 ). The results showed that the fetus intrauterine infection (the fetal heart blood was positive for both HBsAg and anti-HBc IgM) rates were 54.5% (6/11), 4.2% (1/24) and 10.0% (1/10) in group A, B and C respectively (Table 15.3 ). And the positive rate of HBV DNA in fetal hepatic tissue was significantly higher in group A than in group B and C (P<0.005). So, Tang’ s group considered that the fetus born from both HBeAg and HBV DNA positive pregnant women were susceptible to HBV intrauterine infection.

Table 15.3 The relationship between the intrauterine infection rate and the HBV infection status of pregnant women

Group	Number	Positive number in the fetal heart blood			Infection rate (%)
Group	Number	HBsAg	Anti-HBc IgM	HBsAg/anti-HBc IgM	Infection rate (%)
A (positive for HBeAg and/or HBV DNA)	11	2	3	1	54.5
B (negative for HBeAg and HBV DNA)	24	0	0	1	4.2
C (positive for Anti-HBe)	10	0	1	0	10.0

2) HBV markers in the placenta tissues

The fetal tissues were detected by using the dot spot hybridization. The results revealed that the fetal hepatic tissues induced from 5 HBsAg-positive pregnant women were positive for HBV DNA, among which, the placental tissues from 2 cases were also positive for HBV DNA.

3) Leakage of the placenta

Tang’s group reported that both HBeAg and HBV DNA were negative in heart blood of fetus induction delivered by 11 HBeAg-positive pregnant women and 9
HBV DNA positive pregnant women, who had no history of threatened abortion and had integral placentas after parturition. So they concluded that the infiltration of maternal blood to fetal side was not common during gestation period.

Based on those observations, it was suggested that the placenta barrier rupture that induced maternal blood infiltration to fetal side was not correlated to HBV intrauterine infection, but being HBeAg positive, especially being maternal HBV DNA positive, were significantly correlated to HBV intrauterine infection. The positive HBV DNA in placenta tissue suggested that HBV could infect fetus through placental barrier, or HBV could firstly infect placenta tissues and duplicate in placenta tissues, and then, infect fetus [5].

2.2 The researches abroad

Dr. Lucifora, one Italian specialist in gynaecology and obstetrics, published 2 papers about this [6,7]. The group carried out their researches based on the following two assumptions. ① The HBV infection in mother might cause harm to her fetus, such as intrauterine death, premature delivery and neonatal hepatitis. ② It had been generally considered that HBV infection from mother to fetus occurred on the time of delivery and that placenta tissues played key roles in the HBV intrauterine transmission. Lucifora’s group intended to test their own hypothesis that neonatal hepatitis was likely to occur at 5 month after pregnancy, because that placenta consisted of only two layers at this time. It was quite easier for the virus to transmit through this relatively thin placental barrier in spite of low infection rate.

They collected 12 placenta samples from puerperants who carried HBsAg for long-term, and detected HBsAg and HBcAg expressions with immunohistochemical peroxidase-antiperoxidase (PAP) method.

1) HBsAg expression in the placenta tissues

HBsAg located mainly in villus blood vessels, specifically speaking, cytoplasma of vascular endothelial cells. The cytoplasma of Hofbauer cells, very close to the trophoblastic layer, also displayed strong positive reaction. But only a few trophoblastic cells showed weak positive reaction. Only scattered fibroblast cells had positive reaction. Amnion cells were seldom positive for HBsAg.

So it was concluded that HBsAg expression level was high in fetal side of placenta; decidual cells were slightly positive for HBsAg; the maternal blood vessels were absolutely negative for HBsAg.

2) HBcAg expression in the placenta tissues

HBcAg was positive in all 9 placenta samples from puerperants who carried HBsAg for long-term. In chorionic villi tissues, all fetal vascular endothelial cells, fibroblast cells, Hofbauer cells and trophoblastic cells had strong positive reaction. In deciduous membrane tissues, all infiltrating trophoblastic cells, polykaryocytes, megacells and minicells had strong positive reaction too. The subcellular localization patterns of HBcAg in macro-decidual cells included nucleus staining (most predominant), margin staining, and cytoplasm homogenous staining. HBcAg was not detected in maternal vascular endothelial cells.

Therefore, Lucifora’s group considered that HBsAg and HBcAg were positive in the placenta tissues of pregnant women who carried HBsAg asymptomatically for long-term; HBsAg located in cytoplasm, and
HBcAg in various kinds of trophoblastic cells. It was indicated that HBV could infect fetus through placental blood circulation.

3. Molecular epidemiological study

These scholars, including Ohto, Lin, Tang, Lucifora, et al, did valuable researches about mechanism of HBV intrauterine transmission. They obtained enlightening and significant findings. But their conclusions were different, even contradictory to each other, which might be due to the investigation methods and the sample size.

The above-mentioned scholars, except one, were all clinical doctors, whose study methods all belonged to descriptive study category. However, the most advanced and credible method to investigate the causative agents and relative factors of one disease was analytic epidemiological study. HBV intrauterine infection, with low incidence rate and many influencing factors, is a very complicated event. The case-control study should be the most appropriate design for it. This point has been verified through practice worldwide.

Only gold members can continue reading. Log In or Register to continue