Placental infection with human papillomavirus is associated
with spontaneous preterm delivery
L.M. Gomez, Y. Ma, C. Ho, C.M. McGrath, D.B. Nelson and S. Parry1
Center for Research on Reproduction and Women’s Health, University of Pennsylvania School of Medicine,
2000 Courtyard Building, 3400 Spruce Street, Philadelphia, PA 19104, USA
1Correspondence address. Tel: þ1-215-662-6913; Fax: þ1-215-573-5408; E-mail: email@example.com
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 placen-
tal 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 5 0.03). Identi-
fication 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.
Keywords: human papillomavirus; trophoblast; cell invasion; placenta; preterm birth
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 epi-
thelial 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 cer-
vical 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 tro-
phoblast 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 extra-
villous trophoblast cells leads to placental dysfunction and
adverse obstetric outcomes associated with placental dysfunc-
tion, including pre-eclampsia and spontaneous preterm deliv-
ery (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 hypo-
thesized 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 attrib-
uted 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
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Human Reproduction pp. 1–7, 2007
Hum. Reprod. Advance Access published January 8, 2008
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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 tropropho-
blasts, including invasive properties through extracellular matrices
(Appleton et al., 2003). The cells were cultured at 378C with Dulbecco
modified Eagle medium (Gibco BRL, Grand Island, NY, USA) sup-
plemented with 10% fetal bovine serum. All media contained penici-
llin/streptomycin (P/S: 100 U/100 mg/ml) and amphotericin B
In order to reconstitute HPV-16 DNA, digestion of the cloned
plasmid was performed with the restriction enzyme BamHI (Amer-
sham 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) accord-
ing to the instructions provided by the manufacturer. Rapid DNA Lig-
ation 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 mg)
(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). Amplifica-
tion with primers specific for b-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 ml, each reac-
tion contained 1.5 units of Taq DNA polymerase, 10 mM Tris–HCl
(pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 200 mM of each dNTP
(dATP, dGTP, dTTP, and dCTP) and 10 ml of each oligonucleotide.
PCR amplification was performed in a thermocycler (GeneAmp
PCR System 9700, Amersham Biosciences) as follows: 958C ?
5 mindenaturation,(958C ? 1 min,
1.5 min) ? 40 cycles amplification and 748C ? 8 min extension.
Each set of PCR amplifications included positive controls (CaSki cer-
vical cancer cells containing 600 copies of HPV-16, ATCC
CRL-1550) and negative controls (non-transfected HTR-8/SVNeo
cells). A 10 ml aliquot of the reaction volume was subjected to electro-
phoresis in a 2% agarose gel containing ethidium bromide. UV transil-
lumination was used to identify the 152 bp HPV-16 L1 band and a
268 bp b-globin band.
Because visualization of bands in agarose gels is not always sensi-
tive 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 per-
formed in a total volume of 20 ml, consisting of 0.5 ml (2.5 mM) of
forward and reverse primers, 2 ml of template nucleic acid and
10 ml of 2? SYBR Green PCR Master Mix Kit containing AmpliTaq
Gold DNA polymerase, dNTPs with dUTP and optimized buffer com-
ponents (Applied Biosystems, Foster City, CA, USA). The cycling
profiles were programmed as follows: initial denaturation at 958C ?
10 min, followed by 958C ? 15 s, 608C ? 1 min, cycled 40 times.
Each quantification target was amplified in triplicate samples, and
transfection efficiencies were measured at each time point in two sep-
arate 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 b-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. Rela-
tive quantification of HPV-16-L1 gene expression compared with
558C ? 1 min,748C ?
Table I. Oligonucleotides used for PCR analyses
Type-specific HPV primers
TAG TGG GCC TAT GGC TCG TC
ATT TAC TGC AAC ATT GGT AC
GGA ATA CAT GCG CCA TGT GG
CGA GCA GAC GTC CGT CCT CG
TGC TAG TGC TTA TGC AGC AA
ATT TAC TGC AAC ATT GGT AC
AAG GAT GCT GCA CCG GCT GA
CAC GCA CAC GCT TGG CAG GT
van den Brule et al. (1990)
Type-specific HPV probes
CAT TAA CGC AGG GGC GCC TGA AAT TGT GCC
CGC CTC CAC CAA ATG GTA CAC TGG AGG ATA
GCA AAC CAC CTA TAG GGG AAC ACT GGG GCA
TGG TTC AGG CTG GAT TGC GTC GCA AGC CCA
van den Brule et al. (1990)
Internal control primers
GAA GAG CCA AGG ACA GGT AC
CAA CTT CAT CCA CGT TCA CC
TCA CCC ACA CTG TGC CCA TCT ACG A
CAG CGG AAC CGC TCA TTG CCA ATG G
Saiki et al. (1986)
Malarstig et al. (2003)
Gomez et al.
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b-actin gene expression was reported using the 22DDCTmethod (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 22DDCT;
DDCT¼ (CTL12 CTb-actin)timeX2 (CTL12 CTb-actin)time 0(Livak and
In order to further investigate whether HPV could replicate in trans-
fected 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 mg of protein was performed as
described elsewhere (Iwagaki et al., 2003). Anti HPV-16 L1 mouse
IgG2amonoclonal antibody (BD Biosciences Pharmigen, San Diego,
CA, USA) was used as primary antibody at a 1:1000 dilution (1 mg/
ml) and anti-mouse IgG sheep horseradish peroxidase-labeled second-
ary 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.
At 3–15 days after transfection, cells and culture medium were col-
lected 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
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 ml) 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 þ 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 Detec-
tion 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 frac-
tion (supernatant) of the lysate was diluted and subjected to nucleo-
some 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 ?
106cells/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 Inter-
national) were stained with Cell Stain provided by the manufacturer
(Chemicon International) and treated with 10% acetic acid. A
volume of 150 ml 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 preg-
nancy outcomes, we conducted a case–control study in which we col-
lected 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 compli-
cations. 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 3þ or 4þ 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 mem-
branes 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 ml aliquots of the PCR reactions were trans-
ferred to nylon membranes (Amersham Biosciences) using a hybrid-
slot filtration manifold apparatus (GIBCO BRL). The membranes
were hybridized overnight with aqueous hybridization solution
(658C, 0.5 M phosphate buffer, 1 mM EDTA, 6% SDS, 1% BSA,
1% sonicated salmon sperm DNA) containing specific HPV-6, 211,
216 and 218 probes labeled by with 0.05 mCi alpha dCTP (1.5 ?
105c.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 428C). The membranes
were dried and placed in an autoradiograph phosphor screen cassette
for 24 h. Scans were performed using the Storm 860 Optical
HPV infection of the placenta and preterm delivery
Page 3 of 7
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Scanner (Molecular Dynamics, Sunnyvale,CA, USA)according to the
manufacturer’s guide, and results were analysed in a blinded fashion.
Mean OD values and standard errors were calculated and compared
between transfected and control cells using t-tests and analysis of var-
iance (ANOVA). Mean and median values and standard deviations
were used for comparing demographic data between cases and con-
trols. Chi-square tests were utilized to compare rates of HPV exposure
between cases and controls. A P-value of 0.05 was indicative of
The results of our first set of experiments demonstrated that the
HPV-16 plasmid successfully transfected extravillous tropho-
blast cells. HPV-16 L1 DNA sequences were detected in extra-
villous 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 b-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 b-actin; data not shown in figures).
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 analy-
sis, 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 ¼ 0.004 by overall
ANOVA) (Fig. 2A). Rates of apoptosis in transfected tropho-
blast 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 ¼ 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 trans-
fected trophoblast cells through an ECM progressively and sig-
nificantly 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).
Demographic characteristics from our case–control study
are shown in Table II. As expected, the gestational age at deliv-
ery 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
ANOVA. Severe pre-eclampsia cases were more likely to be
nulliparous (68%) compared with spontaneous preterm deliv-
ery cases (23.3%) and controls (23%; P , 0.0001). The same
tendency was observed when comparing the presence of intra-
uterine growth restriction among severe pre-eclampsia (20%)
versus spontaneous preterm delivery cases (0%) and controls
(0%; P ¼ 0.01).
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, 211) and 55% corresponded
P , 0.0001by overall
Figure 1: Transfection of extravillous trophoblast cells with human
(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 b-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
Gomez et al.
Page 4 of 7
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with high risk strains (HPV-16, 218) (Table III). PCR pro-
ducts were sent to the DNA Sequencing Facility at the Univer-
sity 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 ¼ 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 ¼ 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 ¼ 0.02).
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
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
Table II. Demographics and outcomes data for case–control study
Severe PE, n ¼ 48
SPTD, n ¼ 30
Controls, n ¼ 30
GA del (week, range)
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.
HPV infection of the placenta and preterm delivery
Page 5 of 7
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in failed invasion by extravillous trophoblast cells into the
maternal uterine wall, placental dysfunction and adverse preg-
nancy outcomes attributed to placental dysfunction. We found
an association between HPV infection of the placenta and
spontaneous preterm delivery.
ferentiating keratinocytes of the skin, another group of investi-
gators 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
cental infection, which may be a cause of spontaneous miscar-
riage (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
results in placental dysfunction.
In the past several years, evidence has accumulated associat-
ing 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 phys-
iological 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 extravil-
lous 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 com-
plete 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 investi-
gators using HPV-transfected trophoblast cells (Liu et al.,
2001; You et al., 2003). However, the use of appropriate con-
trols [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 metalloprotei-
nases, 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.
We thank our colleagues Zhibing Zhan, PhD and Pedro Ferrand, MD
for their technical help.
This research was supported by the NIH grant HD42100 (S.P.).
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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 ¼ 30)
Severe pre-eclampsia (n ¼ 48)
Spontaneous preterm delivery (n ¼ 30) 154
1Chi-square, compared with controls.
2Low-risk HPV strains (6, 11) ¼ 4; high-risk HPV strains (16, 18) ¼ 2.
3Low-risk HPV strains (6, 11) ¼ 3; high-risk HPV strains (16, 18) ¼ 5.
4Low-risk HPV strains (6, 11) ¼ 6; high-risk HPV strains (16, 18) ¼ 9.
Gomez et al.
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Submitted on April 9, 2007; resubmitted on November 1, 2007; accepted on
November 20, 2007
HPV infection of the placenta and preterm delivery
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