Objective: Human papillomavirus (HPV) vaccination is currently not included in the national vaccination program in Iran. Regional data on the distribution of HPV types in women are important as they can predict the impact of currently available vaccines and help health policy makers to consider all the possibilities with regard to HPV vaccination. Methods: A total of 1,218 Iranian women with normal cervical cytology were included in this study. The presence of the HPV genome was investigated in all specimens by PCR assay, and all HPV-positive samples were genotyped. Results: Totally, HPV was detected in 88 samples (7.2%). According to different geographical regions, the HPV prevalence varied: the highest HPV prevalence was observed in the North (11.7%) and the lowest in the Center (4.5%) of Iran, and this difference reached a statistically significant level (p < 0.05). In this study, the most frequent HPV types were HPV 16, 18, 66, and 11, representing 63.8% of all HPV infections. Conclusion: Based on this study, it is estimated that HPV vaccines could have a great impact on the prevention of cervical cancer in Iran. This study highlights the necessity of introducing educational programs in high schools, robust screening programs, and vaccination in Iran.

Cervical cancer is the 4th most common cancer among women worldwide. Indeed, just in 2012, an estimated 528,000 new cases and 266,000 deaths by cervical cancer occurred globally [1]. The incidence rate of cervical cancer in Iranian women was reported to be around 5:100,000, ranging from 4.1 to 0.4. Analysis of age-specific incidence rates indicates that cervical cancer incidence peaks in women aged 55-65 years [2].

Human papillomavirus (HPV) is an essential trigger in the development of cervical cancer. It is known that almost all cases are associated with high-risk HPV types, including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73 [3,4,5,6,7]. HPV 16 and 18 account for 70.1% of cases worldwide [8]. Three prophylactic HPV vaccines are currently available for cervical cancer prevention. Cervarix is a bivalent vaccine consisting of HPV 16 and 18 types. Two kinds of Gardasil vaccines are available including quadrivalent and ninevalent vaccines; the quadrivalent consisting of HPV 6, 11, 16, and 18 types and the ninevalent containing HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58 types [9,10,11].

HPV vaccination is currently not included in the national vaccination program in Iran. Regional data on the distribution of HPV types in women are important as they can predict the impact of currently available vaccines and help health policy makers to consider all the possibilities with regard to HPV vaccination. The aim of the present study was to assess the overall and type-specific prevalence of HPV and to investigate factors associated with the risk of HPV in Iranian women with normal cervical cytology.

Study Population

This study enrolled 1,218 women (16-60 years) with normal cervical cytology attending private gynecology practices for a routine gynecological examination during 2011-2012. Study subjects were from the West, North, Northwest, and Center of Iran. A ThinPrep Pap test specimen was collected from each participant. Demographic features and information regarding risk factors were collected via a questionnaire. All study subjects gave informed consent, and approval was obtained from the local ethics committee (Tehran University of Medical Sciences).

HPV DNA Detection

DNA was extracted using a QIAamp DNA Mini Kit (Qiagen GmbH, Heidelberg, Germany) according to the manufacturer's instructions. DNA from all samples was amplified by PCR using the PGMY09/11 L1 consensus primer set as described previously [12] along with the HLAdQ-F 5′-GTGGTGTAAACTTGTACCA-3′ and HLAdQ-R 5′-GGTAGCAGCGGTAGAGTT-3′ primer pair to assess the integrity of extracted DNA simultaneously. All HPV-positive samples were amplified by the GP5+/6+ primer pair to obtain an amplicon size around 142-bp of the HPV L1 gene. The first PCR was carried out in a 50-μL reaction mixture including 100-200 ng of DNA template, 3 mM MgCl2, 10 pmol of each primer (PGMY09/11), and 10 pmol of the HLAdQ-F/R primer pair, 50 μM of each dNTP, and 2 U of HotStartTaq DNA polymerase (Qiagen), using the following PCR amplification cycles: an initial 5-min denaturation at 95°C, followed by 45 cycles at 95°C for 30 s, 55°C for 1.5 min, 72°C for 2 min, and a final elongation step at 72°C for 5 min. The second PCR on HPV-positive samples wasdone in a 50-μL reaction mixture including 100-200 ng of DNA template, 2.5 mM MgCl2, 10 pmol of each primer (GP5+/GP6+), 50 μM of each dNTP, and 2 U of HotStartTaq DNA polymerase (Qiagen). PCR amplification cycles included an initial 5-min denaturation at 95°C, followed by 45 cycles at 95°C for 30 s, 50°C for 1 min, 72°C for 1 min, and afinal elongation step at 72°C for 5 min.

Genotyping of HPV

The PCR amplification products were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit and a 3130 Genetic Analyzer Automated Sequencer as specified by Applied Biosystems manuals (Foster City, CA, USA). Nucleotide sequences were edited with BioEdit software and converted to FASTA format. All sequences were compared to other HPV types using the Blast server (http://www.ncbi.nlm.nih.gov/blast/).

Statistical Analysis

Associations between HPV positivity and risk factors were analyzed using multivariate logistic regression models by mutually adjusting for other risk factors. Differences were considered statistically significant for values of p < 0.05.

A total of 1,218 women from the North, West, Northwest, and Center of Iran were included in this study. The mean age of participants was 35.3 years (range: 16-60 years). The presence of the HPV genome was investigated, and all positive samples were subjected to genotyping. Totally, HPV was detected in 88 samples (7.2%).

As shown in Table 1, logistic regression analysis revealed that the risk factors associated with HPV infection were geographic region, age, education, and number of pregnancies (p < 0.05). However, no statistically significant difference was observed between HPV infection and other factors, including occupation, marital status, age at marriage, and oral contraceptive use (p > 0.05). Looking at different geographical regions, the prevalence of HPV varied significantly as the North (11.7%) and the Center (4.5%) of Iran were found to have the highest and lowest HPV prevalence, respectively (p = 0002). The age-specific HPV prevalence was also different. Indeed, in women aged <20 years, HPV prevalence was 11.3%, while it was <9% in the remainder (21-30 years, 7.4%; 31-40 years, 7.0%; 41-50, 5.2%; and >50 years, 8.6%). As indicated, the prevalence of HPV was decreased with increasing age, although a second peak of HPV prevalence was noted in women aged ≥51 years.

Table 1

Multivariate logistic regression analysis of HPV prevalence regarding risk factors in Iranian women with normal cervical cytology

Multivariate logistic regression analysis of HPV prevalence regarding risk factors in Iranian women with normal cervical cytology
Multivariate logistic regression analysis of HPV prevalence regarding risk factors in Iranian women with normal cervical cytology

Looking at the circulating HPV genotypes, the 4 most common types of HPV were found to be HPV 16 (2.09%), 18 (0.9%), 66 (0.83%), and 11 (0.34%), followed by HPV 6, 26, 31, 35, 44, 45, 51, 52, 53, 56, 67, 68, 73, 84, 89, and 90. Regarding geographical regions, HPV 16 was found to be the prominent type in all parts of Iran (ranging from 0.9 to 3.04%).

HPV type-specific prevalence by age revealed that HPV 16, 18, and 66 were more common in both age groups ≤30 and >30 years. However, other common types were HPV 31, 44, 45, 51, and 56 among women aged ≤30 years and HPV 6, 11, 44, and 68 in women aged >30 years.

Stratification of HPV genotypes based on the risk of cancer development [5] including high-risk types (HPV 16, 18, 31, 35, 45, 51, 52, 56, 68, and 73), intermediate-risk types (HPV 26, 53, 66, and 67), and low-risk types (HPV 6, 11, 44, 84, 89, and 90) revealed that most HPV genotypes in our study subjects belonged to the high-risk group, although these rates varied in different geographic parts of Iran (Fig. 1). As shown in Figure 2, high-risk HPV types were more prevalent among those <30 than >30 years, although the difference was not significant (p > 0.05).

Fig. 1

HPV prevalence according to the risk of HPV types among Iranian women with normal cytology, stratified by geographical regions.

Fig. 1

HPV prevalence according to the risk of HPV types among Iranian women with normal cytology, stratified by geographical regions.

Close modal
Fig. 2

HPV prevalence according to the risk of HPV types among Iranian women with normal cytology, stratified by age.

Fig. 2

HPV prevalence according to the risk of HPV types among Iranian women with normal cytology, stratified by age.

Close modal

Implementation of appropriate prevention strategies for cervical cancer is of crucial importance. In this regard, epidemiological data about the types of circulating HPV can be very helpful. The present study allowed us to make a relatively comprehensive estimation of HPV prevalence and distribution of HPV types among women with normal cervical cytology in several geographical regions of Iran.

HPV was detected in 7.2% of the samples (ranging from 4.5% in the Center to 11.7% in the North of Iran). In a global meta-analysis of women with normal cervical cytology, the overall HPV prevalence has been estimated to be 10.4%, although it was different according to the geographical regions [13]. In this regard, the prevalence of HPV was reported to be 22.1% in Africa, 20.4% in Central America and Mexico, 11.3% in Northern America, 8.1% in Europe, and 8% in Asia [8,13].

Globally, with regard to the age-specific HPV prevalence, the highest HPV prevalence was reported in women younger than 25 years of age [13,14,15,16]. In the present study, the highest HPV infection rate was observed among women younger than 21 years.

Alongside with HPV infection, a number of risk factors have been identified as key players in cervical cancer development. In accordance with previous studies [17,18], a significant association was seen between HPV positivity and age, education, and number of pregnancies. However, other factors including occupation, marital status, age at marriage, and oral contraceptive use showed no significant association with HPV infection.

The 4 most frequent HPV types of our study were shown to be HPV 16, 18, 66, and 11, representing 63.8% of all HPV infections. These data are consistent with most studies conducted in Iran and the rest of the world [13,19,20,21,22,23,24], where HPV 16 has been indicated as the most common HPV type, followed by HPV 18 in women with normal cervical cytology. With regard to other HPV types, our data are in agreement with studies from Chile and Southern and Eastern Europe, where HPV 66 was detected at a high frequency [25,26,27,28,29]. Previously, it has been reported that the most prevalent types, in decreasing order, were HPV 16, 18, 6/11, 45, and 31 in Iran [30]. In most previous studies from Iran, HPV-positive samples were often subjected to specific primer genotyping (mainly 6, 11, 16, and 18) but not sequencing or hybridization assays; therefore, the other HPV genotypes have been undetermined in Iran.

Our findings indicated HPV 16 as the most frequent HPV type in all parts of Iran. HPV 16 is the most prevalent type all over the world with the exception of Eastern Africa, Japan, and Taiwan, where HPV 52 was considered to be the most common type [24,28]. The HPV 16 prevalence for HPV-positive women from this study (31.9% of HPV-positive women) was similar to previous reports from Europe, Canada, and the USA [24,31].

The potential impact of prophylactic vaccines for the future protection against cervical cancer can be expected from the fraction of HPV types in women with or without cervical cancer. Regarding the present study, it is estimated that the 3 current HPV vaccines could have a great impact on the reduction of cervical cancer incidence.

In conclusion, this study highlights the necessity of introducing educational programs in high schools, robust screening programs, and vaccination in Iran. Future comprehensive investigations from other areas of Iran are also warranted to estimate the HPV prevalence and HPV type distribution in Iranian women as they will help health policy makers to consider all the possibilities with regard to screening programs and HPV vaccination in Iran.

This study has been funded and supported by the Tehran University of Medical Sciences (grant No. 30266). It has also been part of an MSc thesis supported by the Tehran University of Medical Sciences (grant No. 240/341).

All authors declare that they have no conflict of interests.

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