1. INTRODUCTION:

Cell screening is very effective for early diagnosis of cervical cancer. Lesions identified by screening enable further tests, such as colonoscopy, biopsy, and human papilloma virus (HPV) test^1,2,3.

Cervical cancer is caused by pathogenic factors of the herpes simplex virus II, cytomegalovirus, and trichomonas, which can be simply contracted by sexual habit, further influenced by the number of sexual partners, early sexual experience, smoking, contraceptive pills, and an unhygienic sex life. In addition, it can also occur for internal reasons, such as a decline in the immune response, over expression of oncogene, or inactivation of a repressor gene, which acts as carcinogen⁴.

HPV works as an initiator and promotor of tumorigenesis for cervical cancer⁵.

The most important factor for this cervical cancer is that HPV is contracted through and appears in genital organs. Also, at least 30 different kinds of HPV are contracted via anus and reproductive organs. HPV, a virus with a double-helix structure, infects the epithelium, causing warts, intraepithelial neoplasm, and carcinoma in situ⁶.

The papilloma virus, with species specificity, can infect various animals, including vertebrate and birds. The study of HPV started in the mid-1980s with cervical cancer tissue; HPV now forms more than 70 different virus families. HPV that can progress into malignant tumors can be differentiated into low-risk (LR) (HPV-6, 11, 42, 44) and high-risk (HR) (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) oncogenic types. The LR type is relates to condyloma acuminatum, and the HR type is present in epithelial lesions and can progress into cervical cancer^7,8.

HPV that infects reproductive organs does not have symptoms and cannot be clinically diagnosed. In most diagnoses, infected reproductive organs appear to be positive, and locally developed warts disappear within several months or years.

The histopathological characteristics of HPV infecting reproductive organs are typically condyloma accuminata with exophytic growth, which can be observed with the naked eye. Condyloma can be observed by cervical screening, koilocytes with a nucleophilic zone around epithelial cells can be observed by a light microscope, and dyskaryosis with nuclear enrichment can also be observed⁹.

More than 90% of HPV is detected in pre-cancerous lesions of the cervix and invasive carcinoma tissue¹⁰. Even though diagnostic cytological, histopathological, molecular biological diagnostic test of HPV have been studied by many scholars^11,12,13, the study of the microstructural characteristics of HPV-infected koilocytes and epithelial cells, including dysplasia and activation of the virus inside the nucleus, is insufficient. Therefore, by using transmission electron microscopy, this study identified microstructural characteristics and intracellular activity of HPV from identified condyloma accuminata or koilocytes by cervical screening or smear test of the cervix.

2. MATERIALS AND METHODS:

2.1. Materials:

Among patients visiting H Hospital (in Seoul, Korea) with vaginal inflammatory disease, specimens from 5 patients diagnosed with various types of vaginitis was selected for this study, which gained consent from the patients in advance and then used specimens with diagnosed HPV after consultation with a specialist. In addition, this study was performed after getting approval from the Institution of Ethics Committee appointed by the Korean Ministry of Health and Welfare (assignment number: P01-201405-BS-03).

2.2. Methods:

2.2.1. Specimen collection:

Each specimen was collected from squamous cells and the squamo-columnar junction of the cervix by rotating Roversbrush TM for sampling 5 times clockwise. Then specimens for light microscopy was collected in liquid-based cell preservatives to prevent cytogenesis, and specimens for electron microscopy was immersed in a phosphate buffer (4℃, 0.4 M phosphate buffer, pH 7.4) in 50ml Falcon tubes and stored in the fridge.

2.2.2. Papanicolaou’s stain and light microscope observation analysis:

For HPV specimens stored in liquid-based preservatives, substances obstructing speculum (mucous and RBC) were removed using cell prep and smeared in one layer on glass slides. The smeared slides were fixed with 95% ethanol (Duksan, Korea) and rinsed with alcohol in descending orders of concentration, and the nucleus was dyed with Gill’s hematoxylin (Yeongdong Pharmaceutical, Korea) for 2 minutes. Excess stain was removed with 0.25% hydrochloric acid for 2 minutes, and was neutralized with 0.5% ammonia (NH₄OH) for 30 seconds. Each neutralized agent was rinsed and dehydrated in ascending order of alcohol concentration; the cytoplasm was stained with Orange G6 (Yeongdong Pharmaceutical, Korea) and EA 36 (Yeongdong Pharmaceutical, Korea). Dyed agents were covered with glass after dehydrating and clearing, then observed under a light microscope (BX51, Olympus, Japan).

2.2.3. Transmission electron microscope observation:

To produce samples for an electron microscope, HPV specimens in Falcon tubes were centrifuged (centrifuge, Korea) at 1,000 rpm, and precipitates were fixed in 2.5% paraformaldehyde-glutaraldehyde (4℃, phosphate buffer, pH 7.4) for 1 hour, rinsed twice with phosphate buffer (4℃, 0.4 M phosphate buffer, pH 7.4) for 15 minutes, and fixed with 1% OsO₄(4℃, phosphate buffer) for 1 hour.

Fixed samples were rinsed with the same buffer twice each, dehydrated in ethanol in ascending order of concentration, replaced with propylene oxide, embedded in an Epon-Araldite mixture, and cured in a 60℃ vacuum drying oven (Yamato, Japan) for 36 hours. Each embedded specimen was semi-thinly sectioned with an ultramicrotome (Leica EM UC7, Germany), then dyed with 1% toluidine blue (1% borax) on a hot plate (60℃) for 2 minutes. Each dyed section was cleaned with tap water and observed with a light microscope (Olympus CH30, Japan). To observe the microstructure of each sample, ultra-thin sections were created and attached on copper grid, dyed with uranyl acetate and lead citrate, then observed under a transmission electron microscope (H-7500, Hitachi, Japan) at 100 kV

3. RESULT:

Upon observation of a smear of an HPV-infected specimen with a light microscope, the nucleus of transformed epithelial cells was expanded, the border of the nucleus membrane was unclear, and it contained euchromatin and heterochromatin. Moreover, the ratio of nucleus and cytoplasm was higher than that of normal cells [Figure 1]. HPV-infected superficial cell had parakeratosis of cytoplasm, dyskeratosis of the nucleus, nuclear alignment, poly-nuclearization, and a margin around the nucleus [Figure 1].

Figure 1. Light micrograph of a cervical smear showing so-called koilocytes (circle) in the center. Meganuclei are conspicuous in the center of the koilocyte. Black arrow: superficial cell, White arrow: intermediate cell. Papanicolaou’s stain.

On observation of HPV-infected superficial cervical cells with a transmission electron microscope, koilocytes were clearly seen [Figure 2]. Microstructural characteristics of koilocytes were prominent in the nucleus and cytoplasm. The nucleus of a koilocyte was filled with material of low electron density in the middle—so that it appeared to be empty—and contained heterochromatin along the border of the nuclear membrane. Furthermore, the border of nucleus membrane was unclear and some of it was lost [Figure 2].

The cytoplasm of koilocytes has an irregular nuclear membrane contour, with some empty intervals from the border of the nuclear membrane up to the cellular membrane. Cytoplasmic materials along the cellular membrane contained only fibrous materials, including keratin, with no other observable cellular structure or cell content [Figure 2].

HPV was not observed inside the cytoplasm of koilocytes under high magnification transmission electron microscopy. Cytoplasm was filled with keratin filaments and some vacuoles and ribosomes. A margin formed between the cytoplasm and nucleus contained darkened and aggregated cell debris, which formed from the dissolution of cellular structures in this area [Figure 3].

Figure 2. Transmission electron micrograph of a koilocyte showing a clear halo (H). N: nucleus.

Figure 3. Transmission electron micrograph of a koilocyte, showing the margin of cytoplasm and nucleus (N). H: halo, White arrow: keratin filament.

HPV-infected superficial cells had an unchanged nucleus contour, aggregated heterochromatin on the border of the nucleus close to the nuclear membrane, and low electron density in the center of the nucleus. Activated HPV particles were clearly observed inside the nucleus [Figure 4]. These virus particles were distributed along the border of the nucleus, where chromatins were aggregated, and were dense in the center of the nucleus [Figure 4]. In Figure 4, HPV particles are not observed in the cytoplasm.

The nucleus of HPV-infected cells discontinuously aggregated heterochromatins along the nuclear membrane, and HPV particles existed along aggregated heterochromatins. In addition, there were lots of HPV particles on the discontinuous border of chromatins in the center of the nucleus [Figure 5].

On the transmission electron microscope observation (magnified in Figure 5), HPV particles are aggregated on the isolated chromatins in the center of the nucleus [Figure 6]. These virus particles are spherical, with a diameter of 50 nm [Figure 7].

Figure 4. Transmission electron micrograph of vaginal epithelial cell showing nucleus (N) with HPV particles(white arrow) in the nucleoplasm.

Figure 5. Transmission electron micrograph of HPV in the nucleus (N) of epithelial cells. Fully formed virions (arrow) located in the nucleoplasm of an infected squamous cell. C: cytoplasm, Hc: heterochromatin.

Figure 6. Magnified transmission electron micrograph of figure 5. Note the presence of HPV(arrow) in the center of the nucleus. A small round virus can be seen in the center of the nucleoplasm.

Figure 7. Transmission electron micrograph showing the appearance of the pure HPV particles (arrows), which were icosahedral, with a diameter of 50 nm.

4. DISCUSSION:

Cervical cancer is the most frequent cancer of women, infecting more than 500,000 people per year and causing deaths of 300,000 people^14,15. Most cases of cervical cancer are due to HPV infection. HPV has a strong virulence factor that can cause cervical intraepithelial neoplasia (CIN) and invasive cancer¹⁶. A papanicolaou smear of HPV-infected superficial cells in a light microscope show parakeratosis of the cytoplasm, dyskeratosis of the nucleus, and formation of koilocytes with a nucleophilic zone around the nucleus^9,17,18. The smeared HPV-infected specimen in this study contained distinctive koilocytes, alignment of nucleus, unclear contour of the nuclear membrane, and aggregated heterochromatins. Furthermore, the ratio of nucleus and cytoplasm was higher than that of a normal cell.

Koilocytic atypia means a microscopically large superficial cell, relatively small and irregular under a stained nucleus, and a clear area around the nucleus that responds as negative to a stain for glycogen. Cells with such characteristics are called “halo” cells, thus “koilocyte” as the characteristic term for HPV infection^19,20. Infected superficial cells die because of proliferation and replication of the virus, resulting in irregular cytological atypia²¹. In this study, both koilocytes and multi-nucleated giant cells were well observed under a light microscope. Under the transmission electron microscope, koilocytes form a big halo around the nucleus and nucleus are aligned. Moreover, the center of nucleus is filled with materials of low electron density, just like an empty nucleus.

With these results, the formation of koilocytes is identified as cytological atypia, occurring with the invasion and proliferation of the virus, followed by death of the host cell, and escape of the virus from the host cells.

In this study, superficial cells, inactive because of the HPV infection, were observed to be filled with keratin filaments and some vacuoles and ribosomes. This state is not easily distinguishable under light microscope, but can be easily detected under a transmission electron microscope.

HPV is one of the many viruses in the Papovaviridae family. Normally, a Papovaviridae virus has a diameter of approximately 40-60 nm and has an icosahedral spherical structure when pure culture was stained for electron microscope with negative stain and shadowing cast^22,23.

In this study, HPV particles were clearly observed inside the nuclei of superficial cells that were infected with actively proliferating and replicating HPV. These virus particles are spherical, with a diameter of approximately 50 nm, and were observed to be dense near the nuclear membrane with aggregated chromatins and in the center of the nucleus. These results show characteristic features of HPV particle. In result, as viruses proliferate and activate in infected superficial cells, they escape from the host cell and form koilocytes, which are cytological atypia that form after activation and escape of the virus

5. CONCLUSION:

Koilocytes and multi-nucleated superficial cells of Papanicoliou stained cervical specimens were observed under a light microscope. The cytoplasm of koilocytes under a transmission electron microscope created halos (empty margin), maintaining a regular distance from the border of the nuclear membrane toward the cellular membrane. The cytoplasm of the koilocytes was filled with keratin filaments, and no organelles were observed. Furthermore, the nuclei of koilocytes were filled with material of low electron density in the center, resembling empty sacs, and the border of the nuclear membrane was unclear. HPV was not observed at all in the cytoplasm of koilocytes, and some substance similar to a virus particle was observed in the cytoplasm near the transformed cell membrane. The superficial cells with activated virus were filled with keratin filaments, and some vacuoles and ribosomes were observed. Activated HPV particles were clearly observed inside the nucleus. These virus particles are spherical, with a diameter of 50 nm and exist near the nuclear membrane and in the center of the nucleus. In conclusion, when the virus infects a superficial cell, invades its nucleus, proliferates, and escapes from the host cell, that superficial cell goes under cytological atypia to koilocyte.

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Received on 15.02.2019 Modified on 19.04.2019

Research J. Pharm. and Tech 2019; 12(9):4305-4309.

DOI: 10.5958/0974-360X.2019.00740.6

Ultrastructural Characteristics of HPV in Women’s Vaginal Cells