Human Papillomavirus Infections in Women With and Without Squamous Cell Abnormalities of the Uterine Cervix

Background: Human papillomavirus infections are one of the most common sexually transmitted infections with viral aetiology. The aim of the study was to confirm the existence of an association between human papillomavirus infection and squamous cell abnormalities of the uterine cervix. Methods: Cohort study, conducted in the period from January 2017 to June 2018 of 768 sexually active women, age groups of 20 to 59 years, divided into two groups: examined and control, who came to their annual gynaecological exam at University Clinic for Gynaecology and Obstetrics in Skopje. In all patients was done human papillomavirus-deoxyribonucleic testing. Human papillomavirus detection and typing was done using a polymerase chain reaction and reverse hybridisation. Results: Data analysis showed an association between human papillomavirus infection and squamous cell abnormalities of the uterine cervix (p=0.00001). Human papillomavirus infection was detected in 22.91% of all patients, in 75.00% of patients with abnormal cervical cytology and in 12.50% of patients with normal cervical cytology. A single human papillomavirus infection was detected in 13.67% of all patients (in 59.66% of human papillomavirus positive patients). Mixed human papillomavirus infection was detected in 9.24% of all patients (in 40.34% of human papillomavirus positive patients). Human papillomavirus type 16 was the most common genotype with 40.91%. Conclusion: This study confirmed that there is an association between human papillomavirus infection and squamous cell abnormalities of the uterine cervix and the young population under the age of 30 years is the most affected.


INTRODUCTION
Human papillomavirus (HPV) infections are one of the most common sexually transmitted infections with viral aetiology.¹ Epidemiological studies show that 50% of women are infected with HPV during the first two years of the onset of sex life, about 80% of sexually active women by the age of 50 will receive genital HPV infection and about 20% of women are infected with one or more HPV genotypes.² The prevalence of genotype varies depending on the geographical regions. In Europe and North America, HPV-16 is still the most common high-risk genotype.³ Young population of women, from 18 to 25, has the highest rate of HPV infection. After the 25th year of life, the incidence of HPV infection is reduced to reach its second highest level after 45 years of life.⁴ The most common risk factor for squamous cell abnormalities of the uterine cervix is infection with HPV, especially with highrisk HPV genotypes. Only persistent, high-risk HPV infection represent a major risk factor for squamous cell abnormalities of the uterine cer-

Design of the study:
The study is a cohort study.
Material: The material consists of 768 patients aged 20 to 59, divided into two groups: examined and control.
The examined group included 128 sexually active women with an abnormal cervical cytological finding, i.e. a finding of a Papanicolaou (PAP) test that showed a squamous cell abnormalities of the uterine cervix. The control group included 640 sexually active women with a normal cervical cytology finding, i.e. a PAP test.

Exclusion criteria:
The study did not include: pregnant women, women with previous surgery of the uterine cervix (cervical vaporisation, carbon dioxide laser vaporization and total abdominal hysterectomy) and also previous abnormal cytological and histopathological findings of the uterine cervix.

Period of realisation:
The study was conducted in the period from January 2017 to June 2018.

Location of the study:
The study was conducted at the University Clinic for Gynaecology and Obstetrics in Skopje, the Republic of North Macedonia.

Methods of examination:
In all women we vix.⁵ Deoxyribonucleic acid (DNA) from HPV has been found in 99.7% of cases of cervical carcinoma.⁶ Detection of HPV can be done using two methods, the first one is direct hybridisation or in situ hybridisation, and the other one is amplification or polymerase chain reaction (PCR).⁷ The aims of the study were: to confirm the existence of an association between HPV-DNA infection and squamous cell abnormalities of the uterine cervix; to detect and typing HPV genotypes, which are the most common causes of squamous cell abnormalities of the uterine cervix; to determine the prevalence of HPV-DNA infection and to determine the most affected age group.
have done the human papillomavirus-deoxyribonucleic acid (HPV-DNA) testing.
Cytological analysis: All samples for cytological analysis were taken using the Thin Prep PAP test and were analysed in the cytological laboratory at the University Clinic for Gynaecology and Obstetrics in Skopje by a cytopathologist. Cytological results were classified according to the revised Bethesda classification⁸ʼ⁹ such as: atypical squamous cells of undetermined significance (ASC-US); atypical squamous cellscannot e clude a high-grade squamous intraepithelial lesion (ASC-H); low-grade squamous intraepithelial lesion, LSIL (cervical intraepithelial neoplasia grade 1, CIN 1); high-grade squamous intraepithelial lesion, HSIL (cervical intraepithelial neoplasia grade 2, CIN 2, cervical intraepithelial neoplasia grade 3, CIN 3, carcinoma in situ, CIS) and invasive squamous cell carcinoma (ISCC).
HPV-DNA testing: Samples from the cervical biopsy for HPV-DNA testing were taken and analysed at the University Clinic for Gynaecology and Obstetrics in Skopje at the Laboratory for HPV testing. HPV detection and typing were used to test the use of multiple polymerase chain reaction (Multiplex PCR) and reverse hybridisation. The results of the HPV DNA test were analysed and demonstrated based on the finding of the presence or absence of DNA from HPV and the specified genotype.¹⁰ First step in HPV testing was the isolation of DNA from the collected cells from the cervical biopsies. For isolation of DNA series of three paraffin cuts were prepared. Cuts were incubated in 1 ml of xylene, 5 minutes at 55℃, and centrifuged at 10 000 G for five minutes at room temperature. The same procedure was repeated two more times. After careful removal of the remains of xylene, the samples were briefly incubated twice in 1 ml of 100% ethanol, and centrifuged for 5 minutes at room temperature. After removal of ethanol, short drying followed in air and incubation overnight in buffer with freshly added proteinase K at 55℃. The second step was the detection of DNA in HPV by using polymerase chain reaction (PCR). To verify the quality and integrity of the isolated DNA, actually of a present inhibitor, for each sample a reaction of multiplication of the specific primers for beta globin PC04 and GH20 was first made. Three pairs of primers were used, common to a larger number of HPV types: degenerate primers My09/My11 and CPI/CPII G and Gp5+/6+. The samples were carried through all reactions with primers specific to high-risk and low-risk HPV genotypes. The third step was genotyping by using reverse hybridisation. It is a method that is based on the hybridisation of specific DNA probes that are immobilised on nitrocellulose or nylon tapes. It is a set of primers (SPF 10) with aim-propagation of the L1 gene on the viral DNA. The product of amplification with SPF promers is the size of 65 bp, and allows detection of 25 new genotypes. Denatured biotinylated PCR products are hybridized with specific oligonucleotide probes that are immobilised as parallel lines on membrane strips. After hybridisation and washing with streptavidin, alkaline phosphatase is added, which binds to the biotinylated hybrids formed previously. Incubation with BCIP (5-bromo-4-chloro-3-indolyl-phosphate)/NBT (nitro blue tetrazolium) chromogen The distribution of patients by age groups is shown in Table 1. According to the t-test, the percentage difference between the average ages between the two groups was statistically non-significant with p>0.05 (Table 1).

RESULTS
HPV-DNA infection was detected in 22.91% of all patients, in 75.00% of patients with abnormal cervical cytology and in 12.50% of patients with normal cervical cytology. There was a statistically significant relationship between the modalities of HPV-DNA positive and HPV-DNA negative between the examined and the control group (chi-square test=67.1329, р<0.00001, р<0.05). Data analysis showed: an association gives purple precipitate and the results are interpreted visually.
Statistical analysis: Data were analysed by a specific software for data-bases (Excel). Statistical analysis of the established statistical series was made with the statistical program Statistical Package for Social Sciences (SPSS), version 23.0. The structure of statistical series with attribute sings was analysed with determining the proportions and rates. The structure of numerical signs was analysed by determining the measures of central tendency (arithmetical mean) and measures of dispersion (standard deviation). Analysis of the relationship (the existence of association) between two sets of attribute variables was performed using the Chi-square test and Fisher exact test. Testing the differences between the comparing groups (their distributions, arithmetic environments and proportions) was done using Student's t-test and Chi-square test. Statistical significance was defined as a p value <0.05.
between HPV-DNA infection and squamous cell abnormalities of the uterine cervix (Chi-square test=235.8722, p=0.00001, p<0.05); an association between the oncogenic potential of the virus and squamous cell abnormalities of the uterine cervix (Fisher exact test=0.0027, p<0.05); an increase in the presence of HPV-DNA infection in parallel with an increase in the cytological grade of cervical lesion (Chi-square test=10.7682, p=0.029296, p<0.05), and an association between the oncogenic potential of the virus and cytological grade of cervical lesion (Chi-square test=16.6967, p=0.000044, p<0.05) ( In situ SCC (n=15)     Table 6. Among high-risk HPV-DNA genotypes, HPV-16 was the most common (40.91%). Among the low-risk HPV-DNA genotypes, the most common was HPV-6 (7.96%), HPV-16 was most common in patients with high-grade squamous intraepithelial lesion and invasive squamous cell carcinoma, while HPV-6 in patients with low-grade squamous intraepithelial lesion ( Table 6).

DISCUSSION
HPV infections are widespread among people, occur with almost every person in both sexes at a certain period of their life, are usually short-lived, asymptomatic, spontaneously disappearing and only a small percentage of them need treatment.¹¹ Seventy-five percentage of the sexually active population, in the course of their lives, was in contact with one or more HPV genotypes.¹² Depending on the geographical affiliation, the study population and the method used, the frequency of HPV genotypes in various cervical lesions varies considerably. In 1996, the World Health Association recognized the importance of HPV for cervical cancer.⁶      It is confirmed by this study that: there is an association between HPV-DNA infection and squamous cell abnormalities of the uterine cervix; the young population under the age of 30 years is the most affected and the HPV-16 is the most common genotype in our environment.