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. 2000 Aug;74(15):6808-20.
doi: 10.1128/jvi.74.15.6808-6820.2000.

Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis

S Fisher  1 , O Genbacev, E Maidji, L Pereira
Affiliations

Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis

S Fisher et al. J Virol. 2000 Aug.

Abstract

Human cytomegalovirus (CMV) is the leading cause of prenatal viral infection. Affected infants may suffer intrauterine growth retardation and serious neurologic impairment. Analysis of spontaneously aborted conceptuses shows that CMV infects the placenta before the embryo or fetus. In the human hemochorial placenta, maternal blood directly contacts syncytiotrophoblasts that cover chorionic villi and cytotrophoblasts that invade uterine vessels, suggesting possible routes for CMV transmission. To test this hypothesis, we exposed first-trimester chorionic villi and isolated cytotrophoblasts to CMV in vitro. In chorionic villi, syncytiotrophoblasts did not become infected, although clusters of underlying cytotrophoblasts expressed viral proteins. In chorionic villi that were infected with CMV in utero, syncytiotrophoblasts were often spared, whereas cytotrophoblasts and other cells of the villous core expressed viral proteins. Isolated cytotrophoblasts were also permissive for CMV replication in vitro; significantly, infection subsequently impaired the cytotrophoblasts' ability to differentiate and invade. These results suggest two possible routes of CMV transmission to the fetus: (i) across syncytiotrophoblasts with subsequent infection of the underlying cytotrophoblasts and (ii) via invasive cytotrophoblasts within the uterine wall. Furthermore, the observation that CMV infection impairs critical aspects of cytotrophoblast function offers testable hypotheses for explaining the deleterious effects of this virus on pregnancy outcome.

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Figures

FIG. 1
FIG. 1
(A) Diagram of a longitudinal section that includes a floating and an anchoring chorionic villus at the fetal-maternal interface near the end of the first trimester of human pregnancy (10 weeks of gestational age) (modified from references and 66). The anchoring villus (AV) functions as a bridge between the fetal and maternal compartments, whereas the floating villus (FV), containing macrophages (MØ, Hofbauer cells) and fetal blood vessels, is bathed by maternal blood. Cytotrophoblasts in AV (zone I) form cell columns that attach to the uterine wall (zones II and III). Cytotrophoblasts then invade the uterine interstitium (decidua and first third of the myometrium; zone IV) and maternal vasculature (zone V), thereby anchoring the fetus to the mother and accessing the maternal circulation. Zone designations mark areas in which cytotrophoblasts have distinct patterns of stage-specific antigen expression, including integrins and HLA-G. Decidual granular leukocytes (DGLs) and macrophages (MØ) in maternal blood and fetal capillaries in villous cores are indicated in panels A and B. Areas proposed as sites of natural CMV transmission to the placenta in utero are numbered 1, 2, and 3. (B) Diagram of a uterine (spiral) artery in which endovascular invasion is in progress (10 to 20 weeks of gestation). Endometrial and then myometrial segments of spiral arteries are modified progressively. In fully modified regions (a), the vessel diameter is large. Cytotrophoblasts (CTBs) are present in the lumen and occupy the entire surface of the vessel wall. A discrete muscular layer (tunica media) is not evident. (b) Partially modified vessel segments. Cytotrophoblasts and maternal endothelium occupy discrete regions of the vessel wall. In areas of intersection, cytotrophoblasts appear to lie deep in the endothelium and in contact with the vessel wall. (c) Unmodified vessel segments in the myometrium. Vessel segments in the superficial third of the myometrium will become modified when endovascular invasion reaches its fullest extent (about midgestation), while deeper segments of the same artery will retain their normal structure.
FIG. 2
FIG. 2
Culture models for studying CMV infection of anchoring villus explants and differentiating cytotrophoblasts (CTBs). (A) Diagram of an anchoring villus explant attached to a Matrigel substrate via cytotrophoblasts that migrate from the cell columns. (B) Diagram of purified cytotrophoblasts cultured on Matrigel. The cytotrophoblast stem cells aggregate, invade the matrix, and express stage-specific molecules, including integrins and HLA-G. Cultured cytotrophoblasts mimic the differentiation phenotype and morphology of cell columns formed in placentas in utero. For infection, CMV is added to the medium bathing the explants and cytotrophoblasts.
FIG. 3
FIG. 3
Cytotrophoblasts (CTBs) in villous explants were infected with CMV in vitro. Tissue sections prepared from both floating villi (FV; A and B) and anchoring villi (AV; C and D) were analyzed by double staining with anticytokeratin and anti-CMV IE1/2. (A) Cytokeratin (CK) staining of floating villi showed the multinucleate syncytiotrophoblasts (ST; arrowheads) that cover the villous surface and the underlying villous cytotrophoblast stem cells (CTBv; arrows). (B) CMV IE1/2 proteins were expressed by underlying clusters of infected villous cytotrophoblast stem cells. The inner stromal villous cores (VC) were consistently negative for anti-IE1/2 antibody staining. (C and D) IE1/2 protein expression was also detected in cytotrophoblasts found in the cell columns (CC) of anchoring villi. Insets show infected cytotrophoblasts at higher magnification. As a control, staining was performed as described for the experimental situation except that the primary or secondary antibody was omitted. No staining was detected (data not shown).
FIG. 4
FIG. 4
Cytotrophoblasts (CTBs) and other cells show evidence of natural infection of chorionic villi with CMV in utero. Both floating villi (FV; A to C) and anchoring villi (AV; D) were studied. Tissues were analyzed by using immunolocalization techniques for expression of (A) cytokeratin (CK) and (B to D) CMV IE1/2 proteins. (B) In some cases, clusters of CMV-infected villous cytotrophoblast stem cells (CTBv; arrows) underlying the syncytium (ST, arrowheads) were the only sites of antibody reactivity. More often, numerous cells throughout the villi stained with anti-IE1/2 antibody. (C) In floating villi, nuclei of syncytiotrophoblasts, villous cytotrophoblasts, and stromal components expressed IE1/2 proteins. (D) The same pattern of immunoreactivity was seen in infected anchoring villi. Additionally, cytotrophoblasts in cell columns (CC) stained brightly. As a control, staining was performed as described for the experimental situation except that the primary or secondary antibody was omitted. No staining was detected (data not shown).
FIG. 5
FIG. 5
Purified cytotrophoblasts (CTBs) could be infected with CMV as they differentiated along the invasive pathway in vitro. At 72 h, the cells were stained for expression of (A) cytokeratin (CK) and (B) gB (the major structural glycoprotein in the virion envelope) and IE1/2 proteins. Anti-IE1/2 antibody reacted with the nuclei, and anti-gB antibody showed diffuse cytoplasmic staining. At 96 h, the cells were stained for expression of (C) cytokeratin and (D) gB, which was detected in granules in the cytoplasm.
FIG. 6
FIG. 6
Purified cytotrophoblasts (CTBs) were fully permissive for CMV infection in vitro. Quantitation and comparison of CMV progeny virions produced in cell extracts (intracellular virus) and culture medium (released virions) of purified cytotrophoblasts and human foreskin fibroblasts (HFF) infected in vitro. TCID50, 50% tissue culture infective dose.
FIG. 7
FIG. 7
CMV infection in vitro eventually downregulates cytotrophoblast (CTB) expression of integrin α1. Purified cytotrophoblasts were infected with CMV in vitro as described in Materials and Methods. At 72 h after infection, the cells were fixed and stained for expression of gB and integrin α1. Cytotrophoblasts that did not express gB (A) displayed prominent staining for integrin α1 in a plasma membrane-associated pattern (B). Likewise, cells that stained in a diffuse cytoplasmic pattern for gB (C) also reacted with the anti-integrin antibody (D, cell marked with *). However, when gB was localized in a vesicular pattern, integrin staining was not detected (D). The intense intracellular staining was bleedthrough from anti-gB signal.
FIG. 8
FIG. 8
CMV infection impairs cytotrophoblast invasion in vitro. (Upper panel) Diagram of the assay that assesses the ability of cells to penetrate the Matrigel substrate, migrate through pores in the underlying filter, and emerge on the underside. Purified cytotrophoblasts (CTB) were cultured on Matrigel-coated filters and infected with CMV 12 h later. Staining of the upper surface of the filter (I) for (A) cytokeratin (CK) and (B) IE1/2 proteins expression showed that ∼30% of the cells were infected 48 h after plating. Invasion was quantified by determining the number of cytokeratin-positive cell processes that penetrated the Matrigel and appeared in the pores (marked with arrowheads) that open on the underside of the filter (II). In control cultures (C) many processes were visible, whereas in CMV-infected samples (D) only the pores were visible, indicating a significant reduction in invasion.
FIG. 9
FIG. 9
CMV infection impairs cytotrophoblast expression of HLA-G in vitro. Purified cytotrophoblasts were isolated and infected with CMV as described in Materials and Methods. At 72 h after infection, the cells were stained for gB (A) and HLA-G (B) expression. Cells that did not express gB (black arrows) expressed HLA-G. In contrast, staining for gB (white arrows) was associated with a marked reduction in HLA-G expression.
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