Abstract

BACKGROUND

We sought to determine if human papillomavirus (HPV) infection of extravillous trophoblast cells reduces cell invasion and if placental infection is associated with adverse reproductive outcomes attributed to placental dysfunction.

METHODS

We conducted apoptosis and invasion assays using extravillous trophoblast (HTR-8/SVneo) cells that were transfected with a plasmid (pAT-HPV-16) containing the entire HPV-16 genome. In order to associate HPV infection with reproductive outcomes, we conducted a case–control study to detect HPV DNA in the extravillous trophoblast region of placentas from cases of spontaneous preterm delivery, severe pre-eclampsia requiring delivery at <37 weeks and controls who delivered at term.

RESULTS

Rates of apoptosis were 3- to 6-fold greater in transfected cells than in non-transfected cells or cells transfected with an empty plasmid. Invasion of transfected cells through extracellular matrices was 25–58% lower than that of the controls. HPV was detected more frequently in placentas from spontaneous preterm deliveries than in placentas from controls (P = 0.03). Identification of HPV in placentas from cases of pre-eclampsia was not significantly different to controls.

CONCLUSIONS

HPV infection of extravillous trophoblast induces cell death and may reduce placental invasion into the uterine wall. Thus, HPV infection may cause placental dysfunction and is associated with adverse pregnancy outcomes, including spontaneous preterm delivery.

Introduction

Human papillomavirus (HPV) is a small double stranded DNA virus. Currently, ≥100 different strains have been identified, and most of the papillomaviruses demonstrate tropism for epithelial cells (Huang et al., 2004). Consequently, the pathogenic role of HPV in epidermal and genital warts has been studied extensively, and HPV is considered to be the main cause of cervical cancer (Koutsky and Wolner-Hanssen, 1989; Unger and Duarte-Franco, 2001). However, infection of placental cells by HPV and the effects of placental infection have been studied in less detail.

Two groups of investigators reported detection of HPV in trophoblast tissue from early pregnancy losses, and HPV was more prevalent in spontaneous abortions than in elective terminations of pregnancy (Hermonat et al., 1997, 1998; Malhomme et al., 1997). More recently, the genomes of four different HPV types (11, 16, 18, 31) were shown to undergo complete life cycles in a trophoblast cell line (3A trophoblasts), and preliminary data demonstrate that HPV-31 decreases trophoblast cell number and cell adhesion in an in vitro system (Liu et al., 2001; You et al., 2003).

Extravillous, or invasive, trophoblast cells mediate placental attachment to the maternal uterine wall and are responsible for establishing a high-flow, low-resistance maternal circulation supplying the placenta and fetus. Failed invasion by extravillous trophoblast cells leads to placental dysfunction and adverse obstetric outcomes associated with placental dysfunction, including pre-eclampsia and spontaneous preterm delivery (Germain et al., 1999; Kim et al., 2002, 2003).

Infection of invasive trophoblast cells by HPV and the effects of such infection have not been reported. We hypothesized that human papillomavirus induces pathologic changes that impair trophoblast invasion into the uterine wall, which may result in adverse obstetric outcomes due to placental dysfunction. Thus, we sought to determine if: (i) infection of invasive trophoblasts by HPV induces cell death and/or reduces cell invasion; and if (ii) placental infection with HPV is associated with adverse obstetric outcomes attributed to placental dysfunction, including spontaneous preterm delivery and severe pre-eclampsia.

Materials and Methods

Transfection of the entire HPV-16 genome

We transfected immortalized first trimester trophoblast cells (HTR-8/SVneo) with a cloned plasmid, pAT-HPV-16, which contains the entire HPV-16 genome (Bubb et al., 1988). The HTR-8/SVneo cells were generously provided by C. H. Graham (Queen’s University, Ontario, Canada) and the pAT-HPV-16 plasmid was provided by R. Schlegel (Georgetown University, Washington, DC, USA). HTR-8/SVneo cells originally were obtained from explant cultures of human first-trimester placenta (8–10 weeks of gestation) and immortalized by transfection with a cDNA construct that encodes the SV40 large T antigen (Graham et al., 1993). HTR-8/SVneo cells exhibit phenotypic characteristics of extravillous troprophoblasts, including invasive properties through extracellular matrices (Appleton et al., 2003). The cells were cultured at 37°C with Dulbecco modified Eagle medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum. All media contained penicillin/streptomycin (P/S: 100 U/100 mg/ml) and amphotericin B (2.5 µg/ml).

In order to reconstitute HPV-16 DNA, digestion of the cloned plasmid was performed with the restriction enzyme BamHI (Amersham Biosciences, Piscataway, NJ, USA). Plasmid bands from agarose gels were isolated at the proper size (7000 kB) using the QIAEX II Gel extraction kit (QIAGEN, Valencia, CA, USA) according to the instructions provided by the manufacturer. Rapid DNA Ligation Kits (Roche Diagnostics, Indianapolis, IN, USA) were used to ligate and recircularize the entire HPV-16 genome. Then, 1 × 106 HTR-8/SV neo cells were transfected with the pAT-HPV-16 plasmid using FuGENE 6 (Roche Diagnostics) as a vehicle (3:1 µg) (Liu et al., 2001).

Assessment of HPV replication in extravillous trophoblast cells

The HPV genome contains two major consensus regions that are similar among strains. The early region is expressed in latent infected cells, while late region expression indicates viral replication (Villa et al., 2002; Huang et al., 2004). The late-1 (L1) region encodes the capsid protein i.e. essential for viral assembly; therefore, we attempted to identify L1 DNA and protein as markers of viral replication.

In order to confirm that HPV-16 could replicate in the extravillous trophoblast, transfected cells were harvested every 3 days for DNA detection of a portion of the HPV-16 L1 gene. DNA was extracted using High Pure PCR Template Preparation Kit (Roche Diagnostics) according to manufacturer instructions. A 152 base pair (bp) fragment of the HPV-16 L1 region was amplified by PCR using vdB-16-U and vdB-16-D primers (Table I) (van den Brule et al., 1990). Amplification with primers specific for β-globin (S-GH20 and SPCO04) was used to confirm the presence of intact cellular DNA (Table I) (Saiki et al., 1986). Reactions were completed with Ready-To-Go PCR beads (Amersham Biosciences). In a final volume of 25 µl, each reaction contained 1.5 units of Taq DNA polymerase, 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP (dATP, dGTP, dTTP, and dCTP) and 10 µl of each oligonucleotide. PCR amplification was performed in a thermocycler (GeneAmp PCR System 9700, Amersham Biosciences) as follows: 95°C × 5 min denaturation, (95°C × 1 min, 55°C × 1 min, 74°C × 1.5 min) × 40 cycles amplification and 74°C × 8 min extension. Each set of PCR amplifications included positive controls (CaSki cervical cancer cells containing 600 copies of HPV-16, ATCC CRL-1550) and negative controls (non-transfected HTR-8/SVNeo cells). A 10 µl aliquot of the reaction volume was subjected to electrophoresis in a 2% agarose gel containing ethidium bromide. UV transillumination was used to identify the 152 bp HPV-16 L1 band and a 268 bp β-globin band.

Table I.

Oligonucleotides used for PCR analyses

Oligonucleotides Sequence 5′-3′ Ref. 
 Type-specific HPV primers van den Brule et al. (1990) 
vdB-6-U TAG TGG GCC TAT GGC TCG TC  
vdB-6-D ATT TAC TGC AAC ATT GGT AC  
vdB-11-U GGA ATA CAT GCG CCA TGT GG  
vdB-11-D CGA GCA GAC GTC CGT CCT CG  
vdB-16-U TGC TAG TGC TTA TGC AGC AA  
vdB-16-D ATT TAC TGC AAC ATT GGT AC  
vdB-18-U AAG GAT GCT GCA CCG GCT GA  
vdB-18-D CAC GCA CAC GCT TGG CAG GT  
 Type-specific HPV probes van den Brule et al. (1990) 
HPV-6 CAT TAA CGC AGG GGC GCC TGA AAT TGT GCC  
HPV-11 CGC CTC CAC CAA ATG GTA CAC TGG AGG ATA  
HPV-16 GCA AAC CAC CTA TAG GGG AAC ACT GGG GCA  
HPV-18 TGG TTC AGG CTG GAT TGC GTC GCA AGC CCA  
 Internal control primers  
β-Globin/PCO04 GAA GAG CCA AGG ACA GGT AC Saiki et al. (1986) 
β-Globin/PCO04 CAA CTT CAT CCA CGT TCA CC  
β-Actin forward TCA CCC ACA CTG TGC CCA TCT ACG A Malarstig et al. (2003) 
β-Actin reverse CAG CGG AAC CGC TCA TTG CCA ATG G  
Oligonucleotides Sequence 5′-3′ Ref. 
 Type-specific HPV primers van den Brule et al. (1990) 
vdB-6-U TAG TGG GCC TAT GGC TCG TC  
vdB-6-D ATT TAC TGC AAC ATT GGT AC  
vdB-11-U GGA ATA CAT GCG CCA TGT GG  
vdB-11-D CGA GCA GAC GTC CGT CCT CG  
vdB-16-U TGC TAG TGC TTA TGC AGC AA  
vdB-16-D ATT TAC TGC AAC ATT GGT AC  
vdB-18-U AAG GAT GCT GCA CCG GCT GA  
vdB-18-D CAC GCA CAC GCT TGG CAG GT  
 Type-specific HPV probes van den Brule et al. (1990) 
HPV-6 CAT TAA CGC AGG GGC GCC TGA AAT TGT GCC  
HPV-11 CGC CTC CAC CAA ATG GTA CAC TGG AGG ATA  
HPV-16 GCA AAC CAC CTA TAG GGG AAC ACT GGG GCA  
HPV-18 TGG TTC AGG CTG GAT TGC GTC GCA AGC CCA  
 Internal control primers  
β-Globin/PCO04 GAA GAG CCA AGG ACA GGT AC Saiki et al. (1986) 
β-Globin/PCO04 CAA CTT CAT CCA CGT TCA CC  
β-Actin forward TCA CCC ACA CTG TGC CCA TCT ACG A Malarstig et al. (2003) 
β-Actin reverse CAG CGG AAC CGC TCA TTG CCA ATG G  

Because visualization of bands in agarose gels is not always sensitive for the detection of low copy number after transfection, real-time PCR was performed. The same set of primers was used to amplify the target region of the HPV-L1 gene. Individual reactions were performed in a total volume of 20 µl, consisting of 0.5 µl (2.5 µM) of forward and reverse primers, 2 µl of template nucleic acid and 10 µl of 2× SYBR Green PCR Master Mix Kit containing AmpliTaq Gold DNA polymerase, dNTPs with dUTP and optimized buffer components (Applied Biosystems, Foster City, CA, USA). The cycling profiles were programmed as follows: initial denaturation at 95°C × 10 min, followed by 95°C × 15 s, 60°C × 1 min, cycled 40 times. Each quantification target was amplified in triplicate samples, and transfection efficiencies were measured at each time point in two separate experiments. The same positive and negative template controls for each master mix and the DNA standard from CaSki cells in 1:1, 1:10, 1:100 and 1:1000 dilutions were included in the experiments. Amplification of the house keeping gene β-actin was utilized as endogenous control (Table I) (Malarstig et al., 2003). The ABI PRISM7900 HT Sequence Detection System DNA LightCycler (Applied Biosystems) was used for amplification and detection. Relative quantification of HPV-16-L1 gene expression compared with β-actin gene expression was reported using the 2−ΔΔCT method (Livak and Schmittgen, 2001). Briefly, the threshold cycle (CT) indicated the fractional cycle number at which the amount of amplified target began to increase logarithmically according to SDS2.2 software (Applied Biosystems). At each time point, the relative amount of HPV-16-L1 gene expression was determined by calculating the 2−ΔΔCT; ΔΔCT = (CTL1CTβ-actin)timeX − (CTL1CTβ-actin)time 0 (Livak and Schmittgen, 2001).

In order to further investigate whether HPV could replicate in transfected HTR-8/SVneo cells, we also sought to detect HPV-16 E7 DNA sequences by quantitative PCR. Primers (F16E7, AGC TCA GAG GAG GAG GAT GAA; R16E7 GGT TAC AAT ATT GTA ATG GGC TC) and PCR conditions were published previously (Moberg et al., 2003).

Culture medium from transfected cells was collected every 3 days post-transfection for the detection of HPV-16 L-1 protein. Total protein was isolated at the time of medium collection and standard western blot analysis using 20 µg of protein was performed as described elsewhere (Iwagaki et al., 2003). Anti HPV-16 L1 mouse IgG2a monoclonal antibody (BD Biosciences Pharmigen, San Diego, CA, USA) was used as primary antibody at a 1:1000 dilution (1 µg/ml) and anti-mouse IgG sheep horseradish peroxidase-labeled secondary antibody (Amersham Biosciences) was utilized at 1:1000 dilution (McLean et al., 1990). HPV-16 L1 protein was detected in radiographs after using the Enhanced Chemiluminescence Western Blotting Analysis System (Amersham Biosciences), and protein levels were measured at each time point in two separate experiments.

Functional assays

At 3–15 days after transfection, cells and culture medium were collected to perform functional assays in order to assess HPV effects. These assays included cell viability, apoptosis and invasion assays. Appropriate negative controls for these experiments consisted of non transfected HTR-8/SVneo cells, cells treated with the vehicle FuGENE 6 alone, and cells transfected with an inert plasmid [the pAT plasmid containing a green fluorescent protein (GFP) transgene]. All experiments were conducted in triplicate in two separate experiments.

Cell viability based on lactate dehydrogenase (LDH) release was measured using Cyto Tox 96 Non-Radioactive Cytotoxicity Assay kit according to the protocol recommended by the manufacturer (Promega, Madison, WI, USA) (Decker and Lohmann-Matthes, 1988). Briefly, after collecting cell culture medium, transfected cells and cells used as negative controls were lysed and equal volumes (50 µl) of medium and lysis buffer (containing LDH released from lysed cells) were transferred to separate 96 well plate wells. After the addition of a reconstituted substrate mix provided with the kit, an enzymatic reaction occurred and absorbance was recorded using a microplate reader at 490 nm. Results were obtained after subtracting background values; the absorbance of lysis buffer/absorbance of lysis buffer + cell culture medium ratio was calculated to determine the percentage of cells that remained viable at each time point (Arechaveleta-Velasco et al., 2006).

Apoptosis was detected in transfected cells using Cell Death Detection ELISA (Roche Diagnostics) based on mono- and oligo-nucleosome release in the cytoplasm. Transfected and negative control cells were harvested at various time points and pellets were resuspended and lysed with incubation buffer included in the kit. After centrifugation (15 000 r.p.m. for 10 min), the cytoplasmic fraction (supernatant) of the lysate was diluted and subjected to nucleosome detection by immunoassay (absorbance measured at 405 nm). Average optical density (OD) values (after background subtraction) from transfected and negative control cells were divided by OD values from non transfected cells at day 0, and the ratio was expressed as a percentage.

Invasion of transfected trophoblast cells through an extracellular matrix (ECM) was determined using the Cell Invasion Assay Kit (Chemicon International, Temecula, CA, USA) according to the protocol provided by the manufacturer. In these experiments, 1 × 106 cells/ml of transfected and negative control cells were placed in invasion chambers at different time points after transfection. After 48 h, cells that invaded through the ECM Matrigel (Chemicon International) were stained with Cell Stain provided by the manufacturer (Chemicon International) and treated with 10% acetic acid. A volume of 150 µl of the dye/solute mixture was transferred to 96-well plates and invasion was measured by colorimetric reading at OD 560 nm. Levels of invasion were determined by comparing average OD values of transfected cells to those of non-transfected cells at day 0 (Arechaveleta-Velasco et al., 2006).

Case–control study design

In order to associate HPV infection of the placenta with adverse pregnancy outcomes, we conducted a case–control study in which we collected placentas from 108 subjects. Cases were defined as women who developed severe pre-eclampsia requiring delivery before 37 weeks’ gestation and women who underwent spontaneous preterm delivery before 37 weeks’ gestation subsequent to preterm premature rupture of the membranes and/or idiopathic preterm labor. Controls included women who delivered at term with no obstetrical or medical complications. Medical records were reviewed by a certified perinatologist (S.P.) before subjects were included in the study. This study was approved by the Office of Regulatory Affairs at the University of Pennsylvania (protocol number 700943), and informed consent was obtained from all subjects. Criteria for severe pre-eclampsia included blood pressure 160/110 mmHg sustained for ≥6 h, and proteinuria of 5 gm in a 24-h urine specimen or 3+ or 4+ on two random urine samples (American College of Obstetricians and Gynecologists, 2002). Women with sexually transmitted diseases during the index pregnancy were excluded from the cohort. DNA was extracted from extravillous regions of placentas from all subjects. The extravillous region, or ‘membrane roll,’ is the region where the free fetal membranes attach to the placenta and includes the fetal membranes, maternal deciduas and placental tissues. This region was selected because it likely contains the lowest proportion of villous trophoblast cells and the highest proportion of extravillous trophoblast cells embedded in the maternal decidua. We detected HPV DNA in the extravillous region by PCR using individual primers to amplify a sequence within the HPV L1 region of high-risk types 16 and 18 and low-risk types 6 and 11 (Saiki et al., 1986). Positive controls for these experiments were DNA extracted from CaSki cervical cancer cells, whereas negative controls included DNA extracted from non-transfected HTR-8/SVneo cells. In order to increase the sensitivity of detection of the amplified DNA, a slot-blot method was employed. Briefly, 5 µl aliquots of the PCR reactions were transferred to nylon membranes (Amersham Biosciences) using a hybrid-slot filtration manifold apparatus (GIBCO BRL). The membranes were hybridized overnight with aqueous hybridization solution (65°C, 0.5 M phosphate buffer, 1 mM EDTA, 6% SDS, 1% BSA, 1% sonicated salmon sperm DNA) containing specific HPV-6, −11, −16 and −18 probes labeled by with 0.05 mCi alpha dCTP (1.5 × 105 c.p.m./ml) (Saiki et al., 1986). Primer pairs and probes sequences are shown in Table I. After hybridization, unbound probe was removed from the membranes with 2–10 min washes under moderate stringency conditions (0.2× SSC/1% SDS at 42°C). The membranes were dried and placed in an autoradiograph phosphor screen cassette for 24 h. Scans were performed using the Storm 860 Optical Scanner (Molecular Dynamics, Sunnyvale, CA, USA) according to the manufacturer’s guide, and results were analysed in a blinded fashion.

Statistical analysis

Mean OD values and standard errors were calculated and compared between transfected and control cells using t-tests and analysis of variance (ANOVA). Mean and median values and standard deviations were used for comparing demographic data between cases and controls. Chi-square tests were utilized to compare rates of HPV exposure between cases and controls. A P-value of 0.05 was indicative of statistical significance.

Results

The results of our first set of experiments demonstrated that the HPV-16 plasmid successfully transfected extravillous trophoblast cells. HPV-16 L1 DNA sequences were detected in extravillous trophoblast cells by conventional PCR beginning at day 9 and lasting until day 39 (data not shown in figures). Using quantitative real-time PCR, DNA sequences were shown to peak at days 15–27 (18.2 mean fold change at day 15, 311 mean fold change at day 21 and 54 mean fold change in HPV-16 L1 DNA sequences at day 27, relative to β-actin) (Fig. 1A). In order to confirm that these results indicate HPV DNA replication in HTR-8/SVneo cells, we also observed that HPV-E7 DNA sequences were detected from 6 to 33 days after transfection with the HPV-16 plasmid (10.7 mean fold change at day 9, 124 mean fold change at day 12 and 24 mean fold change in HPV-16 L1 DNA sequences at day 27, relative to β-actin; data not shown in figures).

Figure 1:

Transfection of extravillous trophoblast cells with human papillomavirus plasmid

(A) HPV-L1 DNA sequences were detected by quantitative PCR in transfected extravillous trophoblast (HTR-8/SVneo) cells. The mean fold change in HPV-L1 DNA levels divided by β-actin DNA levels (±SE) was measured along the Y-axis, while days after transfection were depicted along the X-axis. (B) Secretion of HPV-L1 protein into culture medium by transfected HTR-8/SVneo cells was detected by Western Blot (band at 64 kDa). Densitometric analysis of western blots demonstrated that L1 protein was not detected in culture medium 3 days after transfection, and levels were greatest between 18 and 36 days after transfection

Figure 1:

Transfection of extravillous trophoblast cells with human papillomavirus plasmid

(A) HPV-L1 DNA sequences were detected by quantitative PCR in transfected extravillous trophoblast (HTR-8/SVneo) cells. The mean fold change in HPV-L1 DNA levels divided by β-actin DNA levels (±SE) was measured along the Y-axis, while days after transfection were depicted along the X-axis. (B) Secretion of HPV-L1 protein into culture medium by transfected HTR-8/SVneo cells was detected by Western Blot (band at 64 kDa). Densitometric analysis of western blots demonstrated that L1 protein was not detected in culture medium 3 days after transfection, and levels were greatest between 18 and 36 days after transfection

Results from western blot analysis showed that the HPV-16 plasmid could replicate in transfected HTR-8/SVneo cells, as the L1 capsid protein was detected in culture medium from day 15 until day 42 (Fig. 1B). By means of densitometric analysis, bands were shown to be strongest between days 18 and 36. Detection of HPV protein in supernatant lagged behind DNA sequences in cells because of the different kinetic patterns of DNA (measures virus replication in cells) and protein expression (measures virus presence in supernatant).

At 3–15 days post-transfection, the viability of transfected trophoblast cells was consistently and significantly reduced compared with non-transfected cells (negative controls), cells treated with the vehicle FuGENE alone and cells transfected with an empty plasmid (GFP) from day 3 (72.3% viable, 95.6, 99.6 and 98.3%, respectively) through day 15 (50.7, 83.8, 84.3 and 86.8%, respectively; P = 0.004 by overall ANOVA) (Fig. 2A). Rates of apoptosis in transfected trophoblast cells were 3-fold (2.4–3.7) and 5.8-fold (5.6–5.9) greater compared with negative controls at 3 and 12 days, respectively (P = 0.003 by overall ANOVA) (Fig. 2B). At 3 days, 22.6% of transfected HTR-8/SVneo cells were apoptotic, compared with 1.5–8.3% of controls. At 12 days, 51.4% of transfected HTR-8/SVneo cells were apoptotic, compared with 5.9–15.2% of controls. Meanwhile, invasion of transfected trophoblast cells through an ECM progressively and significantly decreased from day 3 until day 15 after transfection (25.2–57.6% lower than negative controls; P < 0.0001 by overall ANOVA) (Fig. 2C).

Figure 2:

Functional assays demonstrating adverse effects of human papillomavirus in extravillous trophoblast cells

(A) The viability of HTR-8/SVneo cells transfected with the pAT-HPV-16 plasmid was compared with negative controls, including HTR-8/SVneo cells that were not transfected (NEG-HTR), treated with the vehicle FuGENE-6 alone (NEG-FuGENE) or transfected with the same plasmid containing a GFP transgene (NEG-GFP). Cell viability was measured by LDH release assays and expressed as a percentage along the Y-axis. (B) Apoptosis of transfected trophoblast cells was measured by nucleosome release (Cell Death Detection ELISA, Roche Diagnostics) assays and was compared with negative controls. The ratio of apoptotic cells (indicated on the Y-axis) was determined by dividing average optical densities of experimental samples by average optical densities of non-transfected cells at day 0. (C) Invasion of transfected trophoblast cells through an ECM was measured using Cell Invasion Assay Kits (Chemicon International) and was compared with negative controls. Levels of invasion were indicated on the Y-axis and were determined by spectrophotometric measurement of labeled cells that invaded through the ECM

Figure 2:

Functional assays demonstrating adverse effects of human papillomavirus in extravillous trophoblast cells

(A) The viability of HTR-8/SVneo cells transfected with the pAT-HPV-16 plasmid was compared with negative controls, including HTR-8/SVneo cells that were not transfected (NEG-HTR), treated with the vehicle FuGENE-6 alone (NEG-FuGENE) or transfected with the same plasmid containing a GFP transgene (NEG-GFP). Cell viability was measured by LDH release assays and expressed as a percentage along the Y-axis. (B) Apoptosis of transfected trophoblast cells was measured by nucleosome release (Cell Death Detection ELISA, Roche Diagnostics) assays and was compared with negative controls. The ratio of apoptotic cells (indicated on the Y-axis) was determined by dividing average optical densities of experimental samples by average optical densities of non-transfected cells at day 0. (C) Invasion of transfected trophoblast cells through an ECM was measured using Cell Invasion Assay Kits (Chemicon International) and was compared with negative controls. Levels of invasion were indicated on the Y-axis and were determined by spectrophotometric measurement of labeled cells that invaded through the ECM

Demographic characteristics from our case–control study are shown in Table II. As expected, the gestational age at delivery and birthweight were lower in cases than in controls: 32 weeks (26–36 weeks) for severe pre-eclampsia and 29 weeks (21–36 weeks) for spontaneous preterm delivery cases versus 39 weeks (37–42 weeks) for controls; P < 0.0001 by overall ANOVA; 1588 ± 469 gm for severe pre-eclampsia and 1447 ± 879 gm for spontaneous preterm delivery versus 3344 ± 527 gm for controls; P < 0.0001 by overall ANOVA. Severe pre-eclampsia cases were more likely to be nulliparous (68%) compared with spontaneous preterm delivery cases (23.3%) and controls (23%; P < 0.0001). The same tendency was observed when comparing the presence of intrauterine growth restriction among severe pre-eclampsia (20%) versus spontaneous preterm delivery cases (0%) and controls (0%; P = 0.01).

Table II.

Demographics and outcomes data for case–control study

 Severe PE, n = 48 SPTD, n = 30 Controls, n = 30 P-value1 
Age (year ± SD) 25.9 ± 7.2 28.1 ± 6.5 26.3 ± 6.6 0.42 
Nulliparity 68.7% 23.3% 23.0% <0.0001 
GA del (week, range) 32 (26–36) 29 (21–36) 39 (37–42) <0.0001 
BW (gm) 1588 ± 469 1447 ± 879 3344 ± 527 <0.0001 
IUGR 20% 0.01 
 Severe PE, n = 48 SPTD, n = 30 Controls, n = 30 P-value1 
Age (year ± SD) 25.9 ± 7.2 28.1 ± 6.5 26.3 ± 6.6 0.42 
Nulliparity 68.7% 23.3% 23.0% <0.0001 
GA del (week, range) 32 (26–36) 29 (21–36) 39 (37–42) <0.0001 
BW (gm) 1588 ± 469 1447 ± 879 3344 ± 527 <0.0001 
IUGR 20% 0.01 

PE, pre-eclampsia; SPTD, spontaneous preterm delivery; GA del, gestational age at delivery; BW, birthweight; IUGR, intrauterine growth restriction (birthweight less than the 10th percentile for gestational age).

1P-value by overall ANOVA.

Among subjects in the case-control study, HPV DNA was identified in the extravillous region of 29/108 (26.9%) placentas. Approximately 45% of HPV DNA corresponded with low-risk strains (HPV-6, −11) and 55% corresponded with high risk strains (HPV-16, −18) (Table III). PCR products were sent to the DNA Sequencing Facility at the University of Pennsylvania, where the sequences of low- and high-risk HPV strains were confirmed. There were no differences in detection of individual HPV types among the three groups (controls, spontaneous preterm delivery and severe pre-eclampsia). Identification of HPV DNA in extravillous regions of placental samples from cases of severe pre-eclampsia was not significantly different from that of controls (8/48 versus 6/30; P = 0.71) (Table III). However, HPV DNA was detected more frequently in the extravillous trophoblast region of placentas from spontaneous cases (15/30) than from controls (6/30; P = 0.03). In the subset of women who underwent spontaneous preterm delivery remote from term (≤34 weeks’ gestation), HPV DNA also was detected more frequently than among controls (12/22 versus 6/30; P = 0.02).

Table III.

HPV L1 sequences detected in extravillous trophoblast region of placentas from cases and controls

Group HPV present HPV absent P-value1 
Controls (n = 30) 62 24 — 
Severe pre-eclampsia (n = 48) 83 40 0.71 
Spontaneous preterm delivery (n = 30) 154 15 0.03 
Group HPV present HPV absent P-value1 
Controls (n = 30) 62 24 — 
Severe pre-eclampsia (n = 48) 83 40 0.71 
Spontaneous preterm delivery (n = 30) 154 15 0.03 

1Chi-square, compared with controls.

2Low-risk HPV strains (6, 11) = 4; high-risk HPV strains (16, 18) = 2.

3Low-risk HPV strains (6, 11) = 3; high-risk HPV strains (16, 18) = 5.

4Low-risk HPV strains (6, 11) = 6; high-risk HPV strains (16, 18) = 9.

Discussion

Our results indicate that HPV-16 can replicate in extravillous trophoblast cells, induce cell death and reduce cell invasion through an ECM. These effects of HPV infection may result in failed invasion by extravillous trophoblast cells into the maternal uterine wall, placental dysfunction and adverse pregnancy outcomes attributed to placental dysfunction. We found an association between HPV infection of the placenta and spontaneous preterm delivery.

Although HPV was previously known to replicate only in differentiating keratinocytes of the skin, another group of investigators utilized the same plasmid used in our experiments to demonstrate de novo replication of the HPV-16 genome in a noninvasive trophoblast cell line (3A trophoblast cells) and defective 3A trophoblast endometrial cell adhesion (Liu et al., 2001; You et al., 2003). These findings support the hypothesis that local spread of HPV from the genital tract may result in placental infection, which may be a cause of spontaneous miscarriage (Hermonat et al., 1997). In our experiments, the rates of HTR-8/SVneo cell viability and invasion were ∼50% lower in HPV-16 transfected cells compared with controls, whereas rates of apoptosis were significantly greater in transfected cells using a mono- and oligo-nucleosome release apoptosis assay. Our observations demonstrate that HPV infection can impair extravillous trophoblast invasion into the maternal uterine wall, possibly by causing increased rates of trophoblast cell death, revealing novel mechanisms by which viral infection results in placental dysfunction.

In the past several years, evidence has accumulated associating placental dysfunction with spontaneous preterm delivery. Other investigators found that women who delivered preterm after idiopathic preterm labor had higher rates of placental ischemia and abnormal placentation (defined as failure of physiological transformation of maternal spiral arteries resulting in reduced blood flow to the placental intervillous space) than controls (Germain et al., 1999; Kim et al., 2002, 2003). Most recently, a large multi-center collaboration of investigators demonstrated that decreased first trimester maternal serum levels of pregnancy associated plasma protein A, which is a protease produced by trophoblast cells, were associated with a significantly increased risk of preterm premature rupture of membranes and preterm delivery (Dugoff et al., 2004). These findings support the hypothesis that spontaneous preterm delivery, at least in part, has its origins in abnormal placental function at the beginning of pregnancy. The results of our case–control study also support this hypothesis and suggest that HPV infection of the placenta is a cause of failed placental invasion leading to placental dysfunction. However, the exact mechanism by which HPV infection alters trophoblast gene expression and increases cell death has not been elucidated.

Our study had some limitations. First, the results of in vitro experiments using transformed cell lines must be interpreted with caution when applying these results to primary extravillous trophoblast cells and in vivo conditions. Additionally, we did not perform our in vitro experiments with wild-type HPV because of lack of availability, but the pAT-HPV-16 plasmid was shown by other investigators to display the complete life cycle of HPV-16 in cultured trophoblast cells, and we detected de-novo production of HPV L1 protein in cell culture medium after HTR-8/SVneo cells were transfected with the pAT-HPV-16 plasmid. The pathological effects that we observed following HPV transfections (i.e. decreased viability and invasion) may have resulted in part from cell detachment from the culture plates, which was observed by other investigators using HPV-transfected trophoblast cells (Liu et al., 2001; You et al., 2003). However, the use of appropriate controls [plasmid containing another transgene, vehicle (FuGENE) without plasmid DNA and non-transfected cells] permitted us to conclude that the pathological effects we observed after transfection with the pAT-HPV-16 plasmid (i.e. decreased cell viability, increased rates of apoptosis and decreased cell invasion) were the result of expression of the HPV-16 genome in HTR-8/SVneo cells. In our case–control study, DNA was extracted from the extravillous, or ‘membrane roll,’ region of the placenta. Although the detection of HPV DNA in this region is informative, in situ PCR or laser capture dissection techniques may be useful in future studies to identify which cells are infected with HPV. Finally, the women comprising our case–control study group were largely African-American, so our observations will require validation in other cohorts before they can be generalized.

Collectively, our data indicate that HPV is able to infect and replicate in invasive trophoblast cells and that infection by HPV induces pathological sequelae that are associated with placental dysfunction and spontaneous preterm delivery. Potential mechanisms by which viral infections may induce failed invasion and placental dysfunction, including altered expression of cell adhesion molecules, matrix metalloproteinases, proinflammatory cytokines and major histocompatibility antigens, need to be explored. However, our findings provide a solid scientific basis for the continued critical investigation of the role of HPV and other common viruses in pregnancy complications related to placental dysfunction.

Funding

This research was supported by the NIH grant HD42100 (S.P.).

Acknowledgements

We thank our colleagues Zhibing Zhan, PhD and Pedro Ferrand, MD for their technical help.

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