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Biology of Reproduction 67, 1572-1579 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Syncytiotrophoblast Is a Barrier to Maternal-Fetal Transmission of Herpes Simplex Virus1

Hideki Koia, Jian Zhanga, Antonis Makrigiannakisa, Spiro Getsiosb, Colin D. MacCalmanb, Jerome F. Strauss, IIIa, and Samuel Parry2,a

a Center for Research on Reproduction and Women's Health, University of Pennsylvania, Philadelphia, Pennsylvania 19104 b Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Herpes simplex virus (HSV)-1 has been discovered in placental tissue from spontaneous miscarriages, but reports of transplacental transmission and fetal infection are extremely rare. Previously, we demonstrated that the villous syncytiotrophoblast, which forms a continuous layer between the maternal and fetal circulation, is resistant to HSV entry. Here, we tested our hypothesis that the villous syncytiotrophoblast prevents transplacental transmission of HSV secondary to decreased expression of HSV entry mediators (HveA, HveB, and HveC). In addition, we investigated the ability of HSV to infect extravillous trophoblast cells, which mediate placental attachment to the uterine wall, and the expression of HSV receptors in these cells. We performed fluorescence-activated cell sorting (FACS) analyses and immunostaining to demonstrate that HveA, HveB, and HveC were not expressed in third-trimester villous trophoblast cells. Consequently, villous explants obtained from third-trimester placentas were resistant to infection by a recombinant HSV-1 vector, HSV-1 KOS, but approximately 20% of mesenchymal cells within the villous core were infected when villous explants were pretreated with trypsin to disrupt the villous trophoblast layer. Conversely, FACS analysis and immunostaining demonstrated that extravillous trophoblast cells expressed HveA, HveB, and HveC, and these cells were efficiently infected by HSV vectors. Infection of extravillous trophoblast cells by HSV-1 was not reduced when the cells were pretreated with an antibody against HveA but was partially reduced when the cells were pretreated with antibodies directed against HveB and HveC. Thus, the decreased expression of herpesvirus entry mediators in villous syncytiotrophoblast prevents placental villous infection, thereby limiting maternal-fetal transmission of HSV.

placenta, pregnancy, syncytiotrophoblast, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the United States, approximately 1500–2000 cases of neonatal infection with herpes simplex virus (HSV) occur each year, and the risk of death or serious sequelae is at least 50% [1]. Most cases are acquired perinatally from an infected maternal lower genital tract [2, 3]. Transplacental transmission resulting in congenital HSV occurs much less frequently, although congenital infection during the first two trimesters has been associated in isolated reports with fetal anomalies, including microcephaly, intracranial calcifications, and chorioretinitis [4–6].

The infrequency with which congenital HSV-1 and -2 infection occurs may be attributed to several factors, including 1) the prevalence in reproductive-aged women of protective antibodies against HSV, 2) the fact that viremia is not a cardinal feature of HSV infections, and 3) the possibility that the placenta functions as a physical barrier preventing vertical transmission of HSV from the maternal to the fetal circulation. However, primary maternal infections with HSV-1 and -2 are common during pregnancy and are associated with an increased risk of miscarriage, presumably as a result of placental dysfunction [2, 3, 7, 8]. Involvement of visceral organs may complicate primary HSV-1 or -2 infections [9, 10]. Taken together, these observations suggest that the placenta is exposed to HSV-1 and -2 during primary infection in some women, but that the placenta generally prevents vertical transmission of the virus. Unfortunately, the mechanisms by which the placenta resists or permits transmission of HSV and other viruses from the maternal to the fetal circulation are an underdeveloped area of research.

Development of the placenta is largely dependent on the differentiation of trophoblast cells along two pathways [11, 12]. In one pathway, a subset of undifferentiated cytotrophoblast cells in anchoring placental villi invades maternal tissues and blood vessels within the decidua and myometrium. These intermediate, or extravillous, trophoblast cells engage in a dialogue with maternal cells and probably mediate suppression of the maternal immune system against the semiallogeneic conceptus [13, 14]. The inflammatory response to HSV infection at the maternal-fetal interface has been postulated to abrogate normal placentation and to predispose both the mother and fetus to adverse reproductive outcomes, including miscarriage, fetal growth restriction, and preeclampsia [8, 13, 15]. In the other pathway of placental development, mononucleated cytotrophoblast cells within the placental villi terminally differentiate into the multinucleated syncytiotrophoblast. As pregnancy progresses, cytotrophoblast cells become more sparse within the placental villi, and the syncytiotrophoblast forms the only continuous layer separating the maternal intervillous space and the fetal capillary endothelium. Importantly, superficial breaks in this barrier do not appear to be significant factors in the vertical transmission of viruses [16].

We previously reported that the villous syncytiotrophoblast cannot be transduced by HSV-1 vectors and is resistant to entry by HSV-1 [17]. Consequently, we hypothesized that the syncytiotrophoblast layer prevents transplacental transmission of HSV secondary to decreased expression of HSV receptors.

Attachment of HSV-1 and -2 to target cells is mediated by heparan sulfate, and entry is regulated by three recently identified herpesvirus entry mediators (HveA, HveB, and HveC) [18–20]. Binding of HSV to heparan sulfate occurs primarily through an interaction with virion glycoprotein C, whereas entry is mediated by interactions between virion glycoprotein D and the cellular entry mediators [21, 22]. Heparan sulfate is a ubiquitous glycoprotein on cell surfaces, and enzymatic removal of heparan sulfate from cells reduces the level of HSV attachment and infection by approximately 85%. However, in no case has removal of heparan sulfate resulted in complete loss of viral attachment or infectivity.

The first identified mediator of HSV entry, HveA (originally designated HVEM, for herpesvirus entry mediator), is a member of the tumor necrosis factor/nerve growth factor-receptor family [19]. It is the principal receptor for entry of HSV-1 and -2 into human lymphoid cells, but not into other cell types [19]. The poliovirus receptor-related protein 2 has been designated HveB (or nectin-2) [20]. It has no poliovirus-receptor activity, but it mediates the entry of HSV-2 and mutant strains of HSV-1 that cannot use HveA as a coreceptor [20]. A member of the immunoglobulin superfamily, HveC (or nectin-1) is also a poliovirus receptor-related protein [21]. It is expressed in human cells of epithelial and neuronal origin, and it appears to be the primary receptor allowing infection of these tissues with HSV-1 and -2 [21, 23, 24].

Here, we describe expression of the herpesvirus entry mediators within placental cells and present evidence that the syncytiotrophoblast, which does not express the entry mediators, limits vertical transmission of virus from the maternal to the fetal circulation. In addition, we report that HSV-1 infects extravillous trophoblast cells, which express all three entry mediators. Infection of these cells may prevent normal placental attachment to the uterine wall, predisposing women to miscarriage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Trophoblast Cells

Extravillous trophoblast cells were isolated from first-trimester villous tissue using a modification of the method originally described by Graham et al. [25, 26]. Briefly, finely minced chorionic villi were cultured at 37°C in Dulbecco modified Eagle medium (DMEM; Gibco BRL, Grand Island, NY) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS). Extravillous trophoblast cells, which outgrew from attached villous fragments, were separated from villous trophoblast cells on the 10th to 12th day of culture. The isolated extravillous trophoblast cells were then cultured and propagated in DMEM containing 10% FBS.

The extravillous trophoblast cells used in these experiments were characterized by immunostaining for cytokeratin filaments 8 and 18 and human leukocyte antigen (HLA)-G, which are trophoblast cell markers, and for HLA-A, -B, and -C, which are not expressed by extravillous trophoblast cells [27]. Monoclonal antibodies against cytokeratin 8 and 18 and HLA-A, -B, and -C are commercially available, whereas monoclonal antibody against HLA-G was generously provided by Joan Hunt (University of Kansas Medical Center, Kansas City, KS). More than 95% of isolated trophoblast cells stained positively for cytokeratin 8 and 18 and HLA-G, whereas fewer than 10% of the cells stained positive for classic HLA antigens (HLA-A, -B, and -C). We also performed dual-sorting fluorescence-activated cell sorting (FACS) analyses of HSV-infected extravillous trophoblast cells using primary antibodies against HLA-G (phycoerythrin) and HSV (fluorescein isothiocyanate, based on an HSV-KOS strain labeled with green fluorescent protein). The FACS analyses indicated that more than three-fourths of the cells infected by HSV in primary cultures expressed HLA-G (data not shown). These results are similar to those obtained by other investigators using the same primary cultures, and they confirm the relative purity of the extravillous trophoblast cell preparations [25, 26].

Primary cytotrophoblast cells were isolated from third-trimester human placentas using a modification of the protocol originally described by Kliman et al. [17, 28]. After isolation, the cells were cultured at 37°C in DMEM containing 10% heat-inactivated FBS. The cytotrophoblast cells spontaneously differentiated into syncytiotrophoblast within 48–72 h in these conditions [17, 29]. These cells were characterized by immunostaining for cytokeratin filaments 8 and 18, which are trophoblast cell markers, and for HLA-A, -B, and -C, which are not expressed by trophoblast cells. More than 95% of the cells expressed cytokeratin 8 and 18, whereas fewer than 5% expressed HLA-A, -B, and -C (data not shown). Syncytialization was confirmed by direct visualization before and after immunostaining with desmoplakin to delineate cell boundaries and by increased progesterone secretion into the cell culture medium (radioimmunoassay).

Transformed human choriocarcinoma (BeWo and JEG-3) cells were cultured at 37°C in DMEM supplemented with 10% FBS. Chinese hamster ovary (CHO-K1) cells, which served as negative controls for viral receptor expression, were cultured in Ham F12 medium containing 10% FBS. The CHO-K1 cells were transfected with HveA cDNA (CHO-HveA cells) inserted in a pBEC10 expression vector that was complexed with a nonliposomal lipid reagent (FuGENE 6; Roche Molecular Biochemicals, Indianapolis, IN). The plasmid was generously provided by Patricia G. Spear (Northwestern University, Chicago, IL). The CHO-HveA cells served as a positive control for HveA expression. The CHO-K1 cells were similarly transfected with plasmids containing HveB and HveC cDNA and used as positive controls for HveB and HveC expression, respectively.

The use of human tissues was approved for this research by the Institutional Review Board at the University of Pennsylvania.

Viral Strains

We used recombinant HSV-1(KOS)tk12 and HSV-1(RID1)tk12 viral strains that contained the ß-galactosidase reporter gene in place of the HSV thymidine kinase gene. The mutant virus HSV-1(KOS)tk12 cannot use HveB and HSV-1(RID1)tk12 cannot use HveA as a coreceptor for viral entry [20]. The construction of these replication-deficient viruses is described elsewhere [20]. Replication-deficient HSV-2 strains have not been developed, but entry mechanisms into target cells are similar for most strains of HSV-1 and -2 [30].

Antibodies Against Viral Receptors

Rabbit polyclonal antibodies against HveA (6.4 mg immunoglobulin/ml) and HveB (3.4 mg immunoglobulin/ml) and murine monoclonal antibody against HveC (2.9 mg immunoglobulin/ml) were generously provided by Gary H. Cohen and Roselyn J. Eisenberg (University of Pennsylvania Schools of Dental Medicine and Veterinary Medicine) [20, 21]. For flow cytometry, we used 50 µl of each primary antibody diluted 1:100 (v/v) in PBS. For immunostaining of placental sections, we diluted polyclonal antibodies against HveA and HveB 1:500 in PBS, and monoclonal antibodies against HveC were diluted 1:40.

FACS Analysis

Cells were harvested and treated with normal goat serum to block nonspecific binding, after which the cells were incubated with antibodies against HveA, HveB, or HveC for 30 min at 4°C. The cells were then incubated with fluorescein-conjugated goat antibody to rabbit or mouse immunoglobulin (heavy- and light-chain specific), and fluorescence intensity was detected by an EPICS XL flow cytometer (Coulter Corporation, Hialeah, FL).

Immunohistochemistry

Permanent paraffin-embedded sections from first- and third-trimester placentas were deparaffinized and incubated at room temperature for 30 min with normal goat serum to block nonspecific binding. The sections were then incubated at room temperature for 30 min with primary antibodies against HveA, HveB, or HveC. Immunoreactivity was demonstrated using avidin and biotinylated horse radish peroxidase complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Reaction product was developed with diaminobenzidine tetrahydrochloride, and sections were counterstained with methyl green solution and examined by light microscopy.

Infection of Trophoblast Cells

Primary trophoblast cells and transformed cell lines were plated on 96-well plates (1 x 104 cells/well) and incubated overnight. Twenty-four hours later, the culture medium was changed and the cells exposed to HSV-1(KOS)tk12 or HSV-1(RID)tk12 at 20 pfu/cell for 90 min at 4°C and then overnight at 37°C. Infection efficiencies were quantitated by measuring ß-galactosidase activity, which was reported as absorbance at 420 nm ± SD [17].

Competition Experiments

Competition assays were performed using antibodies to block HveA, HveB, and HveC before infecting primary trophoblast cells with HSV-1(KOS)tk12 or HSV-1(RID)tk12. Cells (1 x 104 cells/well in 96-well plates) were incubated at room temperature for 30 min with 50 µl of rabbit antiserum containing polyclonal antibodies against HveA and HveB at serial dilutions of 1:20 to 1:160, or 320 to 21 µg/ml of medium. Monoclonal antibody against HveC (Mab CK41) was used at serial dilutions of 1:20 to 1:160, or 145 to 18 µg/ml of medium. After pretreatment with blocking antibodies, the cells were infected with HSV-1(KOS)tk12 or HSV-1(RID)tk12 at 20 pfu/cell, and ß-galactosidase activities were measured as described previously [17].

Infection of Placental Villous Explants

Villous tissues obtained from normal third-trimester placentas were rinsed thoroughly with saline, and small villous fragments (size, ~1 mm2) were dissected from floating villi. Some of the explants were digested with trypsin (Sigma) and deoxyribonuclease-1 (Sigma) in Hanks balanced salt solution at 37°C for 5–20 min to disrupt the syncytial layer. Two explants were plated per well on 96-well plates in 50 µl of DMEM containing 10% FBS and incubated at 37°C overnight with HSV-1(KOS)tk12 (3 x 106 pfu/well). After rinsing, the explants were fixed with 4% paraformaldehyde and stained with a solution containing X-gal, which is a ß-galactosidase substrate [17]. Infection efficiency was determined by counting the number of blue-stained cells in the explants under phase-contrast microscopy. The effects of enzymatic digestion on the villous syncytial layer were assessed by immunostaining the explants with anti-cytokeratin-18 antibody.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FACS Analysis Detected HSV-Receptor Proteins in Extravillous Trophoblast Cells

To determine the level of HveA, HveB, and HveC protein on extravillous and villous trophoblast cells, indirect immunofluorescence assays were performed (Fig. 1). All three receptors were detected in CHO-K1 cells transiently transfected with the corresponding entry mediator cDNA and on 79.4–100% of BeWo and JEG-3 cells (data not shown). Positive shifts were seen in binding of antibodies against HveA, HveB, and HveC to primary extravillous trophoblast cells, but all three receptors were found on the surface of fewer (11.9% for HveC to 97.3% for HveB) extravillous trophoblast cells than transfected CHO-K1 cells or transformed choriocarcinoma cells. The receptors were not detected in villous trophoblast.



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  		FIG. 1. Flow-cytometric analysis of herpesvirus entry mediator (HveA, HveB, and HveC) expression on trophoblast cells. Fluorescence intensity was measured in cells that were incubated with antibodies against the HSV entry mediators and fluorescein-conjugated secondary antibody (shaded curves) and in cells that were incubated with fluorescein-conjugated secondary antibody alone (open curves). The y-axis indicates cell number, and the x-axis indicates logarithm of fluorescence intensity. In the first row, CHO-K1 cells were transfected with HveA, HveB, or HveC cDNA before incubation with antibodies against the same receptor (positive controls). The second row demonstrates results from extravillous trophoblast cells (EVT), and the third row demonstrates results from villous trophoblast (VT)

Immunohistochemistry Demonstrated an Absence of HSV Receptors in Villous Trophoblast

Antibodies were used for immunostaining to detect HveA, HveB, and HveC antigens in sections of third-trimester placental villi (Fig. 2). We were unable to detect HveA, HveB, or HveC protein in villous trophoblast cells. However, mesenchymal cells within the placental villi expressed HveA, HveB, and HveC antigens. Negative and positive controls (CHO-K1 cells and transfected CHO-K1 cells, respectively) stained as expected (data not shown).



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  		FIG. 2. Immunostaining for herpesvirus entry mediators (HveA, HveB, and HveC) within placental villi. In third-trimester placentas, HveA, HveB, and HveC were not detected in villous trophoblast cells (arrowheads). However, HveA, HveB, and HveC protein was detected in mesenchymal cells within the villous core (long arrows)

In sections from first-trimester placentas, HveA, HveB, and HveC were detected in extravillous trophoblast cell columns, but the HSV entry mediators were not detected in villous trophoblast cells (Fig. 3). A negative control using secondary antibody alone (to exclude nonspecific binding) yielded results as expected (Fig. 3A).



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  		FIG. 3. Immunostaining for HSV entry mediators (HveA, HveB, and HveC) in histologic sections from first-trimester placentas. Negative control (A) used secondary antibody alone to exclude nonspecific binding. The HveA (B), HveB (C), and HveC (D) were not detected in villous trophoblast cells. However, HveA, HveB, and HveC protein was detected in extravillous trophoblast cell columns

Infection of Trophoblast Cells with Recombinant HSV Strains

Previously, we demonstrated that villous syncytiotrophoblast was resistant to infection by HSV because of failed viral entry [17]. Here, extravillous trophoblast cells and transformed choriocarcinoma cell lines were infected with HSV-1 KOS and HSV-1 RID, each containing the Escherichia coli lacZ reporter gene (Fig. 4). Virus entry was determined 24 h after viral infection by measuring ß-galactosidase activity as relative light units. The CHO-K1 cells were not infected by HSV-KOS and HSV-RID, but CHO-K1 cells that were transfected with HveA cDNA (CHO-K1/HveA cells) were efficiently infected by HSV-KOS. As expected, HSV-RID, which is a mutant form of HSV-KOS that cannot bind to HveA, did not infect CHO-K1/HveA cells. Both vectors efficiently infected extravillous trophoblast cells, BeWo cells, and JEG-3 cells. These findings demonstrate that extravillous trophoblast cells are susceptible to infection by HSV.



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  		FIG. 4. Infection of CHO-K1 cells, HveA-transfected CHO-K1 cells (CHO-HveA), extravillous trophoblast cells (EVT), BeWo cells, and JEG-3 cells with recombinant HSV vectors. Viral entry was quantitated by measuring average ß-galactosidase activities, which were recorded along the y-axis as relative light units compared to the infection efficiency of HveA/CHO-KI cells by HSV-1(KOS)tk12. White bars indicate infection with HSV-1(KOS)tk12; black bars indicate relative infection efficiency with HSV-1(RID)tk12

Competition Experiments Indicated That HSV Binds to HveB and HveC on Extravillous Trophoblast Cells

Antibodies that block HveA, HveB, and HveC receptors were used to pretreat extravillous trophoblast cells and CHO-K1 cells before infecting the cells with HSV-KOS or HSV-RID. Each competition experiment was performed in triplicate, and mean infection efficiencies were calculated.

Antibodies against HveA and HveC efficiently blocked the infection of CHO-K1/HveA and CHO-K1/HveC cells, respectively, by HSV-KOS, whereas antibodies against HveB blocked the infection of CHO-K1/HveB cells by HSV-RID (data not shown). However, pretreatment with antibodies against HveA did not block the infection of extravillous trophoblast cells by HSV-KOS or HSV-RID (Fig. 5, A and B). These results parallel the findings of previous investigators using the same antibodies and control cell types, and they indicate that the antibodies are functional and specific for their respective entry mediators [19–22]. In addition, these findings provide evidence that entry of HSV into extravillous trophoblast cells is independent of HveA.



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  		FIG. 5. Competition experiments demonstrate that pretreatment with antibodies directed against HveB and HveC partially inhibited herpesvirus infection of extravillous trophoblast cells. The cells were pretreated with serial dilutions of antibodies (1:20 to 1:160) and exposed to HSV-1(KOS)tk12 (black circles) or HSV-1(RID)tk12 (white circles). Herpesvirus entry was assessed by comparing ß-galactosidase activity in the pretreated cells with ß-galactosidase activity in the untreated cells. Baseline infection efficiencies were recorded as 1 in the untreated cells and as fractions of the baseline activity in the pretreated cells. A and B) Pretreatment with antibody directed against HveA did not inhibit herpesvirus infection of extravillous trophoblast cells. C and D) Pretreatment with antibodies against HveB efficiently reduced the infection efficiency of extravillous trophoblast cells by HSV-RID but not by HSV-KOS. E and F) Pretreatment with antibodies against HveC partially reduced infection of extravillous trophoblast cells

Pretreatment with antibodies against HveB efficiently blocked the infection of extravillous trophoblast cells by HSV-RID in a dose-response fashion, but it did not affect viral entry by HSV-KOS, which does not attach to HveB (Fig. 5, C and D). Meanwhile, pretreatment with antibodies against HveC partially blocked infection of extravillous trophoblast cells by both HSV strains (Fig. 5, E and F). Thus, the greater efficiency with which antibodies against HveB blocked infection compared to antibodies against HveC parallels the increased cell-surface expression of HveB (97.3% of extravillous trophoblast cells) compared to HveC (11.9%). However, these findings suggest that both HveB and HveC are functional receptors mediating entry of HSV-1 into extravillous trophoblast cells.

Disruption of the Villous Trophoblast Layer Permits Entry of HSV-1 into the Placental Villous Core

Immunostaining of placental villous explants with anti-cytokeratin-18 antibody demonstrated disruption of the villous trophoblast layer after treatment with trypsin (Fig. 6, A–C). Placental villous explants were not infected (zero to one blue-stained cells observed per explant) by HSV-KOS (Fig. 6D). However, pretreatment of the villous explants with trypsin/DNase for 5–20 min permitted entry of HSV-KOS into cells (presumably mesenchymal cells) within the villous core (0–65 blue-stained cells observed per explant). Each experiment was performed at least 10 times, and the median number of blue-stained cells is illustrated in Figure 6D.



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  		FIG. 6. Entry of herpesvirus into placental villous explants is restricted by the villous trophoblast layer. Immunostaining for cytokeratin 18 at 0 min (A), 5 min (B), and 20 min (C) after trypsinization demonstrates disruption of the trophoblast layer. The number of blue-stained cells in each explant (D) was recorded for controls, for explants at 5–20 min after trypsinization, and for explants treated with Hanks balanced salt solution (HBSS) for 20 min (negative controls). The number (mean ± SD) of blue-stained cells visualized in explants treated with trypsin or HBSS is demonstrated in the bar graph. The number of blue-stained cells was significantly greater in villi pretreated with trypsin for 5–20 min


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Vertical transmission of viruses across the placenta may result in congenital infection and profound neonatal sequelae. Fortunately, clinical observations suggest that the placenta functions as a relatively effective barrier against maternal-fetal transmission of viral pathogens. For example, in untreated mothers infected with the human immunodeficiency virus, the rate of vertical transmission to the fetus is only 25% [31]. The rate of maternal-fetal transmission of parvovirus B19, which causes severe fetal anemia, is estimated to be approximately 33% [32]. Reports of transplacental HSV-1 and -2 transmission are anecdotal in nature, despite the relatively high incidence of primary maternal infection during pregnancy [3, 33]. Here, we present evidence that the expression of HSV entry mediators in placental trophoblast cells is differentiation-dependent. Extravillous trophoblast cells express all three HSV entry mediators and are susceptible to infection by HSV-1, which may result in impaired placental invasion and miscarriage [8]. However, villous syncytiotrophoblast does not express HveA, HveB, or HveC. Consequently, villous trophoblast cells are resistant to infection by HSV-1 and may serve as a barrier that prevents vertical transmission of HSV.

The villous syncytiotrophoblast is in direct contact with maternal blood and forms a continuous layer between the maternal and fetal circulation. Importantly, lateral intercellular spaces do not exist within the syncytiotrophoblast layer. Therefore, potential maternal pathogens must traverse the villous syncytiotrophoblast before reaching the fetal circulation. Because the villous syncytiotrophoblast is relatively resistant to entry by several viruses, we proposed that the syncytiotrophoblast forms an effective barrier to viral pathogens [12, 17, 34]. The villous explant experiments presented here demonstrate that the trophoblast layer prevents HSV-1 from entering the villous core, which contains stromal tissue and the fetal villous capillary. Decreased expression of the HSV entry mediators and the functional importance of HveB and HveC in mediating the entry of HSV-1 into extravillous trophoblast cells suggest a mechanism by which the villous syncytiotrophoblast is resistant to HSV-1.

The relevance of viral infection to impaired placental invasion has not been determined in vivo, although we have postulated that viral infection of extravillous trophoblast cells may be one cause of shallow invasion, placental dysfunction, and adverse reproductive outcomes. In support of this hypothesis, Liu et al. [35] demonstrated a relationship between placental pathology, placental viral infection (including HSV-2), and fetal growth restriction. Schust et al. [8] proposed a mechanism by which HSV infection may cause early miscarriage [8]. They reported that infection of extravillous trophoblast cells by HSV-1 or -2 blocks the expression of cell surface HLA-G molecules that prevent immune rejection of the fetal semiallograft. Although we do not present evidence that HSV-1 infection causes placental dysfunction, we have demonstrated that among trophoblast cells, extravillous trophoblast cells are uniquely susceptible to HSV infection. These findings support the observations of Norskov-Lauritsen et al. [36], who showed that mononuclear trophoblast cells are susceptible to HSV-induced cytopathic effects.

In the present study, we demonstrated that extravillous trophoblast cells express HveA, HveB, and HveC and are susceptible to infection by HSV-1. Our competition experiments demonstrated that HveB and HveC are functional receptors that regulate HSV-1 entry into extravillous trophoblast cells, although incomplete blockage of infection indicates that other entry mechanisms may exist in trophoblast cells. It is interesting that HveB was expressed most abundantly on the surface of extravillous trophoblast cells, and that antibodies against HveB most efficiently blocked HSV-1 entry into these cells. These observations should stimulate additional studies using strains of HSV-2, which are reported to be more dependent than HSV-1 strains on HveB for viral entry [20].

We believe our findings demonstrate that extravillous trophoblast cells are susceptible to infection by HSV, but that the villous syncytiotrophoblast forms an effective barrier preventing transplacental transmission of HSV to the fetus. The differential expression of HveC and HveB is correlated with the susceptibility of trophoblast cells to infection by HSV. These observations suggest that a better understanding of placental biology will be critical for developing strategies to reduce the incidence of congenital viral infection. Additionally, the pathologic effects of placental viral infection and its potential impact on placental function need to be elucidated.


   FOOTNOTES
 
1 Supported by a Women's Reproductive Health Research Award from the National Institute of Child Health and Human Development (S.P.) and the Bill and Melinda Gates Foundation (J.F.S.). Back

2 Correspondence: Samuel Parry, Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, 1352 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6142. FAX: 215 573 5408; parry{at}mail.med.upenn.edu Back

Received: 11 February 2002.

First decision: 8 March 2002.

Accepted: 19 June 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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