Archives o f

Arch Gynecol Obstet (1992) 252:25-30

Gynecology and Obstetrics

© Springer-Verlag1992

Oral contraceptives and human papiHomavirus infection in cervical intraepithelial neoplasia G. Gitsch 1, C. Kainz 1, M. Studnicka 2, A. Reinthaller 1, G. Tatra 1, and G. Breitenecker 3 1Second Department of Obstetrics and Gynecology, 2Institute of Medical Statistics and Documentation, and 3Department of Pathology, Unit of Gynecopathology, University of Vienna and Medical School, Spitalgasse 23, A-1090 Vienna, Austria Received January 17, 1992/Accepted May 27, 1992

Summary. We report about 142 patients from whom colposcopically directed cervical punch biopsies were taken which showed condylomatous lesions with or without cervical intraepithelial neoplasia (CIN). Fiftysix (39.4%) of these women used oral contraceptives (OC) for at least two years before examination. We used D N A in situ hybridization on all biopsies for detection of human papillomavirus (HPV)-DNA. Among OC users a significant trend towards higher HPV infection rates in high grade CIN (odds ratio 2.9, P < 0.05) was found, whereas non-users of oral contraceptives had the highest HPV infection rate in condylomatous lesions without CIN (odds ratio 0.5, P < 0.05). Thus in OC users HPV infection was about 24 times more likely in CINIII as in condyloma, whilest among non-users the trend was the other way round (7-fold likelyhood of HPV positivity in condyloma compared to CIN III). Other known risk factors for cervical carcinoma did not influence HPV infection rates in either group. Key words: Oral contraceptives - Human papillomavirus- Cervical intraepithelial neoplasia Introduction Many factors play a role in natural history of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (Reid et al. 1982, Reid et al. 1987, Sebastian 1987). Epidemiologic studies have shown that promiscuity and low social status are important in the development of CIN (Naguib et al. 1966, Reeves et al. 1989, Gitsch et al. 1991). Furthermore several studies have shown an association between smoking, oral contraceptives (OC) and parity on the one hand and cervical cancer on the

Correspondence to: Dr. G. Gitsch (address see above)

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G. Gitsch et al.

o t h e r ( L y o n et al. 1983, G r e e n b e r g et al. 1985, W i n k e l s t e i n 1986, W H O 1985, B r i n t o n et al. 1986 a n d 1987, B e r a l et al. 1988). D u r i n g t h e last d e c a d e t h e i m p o r t a n c e of H P V in C I N has b e e n r e c o g n i z e d ( R e i d e t al. 1987). C e r t a i n s t r a i n s o f H P V D N A a r e a p p a r e n t l y a s s o c i a t e d w i t h C I N a n d invasive cervical c a n c e r a n d this has p r o d u c e d efforts to d e t e c t p r e c a n c e r o u s l e s i o n s with a s s o c i a t e d H P V i n f e c t i o n as e a r l y as p o s s i b l e ( R e i d et al. 1982). T h e n a t u r a l h i s t o r y o f H P V - a s s o c i a t e d C I N d e p e n d s o n t h e g r a d e o f t h e l e s i o n a n d o n t h e virus t y p e (Syrjfinen et al. 1985, Z u r H a u s e n 1986, M e a n w e l l e t al. 1987). H P V t y p e s 16, 18, 31, 33 h a v e b e e n a s s o c i a t e d w i t h C I N I I I a n d i n v a s i v e cervical c a n c e r , w h e r e a s H P V 6 a n d 11 w e r e a s s o c i a t e d w i t h l e s s e r g r a d e s o f C I N a n d also c o n d y l o m a w i t h o u t a t y p i a . T h e r e also s e e m s to b e an i n t e r a c t i o n b e t w e e n o r a l c o n t r a c e p t i v e s a n d H P V i n f e c t i o n in w o m e n with cervical c a n c e r . H i l d e s h e i m et al. (1990) f o u n d a 2.3 f o l d i n c r e a s e d risk o f H P V p o s i t i v i t y in c a r d n o m a t o u s tissue o f O C u s e r s as c o m p a r e d to n o n - u s e r s . T h i s p r o m p t e d us to e v a l u a t e t h e p r e v a l e n c e o f H P V in tissue f r o m 56 O C u s e r s a n d 86 n o n - u s e r s w i t h C I N o r c o n d y l o m a t a . I n situ h y b r i d i z a t i o n was u s e d for D N A d e t e c t i o n r a t h e r t h a n t h e s o u t h e r n b l o t t e c h n i q u e w h i c h o f t e n is n o t satisfying in v e r y s m a l l tissue s p e c i m e n s o r t h e p o l y m e r a s e c h a i n r e a c t i o n w h i c h is t o o sensitive, giving false p o s i t i v e s d u e to c o n t a m i n a t i o n .

Material and methods Patients

We studied 142 patients who were referred to our colposcopy clinic because of cervical smears indicating CIN and/or abnormal colposcopy findings. The mean age was 32 years, 56 (39.4%) used hormonal contraception for at least two years before examination, 97 (68.3%) were smokers, 69 (49%) were nullipara and 33 (23.2%) were prostitutes. In situ hybridization

All biopsy specimen were examined by in situ hybridization according to the method described by H6fler 1987. Five micron thick sections of f0rmalin-fixed and paraffin-embedded biopsies were mounted on amino-alcylisane pretreated slides, deparaffinizised and then incubated for 10 min in 0.05 % pronase in PBS (phosphate buffered saline) at 37° C. Biotin-marked DNA-probes for HPV 6/ 11, 16/18 and 31/33 were added (Enzo Biochem. Inc. New York, USA) and glass covered. Denaturation was performed in a microwave (HM 146, Elektra Bregenz, Schwaz, Austria) for 2 rain at 240 Watts and 6 min at 120 Watts (microwave frequency: 2450 MHz). Hybridization for 60 rain at room temperature was followed by rinsing in PBS and the subsequent use of mouse-a-biotin (1 : 100 dilution in PBS) (Dakopatts, Carpintiera, USA). The incubation time for mouse-a-biotin was 30 min at room temperature. Then the Calf intestinal Alkaline Phosphatase and mouse monoclonal Anti-Alkaline Phosphatase staining (APAAP) method was used: we applied bridge antibody rabbitanti-mouse immunoglobulin (Dakopatts, Carpintiera, USA), 1:30 dilution in PBS, and incubated for 30 rain at room temperature. After rinsing in PBS we added the APAAP-complex (1 : 50 in PBS) (Dakopatts, Carpintiera, USA) and stored for 30 rain at room temperature. As a substrate we used 2% 3-amino-9-ethylcarbazole and 3% HzOz in 0.05 M acetate buffer, rinsed in destilled water and counterstained using hematoxylin for 30 s.

Cervical lesions: oral contraceptives and HPV infection

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Statistics

Relative risk was derived from odds ratios (OR). OR and two tailed P-values were calculated to express the relationship between CIN, HPV infection, OC-use and other risk factors. Adjustment for confounding variables was achieved by means of logistic regression analysis (Cornfield 1956), and adjusted OR's derived. The test for trend (Cochran 1954) was applied to assess decrease or increase in risk by grade of CIN as associated with HPV infection and OC-use (Cornfield 1962). Statistical analysis was calculated with the software package E G R E T (Epidemiological Graphics, Estimation and Testing Package, (c) 1985-1989, SERC and Cytel, USA).

Results

HPV was detected in 48% of 142 patients. No significant difference was found between OC users and non-users. Amongst the OC users a significant trend to higher HPV infection rates in high grade CIN became evident (test for trend: unadjusted P-value < 0.02). The relative risk, as estimated by odds ratio, for HPV positivity was 1.99 fold in CIN I lesions, 5.42 fold in CIN II lesions and infinitivly higher in CIN III lesions when compared to HPV positivity in condylomas without atypia (Table 1). In OC non-users the results were the other way round, the highest HPV infection rate being found in condylomas without atypia and mild dysplasia. Before adjusting for confounding variables the trend was not significant (test for trend: unadjusted P-value < 0.1) (Table 2). The adjusted trend and odds ratios for HPV positivity in different grades of cervical lesions were adjusted for cervical cancer risk factors namely sexual activity, smoking, parity and age. The resultant trend for increased HPV Table 1. HPV infection rate in cervical lesions of OC users (n = 56)

Cervical lesion

HPV positive n

HPV positive %

Condyloma CIN I CIN II CIN III

4/17 11/29 5/8 2/2

24 38 62 100

Crude odds ratio

P-value

1.99 5.42 infinitive

0.32 0.06 0.03

Test for trend: P = 0.013 Table 2. HPV infection rate in cervical lesions of OC non-users (n = 86)

Cervical lesion

HPV positive n

HPV positive %

Condyloma CIN I CIN II CINIII

18/30 20/33 6/1735 2/6

60 61 0.36 33

Test for trend: P = 0.077

Crude odds ratio

P-value

1.03 0.11 0.33

0.96 0.24

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Table 3. HPV infection rate in cervical lesions of OC users and non-users adjusted for confounding variables

OCuser

CIN(by grade)

OC non-user

P-value

Adjustedodds ratioa

P-value

Adjustedodds ratioa

0.022

2.88

0.024

0.52

a The overall OR expressing the linear trend as present by stage of CIN Table 4. HPV typingin 142 biopsies of cervical lesions of OC users (n = 56) and non-users (n = 86)

Cervical lesions

HPV types 6/11

16/18 31/33

6/11 + 16/18

6/11 + 31/33

16/18+ 31/33

o c users Condylorna CINI CIN II CIN III

1 4 0 0

1 2 1 0

2 4 2 0

0 0 0 0

0 0 0 1

0 1 2 1

OC non-users Condyloma CIN I CIN II CIN III

5 4 1 0

3 4 4 1

5 6 1 1

0 2 0 0

2 1 0 1

3 3 0 1

positivity with grade of CIN stayed significant in O C users (P < 0.05, O R : 2.88). After adjustment for confounding variables the inverse trend in n o n - O C users also became significant (P < 0.05, O R : 0.52). Trends were calculated for each of the variables adjusted for the others and were similar for all potential risk factors (Table 3). Distribution of H P V types 6/11, 16/18 and 31/33 was similar in O C users and non-users (Table 4).

Discussion

The interaction between steroid hormones, H P V and the generation of cervical cancer has been subject of several biological and clinical investigations. Cervical tissue has h o r m o n e receptors and administration of hormones can result in histological alterations (Gall et al. 1969, Ford et al. 1983). There might be an interaction between oral contraceptives and H P V in cervical cancer since the transcriptional region of H P V 16 D N A contain h o r m o n e recognition elements (Gloss et al. 1987). In addition Pater et al. reported the oncogenic transformation of baby rat kidney cells in vitro with a combination of H P V 16 D N A , but not H P V 11 D N A , and the activated form of the human h-ras oncogene in the presence of dexamethasone (Pater et al. 1990). In a second report the same group provided evidence that the oncogenic transformation of baby rat kidney

Cervical lesions: oral contraceptives and HPV infection

29

cells by H P V 16 D N A plus ras oncogene was dependent on the presence of progesterone or its derivate, norgestrel. They also observed oncogenic transformation with H P V 16 D N A plus ras in the presence of ethanol soluble extracts from two brands of commonly used contraceptive tablets (Pater et al. 1988). This indicates that oral contraceptive use may be a risk factor for high grade CIN and invasive cancer, possibly by enhancing the expression of the virus and increasing the likelihood that H P V will induce transformation of cervical epithelial cells. However, we have to take in account that O C users could be at more risk of infection due to increased sexual activity. Other risk factors for cervical cancer such as smoking and parity showed no significant interaction with H P V infection. Hildesheim et al. reported that oral contraceptive use was associated with an increased risk of positivity to HPV. W o m e n with invasive cervical cancer who were recent contraceptive users were at a 2.3 fold increased risk of H P V positivity compared to non-users. Long term users were at a 2.9 fold increased risk of H P V positivity compared with non-users. However, it was not possible to determine the independent effects of recent and long term users because of the high correlation (r = 0.95) of these two variables (Hildesheim et al. 1990). Our data also show that H P V positivity in high grade cervical dysplasia is significantly increased in OC users as compared to non-users. In 1977 Stern et al. have suggested that oral contraceptives may accelerate the progression of preinvasive lesions to invasive lesions (Stern et al. 1977). This suggestion is supported by our finding a significant increase of H P V positivity in advanced stages of CIN when oral contraceptives are used. The high H P V detection rate in low grade CIN in non-OC-users might suggest that H P V positive dysplasia in such patients remains stable or regresses spontaneously. In the presence of H P V infection O C usage seems to make progression from low grade to high grade dysplasia more likely with resultant increases in H P V positivity in more advanced stages of CIN. This hypothesis is supported by the fact the incidence of "high risk" H P V types 31/33 and 16/18 was nearly equal in O C users and non-users. Our results suggest that oral contraceptives enhance the possibility of neoplastic change due to H P V infection.

Acknowledgements. The authors thank Mrs. Andrea Steiner for providing excellent technical assistance. The study was supported by the "Medizinisch-wissenschaftlicherFonds des Bfirgermeisters der Bundeshauptstadt Wien".

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Oral contraceptives and human papillomavirus infection in cervical intraepithelial neoplasia.

We report about 142 patients from whom colposcopically directed cervical punch biopsies were taken which showed condylomatous lesions with or without ...
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