BACKGROUND: There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes. METHODS: Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV. RESULTS: There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23–55 years. All samples were β-globin positive. HPV was not detected in any of the paired samples. CONCLUSION: Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none

AL Fuessel Haws

C Worda

CJ Tseng

D Deng

Daisy R Lee

David C Swan

DH Watts

Diane Roulston

Elizabeth R Unger

EM Smith

F Pakarian

Joanne M Bailey

M Favre

M Puranen

Mack T Ruffin

MH Puranen

MH Schiffman

MJ Silverberg

ML Gillison

N Munoz

PE Gravitt

Ruth Ann Tucker

S Syrjanen

S Xu

X Wang

English

PubMed

Springer - Publisher Connector

Human papillomavirus in amniotic fluid

Crossref

BioMed CentralBMC Pregnancy and Childbirth
ssOpen AcceResearch article
Human papillomavirus in amniotic fluid
Mack T Ruffin IV*1, Joanne M Bailey2, Diane Roulston3, Daisy R Lee4, 
Ruth Ann Tucker4, David C Swan4 and Elizabeth R Unger4
Address: 1Department of Family Medicine, University of Michigan, 1018 Fuller St., Ann Arbor, MI 48109-0708, USA, 2Department of Obstetrics 
and Gynecology, University of Michigan, Nurse Midwives, F4835 Mott 0264, Ann Arbor, MI 48109-0264, USA, 3Department of Pathology, 
University of Michigan, 1301 Catherine Road, Ann Arbor, MI 48109-0602, USA and 4Division of Viral and Rickettsial Diseases, Centers for Disease 
Control and Prevention, 1600 Clifton Rd. NE B6109, Atlanta, GA 30329, USA
Email: Mack T Ruffin* - mruffin@umich.edu; Joanne M Bailey - jabailey@umich.edu; Diane Roulston - droulstn@umich.edu; 
Daisy R Lee - del5@cdc.gov; Ruth Ann Tucker - rat1@cdc.gov; David C Swan - dswan@cdc.gov; Elizabeth R Unger - eru0@cdc.gov
* Corresponding author    
Abstract
Background: There is evidence to suggest that human papillomavirus (HPV) can cross the
placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be
detected in amniotic fluid from women with intact amniotic membranes.
Methods: Residual amniotic fluid and cultured cell pellets from amniocentesis performed for
prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were
used in a nested polymerase chain reaction assay for HPV.
Results: There were 146 paired samples from 142 women representing 139 singleton pregnancies,
2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African
American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23–55 years.
All samples were β-globin positive. HPV was not detected in any of the paired samples.
Conclusion: Given the age range, race, and ethnicity of the study population, one would anticipate
some evidence of HPV if it could easily cross the placenta, but there was none.
Background
Infection with human papillomaviruses (HPV) is causal
in the development of genital and oral cancers [1,2]. HPV
infection is considered a sexually transmitted infection
among adolescents and adults [3]. Detection rates of HPV
infection by polymerase chain reaction (PCR) have
ranged widely between 1% and 20% in newborns of preg-
nant women without apparent infection in their cervix [4-
7] and between 5% and 72% in women with HPV-related
cervical diseases diagnosed during pregnancy [8]. Many
investigators believe that infants are exposed to HPV and
infected during vaginal delivery [9,10]. In contrast, some
investigators have suggested that vertical transmission is
possible without evidence of vaginal or cervical secretion
contact of the fetus [11-16]. However, the number of
women studied to confirm placental transmission of HPV
has been limited.
The goal of this study was to determine if HPV could be
detected in amniotic fluid from women with intact mem-
branes.
Published: 04 September 2006
BMC Pregnancy and Childbirth 2006, 6:28 doi:10.1186/1471-2393-6-28
Received: 22 February 2006
Accepted: 04 September 2006
This article is available from: http://www.biomedcentral.com/1471-2393/6/28
© 2006 Ruffin et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), 
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 3
(page number not for citation purposes)
BMC Pregnancy and Childbirth 2006, 6:28 http://www.biomedcentral.com/1471-2393/6/28Methods
Human subject approval from the University of Michigan
was obtained to use residual amniocentesis samples that
were to be discarded. To be eligible for the study, pregnant
women had to be undergoing clinically indicated amnio-
centesis and have intact membranes. The only informa-
tion on the women available to the investigators was age,
race/ethnicity, and number of fetuses present at time of
sampling. All clinical assays related to chromosomal
assessment of the fetus had been completed, and the
residual amniotic fluid and amniotic cell culture pellets
were retrieved from the cytogenetics laboratory.
Amniotic fluid samples (8–12 ml) were spun at 4500 rpm
for 30 minutes. After decanting the supernatant fluid, the
pellet was suspended in 150 μL of TrisEDTA buffer. The
cultured cell pellets were resuspended in 200 μL PBS. A
150 μL volume of each sample was extracted using the
Roche MagNA Pure LC instrument and the DNA isolation
kit I resulting in a final volume of 150 μL.
We used a direct PCR method followed by a nested PCR
method to detect HPV. The Roche line blot assay, based
on L1 consensus PCR with biotinylated PGMY09/11
primer sets and β-globin as an internal control for sample
amplification [17,18] was used as previously described,
with 10 μL extract in each 50 μL reaction. All samples were
HPV gel-band-negative and Roche Prototype Strip Assay-
negative after 40 cycles (reagents provided as a gift from
Roche Molecular Systems, Inc., Pleasanton, CA).
For the nested reaction, five microliters of each L1 ampli-
con was added to a PCR reaction mix containing GP5+/
GP6+ [19] primers (1 μM each) and run for an additional
40 cycles under the following conditions: 40 cycles of
94°C for 45 s, 48°C for 4 s, 38°C for 30 s, 42°C for 5 s,
66°C for 5 s, and 71°C for 1.5 min. This was followed by
a final extension of 10 min at 72°C. Fifteen μL from each
sample was analyzed on a 2% agarose gel.
The HPV assays were repeated using separate extractions,
increased volumes of extracts in the PCR assays and re-
amplification of L1 consensus PCR products with PGMY
primers.
The data was analyzed using SPSS 11 with frequency dis-
tributions of study participants by age, race, and ethnicity
by HPV status.
Results
The residual amniotic fluid and cell pellets were collected
from 142 women. There were 146 paired samples repre-
senting 138 singleton pregnancies, 2 twin pregnancies, 1
triplet pregnancy, and 1 singleton pregnancy with an egg
donor. The women's average age was 35.2 years with a
range from 23–55 years. The race and ethnicity was 78%
Caucasian, 5% African American, 14% Asian, and 2% His-
panic. No other data were available on the women. No
HPV was detected in any of the amniotic fluid or pellet
cell samples. All samples were β-globin positive. There
was no evidence of technical barriers to detecting HPV in
samples if it was present.
Discussion
In this study, no HPV was detected in amniotic fluid or
culture cell pellets from 142 pregnant women with intact
amniotic membranes. Other investigators have reported
the upper 95% confidence interval for detection of perina-
tal transmission from women with any evidence of genital
HPV is only 2.8% [7]. Worda and colleagues found no
HPV in amniotic fluid taken just prior to a cesarean deliv-
ery in 153 Austrian women with intact membranes [10].
With 142 amniotic samples, we have 80–85% power to
detect prevalence of HPV from 1% to 10%. Therefore, the
lack of detecting HPV in the amniotic samples is not due
to inadequate number of samples.
This study was not able to actually determine the HPV sta-
tus of the study participants or their risk for having HPV.
It is possible that none of the women had ever been
infected or were not currently infected with HPV. Other
studies addressing the issue of vertical transmission of
HPV have reported that 12–36% of pregnant women
asymptomatic for HPV were positive for HPV
[8,10,16,20]. The population characteristics of these stud-
ies were similar to this study with a reported age range of
18–45 years and primarily Caucasian women except stud-
ies conducted in Asian countries [8,10,16]. The similari-
ties between our study population and others suggest that
12–36% of the pregnant women should have been
infected with HPV [6,8,10,11,16,20,21]. Yet, no HPV was
detected in the amniotic fluid.
Conclusion
Perinatal transmission of HPV clearly does occur; juvenile
onset recurrent respiratory papillomatosis is a docu-
mented rare outcome of infection with HPV. This occurs
in 7 of every 1000 infants born to a mother with genital
warts [22]. Some investigators have suggested that infants
are exposed and infected by HPV during a vaginal delivery
[9]. Studies have documented that there is poor concord-
ance between parents' genital HPV and newborns' oral/
genital HPV [20]. In contrast, some investigators have sug-
gested that vertical transmission is possible without evi-
dence of vaginal or cervical secretion contact of the fetus
[11-15]. However, this study and others suggest that verti-
cal transmission without exposure to vaginal or cervical
secretions is extremely unlikely [8,10].Page 2 of 3
(page number not for citation purposes)
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Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
MR and JB participated in concept development, study
design, implementation, and coordination; data analysis,
and drafting the manuscript. DR collected the data and
coordinated the sample numbers with demographic data.
DRL, RAT, DCS, and ERU participated in the HPV assays
and drafting the manuscript.
Acknowledgements
The authors thank Kathleen Crawford for assistance in retrieving anony-
mous materials from the cytogenetics laboratory and Miriam Lawson, 
Sonya Patel, and Juanita Onyekwuluje for assistance with laboratory testing.
Drs. Ruffin and Bailey were supported by NCI Grant CA080846. The fund-
ing agency had no role in the study design, data collection, analysis, inter-
pretation of the data, writing of the manuscript or the decision to submit 
the manuscript for publication.
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Pre-publication history
The pre-publication history for this paper can be accessed
here:
http://www.biomedcentral.com/1471-2393/6/28/prepubPage 3 of 3
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Abstract Background There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes. Methods Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV. Results There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23–55 years. All samples were β-globin positive. HPV was not detected in any of the paired samples. Conclusion Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none.</p

Swan David C

Tucker Ruth

Lee Daisy R

Roulston Diane

Bailey Joanne M

Ruffin Mack T

Unger Elizabeth R

Directory of Open Access Journals

BMC Pregnancy and Childbirth

Abstract
              
                Background
                There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes.
              
              
                Methods
                Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV.
              
              
                Results
                There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23–55 years. All samples were β-globin positive. HPV was not detected in any of the paired samples.
              
              
                Conclusion
                Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none.http://deepblue.lib.umich.edu/bitstream/2027.42/112696/1/12884_2006_Article_95.pd

Ruffin, Mack T

Bailey, Joanne M

Roulston, Diane

Lee, Daisy R

Tucker, Ruth A

Swan, David C

Unger, Elizabeth R

Deep Blue Documents at the University of Michigan

Apple RJ: Improved amplification of genital human papillomaviruses.

Cancer associated human papillomaviruses: perinatal transmission and persistence.

CC: Perinatal transmission of human papillomavirus in infants: relationship between infection rate and mode of delivery. Obstet Gynecol

Clinical observation on vertical transmission of human papillomavirus.

CM: Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.

DA: Low risk of perinatal transmission of human papillomavirus: results from a prospective cohort study.

Eppel W: Prevalence of cervical and intrauterine human papillomavirus infection in the third trimester in asymptomatic women.

Human papillomavirus and cancer: the epidemiological evidence.

Human papillomavirus infections in children: the potential role of maternal transmission. Crit Rev Oral Biol Med

KV: Condyloma in pregnancy is strongly predictive of juvenile-onset recurrent respiratory papillomatosis. Obstet Gynecol

Ling X: Asymptomatic genital infection of human papillomavirus in pregnant women and the vertical transmission route. J Huazhong Univ Sci Technolog Med Sci

Maternal-fetal transmission of human papillomavirus.

MM: Epidemiologic evidence showing that Human Papillomavirus infection causes most cervical intraepithelial neoplasia.

Pao CC: Possible transplacental transmission of human papillomaviruses.

S: A possible vertical transmission of human papillomavirus genotypes associated with epidermodysplasia verruciformis.

Sidransky D: Evidence for a causal association between human papillomavirus and a subset of head and neck cancers [see comments].

Syrjanen S: Transmission of genital human papillomavirus infections is unlikely through the floor and seats of humid dwellings in countries of high-level hygiene.

Syrjanen S: Vertical transmission of human papillomavirus from infected mothers to their newborn babies and persistence of the virus in childhood.

Syrjanen SM: Exposure of an infant to cervical human papillomavirus infection of the mother is common.

Turek LP: Human papillomavirus prevalence and types in newborns and parents: concordance and modes of transmission. Sex Transm Dis

Turek LP: Perinatal transmission and maternal risks of human papillomavirus infection. Cancer Detect Prev

Tyring SK: Nested PCR with the PGMY09/11 and GP5(+)/6(+) primer sets improves detection of HPV DNA in cervical samples.

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Human papillomavirus in amniotic fluid

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