LITHUANIAN UNIVERSITY OF HEALTH SCIENCES FACULTY OF MEDICINE
DEPARTMENT OF OBSTETRICS AND GYNECOLOGY
Maria Epifaniou
INTRAEPITHELIAL LESIONS OF THE CERVIX:
COMPARISON OF CYTOLOGIC, HISTOLOGIC AND COLPOSCOPIC FINDINGS AND THE ASSOCIATION WITH
THE HPV TEST
MASTER THESIS
of Integrated Studies of Medicine
Kaunas – 2021
Supervisor: Lect. Artūras Sukovas
Table of Contents
SUMMARY ... 4
SANTRAUKA ... 5
ETHICS COMMITTEE APPROVAL ... 6
ACKNOWLEDGEMENTS ... 7
CONFLICT OF INTEREST ... 8
ABBREVATIONS LIST ... 9
INTRODUCTION ... 10
AIM AND OBJECTIVES ... 11
1. LITERATURE REVIEW ... 12
1.1. Cervix ... 12
1.1.1. Anatomy ... 12
1.1.2. Histology ... 13
1.2. Human Papillomavirus ... 14
1.2.1. Background and Epidemiology ... 14
1.2.2. Pathogenesis ... 14
1.2.2.1. Biology – HPV Genome ... 14
1.2.2.2. The HPV life cycle ... 15
1.2.3. Classification ... 16
1.2.3.1. Low risk HPV ... 16
1.2.3.2. High risk HPV ... 16
1.2.3.3. Cutaneous HPV ... 17
1.2.4. Transmission ... 17
1.2.5. Risk Factors ... 17
1.2.6. Signs and Symptoms ... 18
1.2.7. Diagnosis and Laboratory Screening ... 19
1.2.7.1. Cytology ... 19
1.2.7.2. HPV – DNA test ... 24
1.2.7.3. Colposcopy ... 24
1.2.7.3.1. Normal colposcopy Finding ... 25
1.2.7.3.2. Abnormal Findings ... 26
1.2.7.4. Histopathology ... 27
1.2.8. Factors that affect diagnostic discrepancy ... 28
1.2.9. Management ... 29
2. METHODS ... 31
3. RESULTS ... 33
3.1. Frequency and percentage distribution for all variables used in this study ... 33
3.2. Percentage distribution of punch biopsy deriving results, between tested and not tested for HPV . 35 3.3. Association between Papanicolaou Test deriving results, between HPV type deriving results ... 36
3.4. Association between punch biopsy deriving results and HPV type deriving results ... 37
3.5. Association between Papanicolaou test deriving results and punch biopsy deriving results ... 38
3.6. Association between colposcopy deriving results, between Papanicolaou test deriving results ... 39
3.7. Association between punch biopsy deriving results and colposcopy deriving results. ... 41
3.8. Association between punch biopsy deriving results and conization biopsy deriving results. ... 42
6. PRACTICAL RECOMMENDATIONS: ... 46
7. CONCLUSIONS ... 47
Bibliography ... 48
ANNEXES ... 54
INTRAEPITHELIAL LESIONS OF THE CERVIX: COMPARISON OF CYTOLOGIC, HISTOLOGIC AND COLPOSCOPIC FINDINGS
AND THE ASSOCIATION WITH THE HPV TEST
Maria Epifaniou Master Thesis SUMMARY
The aim of this study, is to evaluate the cervical intraepithelial neoplasia and the association between the cytology, colposcopy, punch biopsy and conization biopsy results and the association with the HPV test.
The main objectives of the study are to evaluate: the association between Human papillomavirus and cytological findings; the association between Human papillomavirus and punch biopsy findings; the association between cytological findings and colposcopy findings; the association between punch biopsy and colposcopy findings; and the association between punch biopsy findings and conization biopsy findings.
This is a retrospective study conducted by reviewing personal files in the hospital information system. The study took place in the Hospital of Lithuanian University of Health Sciences (LSMU) Kauno klinikos in the department of Obstetrics and Gynecology. They were 174 patients who arrived to LSMU KK gynecological department for cervix conization procedure, and their ages were between 21 – 63 (mean 37
!8.596), during the period of
2020.01.03 to 2020.12.30.From the personal files of the patients, the data that was selected was the age of the patient, the primary cytology results, the punch biopsy results after the cytology and then the grade of the lesion that was established. Also, the data concerning the HPV test and the colposcopy findings were obtained. Lastly the biopsy results after conization were obtained.
There is a significant association between the Papanicolaou test deriving results and HPV type deriving results, X
2(30)= 61.575, p <.001, Cramer’s V=.320. There is a significant association between the punch biopsy and Papanicolaou test results, X
2(10)=22.408, p =.020, Cramer’s V=.254.
There is a significant association between the colposcopy and Papanicolaou test results,
X
2(15)=80.703, p <.001, Cramer’s V=.393. There is a significant association between the punch
biopsy and colposcopy deriving results, X
2(6)=19.691, p =.003, Cramer’s V=.238. There is a
significant association between the punch biopsy and conization biopsy deriving results,
X
2(6)=31.518, p <.001, Cramer’s V=.301.
GIMDOS KAKLELIO INTRAEPITELINIAI POKYČIAI:
CITOLOGINIŲ, HISTOLOGINIŲ IR KOPLOSKOPINIŲ POKYČIŲ PALYGINIMAS IR RYŠYS SU ŽPV TYRIMO REZULTATU
Maria Epifaniou Master Thesis
SANTRAUKA
Šio tyrimo tikslas – nustatyti gimdos kaklelio citologijos, kolposkopijos, biopsijos ir konizacinės biopsijos rezultatų ryšį bei ryšį tarp ŽPV tyrimo rezultato ir citologijos bei biopsijos rezultatų. Pagrindiniai tyrimo tikslai - įvertinti ryšį tarp žmogaus papilomos viruso ir citologinių tyrimų rezultatų, ryšį tarp ŽPV ir biopsijos rezultatų, ryšį tarp citologinių tyrimų rezultatų ir kolposkopijos rezultatų, ryšį tarp biopsijos rezultatų ir kolposkopijos rezultatų bei ryšį tarp biopsijos rezultatų ir konizacinės biopsijos rezultatų.
Tai retrospektyvus tyrimas, atliktas analizuojant ligos atvejus. Tyrimas vyko Lietuvos sveikatos mokslų universiteto ligoninės (LSMU) Kauno klinikų Akušerijos ir ginekologijos klinikoje. Analizuotos 174 pacientės, atvykusios į LSMU KK Ginekologijos skyrių gimdos kaklelio konizacijai, jų amžius buvo nuo 21 iki 63 metų (vidurkis 37 ± 8.596), laikotarpiu nuo 2020.01.03 iki 2020.12.30. Iš pacienčių elektroninių ligos istorijų buvo atrinkti šie duomenys: pacienčių amžius, pirminio citologinio tyrimo rezultatai, ar po citologinio tyrimo joms buvo atlikta biopsija, ir kokio laipsnio pažeidimai nustatyti. Taip pat buvo išrinkti duomenys apie ŽPV tyrimą, ir kolposkopijos rezultatus ir gimdos kaklelio konizacijos histologinio tyrimo rezultatai.
Yra reikšmingas ryšys tarp gimdos kaklelio citologinio tyrimo rezultatų ir ŽPV tyrimo rezultatų, X
2(30)= 61.575, p <.001, Cramer's V=.320. Yra reikšmingas ryšys tarp biopsijos ir gimdos kaklelio citologinio tyrimo rezultatų, X
2(10)=22.408, p =.013, Cramer's V=.254. Yra reikšmingas ryšys tarp kolposkopijos ir gimdos kaklelio citologinio tyrimo rezultatų, X
2(15)=80.703, p <.001, Cramer's V=.393. Yra reikšmingas ryšys tarp biopsijos ir kolposkopijos rezultatų, X
2(6)=19.691, p =.003, Cramer's V=.238. Yra reikšmingas ryšys tarp biopsijos ir konizacinės biopsijos rezultatų, X
2(6)=31.518, p <.001, Cramer's V=.301.
Raktažodžiai: Žmogaus papilomos virusas, kolposkopija, citologija, biopsija,
patohistologinis tyrimas.
ETHICS COMMITTEE APPROVAL
Title: “ Association between cytological findings, Human Papilloma virus infection, histology and colposcopy”.
Number of issued approval: BEC-MF-177
Date of issued approval: 2019-12-18
Follow Annex 1
ACKNOWLEDGEMENTS
I would like to express my gratitude to my supervisor, Lect. Artūras Sukovas, for the
valuable supervision and guidance throughout this study. I would also like to thank Hercules
Aristidou for providing advice for the statistical analysis. Finally, I would like to thank my family
and friends for supporting me during the compilation of this study.
CONFLICT OF INTEREST
No conflict of interest is declared.
No funding sources were applied.
ABBREVATIONS LIST
AGC – Atypical Glandular cells AIS – Adenocarcinoma in situ
ASC-H – Atypical Squamous Cells, cannot exclude HSIL
ASC-US – Atypical Squamous Cells of undetermined significance CIN – Cervical intraepithelial neoplasia
DNA – Deoxyribonucleic acid E – Early
HPV – Human Papillomavirus
hrHPV – High risk Human Papillomavirus
HSIL – High grade squamous intraepithelial lesions L – Late
LCR – long control region
LSIL – Low grade squamous intraepithelial lesions ORFs – Open reading frames
Pap test – Papanicolaou test
RCI – Reid Colposcopy index
TZ – Transformation zone
X
2-test – Chi-square test
INTRODUCTION
Worldwide, cervical cancer is estimated to be the fourth most common cancer that is diagnosed, as well as the fourth leading cause of death due cancer among women [1]. In Lithuania, cervical cancer is ranked in the fourth place as the most frequent cancer among Lithuanian women [2].
Human papillomavirus is a sexually transmitted disease which increases the risk of the development of cervical intraepithelial neoplasia. Cervical intraepithelial neoplasia is the abnormal cell growth on the surface of the cervix. The progression of the disease from the infection to cervical intraepithelial neoplasia or to carcinoma is associated with the human papillomavirus genotype that causes the infection. Human papillomavirus genotype 16 is found to be the most carcinogenic and genotype 18 to be the second most carcinogenic respectively accounting for 55 – 60% and 10 – 15% of cervical cancers worldwide. Risk factors such as the number of the sexual partners, smoking and people with immunocompromised systems can increase the risk for the development of cervical intraepithelial neoplasia and the persistent infection of human papillomavirus [3].
Human papillomavirus infection may be asymptomatic or may remain dormant for a very long time. As preventive measures, there are screening programs in place and the cervical screening program in Lithuania includes women from 25 to 60 years of age. The screening programs involve a cervical cytology which is the Papanicolaou test every 3 years in case of negative results, whereas in case of positive results, colposcopy examination, cervical inspection, HPV test and biopsy are done. Cervical intraepithelial neoplasia can progress to cervical cancer if it is left untreated. Currently the treatments options that are available are cryotherapy, cold knife conization and large loop excision of the transformation zone [4].
Therefore, the aim of the study was to examine the association between the cytology,
colposcopy, punch biopsy and conization biopsy results and their association with the HPV test.
AIM AND OBJECTIVES
The aim of the study was to find the association between the cytology, colposcopy, punch biopsy and conization biopsy results and the association with the HPV test.
Objectives:
1. To determine the association between Human papillomavirus and cytological findings.
2. To determine the association between Human papillomavirus and punch biopsy findings.
3. To determine the association between cytological findings and colposcopy findings.
4. To determine the association between punch biopsy findings and colposcopy findings.
5. To determine the association between punch biopsy findings and conization biopsy findings.
1. LITERATURE REVIEW
1.1. Cervix 1.1.1. Anatomy
The uterus consists of the body and the lower part which is the cervix. The cervix is a cylinder-shaped fibromuscular organ, which is 2 – 4 cm in long and 2.5 cm in diameter. Laterally to the cervix lies the parametrium which is cellular connective tissue. Approximately 1 cm of the ureter passes laterally to the supravaginal cervix. Posteriorly, the cervix is covered by the peritoneum of the pouch of Douglas [5]. The endocervical canal has a fusiform shape and connects the uterine cavity with the vagina [6]. The cervix is located between the base of the bladder and rectum and projects approximately 2 cm into the upper vagina cavity. The cervical opening leads to the vagina through an orifice which is called the external os and the opening into the uterine cavity is called the internal os [7]. The cervix is supported by the uterosacral and cardinal ligaments, and stretches between the posterior and lateral portions of the cervix and the bony pelvis walls. Portio vaginalis (or ectocervix) is the lower half of the cervix, which protrudes into the vagina through the anterior wall of the cervix. The remaining upper half remains above the vagina and is also known as the supravaginal portion of the cervix [8]. The vascular supply of the cervix derives from the descending branch of the uterine artery which originates from the anterior division of the internal iliac artery. The descending branch of the uterine artery branches into the upper vagina and anastomoses with the ascending vaginal artery to form the median longitudinal arteries and the azygos arteries of the vagina [9, 10].The uterine vein goes along with the artery and drains into the internal iliac vein [11, 12].
Figure 1: Uterus and Adnexa,(Source: Atlas of human anatomy, ed 7, Plates 355 and 357)
Figure 2: Female pelvis, median section, view from, the left side. The uterus lies above the urinary bladder. It's fundus and
13 1.1.2. Histology
The cervical lining consists of two different types of epithelium which are the simple columnar and the stratified squamous non keratinizing. The endocervix, also known as the cervical canal, is lined by simple columnar epithelium which has cells secreting mucous. The simple columnar cells have basal nuclei and mucinous cytoplasm. The ectocervix is lined by stratified squamous non keratinizing epithelium which continues with the vaginal epithelium. The cervical stroma consists of muscular, fibrous and elastic tissue [13].
The borderline between the simple columnar epithelium of the endocervix and the stratified squamous non keratinizing epithelium of the ectocervix is called the squamocolumnar junction.
Remotely to the squamocolumnar junction, the immature metaplastic epithelium is located. The immature metaplastic epithelium can mature under the influence of various cervical infections, trauma and chronic irritation.
The transformation zone (TZ) is located in the middle of the original squamocolumnar junction and the current squamocolumnar junction. The original squamocolumnar junction is localized where the endocervical tissue spreads out from the cervical os in newborns and young adulthood, also known as ectropion or cervical eversion. Within the transformation zone, at the squamocolumnar junction, there is an abrupt change from the simple columnar epithelium to the stratified squamous epithelium. The transformation zone is the area which is favorable for metaplastic changes. The metaplasia in the transformation zone is the substitution of the columnar epithelium by the stratified squamous non keratinizing epithelium, increasing the risk of development of cervical dysplasia and carcinoma. The squamous metaplasia is increased during fetal development, the menarche and during pregnancy. For the Papanicolaou smear test the area of sampling is the transformation zone [14].
Figure 3: A. The cervical canal (CC) mucosa continues with endometrium and it is lined with a simple columnar epithelium (SC). The endocervical mucosa incorporates cervical mucous glands (arrows). At the place where the cervical canal opens into the vagina (V), at the external os, where the squamocolumnar junction (J) is located, which is a junction between columnar epithelium, and the stratified squamous epithelium (SS). The stratified squamous epithelium covers the ectocervix and the vagina. Fibromuscular tissue (F) is the main tissue found in the deeper cervical wall. (X15; H&E) B. The Squamocolumnar junction is seen more clearly. (X50; H&E). (Source: Chapter 22 The Female
Reproductive System, Mescher AL. Junqueira’s Basic Histology: Text and Atlas, 15e; 2018. Available at:
https://accessmedicine.mhmedical.com/content.aspx?sectionid=190287163&bookid=2430 Accessed: March 19, 2021)
1.2. Human Papillomavirus
1.2.1. Background and Epidemiology
The Human papillomavirus (HPV) is one of the most frequent sexually transmitted diseases [15]. Sexually transmitted infections are infections that spread from one person to another through sexual intercourse [16]. Sexual intercourse includes the vaginal, anal, penile and oral sex. As a result, the transmission of the HPV is caused by skin to skin contact with an infected person. In addition, humans are exclusively the reservoir for HPV [17]. It is estimated that about 50% of new infections involve individuals between the ages of 15 to 24 years of age [18]. By 2020, it was established that there are in excess of 100 HPV types [19]. Papillomaviruses are deoxyribonucleic acid (DNA) viruses, double stranded, which have a circular genome of roughly 8kb and a non – enveloped icosahedral capsid [20]. The human papillomavirus is well known for the oncogenic characteristics it has to the human body [21]. The HPV tends to cause cancer of the cervix, vulva, vagina, anus, penis and oropharynx [20]. Also, HPV can lead to benign diseases, such as recurrent respiratory papillomatosis and genital warts [22]. Cervical cancer is the fourth most common cancer which affects women, as well as the fourth cause of death [23].
1.2.2. Pathogenesis
The HPV infection takes place in the stratified squamous epithelium in the basal layer. The infection triggers the cellular proliferation in the epithelium and the cells which are infected may present a broad spectrum of changes which may range from benign dysplasia to dysplasia and lastly to invasive carcinoma. Predisposition to cervical cancer can be due to persistent infection that is mediated by the E6 and E7 oncoproteins [24].
1.2.2.1. Biology – HPV Genome
Human papillomaviruses are a part of the family of Papillomaviridae, with a diameter of 52
– 55 nm and they are composed of 72 pentameric capsomers. HPVs are circular, double – stranded
DNA viruses, with non – enveloped icosahedral capsid and a genome with approximately 8000 base
pairs. They are organized into three functional regions, according to their functional properties and
location [25]. HPVs are exclusively replicate and assembled in the nucleus. HPVs replicate entirely
in keratinocyte, which means they infect surface tissues composed of squamous epithelium and skin
[26]. The functional regions are the early (E), and the late (L) region, which are referred to as
opening reading frames (ORFs) that are transcribed from only one DNA – strand and the third region
is the long control region (LCR), differently known as translation units or non – coding region or upstream regulatory region.
The E region consists of six ORFs which are E1, E2, E4, E5, E6, E7, genes which are expressed during the onset of the infection. E1 and E2 are accountable for the replication of the HPV, E2 is also responsible for the DNA transcription. Furthermore, E4 is in charge of the maturation and the release of the viral particles and E5, E6, E7 are in charge of the cell transformation [24, 27]. Also, E6 and E7 genes are responsible for immobilization and they encode proteins related to malignant lesions [24]. The L gene region consists of L1 and L2 ORFs which are expressed during the final stages of viral production and are responsible for encoding structural proteins, the major and the minor capsid proteins. In addition, the long control region is a non – coding part which contain cis components essential for viral replication and transcription [27].
1.2.2.2. The HPV life cycle
The human papillomavirus life cycle is associated with the differentiation of the keratinocyte cells of the host and it is defined by different phases of replication. The HPV infects the host cells by gaining access from small abrasions and wounds in the epithelium leading to a persistent and long term infection. For successful entry of the HPV into the cell there is an interaction with heparan sulphate proteoglycans, a surface cell on the basal lamina. Afterwards endocytosis occurs which is facilitated by clathrin or caveolin according to the type of HPV. In the course of non – productive infection stage, in the undifferentiated basal cells, a low number of double stranded circular viral DNA genomes, which are estimated to be around 50 to 100 copies per cell, are preserved. Therefore, a long – term viral DNA reservoir is created. The infected cells transit the differentiation process and the productive stage begins. During the productive stage of the viral life cycle there is an expression of the late viral genes L1, L2 and activation of the late promoters E4 and E5. Along with
Figure 4: Structure and organization of HPV16 genome (Source: Pal A, Kundu R. Human Papillomavirus E6 and E7: The cervical cancer hallmarks and targets for therapy. Front Microbiol [Internet]. 2020;10. Available from:
http://dx.doi.org/10.3389/fmicb.2019.03116)
the L1, L2, E4, E5 there are increased levels of E1 and E2 and as a result, thousands of copies per cell are produced from the viral gene amplification. L1 and L2 immunogenic capsid proteins are exclusively highly expressed in differentiated suprabasal cells. Subsequently, the assembly and release of the virion is restricted in the topmost epithelium layer. In the basal and parabasal layers of the epithelium, oncoproteins E6 and E7 target p53 and retinoblastoma (pRb) tumor suppressor proteins to slow down the cell differentiation and to allow human papillomavirus to replicate [28].
1.2.3. Classification
Exceeding 200 HPV types have been identified and are categorized based on the DNA sequence similarities into 5 genera which are α, β, γ, μ and ν. Nearly 40 out of the 200 tend to infect genital area [29]. Based on the International Agency for Research on Cancer (IARC), 12 types have been found to be carcinogenic to humans, one of which is HPV16, which is the most carcinogenic type known. HPV types have been categorized into 3 classes, which are low risk, probable high risk and lastly high risk, based on the risk that they induce for the transformation of the normal epithelial cells of cervix uteri to an invasive squamous cell carcinoma after a persistent infection of HPV [30].
1.2.3.1. Low risk HPV
The low risk HPV types are 6, 11, 40, 42, 43, 44, 54, 61, 70, 81, and CP6108 [31]. Several low risk HPV types are known to cause warts around or on the anogenital area in both sexes. Women might also have warts in the vagina and cervix. Moreover, warts may appear in the throat and mouth.
However low risk HPV types might also be asymptomatic. HPV6 and HPV11 are well known to be low risk types causing approximately 90% of the respiratory papillomatosis and benign anogenital warts [32, 33].
1.2.3.2. High risk HPV
As already mentioned, high risk HPV (hrHPV) types such as 16, 18, 31, 35, 39, 45, 51, 52,
56, 58, 59, 68, 73 and 82 are known to be carcinogenic [34]. HPV types 26, 53 and 66 are considered
to be probably high risk [35]. Furthermore, HPV types 16 and 18 are widely known to be
carcinogenic, causing more than 70% of cervical cancers. Oncogenic types of HPV with persistent
infection are closely associated with high grade cervical intraepithelial lesions, as well as cancers in
region of vagina, vulva, cervix, anus, penile and oropharynx [36, 37].
1.2.3.3. Cutaneous HPV
Types 1, 2, 27 and 57 of HPV are identified to cause most of the cutaneous warts which are non – genital warts and are not sexually transmitted. Below the age of 12, children may get infected with HPV1 causing plantar warts. The common hand warts are cause by HPV2 and HPV27 and in adults older than 21 years of age, HPV57 tends to cause common and plantar warts [38].
1.2.4. Transmission
Human papillomavirus is considered to be one of the most common sexually transmitted infections that a sexually active woman can acquire. An infected partner is required to come into sexual contact with the other partner and through the epithelium’s micro abrasions the transmission of the HPV is established. With reference to sexual intercourse, this includes anal, vaginal and oral routes. Moreover, HPV transmission can be from oral – genital contact and from genital to genital contact.
Non – sexual modes of HPV transmission also exist. Auto inoculation is non sexual mode of transmission, which occurs when an infected person transfer the HPV by touching the infected area on their body and then touching different area. Also, HPV can be transmitted via fomites which is the transfer of the virus via different objects [39]. Lastly, vertical transmission or perinatal transmission, is when there is transmission of the HPV from the mother to the fetus during the vaginal birth. As the fetus passes through the birth canal, comes into contact with the already infected epithelium [40].
1.2.5. Risk Factors
The fundamental risk factor for the transmission of the HPV infection is related to the sexual
activity of an individual. In females, the age of which sexual intercourse begins, as well as the
number of partners they have is crucial. Another key factor is the behavior of their partner towards
their previous partners and the number of these. People who have a weakened immune system, like
human immunodeficiency virus (HIV) or following transplant surgery, are more susceptible to
contract the HPV [41]. Also, for women who are already infected with HPV and use oral
contraceptive pills there is a higher risk in the development of cervical cancer [42]. People who
smoke increase their chances of becoming infected with HPV [43, 44].
1.2.6. Signs and Symptoms
Human papillomavirus infection may be asymptomatic or may remain dormant for a very long time till the first signs and symptoms are presented. The incubation period may range from 3 weeks to 8 months or even years from the infection incidence and the presentation of clinical signs.
Different strains of HPV tend to lead to different types of symptoms. Depending on the HPV risk, if it is low risk HPV or high risk HPV, they can respectively cause warts (genital or skin) or benign lesions in the uterus which will probably advance in malignancy.
Low risk HPV triggers the production of anogenital warts which are located in vagina, the labia majora and on the internal surfaces of the anus and vagina. Genital warts are also known as condyloma acuminatum and are associated with HPV types 6 and 11. Nevertheless, HPV types 54, 43, 43, 42, 40 and 2 are also known to cause genital warts. Based on the morphologic features of the warts, these can be classified into four categories which are: condylomata acuminata; keratotic;
smooth papular and flat warts. Genital warts are small, raised skin growths or flat, which their color may vary from pink to flesh color. Warts can be found to be in groups or singularly. Condylomata acuminata usually present as a cluster having the appearance of a cauliflower. The size and number of the warts tend to increase during pregnancy and in immunocompromised patients. Vulvar warts may cause discomfort and painful intercourse. In addition, vaginal warts may cause vaginal bleeding or discharge or even birth canal obstruction. In the case of intra – anal and perianal warts, these might lead to bleeding during defecation, pain and pruritus [45].
High risk HPV oncogenic types 16 and 18 are responsible for the cervical dysplasia which
afterwards advances into cervical cancer. In the early stages of the cervical cancer, usually no
symptoms are presented. In some cases, the complaints a woman might have are increased vaginal
discharge, abnormal vaginal bleeding and painful sexual intercourse. The abnormal bleeding will
manifest as prolonged and heavier bleeding during menstrual cycle, after sexual intercourse and
postmenopausal bleeding [46].
1.2.7. Diagnosis and Laboratory Screening
The clinical manifestation of low risk HPV, such as warts, is diagnosed by clinical examination, inspection of the macroscopic changes in the infected area and also a biopsy might be considered in the case of unclear diagnosis. In the cases that genital intraepithelial lesions are suspected, more tests are needed such as cytology, then colposcopy and further histological confirmation for staging [47].
The cervical screening program in Lithuania includes women from 25 to 60 years of age.
Firstly, the women undergo the Papanicolaou test, a cytology test. If the results are negative, women have to repeat the Papanicolaou test in 3 years. Women who receive an abnormal result will need to do an HPV test. When the Papanicolaou test reveals low squamous intraepithelial lesion or atypical squamous cells of undetermined significance and the HPV test is negative, the cytology test should be repeated in 6 – 12 months. In cases where the HPV test was positive, the patient is referred for a colposcopy. When the Papanicolaou test reveals high grade squamous intraepithelial lesion or atypical squamous cell cannot exclude high grade squamous intraepithelial neoplasia, the patients are referred for colposcopy, regardless of the HPV test results. After the results of the colposcopy are established according to degree, it is decided if a biopsy is needed. In cases of normal or low- grade lesions, the screening test is repeated in one year, on the other hand if colposcopy results reveal high grade lesions, a biopsy is indicated [48].
1.2.7.1. Cytology
The Papanicolaou test (Pap test) evaluates the morphological changes of cervical cells under cytological examination for the early detection of atypical or precancerous cells. The samples are from the cervix and are obtained by a gynecologist by the following procedure. The patient lies on her back on the gynecological table, spreads the legs apart and place them in the foot holds. Then, the doctor opens the vulva gently and inserts the speculum into the vagina. The speculum is turned horizontally so the cervix is visualized. The doctor examines the vagina walls and the cervix and checks if any discharge is presented. With the help of a pair of forceps and a cotton swap, the doctor will clean any mucus and discharge from the cervix. Afterwards, a cytobrush is inserted at the epithelial transitional zone of the endocervical canal. The cytobrush is rotated 360 degrees, 3 times, and then it is removed. Then the doctor spreads the collected material in a sweeping motion in a slide and from a distance of 30 cm, sprays a fixative solution. Finally, the doctor removes the speculum and labels the slide with the patient’s information and fills out the request for the cytology [49].
The results on the reports of a Pap test are in accordance to the Bethesda system. Firstly, the
specimen adequacy needs to be checked to see if it is satisfactory or unsatisfactory. In case of
unsatisfactory specimen adequacy, the test needs to be repeated in 3 months. Results from the Pap test may be normal or abnormal. Normal Pap test results illustrate that there are no intraepithelial lesions or a malignancy identified and the test is negative. In case where abnormal Pap test results are found it means that the test is positive. Under these circumstances abnormal findings include atypical squamous cells of undetermined significance (ASC-US), atypical glandular cells (AGC), low grade squamous intraepithelial lesion (LSIL), atypical squamous cell – cannot exclude HSIL (ASC-H), high grade squamous intraepithelial lesion (HSIL), adenocarcinoma in situ (AIS) and cervical cancer. In ASC-US, abnormal cells have been identified but the cause is unclear, for example if its due to infection or precancerous. On the other hand, with ASC-H, there are abnormal cells but with a higher probability of being precancerous. In LSIL, there is mild dysplasia or cervical intraepithelial neoplasia (CIN) 1 and in HSIL there is moderate and severe dysplasia as well as CIN2 or CIN3 [50].
In atypical squamous cells of undetermined significance, the changes insinuate LSIL with the nuclei size having increased from 2.5 to 3 times compared with normal intermediated squamous cells and the nucleus to cytoplasm area ratio is increased slightly. Also, the changes can be with or without nuclear shape being irregular, or with irregular distribution of the chromatin and mild nuclear hyperchromasia. Koilocytes are resembled by the cytoplasmic vacuoles or halos which are poorly formed. Furthermore, the cytoplasm may show parakeratosis which is when the cells have eosinophilic or organophilic cytoplasm with pyknotic nuclei [51].
Atypical glandular cells are arranged in strips or sheets with nuclear crowding and pseudo stratification. The nuclei size is enlarged by 3 to 5 times compared to the normal intermediated squamous cells. There is an increased ratio between nucleus to cytoplasm, as there is abundant
Figure 6: ASC-US. Intermediate cells with x2-3 times nuclear enlargement compared to normal intermediate squamous cell nucleus. There are binucleated cells which are rare. Also, there is nuclear hyperchromasia and irregularity that do not meet the diagnostic criteria of LSIL (Source: Nayar R, Wilbur D, editors. The Bethesda system for reporting cervical cytology: Definitions,
criteria, and explanatory notes. 3rd ed. Cham, Switzerland: Springer International Publishing; 2015)
cytoplasm. The nuclei are mildly hyperchromatic with mildly irregular distribution of the chromatin.
Nucleoli and mitotic activity are rarely presented [52].
In atypical squamous cells – cannot exclude HSIL, the changes that can be found are the distribution of the cells which can be singular or in groups of maximum 10 cells in strands of mucus.
The nucleus is 1.5 to 2.5 times bigger than in a normal cell and the general size of the cell is the same as the metaplastic cells. In case of ASC-H, the ratio of nucleus to cytoplasm might resemble that of HSIL. Moreover, in ASC-H involves atypical immature cells, with distinctly atypical repair and crowded sheets of cell with severe atrophy. There is also post-radiation changes in regarding residual or recurrent carcinoma.
In low squamous intraepithelial lesion, the cells can be found in cluster, in a single cell or in sheets. The cytological changes are usually restricted to cytoplasm, which can be superficial squamous or with mature intermediate squamous cell type cytoplasm. Generally, the cell size is large with well-established mature cytoplasm. The nucleus size is significantly increased by 3 times in area compared with the normal intermediate nuclei. Even so the nuclear to cytoplasmic ratio is increased by very little. In LSIL, the cell’s nuclei can be monochromatic but mostly are found to be hyperchromatic with the size being inconsistent. The distribution of the chromatin is even; however, the nuclear membranes contour is variable. In addition, multinucleation and binucleation are usually present. In LSIL, cells should illustrate nuclear abnormalities in cells with dense eosinophilia and koilocytosis being considered diagnostic. On the other hand, clearing in the lack of nuclear abnormalities or perinuclear halos are not suitable for the diagnosis of LSIL.
Figure 7: ASC-H. A group of immature metaplastic atypical cells with high nuclear to cytoplasmic ratio, enlarged nuclei, irregular nuclear contour and coarse chromatin. The cytologic changes are worrisome, however insufficient to meet the criteria
of HSIL. (Source: Nayar R, Wilbur D, editors. The Bethesda system for reporting cervical cytology: Definitions, criteria, and explanatory notes. 3rd ed. Cham, Switzerland: Springer International Publishing; 2015)
In high squamous intraepithelial lesions, the cells tend to be smaller with less mature cytoplasm compared to LSIL cells. In HSIL, the cells are distributed in syncytial – like aggregate pattern resulting in hyperchromatic crowded groups, but they can also be in sheets or in singly.
HSIL cells and LSIL cells have the same nuclear enlargement, but the HSIL cells have a decreased cytoplasmic area which results in an increase of the nuclear to cytoplasm ratio. Overall, the nuclei are hyperchromatic and they do not have nucleolus, the chromatin is equally divided and the nuclear membrane contour is distributed unevenly resulting in the presentation of grooves or prominent indentations.
Figure 9: HSIL. Dysplastic cells in hyperchromatic crowded group or in a syncytial cluster. (Source: Nayar R, Wilbur D, editors.
The Bethesda system for reporting cervical cytology: Definitions, criteria, and explanatory notes. 3rd ed. Cham, Switzerland:
Springer International Publishing; 2015)
Figure 8: A. LSIL. Mature squamous cells with nucleus enlargement with variable nuclear membranes and chromatin. Perinuclear cavitation or koilocytosis are characteristic for HPV infection. B. Nuclear area. Intermediate squamous cell’s nuclear area is approximately 35μm2, ASC-US cells have nuclear area of approximately 100 μm2 and LSIL ~150-175 μm2 (Source: Nayar R, Wilbur
D, editors. The Bethesda system for reporting cervical cytology: Definitions, criteria, and explanatory notes. 3rd ed. Cham, Switzerland: Springer International Publishing; 2015)
In endocervical adenocarcinoma in situ, the cells can be arranged in clusters, pseudostratified strips, rosettes, sheets or in peripheral feathering manner with nuclear crowding. The nuclei shape can be elongated or oval and enlarged. Also, the nuclei are evenly hyperchromatic with coarsely granular chromatin and the nucleoli are small or undistinguishable. Frequently apoptotic bodies and mitotic activity are found in a clean background.
The endocervical adenocarcinoma presentation is the same as in AIS with some additional findings. The endocervical adenocarcinoma is composed by a profuse number of abnormal cells which have a columnar presentation. The nuclei are enlarged, elongated with a pleomorphic appearance, with nuclear clearing and uneven distribution of the chromatin as well as nuclear membrane irregularities. The cytoplasm is well vacuolated and the diathesis of necrotic tumor is prominent [51].
Figure 10: A. Endocervical adenocarcinoma in situ. Pseudostratified strip of cells illustrating peripheral feathering, crowding and nuclear enlargement. B. Benign and neoplastic cells. Strip of cells and rosette configuration, nuclear hyperchromasia, increased nuclear to cytoplasmic ratio, crowding, overlapping and feathering. (Source: Nayar R, Wilbur D, editors. The Bethesda system for
reporting cervical cytology: Definitions, criteria, and explanatory notes. 3rd ed. Cham, Switzerland: Springer International Publishing; 2015)
Figure 11: Squamous cell carcinoma, keratinizing. Note the cell's pleomorphism in shape and size, as well as the cytoplasmic keratinization and the background tumor diathesis. (Source: Nayar R, Wilbur D, editors. The Bethesda system for reporting cervical cytology: Definitions, criteria, and explanatory notes. 3rd ed. Cham, Switzerland: Springer International Publishing;
2015)
1.2.7.2. HPV – DNA test
The HPV test is a screening test done by the polymerase chain reaction (PCR) for high risk types of HPV. For this test, the same samples that have been obtained from the cervix during the Pap test are used. Results which are negative illustrate that no HPV DNA was found in the targeted genotypes of the HPV. Positive results in HPV-DNA tests indicate the presence of one or more of the high risk genotypes, such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68. A positive HPV-DNA test shows that later in life there is a higher probability of developing cervical cancer and in accordance with the Pap smear results and the age of the patient, further recommendation are established [53].
1.2.7.3. Colposcopy
A colposcopy is a procedure that uses a modern colposcope with a magnifier x40 to visualize the cervix tissue and the vaginal wall. The colposcopy requires the patient to lie on their back on the examination table, spreading their legs apart and placing them in the leg holders. Then lubrication is applied on the speculum and it is inserted into the vagina, for the successful visualization of the cervix. The doctor then checks and evaluates the surrounding tissue for any signs of inflammation and secretions. A dry swap is then used to remove any secretions from the cervix. The next step is the application of the acetic acid test and the iodine test, which are considered to be essential in the colposcopy examination.
The acetic acid test is the application of 3% acetic acid in the cervix and it is considered positive when there are areas colored in white. In the areas where ectopy is presented, the ectopic columnar epithelium from the deep red color transforms into pale, with shades of pink, to white.
Also, in ectopy, the epithelium has a grapelike appearance which becomes more accentuated, as the villi are enlarged and swollen. When acetic acid is used, in cases of atypical epithelium, the color turns into white. In addition, mosaic and punctation pattern become more evident as well as fine petechiae and partitions as they become more evident against the white epithelium. At the end of the examination a decision has to be made as to whether a biopsy is needed and where the samples should be taken.
For a more objective evaluation of the colposcopy findings there are two score systems in
place, the Reid’s colposcopy index and the Swede score both of which are used to predict the
histology results. During 1980s, Reid R. and Scalzi P. established a scoring system for the
colposcopic evaluation to grade precancerous cervical intraepithelial lesions in a more objective
fashion. The scoring system relies on four different morphological changes that are: the color after
the application of the acetic acid, the lesion margins, the vessels presentation and the iodine staining
range from 0 to 2 points in each category. Then the points are summed up and the final score for the Reid Colposcopy index (RCI) is found. Score in RCI 0 – 2 are likely to be CIN1 in histology, RIC 3 – 4 is likely to be CIN1 or CIN2 and RCI 5 – 8 is likely to be CIN2-3 in histology [55]. The Swede score has the same principle, but in Swede score there are 5 characteristics that are evaluated (acetic acid uptake, lesion margins, blood vessels characteristics, the size of the lesion, Lugol’s iodine results). In Swede score, again each category has a points from 0 – 2 and in the end the sum of the points from all the categories is established. Swede score of 0 – 4 predicts normal or CIN1, 5 – 6 predicts CIN2 or CIN3 and 7 – 10 predicts CIN3 or suspected cancer. [56] Many studies have evaluated this scoring system and they found them to have a significant compliance with the histopathological findings [57, 58].
1.2.7.3.1. Normal colposcopy Finding
Normal colposcopy finding is when the cervix uteri original squamous epithelium is smooth and no gland openings are presented, this differentiates it from the normal squamous epithelium which derives from metaplasia. The normal squamous epithelium which derives from the metaplasia is covered by retention cysts and gland openings, and as a result it reveals that the area was initially occupied by columnar epithelium. During the reproductive period, the original squamous epithelium has a reddish color that fluctuates form pale to deep pink depending on the different stages of the menstrual cycle. During the iodine test, the original squamous epithelium is stained brown, which indicates its glycogen content.
The atrophic squamous epithelium is a result of the lack of estrogen after menopause, consequently causing the squamous epithelium to become thin, lack glycogen and diminishing the blood supply from the stromal layers. The consequences of these changes are the distinct appearance of the network of capillaries due to the pale epithelium. During the iodine test, there is evident irregular uptake and stippled appearance because of the loss of glycogen and epithelial thinning. In cases of complete loss of glycogen in older women, the epithelium presents an even light brown to yellow color. The terminal vessels are more susceptible to minor trauma, which may result in subepithelial hemorrhages and erosions as the epithelium is thin and fragile.
The original squamocolumnar junction is located in the external os. The squamocolumnar
junction is the junction at the columnar epithelium and the squamous epithelium. In young women
the columnar epithelium is located on the ectocervix, with a red appearance surrounding the external
os, and this is called ectopy. Ectopy is presented as a red patch with an exclusive papillary
architecture. During the iodine test the ectopy is not stained with iodine. The mucus secreting
columnar epithelium covers the ectopy. During the acetic acid test, the mucus is removed and the
papillary structure is revealed. Also, the acetic acid causes swelling in the tissue, showing the
papillae as a grapelike. The red patch is substituted with a pink or white color. A sharp steplike view
has the squamocolumnar junction. The initiation of the transformation zone is revealed by the gland openings and the white margin.
The transformation zone is the area among the current squamocolumnar junction and the original squamous epithelium. Across the TZ, Nabothian cysts, gland openings and islands of columnar epithelium can be found. The TZ changes from red color to a grayish white after the application of acetic acid. Metaplastic epithelium of various degrees occupies the transformation zone and the maturity of the metaplastic epithelium can be identified by the application of the iodine intake. There are three different types of the TZ. The type 1 of the TZ is fully visible and it is and it is located fully ectocervically. The type 2 TZ it is totally or partially endocervical however it is visible. The type 3 TZ is totally or partially endocervical and is not entirely visible.
1.2.7.3.2. Abnormal Findings
Abnormal findings in colposcopy are the mosaic, punctation, vascular changes, acetowhite epithelium and leukoplakia. Acetowhite epithelium occurs after the application of 5% of acetic acid.
In the cases where there is a rapid and dense appearance of the acetowhite epithelium, this indicates
a high-grade lesion in comparison with a slow and slight acetowhite change which occurs in a low
grade lesion. Leukoplakia is when there is a white patch, visible to the naked eye before the
application of acetic acid. Leukoplakia can be spotted outside or within the TZ. The leukoplakia
finding cannot be a significant finding in relation to the degree of the lesion. The mosaic term is
used to describe a vascular pattern which forms a polygonal irregular network of tile-like blocks in
appearance. The mosaic pattern can be categorized into fine and coarse, which consequently reflect
low grade and the high grade lesions respectively. Fine mosaic is defined as a fine network of red
pale lines and a coarse mosaic it is defined by its increased irregularity, the network of vessels are
prominent by their intense red color and the larger interpapillary distance. A punctation pattern is
the illustration of dots in a stipple arrangement which indicates the dilated terminal capillaries are
visible on the epithelium surface. Once again, a fine punctation is associated with the low grade
lesions, and the coarse punctation pattern is associated with the high grade lesions. In fine
punctuation the dots are close together and they may appear as white or red. Coarse punctation is
seen when the interpapillary distance is bigger and the vessels caliber is bigger [59].
1.2.7.4. Histopathology
The conclusive diagnosis for the degree of the cervical intraepithelial neoplasia is established by the histopathological examination of tissue which is obtained during the punch biopsy or with the excision specimen from the cervix. After an abnormal Pap smear the patients are referred for colposcopy and the colposcopy directed cervical biopsy. The biopsy sample is assessed under light microscope to reveal the cell changes.
The stratification, maturation and differentiation of the cells and the nuclear abnormalities will reveal if there is a cervical intraepithelial neoplasia and its degree. The degree of the epithelium thickness, which exhibits differentiation and maturation of the cells, is used to determine the grading of the cervical epithelial neoplasia. During the diagnosis, nuclear abnormalities are evaluated. The nuclear abnormalities that can be found are nuclei enlargement, increased nuclear to cytoplasmic
Figure 12: Schematic Illustration of mosaic and punctation pattern (Source: Training of health staff in colposcopy, LEEP and CKC – Trainees’ handbook, World Health Organization, 2017)
Figure 13: Low grade changes in colposcopy. A. Acetonewhite area with irregular margins. B. Fine mosaic pattern. C.
Fine punctations (Source: Training of health staff in colposcopy, LEEP and CKC – Trainees’ handbook, World Health Organization, 2017)
Figure 14: High grade changes in colposcopy. A. Dense acetonewhite area with raised sharp margins. B. Coarse mosaic pattern. C. Coarse punctuation (Source: Training of health staff in colposcopy, LEEP and CKC – Trainees’ handbook,
World Health Organization, 2017)
ratio, nuclear polymorphism, hyperchromasia and the variable sizes of the nucleus. In the parabasal layer there is increased mitotic activity, however when it is located in the superficial layers it indicates higher CIN severity. A biopsy is recommended for patients who had HSIL results in cytology, who tested positive with high risk HPV 16 or 18 and for whom during the colposcopy high grade changes were observed. Histological findings are classified as low grade cervical intraepithelial neoplasia or CIN1 and high grade cervical intraepithelial neoplasia as CIN2 and CIN3 or cervical carcinoma in situ.
In low grade CIN or CIN1, the dysplastic changes are confined to the lower one third of the cervical epithelium thickness. The changes which are noted are nuclear atypia with increased nuclear to cytoplasmic ratio and increased mitotic activity. HPV infection changes may be observed in the full thickness of the epithelium, such as koilocytosis. In CIN2, the cellular dysplastic changes are limited into the lower two thirds of the epithelium thickness. There are atypical cell changes, with increased nuclear to cytoplasmic ratio, pleomorphism and increased mitotic activity. CIN3 affects the upper one third from the entire thickness of the epithelium and the changes are cellular atypia and loss of maturation. In the case of carcinoma in situ, which is in the high grade CIN, this involves the entire thickness of the cervical epithelium [60].
1.2.8. Factors that affect diagnostic discrepancy
The factors that contribute in diagnostic discrepancy are the colposcopy findings evaluation, and the morphological changes that suggest high grade lesions. Visual changes such as dense acetonewhite epithelium, coarse mosaic, coarse punctuation and atypical vessels are not reliable to predict the histological degree of the abnormality. The interpretation of colposcopic findings may be difficult to be evaluated when erosion and severe cervical inflammation are presented, as they might resemble the morphological view of the CIN and invasive cancer. Several diseases such as Herpes simplex may replicate the changes of cervical cancer and for that reason a biopsy sometimes is indicated to distinguish between the two. Also, Syphilis is presented as an ulcer, which might
Figure 15: Histopathologic progression from normal epithelium to LSIL (CIN1), to HSIL (CIN2) and (CIN3) and carcinoma in situ (CIS) (Source: Christopher P. Crum, Emily E. Meserve and William A. Peters. Diagnostic Gynecologic and Obstetric
Pathology, E3, Chapter 13, Pages 298-374)
resemble an invasive cervical cancer and a biopsy again is indicated to distinguish the final diagnosis [55].
Another factor that may contribute to diagnostic discrepancy is the spontaneous regression of the lesion either to normal tissue or to a less severe lesion. The cervical intraepithelial lesions may regress naturally, and this may lead to the observation of a discordant in diagnosis [61].
A punch biopsy sample is obtained from the area with the most abnormal tissue. Several studies suggested to obtain tissue samples from two or more areas as this increases the high grade lesions sensitivity detection [61, 62].
In study conducted by Kim SI et al. (2020), it was established that the colposcopy – directed biopsy inaccuracy was increased in women with HSIL in cytology and 50 years of age or more [63].
In another study, women from 40 years of age and older had decreased sensitivity of the colposcopy directed biopsy in comparison with the endocervical curettage which showed increased sensitivity in CIN2+ lesion detection [64].
1.2.9. Management
There is no definitive treatment for the human papillomavirus infection. With regards to genital warts or low grade CIN, these will regress within six months without any treatment. There are different types of topical creams that can be used in the treatment of genital warts. Example of these topical treatments are podophyllotoxin solution and imiquimod cream, which are applied to the area the warts are presented. Podophyllotoxin solution 0.5% is applied with a swab to the visible warts two times per day for three days, then 4 days without treatment. This cycle can be repeated up to 4 times. Imiquimod 5% cream is applied once per day, 3 times per week for up to 16 weeks.
Around 6 to 10 hours after the application of treatment, the area must be washed with soap and water.
With reference to precancerous lesions, the following procedures can be applied:
cryotherapy, loop electrosurgical excision procedure (LEEP) and conization. Cryotherapy is a procedure which uses liquid nitrogen on the warts and destroys them. This procedure can be repeated every 7 days and up to 7 treatments are allowed. The LEEP is performed under local anesthesia and, with low voltage electrified thin loop wires, genital warts are removed by separating the warts from the skin, as the excision of the precancerous lesions of the cervix. Conization is the procedure which involves the excision of a cone shape tissue from the cervix, usually done under general anesthesia [65, 66].
A good preventive measure is the HPV vaccine which is recommended for ages 9 – 26. The
vaccine is administered in the deltoid region intramuscularly. The routine examination schedule
recommends the vaccination at 11 or 12 years of age before the sexually activity is initiated. For
persons who are 9 to 14 years of age, 2 doses of vaccine are required, with the second dose being 6
months apart from the first one. For persons who are 15 – 26 years of age or who are
immunocompromised three doses are required, with the second dose being 2 months apart from the
first one, and the third dose six months apart from the second dose. At the moment, there are three
licensed HPV vaccines available. The first one is a quadrivalent vaccine named “Gardasil” and
targets HPV types 6, 11, 16 and 18. The Gardasil vaccine provide prophylaxis against genital warts,
cervical precancer and cancerous lesion in both female and males, aged 9 to 26 years. The second
vaccine is bivalent and is called “Cervarix”. It targets the HPV types 16 and 18, which are the high
risk types that might lead to cervical precancerous and cancerous lesions, and it is available only
for females. The third vaccine is nonvalent called “Gardasil 9” that targets HPV types 6, 11, 16, 18,
31, 33, 45, 52 and 58. The Gardasil 9 vaccine provides prophylaxis against genital warts, cervical
precancerous and cancerous lesions as well as vaginal, vulvar and anal cancer in both female and
male between the ages of 9 to 45 years [67].
2. METHODS
This is a retrospective study which was conducted by reviewing personal files in the hospital information system. The study took place in the Hospital of Lithuanian University of Health Sciences (LSMU) Kauno klinikos in the department of Obstetrics and Gynecology. A bioethics approval (BEC-MF-177) was obtained before the beginning of the study. They were 221 patients who arrived to LSMU KK gynecological department for cervix conization procedure, during the period of 2020.01.03 to 2020.12.30.
Patients for whom no files were available in the hospital’s electronic database or the documentation was not understandable, as well as patients who did not have cytology, colposcopy or biopsy results, were excluded from this study. Therefore, the sample size included 174 females and their ages were between 21 – 63 (mean 37
!8.596) out of the 221 female patients.
From the personal files of the patients, the data that was selected was the age of the patient, the primary cytology results, if they had a punch biopsy after the cytology and then the grade of the lesion that was established. Also, the data concerning the HPV test and the colposcopy findings was obtained. Lastly the biopsy results after conization were obtained.
Abnormal Papanicolaou smears were classified according to Bethesda system classification.
Patients who had cervicitis or normal cytological findings were classified in one group as normal.
The punch and conization biopsy results were categorized as normal, low and high grade precancerous lesions and cancer. Normal histopathological results were concluded when no abnormalities were revealed or when inflammation was presented. Low grade lesions were considered when the histopathology results revealed cervical intraepithelial neoplasia – 1 (CIN1).
High grade lesions were considered when the histopathology revealed cervical intraepithelial neoplasia 2 and 3 (CIN2, CIN3) and carcinoma in situ (CIS). When the histopathological results were marked as cervical cancer, in the study it they were marked as cancer, irrespective of grade.
Firstly, it was assessed whether the HPV test had been performed or not. Secondly, for the patients who had the HPV test, the results were marked as negative and the positive cases were categorized as “Other High risk HPV” (hrHPV) types (HPV- 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68), “HPV16, other hrHPV” (HPV16, and any other high risk type except 18) “HPV18, other hrHPV” (HPV18, and any other high risk type except 16), “HPV16 and 18” (HPV16 and 18 only),
“HPV16” and “HPV18” .
Colposcopy results were classified as normal, low or high grade changes and as not
informative. Based on if the pattern was fine or coarse, mosaic and punctation cases were classified
as low or high grade changes respectively. The acetonewhite epithelium and vascular changes were
taken into consideration for the colposcopy grade changes. In addition, during colposcopy the
visibility of the TZ was established. Knowing if the TZ is visible or not, colposcopy can be classified as satisfactory or unsatisfactory. Visible TZ was considered when transformation zone was type 1 and type 2. Type 1 is when the transformation zone is fully visible and it is completely ectocervical.
Type 2 is when the transformation zone is fully visible with an endocervical component, and it may have an ectocervical components as well. TZ type 3, is when transformation zone was not fully visible. In addition, there were cases where the TZ was not described at all.
The collected data from the patients’ files were encoded and entered into a Microsoft Excel spreadsheet. Analysis was carried out on the IBM SPSS Statistics for Mac, version 27 (IBM Corp., Armonk, N.Y., USA). Descriptive statistics were used to determine the frequencies of different levels of the cervical squamous intraepithelial lesions in cytology, primary punch biopsy and during the conization biopsy. Also, descriptive statistics were used to determine the grade of changes during the colposcopy examination and the number of women who were tested for HPV and the HPV Type.
Lastly, descriptive statistics were used for the age groups among the women who participated in the study. Chi-square test (X
2-test) was used to determine the association between different categorical variables.
For the chi-square test to be meaningful it is imperative that each person, item or entity
contributes to only one cell of the contingency table. Furthermore, an assumption of the chi-square
test is that the expected frequencies should be greater than five. Although, it is acceptable in larger
contingency tables to have up to 20% of expected frequencies below 5, the result is a loss of
statistical power (so, the test may fail to detect a genuine effect). Even in larger contingency tables
no expected frequencies should be below one [68, 69]. Fisher’s exact test might be used for those
cases, even though it can be used only for 2 × 2 contingency tables. Phi and Cramer’s V are measures
of the strength of association between two categorical variables. If one of the two categorical
variables contains more than two categories, then Cramer’s V is preferred to phi because phi fails to
reach its minimum value of 0 (indicating no association) in these circumstances. For this study, the
Cramer’s V values <0.2 were considered to be a week association, 0.2 – 0.6 a moderate association
and >0.6 a strong association. In this study p <0.05 was considered to be statistically significant.
3. RESULTS
3.1. Frequency and percentage distribution for all variables used in this study
The age distribution among the participants (Table 1) was: 21.3% were 20 – 29 years of age;
44.3% were 30 – 39 years of age; 24.1% were 40-49 years of age; 9.2% were 50 – 59 years of age and 1.1% were 60 – 69 years of age.
Table 1: Frequency and percentage distribution for the age of participants used in this study.
Age Groups Frequency Percentage
20-29 37 21.3%
30-39 77 44.3%
40-49 42 24.1%
50-59 16 9.2%
60-69 2 1.1%
Total 174 100.0%
In (Table 2) Papanicolaou test results, 3.4% of women had “Normal” results, 11.5%, 17.8%, 19.5%, 46.6% and 1.1% were ASC-US, LSIL, ASC-H, HSIL and AIS respectively.
The punch biopsy results reveal 4.0% of the participants to have “Normal” histological evaluation, 2.9% and 93.1% had LSIL and HSIL, respectively.
The HPV test was performed among 69.0% of the participants while 31.0% did not had the HPV test. From the participants that have been tested for HPV, 6.7% had negative results. Other hrHPV genotypes were identified in 40.0% of women, HPV 16 – other hrHPV were identified in 8.3% women, HPV 18 – other hrHPV were identified in 0.8% and HPV 16 and 18 were found in 1.7% of participants. HPV 16 and HPV 18 were found in 37.5% and 5.0% respectively among the tested women.
During the colposcopy examination the TZ visibility was evaluated. The TZ was not described among 21.3% colposcopies, TZ was visible in 71.8% and not visible in 6.9%, which shows that they were 71.8% satisfactory colposcopies and 6.9% unsatisfactory colposcopies.
Furthermore, the colposcopic findings were 1.1% normal, 25.3% had low grade changes, 71.3% had high grade changes and 2.3% the colposcopy examination was identified as not informative.
The conization biopsy results were 5.7% defined as normal, 5.2% were LSIL, 85.6% were
HSIL and 3.4% were established as cancer.
Table 2: Frequency and percentage distribution for all variables used in this study.
Frequency Percentage (%)
Papanicolaou Test Results
Normal 6 3.4%
ASC-US 20 11.5%
LSIL 31 17.8%
ASC-H 34 19.5%
HSIL 81 46.6%
AIS 2 1.1%
Total 174 100.0%
Punch Biopsy Results
Normal 7 4.0%
LSIL 5 2.9%
HSIL 162 93.1%
Cancer 0 0.0%
Total 174 100.0%
HPV-Test: Tested or Not tested
Tested 120 69.0%
Not tested 54 31.0%
Total 174 100.0%
HPV Type
Negative 8 6.7%
Other hrHPV 48 40.0%
HPV 16. other hrHPV 10 8.3%
HPV 18. other hrHPV 1 0.8%
HPV 16 and 18 2 1.7%
HPV 16 45 37.5%
HPV 18 6 5.0%
Total 120 100.0%
Transformation Zone Visibility in colposcopy
Not Described 37 21.3%
TZ Visible 125 71.8%
TZ not Visible 12 6.9%
Total 174 100.0%
Colposcopy results
Normal 2 1.1%
Low grade changes 44 25.3%
High grade changes 124 71.3%
Not informative 4 2.3%
Total 174 100.0%
Conization Biopsy Results
Normal 10 5.7%
LSIL 9 5.2%
HSIL 149 85.6%
Cancer 6 3.4%
Total 174 100.0%
3.2. Percentage distribution of punch biopsy deriving results, between tested and not tested for HPV
From the women who proceeded for the HPV test, 5.8% women were normal, 4.2% were LSIL and the 90.0% were HSIL in punch biopsy deriving results. (Table 3)
Table 3: Frequency and percentage distribution of punch biopsy deriving results, between tested and not tested for HPV.
HPV-Test Punch Biopsy Results
Normal LSIL HSIL Total
Tested f 7 5 108 120
% 5.8% 4.2% 90.0% 100.0%
Not tested f 0 0 54 54
% 0.0% 0.0% 100.0% 100.0%
Total f 7 5 160 174
% 4.0% 2.9% 93.1% 100.0%
In Figure 16, the percentage distribution of punch biopsy deriving results, between tested and not tested for HPV is illustrated graphically.
Figure 16: Percentage distribution of punch biopsy deriving results, between tested and not tested for HPV.
0% 20% 40% 60% 80% 100%
Tested Not tested Total
5.8%
4.0%
4.2%
2.9%
90.0%
100.0%
93.1%
Punch Biopsy results
HPV-Test
Normal LSIL HSIL
