@2024 Afarand., IRAN

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ISSN: 2251-8215    Sarem Journal of Reproductive Medicine 2017;1(3):113-116

Background
Human papillomavirus (HPV) is recognized as one of the causes of anogenital cancers, including ‎uterine cancer.‎
Previous Studies
About 30-40% of the different types of the virus cause mucosal contamination, especially in the ‎anogenital areas. These viruses are divided into two types of high risk and low risk for cancer. Of ‎course, in some sources, they are categorized into three types of low-risk, medium and high risk. Most ‎papillomavirus infections of the cervix and anogenital areas are of high risk type and can cause ‎cervical cancer (uterine carcinoma). Virusologically, the papillomavirus belongs to the group of naked ‎viruses with 20-fold cadmium, and has eight primary and regulatory genes, two L1 and L2 genes, and ‎delayed expression of the capsid. The gene and capsid protein are common in almost all types of the ‎virus, and in most molecular methods, they are the basis for isolating the virus from the patient's ‎specimen. Of the eight regulatory genes, e6 and e7 genes determine the ability of the virus to convert ‎the lesion to uterine carcinoma. Therefore, the first essential step in detecting the presence of the virus ‎in the cervical sample and other samples obtained from the patient is the isolation of the DNA of the ‎virus. Viral infection is responsible for about one-fifth of human cancers, and HPV (human ‎papillomavirus) is one of the most important oncoviruses, that not only causes damage in the ‎anogenital area, but also in the respiratory tract, lung, mucosa, scalp and neck, and its type 16 and 18 ‎cause cancer. Anogenital infections include warts, condyloma acuminates, cervical mucous membrane ‎lesions (CIN) [1]. Uterine lesions may be present in mild cervical lesions, mild LSIL epileptic lesions, ‎severe HSIL, and uterine carcinoma due to HPV infection [2, 3]. These lesions are classified according ‎to the Bethseda classification as CINII, CINI, CINIII, or CIS.‎ The most common types of HPV are types 6 and 11, but infection with types 16 and 18 is also common ‎‎[4, 5]. Anogenital warts are an infectious and sexually transmitted disease that occurs in both sexes ‎and is a serious clinical problem for women [6].‎ The relationship between the papillomavirus virus and malignant and progressive lesions, as well as ‎the cancer of the uterus, has been well identified. Today, the virus is accurately detected by various ‎methods such as DNA replication of the target with PCR and the Hybrid capture method [2, 5].‎
Aim(s)
The aim of this study was to investigate the presence of human papillomavirus in samples of Iranian ‎patients and the relationship between the type of virus (high risk and low risk) and cervical lesions by ‎molecular methods based on PCR virus samples.‎
Research type

Research society, place and time

Sampling method and number
The present study was performed on 67 cervical cytology samples (LBC) and cervical tissue samples ‎were evaluated using PCR and molecular methods in Sarem Hospital.‎
Used devices&materials
With an extraction kit (QIAGEN), DNA was extracted from samples of cervical, condyloma and uterine ‎cancer. To determine the presence of HPV in a patient's sample, the MY09 / MY11 primer for the L1 ‎capsid gene was used with the following sequences:‎ Forward: 5’-CGTCCMANNGGASACTGATC-3’‎ Reverse: 5’-GCMCAGGGSCATAKAATG-3’‎ C/A=M G/A=N T/A=S T/C=K With the aid of a specific primer of L1 capsid virus, and with observing temperature of different stages ‎of PCR in thermocyler, the Denaturation temperature of 95 ° C, 56 ° C primer annealing and ‎multiplication temperature of 72 ° C, during 35 cycles, followed by electrophoresis in 2% agarose gel ‎was run. After this step, DNA samples from HPV virus were selected based on PCR for typing with a ‎special kit (DNA-Technology JSC, 115478; Moscow, Russia). The reaction mixture was prepared in ‎separate micro tubes, with 10 μl buffer and the same amount of reaction mixture and 5 μL of the ‎sample, or positive or internal control. Based on the order of the kit in 45 cycles with a denaturation ‎temperature of 94 ° C, a primer annealing of 94 ° C and a reproduction temperature of 70 ° C, the PCR ‎reaction was performed and finally, the product was observed with 2% agarose gel, stained with ‎ethidium bromide and interpreted according to the instructions of the kit. Subsequently, positive ‎samples with polyacrylamide gel were isolated for type 18 and some were separated by Real time PCR. ‎Finally, their type was determined and classified according to the type of lesion.‎
Findings by Text
The existence of a 450-bp band signaled the presence of HPV in the sample (Fig. 1).‎ The appearance of the 570 bp band, indicated the presence of HPV type 33, 58 or 67, and the presence ‎of a 642 bp band, signaled the presence of types 16, 31, 35 or H 35 or 52. Also, bands with sizes 285, ‎‎291, 294 and 297 bp were HPV types 18, 45, 39, and 59 respectively (Fig. 2).‎ Of the samples with lesion, the virus was not found in 2 samples (0.3%) despite the clinical signs of ‎endothelium lesion and the effects of alteration of epithelial cells. Thus, out of 67 cases, 65 (97%) of ‎samples were positive in terms of HPV. ‎ Two patients had uterine carcinoma with two commonly high risk infected types and seven cases of ‎severe epithelial lesions were infected with high-risk types. Of the patients with epithelial lesion, only ‎‎2 were infected with low-risk types and the rest were risky. 30 (44.8%) patients had cellular ‎symptoms, of which half of them were classified into high-risk virus types and half of them were ‎infected with low-risk species. From the cytology point of view, 7 samples (10.4%) had no clinical ‎signs and 44 (65.7%) cases were infected with high-risk virus types (Table 1; Chart 1).‎ Most cases of infection were related to virus type 16. Among the samples, a significant number of HPV ‎types 18 and then 52, 39, 33, and 31 were observed. Also, in 2 samples of the uterine carcinoma, the ‎infection was accompanied by two types of viruses, one with type 16 and 31, and another with type 18 ‎and 33.‎
Main comparison to the similar studies
In similar studies, an examination for intra-epithelial uterine neoplasia (CIN III) with on-site ‎hybridization was found on a case of simultaneous infection with types 16 and 18, which suggested ‎that HPV infection was the most important responsible for the cause and effect of malignancy changes, ‎and these two types simultaneously Infect the squamous epithelial cells of the cervix [7]. As the ‎persistent infection with viral carcinogenesis leads to neoplasia or HSIL lesions, determining HPV-‎DNA can be used as a marker for existing infection or future developmental lesions [8]. In the present ‎study, similar to the similar studies by Garland and Tabrizi [8], it was observed that the HPV-DNA test ‎had a higher sensitivity to detect the virus for the prognosis of precancerous lesions (LSIL, HSIL) than ‎cytology.‎ In the present study, 5 out of 125 patients with cervical lesions were positive for HPV-DNA, therefore, ‎cervical ulcer cannot be said to be a specific sign for HPV infection. Ultimately, the combination of Pap ‎smear test with HPV-DNA testing can reduce the intervals of Pap smear testing in a group where HPV-‎DNA is negative and the result of a cystic Pap smear test is normal and therefore more cost-effective ‎‎[8].‎ Based on comparative studies for propagation and hybridization methods, PCR was found to be more ‎sensitive for detecting CIN by 83.33% than Tur e Hybrid Cap with 66.67% [9, 10].‎ In a sensitivity analysis for the determination of severe lesions, it was found that the Hybrid Capture ‎method was incapable of detecting a large number of cases compared to the PCR method [11, 12]. In 12 ‎cases of CIN, 10 cases (83.33%) were positive with PCR. In 7 cases of CIN II and CINIII, all were ‎positive. Six cases of 16 and 18 type papilloma virus and one type 16 case were obtained [11]. The ‎results of that study are consistent with the present study.‎
Suggestions
Repeated studies with more samples and molecular methods are recommended for more accurate ‎diagnosis.‎
Limitations

Conclusions
‎97% of intraepithelial symptoms in cytology of the cervix are associated with the presence of human ‎papillomavirus, and the highest number of viral cervical mucus is of high risk. The most common type ‎of epithelial-infectious cervical virus type is the type 16 human papillomavirus.
Acknowledgments

Conflict of interest

Ethical Permissions

Funding Sources

TABLES and CHARTS

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