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Obstetrics & Gynecology 2000;96:337-341
© 2000 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Human Papillomavirus in the Cervix and Placenta

WOLFGANG EPPEL, MD, CHRISTOF WORDA, MD, PETER FRIGO, MD, MARTIN ULM, MD, ELISABETH KUCERA, MD and KLAUS CZERWENKA, MD

From the Department of Gynecology and Obstetrics, Division of Prenatal Diagnosis and Therapy, Division of Gynecological Endocrinology and Reproductive Medicine, Division of Gynecology, and Department of Pathology, Vienna University Hospital, Vienna, Austria.

Address reprint requests to: Wolfgang Eppel, MD Vienna University Hospital Department of Gynecology and Obstetrics Division of Prenatal Diagnostics and Therapy Waehringer Guertel 18-20 A-1090 Vienna Austria E-mail: wolfgang.eppel{at}univie.ac.at


   Abstract
Top
Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: To determine the prevalence and association of human papillomavirus (HPV) infection in the cervices and placentas of pregnant women.

Methods: Cervical samples were taken from 179 of 226 women who had placental biopsies because of abnormal ultrasound findings or were older than 35 years, to detect HPV infections with hybrid capture II tests. Polymerase chain reaction (PCR) was done on placental tissue of 147 of the 226 women to detect HPV DNA.

Results: We found 44 of 179 women (24.6%, 95% confidence interval 18.3, 31.0) to test positive for HPV in their cervices. Logistic regression analyses showed decreased prevalence of HPV infection with increased maternal age (P = .039). The HPV DNA E6 PCR from the villus tissue was negative in the 147 cases examined. However, a significant contingency coefficient between low-risk HPV infection and elevated risk of chromosome aberration was found ({phi} = V = 0.15, P = .050).

Conclusion: The infection rate of 24.6% in women without clinical symptoms of HPV infection was high, but there seemed to be no virus transmission to the placenta in women with subclinical infections. Low-risk cervical HPV infection might be associated with a slightly higher risk of abnormal fetal karyotype.

Human papillomavirus (HPV) might be linked to many diseases. Papillomaviruses can cause papillomas such as genital (mucosotrophic virus) or skin warts (cutaneous virus), and some types are associated highly with cervical cancer. Those subclinical infections are much more common than previously suspected because they now can be detected by many new assay methods.1

Human papillomavirus infection is more common in pregnant than nonpregnant women, and a faster progression of cervical intraepithelial neoplasia grade into a cervical carcinoma has been reported.2,3 Elevated steroid hormone levels might interact with progesterone and glucocorticoid elements of the virus and activate HPV replication. Raised viral levels and a change in the viral DNA state of the cervix could facilitate dissemination of the virus to other genital regions such as the amniotic cavity.3

In the present study, we looked at prevalence of HPV infection and age distribution in a population that had genetic examinations of their fetuses to exclude disorders. We attempted to detect HPV DNA sequences in their placentas during pregnancy by transabdominal placental biopsy. The results of fetal karyotyping are compared with HPV status of their cervices.


   Materials and Methods
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Abstract
 Materials and Methods
Results
 Discussion
 References
 
From November 1997 through October 1998, 226 consecutive women sent to our department for genetic examinations by chorionic villus sampling (CVS) or placental biopsy gave written informed consent and were entered in the study. The main indications for CVS and placental biopsy were conspicuous genetic disorders, abnormal ultrasound findings, positive maternal serum screenings, failed amniotic cell cultures, age exceeding 35 years, and gestation of more than 20 weeks (Table 1Go). Exclusion criteria were multiple gestation; clinically apparent genital warts; seropositivity for syphilis, human immunodeficiency virus (HIV), and hepatitis B or C viruses; and positive Papanicolaou tests in current pregnancies. In 79 women, we were not able to do HPV polymerase chain reaction (PCR) with cytogenetic examinations because not enough villous material was available. The HPV status of cervices was determined in 179 of 226 women (Table 2Go). Forty-seven did not receive HPV examinations of their cervices because of exclusion criteria4 or refusal of specimen collection.


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  		Table 1. Indications for Placental Biopsy
 

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  		Table 2. Patient Characteristics in Women With Cervical Examinations for HPV (n = 179)
 
Cell specimens from cervices were collected minutes before CVS procedures with the use of swabs with specimen transport medium (Digene Specimen Collection Kit, Digene Corp., Beltsville, MD), were taken to the laboratory, and stored at -20C. For analysis of cell specimens, a second-generation RNA probe assay with Hybrid Capture II technology was used (Digene HPV II Test, Digene Corp.)5 according to the manufacturer’s guidelines.

Villi obtained by placental biopsy were transferred immediately to medium (Ham’s F-10 medium with glutamin + 25% fetal calf serum + 0.5% gentamycin) and brought to the laboratory. Villi were cleaned macroscopically of blood and decidual remnants under sterile conditions to keep the risk of maternal contamination as low as possible. Samples of 1–2 mg of chorionic tissue were collected by CVS.

Direct preparation of placental villi was in accordance with a pattern proposed by Simoni et al6 and modified by the laboratory of the Division of Prenatal Diagnosis and Therapy. For questionable or poorly interpretable karyotype findings, eg, mosaicism or ambiguous structural aberrations, amniocentesis or cord blood biopsies were done.7

The guidelines of Bauer et al8 were used for detection of the HPV E6 region. Cervical cancer cell lines (HTB 35; American Type Culture Collection, Rockville, MD) containing 1–2 copies of HPV 16–DNA/cell and CaSki (CRL 1550, American Type Culture Collection), containing more than 100 copies of HPV 16–DNA/cell, were used as positive controls. Negative controls included breast carcinoma cell lines (MCF-7, HTB 22, American Type Culture Collection) and the elimination of Ampli-Taq polymerase (Perkin Elmer Cetus, Norwalk, CT) in the reaction mixture. As an additional control of specificity, we checked the coamplification in the sample for ß-globulin-genes. Human papillomavirus-DNA E6 primers WD 72 and WD 66 were used.

Statistical data were evaluated with SPSS 8.0 statistical package version 8.0 (Superior Performing Software Systems, Chicago, IL). The variables HPV A (low-risk HPV types: 6, 11, 42, 43, 44), HPV B (high/intermediate-risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68), fetal sex, and karyotype were tested for stochastic independence by {chi}2 test. If independence of two variables is refuted significantly by {chi}2, it is possible to measure the strength of association by {phi} coefficient, which is also called the Pearson product-moment correlation coefficient for the binary data and Cramer’s V coefficient, a measure of association based on {chi}2. Those contingency coefficients take values between -1 and 1. If {phi} and V are equal to 0, there is stochastic independence. Total association is expressed by -1 and 1.9 We also did univariate and multivariate logistic regression analyses to examine whether women’s ages, gestational weeks, marital statuses, pregnancies, abortions, interruptions, years of education, and smoking habits could be prognostic factors for HPV infection. We applied backstep selection, using the Wald statistic for multiple logistic regression analysis. The entry value was 0.05 and the removal value was 0.10. The odds ratios (ORs) and 95% confidence intervals (CIs) were computed as proportional hazards by using univariate and multiple logistic regression analyses. Groups defined by the cutoffs were centered by medians.


   Results
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Abstract
 Materials and Methods
 Results
Discussion
 References
 
The average age of the 226 women was 29.5 years (standard deviation [SD] = 6, range 15.3–46.2 years). The gestational week variable was not distributed normally. In that regard, the Kolmogorov-Smirnov test indicated a highly significant P < .001. The median time of examination was in the 22nd week of pregnancy (median 21.3, minimum 9.6, maximum 31.3).

Cases examined included 214 with normal and 12 with abnormal karyotypes (Table 3Go). The association between female sex and pathologic chromosome set in this study was weak, but statistically significant ({phi} = V = 0.24, P < .001 by {chi}2). Among 179 women who had their cervical HPV statuses determined, 44 (24.6%, 95% CI 18.3, 31.0) had positive HPV findings, whereas no HPV infections were found in 135 (75.4%, 95% CI 69.0, 81.8). Eighteen women (10.1%, 95% CI 5.6, 14.5) were HPV A–positive, and 37 (20.7%, 95% CI 14.7, 26.7) were infected with HPV B. Human papillomaviruses A and B were detected in 11 women (6.2%, 95% CI 2.6, 9.7).


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  		Table 3. Karyotype Abnormalities
 
There was a highly significant correlation between HPV A and HPV B infection. {chi}2 test refuted independence of the two variables (P < .001). {phi} coefficient and Cramer’s V coefficient gave an identical result of 0.33 at P < .001. We also did a multivariate logistic regression analysis to determine whether sociodemographics were prognostic factors for cervical HPV infections (Table 4Go). In the univariate logistic regression analysis, only the association between age and cervical HPV infection was significant (P = .039) (Table 5Go). The group younger than 30 years old had a higher risk for cervical HPV infection than women older than 30 years (OR 1.82, 95% CI 1.0, 3.2). Among 226 chorion biopsies or placental punctures, 147 provided enough material samples for HPV PCR. The HPV DNA E6 PCR was negative in all 147 cases. We also examined whether there was an association between HPV infection and elevated risk of abnormal karyotype. The independence of HPV A and abnormal karyotype was refuted narrowly by {chi}2 test. The result showed a barely significant association (P = .05) between HPV A and abnormal karyotype ({phi} = V = 0.146).


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  		Table 4. Multivariate Analysis of Risk Factors for HPV Infection*
 

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  		Table 5. Age Distribution and HPV Status of the Cervix (n = 179)
 

   Discussion
Top
Abstract
 Materials and Methods
 Results
 Discussion
References
 
The prevalence of HPV infection of the cervix in this cohort of pregnant women was 24.6%. The overall picture presented in the literature on HPV infection rates in pregnant women is inconsistent. Reported rates of HPV infections during pregnancy vary between 5%10 and around 50%.11 That could be owing to several factors, including choice of detection method, differences in risk factors (smoking, promiscuity, low socioeconomic level, infection with other sexually transmitted diseases, history of cervical dysplasia, and genital warts of included women), and designs of the studies. Maternal age seems to be one possible reason for variation. In our study, we found a significant decrease in HPV infection rates in older women, and other authors also observed that. De Roda Husman et al12 found that with increasing age, the prevalence of HPV infection declines in pregnant and nonpregnant women with cytomorphologically normal smears. Tenti et al10 examined a collective of pregnant women with no clinically apparent genital warts and detected higher rates of HPV in younger women. In contrast to our study, the results did not reach statistical significance.12 In that study, they also collected smears at term, whereas our median examination time was the 22nd week of pregnancy, owing to examination methods in our health system (mother-and-child pass).13 Subsequent studies of HPV infections in pregnant women have not been conclusive on influence of duration of pregnancy and HPV status. Some studies found increased incidence of HPV infection with higher gestational age,14 whereas other studies like ours reported no significant correlation between gestational age and varying HPV infection rates.11

Human papillomavirus DNA was not found in any of the 147 chorionic villi or placenta samples collected by transabdominal puncture. A case report of epidermodysplasia verruciformis has been reported, in which HPV types 3, 5, 8, 24, and 36 were found in the woman, the placenta, and amniotic fluid (AF) collected during cesarean delivery. Mononuclear cells in maternal blood were not infected, so it seems that infection was not by hematogenous transmission.15 There are different studies that prove the transmission rate for neonates delivered by cesarean is lower than vaginal delivery.16 Cesarean does not prevent HPV infection of the fetus, which makes the possibility of transmission in utero more likely. Human papillomavirus could be detected in AF of pregnant women with cervical lesions, and Tseng et al17 reported HPV infections in mononuclear cells of women and cord blood of their newborns. Hermonat et al18 were able to detect HPV DNA by using in situ PCR in syncytiotrophoblast cells from spontaneous abortions in HPV-positive pregnant women. Contamination of placental tissues through specimen collection or ascension of HPV could not be excluded.

The route by which HPV infects fetal compartments such as cord blood or AF is unknown. Viremia could be a route, but it has not been documented convincingly for HPV. A local spreading process between the vulva, cervix, and AF could be another possibility.3 During pregnancy, the uterine cavity has many physiologic changes that might allow viral particles to reach the amniotic cavity more easily. At the end of the first trimester, the lower uterine segment unfolds and the amniotic sac comes within anatomic proximity of the cervix, and during premature contractions the cervix shortens significantly.19 Infection of the oocyte or zygote before or soon after implantation and infection by sperm carrying a latent HPV infection are reported.20

In cases affected by HPV A, our study found association between HPV infection of the cervix and abnormal karyotype in the fetus. Human papillomavirus A–positive women had a slightly higher significant risk of pathologic chromosome sets. The effect of HPV in promoting abnormal karyotypes might be owing to HPV DNA integration, causing genomic alterations from gene interruption and loss of chromosome heterozygosity21 that might lead to chromosome aberration. The remaining question is what effect a larger sample has on increased correlations and contingency coefficients.

Two large series of reports on spontaneous abortions showed an overall excess of female over male trisomies,22,23 whereas Hassold et al24 found a sex ratio among abortions with abnormal chromosome sets with a male excess in trisomies 21 and 22 and a female excess in trisomy 9. Hook et al25 reported on about 24,951 fetuses studied prenatally. In their analysis of results in live births, the rate of all structural rearrangements, balanced and unbalanced, was increased in females, although none of the trends was significant.


   Footnotes
 
The authors thank Dr. Ernst Rücklinger helped with statistical analysis.

PII S0029-7844(00)00953-4

Received December 16, 1999. Received in revised form April 14, 2000. Accepted May 11, 2000.


   References
Top
Abstract
 Materials and Methods
 Results
 Discussion
 References
 
1. Beutner KR, Tyring S. Human papillomavirus and human disease. Am J Med 1997;102:9–15.[Medline]

2. Schneider A, Hotz M, Gissmann L. Increased prevalence of human papillomaviruses in the lower genital tract of pregnant women. Int J Cancer 1987;40:198–201.[Medline]

3. Armbruster-Moraes E, Ioshimoto LM, Leão E, Zugaib M. Presence of human papillomavirus DNA in amniotic fluids of pregnant women with cervical lesions. Gynecol Oncol 1994;54:152–8.[Medline]

4. Meisels A, Morin C. The gynecologic smear. In: Meisels A, Morin C, eds. Cytopathology of the uterine cervix. Chicago: ASCP Press, 1990:243–58.

5. Clavel C, Masure M, Bory JP, Putaud I, Mangeonjean C, Lorenzato M, et al. Hybrid Capture II–based human papillomavirus detection, a sensitive test to detect in routine high-grade cervical lesions: A preliminary study on 1518 women. Br J Cancer 1999;80:1306–11.[Medline]

6. Simoni G, Brambati B, Danesino C, Rossella F, Terzoli GL, Ferrari M, et al. Efficient direct chromosome analyses and enzyme determinations from chorionic villi samples in the first trimester of pregnancy. Hum Genet 1983;63:349–57.[Medline]

7. Miny P, Basaran S, Pawlowitzki IH, Horst J, Westendorp A, Niedner W, et al. Validity of cytogenetic analyses from trophoblast tissue throughout gestation. Am J Med Genet 1989;33:136–41.[Medline]

8. Bauer HM, Greer CE, Manos MM. Determination of genital human papillomavirus infection by consensus PCR amplification. In: Herrington CS, McGee JO, eds. Diagnostic molecular pathology. New York: Oxford University Press, 1992:131–52.

9. Goodman LA, Kruskal WH. Measures of association for cross classifications. New York: Springer, 1979.

10. Tenti P, Zappatore R, Migliora P, Spinillo A, Maccarini U, De Benedettis M, et al. Latent human papillomavirus infection in pregnant women at term: A case-control study. J Infect Dis 1997;176:277–80.[Medline]

11. Kemp EA, Hakenewerth AM, Laurent SL, Gravitt PE, Stoerker J. Human papillomavirus prevalence in pregnancy. Obstet Gynecol 1992;79:649–56.[Abstract/Free Full Text]

12. De Roda Husman AM, Walboomers JMM, Hopman E, Bleker OP, Helmerhorst TMJ, Rozendaal L, et al. HPV prevalence in cytomorphologically normal cervix scrapes of pregnant women as determined by PCR: The age-related pattern. J Med Virol 1995;46:97–102.[Medline]

13. Bernaschek G, Stuempflen I, Deutinger J. The influence of the experience of the investigator on the rate of sonographic diagnosis of fetal malformations in Vienna. Prenat Diagn 1996;16:807–11.[Medline]

14. Morrison EAB, Gammon MD, Goldberg GL, Vermund SH, Burk RD. Pregnancy and cervical infection with human papillomaviruses. Int J Gynaecol Obstet 1996;54:124–30.

15. Favre M, Majewski S, De Jesus N, Malejczyk M, Orth G, Jablonska S. A possible vertical transmission of human papillomavirus genotypes associated with epidermodysplasia verruciformis. J Invest Dermatol 1998;111:333–6.[Medline]

16. Tseng CJ, Liang CC, Soong YK, Pao CC. Perinatal transmission of human papillomavirus in infants: Relationship between infection rate and mode of delivery. Obstet Gynecol 1998;91:92–6.[Abstract]

17. Tseng CJ, Lin CY, Wang RL, Chen LJ, Chang YL, Hsieh TT, et al. Possible transplacental transmission of human papillomaviruses. Am J Obstet Gynecol 1992;166:35–40.[Medline]

18. Hermonat PL, Kechelava S, Lowery CL, Korourian S. Trophoblasts are the preferential target for human papilloma virus infection in spontaneously aborted products of conception. Hum Pathol 1998; 29:170–4.[Medline]

19. Eppel W, Schurz B, Frigo P, Kudielka I, Wenzl R, Reinold E. Imaging of premature uterine contraction in vaginal sonography. Z Geburtshilfe Perinatol 1992;196:106–10.[Medline]

20. Lai YM, Yang FP, Pao CC. Human papillomavirus deoxyribonucleic acid and ribonucleic acid in seminal plasma and sperm cells. Fertil Steril 1996;65:1026–30.[Medline]

21. Gallego MI, Lazo PA. Deletion in human chromosome region 12q13–15 by integration of human papillomavirus DNA in a cervical carcinoma cell line. J Biol Chem 1995;270:24321–6.[Abstract/Free Full Text]

22. Boué J, Boué A, Lazar P. Retrospective and prospective epidemiological studies of 1500 karyotyped spontaneous human abortions. Teratology 1975;12:11–26.[Medline]

23. Creasy MR, Crolla JA, Alberman ED. A cytogenetic study of human spontaneous abortions using banding techniques. Hum Genet 1976;31:177–96.[Medline]

24. Hassold T, Chen N, Funkhouser J, Jooss T, Manuel B, Matsuura J, et al. A cytogenetic study of 1000 spontaneous abortions. Ann Hum Genet 1980;44:151–78.[Medline]

25. Hook EB, Schreinemachers DM, Willey AM, Cross PK. Inherited structural cytogenetic abnormalities detected incidentally in fetuses diagnosed prenatally: Frequency, parental-age associations, sex-ratio trends, and comparisons with rates of mutants. Am J Hum Genet 1984;36:422–43.[Medline]





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