In this cross-sectional study, a total of 48 Pap smears were collected over a period of one year (2019) from women referred to the Obstetrics and Gynecology Clinic of Shahid Sadoughi Hospital in Yazd, Iran. In all cases, written informed consent was obtained, and the research protocol was approved by the local ethics committee. Based on the Pap smear results, samples were categorized into two groups: normal (n = 24/48; control) and abnormal (n = 24/48; case). All samples were stored at -20 ºC until further examination. Inclusion criteria included women with normal and abnormal Pap smears who were infected with high-risk HPV (Hr-HPV) types, such as HPV-16 and HPV-18, and were between 15 and 45 years of age. Exclusion criteria included pregnancy, a history of hysterectomy or abnormal cervical epithelial lesions, infection with low-risk HPV types (Lr-HPV), and age not between 15 and 45 years.

Template DNA extraction from the paraffin-embedded blocks was performed as follows: Heated xylene (1 ml; 60˚C) was added to the samples, followed by incubation at 58˚C for 20 minutes and centrifugation at 15,000 rpm for 10 minutes. Ethanol (99%) was added to all samples and incubated at 60°C for 20 minutes. Subsequently, 300 μl of digestion buffer was added and incubated overnight at 58°C. After centrifugation, the supernatant was transferred to another sterile tube. To obtain a pure template, the extraction procedure was supplemented by a phenol/chloroform assay. The concentration and quality of the template DNA were determined using a NanoDrop 1000 spectrophotometer and gel electrophoresis, respectively. The presence of the Hr-HPV genome (HPV-16 and -18) was determined using MolecuTech REBA HPV-ID.

To investigate the methylation pattern of the E6 gene in the Hr-HPV samples, a bisulfite modification technique using the EpiTect Bisulfite Conversion Kit was performed. In brief, sodium bisulfite was prepared according to the kit protocol, and the sample DNA was treated. In the bisulfite DNA modification method, a pair of forward primers with CpG (F2/F3) and without CpG positions (F1) are used (Table 1). For all forward primers, a reverse primer (R) is also used. Each pair of forward and reverse primers was used separately in one tube. The presence of one of the CpG loci results in a negative PCR for that primer pair. Primer F1 generates the desired band in all cases with R. However, the forward primers (F2, F3) are formed without generating the desired band, indicating that the CpG site is present at that sequence point.

The DNA treated with sodium bisulfite was subjected to a PCR reaction. The primer sequences are listed in Table 2. PCR was performed to amplify E6 methylation in HPV-16 and HPV-18 using the following program: Denaturation at 95°C for 8 minutes, 36 cycles of denaturation at 95°C for 35 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds, and final extension at 72°C for 10 minutes. The amplified products were visualized by electrophoresis in 1.5% agarose gels stained with Gel Red™.

In this study, participant data was collected confidentially using questionnaires and incorporating the results of the experiments. All patient data were then entered into SPSS software for statistical analysis. A Student's t-test was performed to compare between groups. A p-value of less than 0.05 was considered statistically significant, indicating a statistically relevant difference between the groups compared.

Table 1

Primer type	Sequence of primers (5'-3')
HPV16- F1	ATGGGAATTTATATGTTGTATGTGAT
HPV16- F2	ATGGGAATCTATATGTTGTATGTGAT
HPV16- F3	ATGGGAATTTATATGCTGTATGTGAT
HPV16- R	CTCCTCCTCTGAGCTATCATTTAATTGCTC
HPV18_F1	ATGATTAAGTTGTGTATATATAGTTTATGT
HPV18_F2	ATGACTAAGTTGTGTATATATAGTTTATGT
HPV18_F3	ATGATTAAGTTGTGTATATACAGTTTATGT
HPV18_R	TTCAAATACCTCTATAAATTCCAATACTG

Table 2

E6 Proto-oncogene	Sequence of primers	Product size (bp)
HPV-16F	5'-ATGGGAATTTATATGTTGTATGTGAT-3'	379
HPV-16R	5'-CTCCTCCTCTAAACTATCATTTAATTACTC-3'
HPV-18F	5'-ATGATTAAGTTGTGTATATATAGTTTATGT-3'	309
HPV-18R	5'-CTCCTCCTCTAAACTATCATTTAATTACTC-3'

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

In this study, the nucleotide pattern at positions 494 and 506 for HPV-16 and 1985 and 2003 for HPV-18 was examined. The mean age of the patients examined was 34.5 ± 7.23 years. The incidence of HPV-16 and HPV-18 was 64.6% and 35.4%, respectively. The incidence of HPV-16 was higher than that of HPV-18 in both groups (cases/controls).

Twenty-four HPV-16 infected samples with abnormal Pap smears were analyzed for CpG methylation patterns in the E6 gene promoter. Of all subjects in this study, 5 cases (10.4%) were found with methylation at position 506 in the HPV-16 E6 gene. Of the 24 samples in each group, only 4 samples in the abnormal Pap smear group and 1 sample in the normal Pap smear group had methylation in the 506 region of the E6 gene promoter of the HPV-16 genotype. It is noteworthy that none of the samples showed methylation at nucleotide site 494 of the promoter of the E6 gene of this genotype. The frequency of HPV-16 DNA methylation at all analyzed CpG sites ranged from 0% to 4.1% in the different subgroups and was not statistically significant in any of them (P = 0.348). The results of DNA methylation by the modified bisulfite method show that the 92, 104, and 106 methylation sites have no methylation after bisulfite treatment, and the cytosine is converted to uracil, with thymine finally being found in the PCR product. Comparison of the methylation rate of 506 HPV-16 nucleotides in normal and abnormal Pap smear samples showed that the methylation rate was 53.5% in normal Pap smear samples and 43.5% in abnormal Pap smear samples (Figure 1, Figure 2 ).

The HPV-18 methylation pattern was analyzed at the CpG site 1985 of the selected E6 gene promoter using 24 pure DNA samples from HPV-18 infected cervical samples with abnormal Pap smears. In 24 samples, non-methylated cytosines at the 1981-2010 nucleotide position were analyzed using methyl primers similar to those used for HPV-16. In total, 10.4% of the samples showed methylation at this position. In each group, only 5 samples in the group of abnormal Pap smears showed methylation in the 1985 region of the E6 promoter of genotype 18 (20.8%). None of the samples showed methylation in the nucleotide locus of the 2003 E6 promoter of this genotype. According to the results, no methylated cytosine was detected in other selected DNA fragments isolated from cervical samples with HPV-18 infection. According to the statistical analysis, there was no significant difference (P ≥ 0.05) in the frequency of HPV-18 DNA methylation at all selected 1985 CpG nucleotide sites in HPV-18 DNA samples isolated from specimens with different degrees of cervical pathology. The results of the methylation modification study showed that no methylation occurred at methylation sites 152 and 159 after bisulfite treatment, and the cytosine was converted to uracil, eventually being incorporated into the thymine PCR product. Comparison of the methylation rate of 1985 HPV-18 nucleotides in normal and abnormal Pap smear samples showed that the methylation rate was 55.8% in normal tissue and 44.2% in abnormal tissue (Figure 3, Figure 4, Figure 5).

In the normal and abnormal Pap smears, methylation at the position of the 506 nucleotide in the CpG region was 54.8% and 45.2% for HPV-16, respectively, but 58.8% and 41.2% for HPV-18 in 1985.

An estimated 71% of cases of uterine cancer are associated with HPV-16 and HPV-18 infections29, 30. Different ethnic groups have varying rates of HPV prevalence and types; infections with HPV-52 and HPV-58 are more common in Asia than on other continents31, 32. Numerous studies establish a link between host genes and hyperimmunity to human papillomavirus and uterine cancer. However, the relationship between the severity of cervical lesions and the methylation rate of different HPV strains is controversial. Research has investigated the relationship between the methylation status of the HPV genome, early and late genes, and disease severity, with some results suggesting a negative association and others suggesting a positive association.

One of the key players in gene silencing is increased methylation in CpG islands. Several genes are impacted by CIHM (CpG island hypermethylation) in a large number of tumors. Examining E6 gene promoter methylation in patients with HPV-16 and HPV-18 Pap smears was the goal of this investigation. Despite the contentious discoveries on the pattern of HPV DNA methylation in cervical disease, the various results are nearly equivalent. The enhancer region's nucleotide 7862 (CpG) has a very low rate of DNA methylation in the majority of earlier studies. These results indicate that all HPV DNA samples that lacked methylation at nucleotide position 7862 were examined in this investigation. There is overlap between the CpG location (7862 nucleotides) and the E2 crossing. The transcription of the oncogenes E6 and E7 has been demonstrated to be repressed by E2 gene products. Consequently, loss of E2 suppression and stable E6/E7 expression can result from methylation of the E2 cross13, 16. Among the several investigations identifying the two proteins E6 and E7 as candidates, it is important to highlight the study by Yan et al., which demonstrated that cellular immune responses to peptides E7 and E6 were strongly related to illness progression. High diversity of cytosine methylation at selected CpG sites and between various amplicons was not found when DNA methylation abundance in specific CpG nucleotides was examined. Cytology is still utilized for the early detection of cervical cancer despite its very low sensitivity. Both hypomethylation and hypermethylation are examples of incompatible DNA methylation in cervical lesions. In high-grade intrauterine neoplasia and cervical carcinoma, only three promoter regions are hypomethylated, while most genes are hypermethylated. The most significant risk factor for cervical cancer and its antecedents is hrHPV infection. Earlier research demonstrated that the HPV-16 E6 protein binds to p53, potentially releasing more Sp1, activating more DNMTs, and altering DNA methylation. Identifying disease-specific gene methylation is a new approach to cancer screening, enhancing early cancer diagnosis as the best diagnostic. Previous research has shown that methylation of certain genes, such as PAX1, SOX1, ZNF582, and POU4F3, in uterine cancer is therapeutically valuable for identifying INCIN3 cancer samples. In the present study, due to the frequency of methylation in patients with HPV-16 and HPV-18 infection, methylation changes in the E6 gene promoter did not play an important role in the development of infection with high-risk HPV-16 and HPV-18 virus types in the study population. No significant association was found between GG genotype and E6 gene polymorphism (P ≥ 0.05), although further studies are needed to confirm this.

In the current study, the methylation pattern in the E6 gene promoter was determined using tissue samples from the cervix of 48 women who were referred to the clinic at Shahid Sadoughi Hospital in Yazd and had abnormal or normal Pap smears, in addition to the usual high-risk HPV types. The results show that there is a poor correlation coefficient for the methylation of nearby CpG sites in HPV-16 and HPV-18 for both the 1985 and the 506 sites. This finding could indicate the existence of methylation boundaries. After considering repeated testing, there was no discernible correlation between the methylation of individual CpG sites and viral load in the diagnostic CIN-II or CIN-III samples or the control group.

CIN-II: Cervical Intraepithelial Neoplasia II, CIN-III: Cervical Intraepithelial Neoplasia III, CIHM: CpG Island Hypermethylation, CpG: Cytosine-phosphate-Guanine, DNMT: DNA Methyltransferase, DNMTs: DNA Methyltransferases, HPV: Human Papillomavirus, HR-HPV: Human High-risk Papillomavirus, hr-HPV: High-risk Human Papillomavirus, LCR: Long Control Region, Lr-HPV: Low-risk Human Papillomavirus, PCR: Polymerase Chain Reaction, SPSS: Statistical Package for the Social Sciences

None.

All authors equally contributed to this work, read and approved the final manuscript.

None.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

In all cases, written informed consent was obtained, and the research protocol was approved by the local ethics committee.

Not applicable.

The authors declare that they have no competing interests.