Use of various contraceptives and human papillomavirus 16 and 18 infections in women with cervical intraepithelial neoplasia

Abstract

The aim of the study was to evaluate the frequency of human papillomavirus (HPV) 16 and 18 infections in patients with cervical intraepithelial neoplasia (CIN) according to the use of various contraceptive methods. In a study group of 1435 patients with histologically proven CIN, among whom 391 (27.2%) used no contraception, 44 (3.1%) used barrier methods, 705 (49.1%) used oral contraceptives and 295 (20.7%) used an intrauterine device (IUD), the presence of HPV infection was evaluated by DNA in situ hybridization. HPV 16 or 18 infection was present in 666 (46.4%) of all patients in 348 (49.4%) patients who used oral contraceptives, in 169 (43.2%) patients with no contraception, in 128 (43.4%) patients with an IUD and in 21 (47.7%) patients who used barrier methods. There were no significant differences in HPV 16 and 18 infection frequencies among all four groups of patients regarding the contraception method used.

Keywords

contraception human papillomavirus 16 and 18 cervical intraepithelial neoplasia

Introduction

Persistent high-risk human papillomavirus (HR-HPV) infection is the most important aetiological agent of cervical intraepithelial neoplasia (CIN) and cervical cancer (CC). Recently, it has been demonstrated that HPV testing increases the detection of CIN.¹ The National Health and Nutrition Examination Survey 2003–2004 in the USA demonstrated the seroprevalences of HPV types 16 and 18 among female subjects to be 15.6% and 6.5%, respectively. Among males, the seroprevalences were lower for each type, with 5.1% observed for HPV 16 and 1.5% for HPV 18.² In patients with CIN3, HPV 16 and 18 are even more prevalent, detected in up to 67.4%.³ In patients with CC, HPV 16 is present in 64.9% and HPV 18 in 12.2%.⁴

Although many prior studies have evaluated contraceptive risk factors for CIN and CC, few have controlled for the presence of cervical HPV infection. HPV infections are closely related to the sexual behaviour of women, which is closely linked with individual women's preferences for contraception modes.

An approximately equal number of studies have reported either an increased risk of HPV infection among oral contraception (OC) users, or failed to establish any such risk.⁵ Moreover, some studies found OC use as protective against incident HPV infections.^6,7

Coker et al.⁸ demonstrated that among HR-HPV-positive women, a longer duration of barrier method use was associated with a reduced risk of CIN. Other studies did not confirm the protective role of condom use for the development of CIN.⁹

The use of an intrauterine device (IUD) might be associated with an increased risk of CIN,⁹ yet in a study performed by Redecha et al.¹⁰ the higher prevalence of HPV (by 6.6%) in women with an IUD as compared with the control group was not statistically significant.

Conflicting results of several studies dealing with contraception use and HPV infection in CIN patients led us to evaluate the impact of various contraception methods used on HPV 16 and 18 infections in patients with CIN. We hypothesized that different contraceptive methods might have an impact on the acquisition of these viruses.

Methods

Study design

The study subjects consisted of 1435 patients who underwent conization of the uterine cervix at the University Clinical Department of Gynecology and Perinatology of the University Clinical Centre Maribor, Slovenia. Inclusion criteria included any woman requiring excisional treatment or conization of the uterine cervix at our center. Ethical Committee approval was obtained for the study and all patients eligible to take part in the study were provided with a patient information leaflet. Informed consent was obtained from all subjects.

Laboratory procedure

On the basis of cytological examination, colposcopy and biopsy, patients with presumed high-grade squamous intraepithelial lesions were referred for excisional treatment or conization of the uterine cervix. Before surgery, cytological material for DNA in situ hybridization was obtained with plastic cytobrushes. Cervical smears were immediately put into an appropriate buffer and transported to the laboratory. Smears were vortexed vigorously. After sedimentation of cells, the cytological smears were prepared. The cells were fixed in cold acetone and hybridization procedures were performed. Biotinylated probes were used (Epignost, Euro Diagnostics, Giessen, Germany). The hybrids were detected using streptavidin peroxidase complex with H₂O₂ as the substrate and DAB as chromogen. A red-brown precipitate in the nuclei of the cells was considered as a positive reaction.

Treatment and histopathological procedure

The surgical procedure was performed under local anaesthesia for excisional treatment (large loop excision of the transformation zone [LLETZ]) or short-term general anaesthesia under aseptic conditions for conization. The collected material was transferred directly to the laboratory where tissue blocks were fixed in 10% buffered neutral formaldehyde, prepared using standard paraffin embedding, sectioned at 5 μm and stained with haematoxylin and eosin. The slides were reviewed by an expert pathologist.

Statistical analysis

Charts were reviewed and the following data were obtained: age, parity, abortions, age at first intercourse, number of lifetime sex partners, contraception use, HPV 16/18 infection and definitive histology. HPV-infected and non-infected groups of patients with different contraceptive use were compared for significant differences using the χ² test. A P value of less than 0.05 was considered to indicate a significant difference.

Results

The key epidemiological characteristics of study patients are shown in Table 1. The average age of all patients was 35.7 ± 9.8 years (mean ± SD). On average, patients had 1.9 ± 1.1 pregnancies, 1.4 ± 1.0 parturitions, 0.6 ± 1.1 terminations of pregnancies and 0.1 ± 0.5 spontaneous abortions (mean ± SD). The mean age at first intercourse was 17.6 ± 1.9 years and the average number of lifetime sex partners was 2.8 ± 2.2 (mean + SD).

Table 1

Demographic characteristics of study participants (n = 1435)

Variable	Contraceptive method (mean ± SD)
Variable	No	OC	IUD	Barrier methods
Age (years)	36.2 ± 10.1	33.5 ± 7.9	39.6 ± 8.2	38.3 ± 11.6
No. of pregnancies	1.9 ± 0.9	2.1 ± 1.2	2.4 ± 1.0	1.5 ± 1.0
No. of abortions	0.5 ± 0.8	0.7 ± 1.5	0.7 ± 0.9	0.6 ± 0.9
No. of deliveries	1.3 ± 0.9	1.3 ± 1.1	1.7 ± 0.8	1.4 ± 0.9
No. of lifetime sex partners	2.8 ± 1.8	3.0 ± 2.4	2.5 ± 2.4	2.9 ± 1.5
Age at first intercourse	17.8 ± 1.9	17.6 ± 1.7	17.7 ± 1.9	18.1 ± 2.0

OC = oral contraceptives; IUD = intrauterine device

According to definitive histology in the study group, there were 25 (1.7%) patients with cervical condylomas, 94 (6.6%) with CIN 1, 301 (21.0%) with CIN 2, 988 (68.8%) with CIN 3, 18 (1.3%) with microinvasive CC (stromal invasion less than 5 mm) and nine (0.6%) with invasive CC.

The frequencies of HPV 16/18 infection in cervical lesions in non-users of contraception are shown in Table 2, in OC users in Table 3, in IUD users in Table 4 and in users of barrier methods in Table 5.

Table 2

HPV 16 and 18 infections in cervical lesions of non-users of contraception (n = 391)

Cervical lesion	HPV 16/18 negative n (%)	HPV 16/18 positive n (%)
Condyloma	1 (0.2)	5 (1.3)
CIN 1	9 (2.4)	10 (2.6)
CIN 2	57 (14.6)	25 (6.4)
CIN 3	153 (39.1)	127 (32.5)
MIC	2 (0.5)	1 (0.2)
CC	0	1 (0.2)
Total	222 (56.8)	169 (43.2)

MIC = microinvasive carcinoma; CC = invasive cervical carcinoma; HPV= human papillomavirus; CIN = cervical intraepithelial neoplasia

Table 3

HPV 16 and 18 infections in cervical lesions of OC users (n = 705)

Cervical lesion	HPV 16/18 negative n (%)	HPV 16/18 positive n (%)
Condyloma	9 (1.3)	3 (0.4)
CIN 1	31 (4.4)	23 (3.3)
CIN 2	75 (10.6)	69 (9.8)
CIN 3	230 (32.6)	248 (35.2)
MIC	9 (1.3)	3 (0.4)
CC	3 (0.4)	2 (0.3)
Total	357 (50.6)	348 (49.4)

MIC = microinvasive carcinoma; CC = invasive cervical carcinoma; HPV= human papillomavirus; OC = oral contraceptive; CIN = cervical intraepithelial neoplasia

Table 4

HPV 16 and 18 infections in cervical lesions of IUD users (n = 295)

Cervical lesion	HPV 16/18 negative n (%)	HPV 16/18 positive n (%)
Condyloma	4 (1.4)	3 (1.0)
CIN 1	9 (3.1)	9 (3.1)
CIN 2	39 (13.2)	30 (10.2)
CIN 3	110 (37.3)	85 (28.8)
MIC	3 (1.0)	0
CC	2 (0.6)	1 (0.3)
Total	167 (56.6)	128 (43.4)

MIC = microinvasive carcinoma; CC = invasive cervical carcinoma; IUD = intrauterine device; HPV = human papillomavirus; CIN = cervical intraepithelial neoplasia

Table 5

HPV 16 and 18 infections in cervical lesions of barrier methods users (n = 44)

Cervical lesion	HPV 16/18 negative n (%)	HPV 16/18 positive n (%)
Condyloma	0	0
CIN 1	1 (2.3)	2 (4.5)
CIN 2	2 (4.5)	4 (9.1)
CIN 3	20 (45.5)	15 (34.1)
MIC	0	0
CC	0	0
Total	23 (52.3)	21 (47.7)

MIC = microinvasive carcinoma; CC = invasive cervical carcinoma; HPV = human papillomavirus; CIN = cervical intraepithelial neoplasia

The difference in the frequency of HPV 16/18 infection between non-users of contraception and OC users was not significant (χ² = 3.561; P > 0.05), neither was it significant between non-users of contraception and IUD users (χ² = 0.001; P > 0.05) nor between non-users of contraception and users of barrier methods (χ² = 0.169; P > 0.05). Similarly, there was no significant difference in HPV 16/18 infection between OC users and IUD users (χ² = 2.739; P > 0.05), OC users and users of barrier methods (χ² = 0.003; P > 0.05), as well as IUD users and users of barrier methods (χ² = 0.143; P > 0.05).

Table 6 summarizes the frequencies of HPV 16/18 infection in patients with CIN regarding the use of various methods of contraception.

Table 6

HPV 16 and 18 infections in patients with CIN regarding various methods of contraception use (n = 1435)

Contraception	HPV 16/18 negative n (%)	HPV 16/18 positive n (%)
No	222 (56.8)	169 (43.2)
OC	357 (50.6)	348 (49.4)
IUD	167 (56.6)	128 (43.4)
Barrier methods	23 (52.3)	21 (47.7)
All	769 (53.6)	666 (46.4)

OC = oral contraceptives; IUD = intrauterine device; HPV= human papillomavirus; CIN = cervical intraepithelial neoplasia; NS = not significant

No: OC, χ² = 3.561; NS

No: IUD, χ² = 0.001; NS

No: barrier methods, χ² = 0.169; NS

OC: IUD, χ² = 2.739; NS

OC: barrier methods, χ² = 0.003; NS

IUD: barrier methods, χ² = 0.143; NS

Discussion

To the best of our knowledge, this is the first study that looks at HPV 16/18 infection in women with CIN according to the use of different contraceptive methods in Slovenia. Uršič-Vrščaj et al.¹¹ studied risk factors in 70 patients with cervical carcinoma and when analysing some already known risk factors, no statistically significant differences could be established for any of the factors studied, except for the age at first birth. Futai et al.¹² demonstrated that 34.0% of healthy patients, 86.4% of patients with CIN 1, 83.9% with CIN 2, 87.7% with CIN 3 and 94.3% with invasive CC had results positive for 39 genotypes of HPV. In our study, we used the identification of HPV genotypes 16 and 18 only, therefore the proportion of HPV infection was considerably lower.

In a study performed by Gitsch et al.¹³ on 142 patients with CIN, HPV infection was detected in 48% of patients. Among these 142 patients, there were 56 (39.4%) patients using OC. In our study HPV 16/18 infection was present in 46.4% of all patients and the proportion of OC users was 49.1%.

Coker et al.¹⁴ examined the influence of barrier methods of contraception on the development of CIN in 103 women with CIN and in 258 patients with normal cervical cytology. They found that barrier methods of contraception were associated with a reduced risk of preinvasive cervical neoplasia with the adjusted odds ratio of 0.5 (95% confidence interval: 0.2–0.9). Using the same study population, Coker et al.¹⁵ explored the somewhat controversial relationship between OC and CIN and found no positive association between OC and CIN. In our study barrier methods were the least frequent method of contraception used, namely in only 44 (3.1%) of all patients. In these patients HPV 16/18 infection was present in 47.7%, so we can confirm the observation of other authors that barrier methods do not protect against HPV infection.¹⁶ In the early 1990s, Thomas et al.¹⁷ performed a prospective, controlled study of condom use in 46 patients with histologically proven CIN 1. They were allocated to either condom use or non-condom use for six months. At the end of the study, they found no difference between the groups with respect to outcome. They concluded that condom usage is not an effective treatment for CIN 1. We did not evaluate the possible protective value of condom use on the development of CIN in a prospective study. Moreover, because of the relatively small number of patients in this group no clear conclusion can be drawn.

Schiff et al.⁹ evaluated contraceptive and reproductive risk factors for CIN in a clinic-based case-control study of 628 American Indian women. Among all contraceptives, they found only IUD use to be associated with CIN. In our study we found no association between IUD use and HPV 16/18 infection. Among IUD users, 43.4% were infected with one or both types of HPV and the infection rate did not differ from other groups of patients.

McFarlene-Anderson et al.¹⁸ demonstrated an increased risk for developing CIN in Jamaican women using OC. Syrjänen et al.⁵ found OC not to be an independent risk factor for CIN or high-risk HPV infection. Moreover, they clearly demonstrated that sexual behaviour was different among OC users, non-OC users and non-users of contraception and that these different risk factors predisposed the women to high-risk HPV, development of high-grade CIN and also influenced the outcome of their cervical disease/high-risk HPV infection, which was similar irrespective of their OC status. We concur with these results, because in our study 49.4% of OC-using patients were infected with HPV 16/18, which was not different from other groups of patients.

Our study has several potential limitations that must be considered in the interpretation of our results. A limiting factor in our study might be the presence of contamination by normal cells in the cervical swabs, which are supposed to collect both normal and premalignant cells from the cervix. Most studies done so far have looked at DNA levels, while the study performed by Gnanamony et al.¹⁹ showed that active replication, as seen by an increasing mRNA transcript level, can be a marker of advancing cervical disease. Another limitation is the relative low number of patients who used barrier methods. Moreover, our HPV data are limited by our collection of a single specimen at study entry. We agree with Schiff et al.⁹ that additional sampling would likely result in a higher proportion of women with HPV infection, but this higher proportion would be probably equally distributed among all groups of patients using different contraceptive methods. Another limitation of our study is the absence of information on women presenting with CIN who did not have conization and therefore were excluded from the study. Moreover, in our study we did not collect data regarding the duration of use of each contraceptive method, which might be important considering that the development of CIN takes years.

In conclusion, our study demonstrated that HPV 16/18 infection is present in nearly half of patients with CIN, and is equally prevalent in contraception users and non-users. Our data show that there is no difference in the frequency of HPV 16/18 infection among women with CIN using various methods of contraception. Moreover, our study confirms the lack of association between contraceptive use and acquisition of HPV 16/18.

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