Journal of Womens Health, Issues and Care ISSN: 2325-9795

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Research Article, J Womens Health Issues Care Vol: 2 Issue: 6

The Correlation of the Lifestyle and Medical Conditions with the Incidence of Micronuclei in the Vaginal Epithelial Cells

Štefica Findri-Guštek1, Višnja Oreščanin2*, Nevenka Kopjar3, Emilija Mlinarić-Missoni4, Ivan Fistonić5 and Ines Krivak Bolanča6
1Findri-Guštek L.t.d., Centre for gynecology, urology and occupational medicine, Ninska 5A, Sesvete, Croatia
2Advanced Energy Ltd., V. Prekrata 43, Zagreb, Croatia
3Institute for Medical Research and Occupational Health, Mutagenesis Unit, Ksaverska cesta 2, Zagreb, Croatia
4Croatian Institute of Public Health, Rockfellerova 7, Zagreb, Croatia
5Private gynecological practice, Preradovićeva 10, Zagreb, Croatia
6”Merkur” University Hospital, Zagreb, Croatia
Corresponding author : Višnja Oreščanin
Advanced Energy Ltd., V. Prekrata 43, Zagreb, Croatia
Tel: +385-1-6535532
E-mail: [email protected]
Received: September 10, 2013 Accepted: November 19, 2013 Published: November 25, 2013
Citation: Findri-Guštek Š, Oreščanin V, Kopjar N, Mlinarić-Missoni E, Fistonić I, et al. (2013) The Correlation of the Lifestyle and Medical Conditions with the Incidence of Micronuclei in the Vaginal Epithelial Cells. J Womens Health, Issues Care 2:6. doi:10.4172/2325-9795.1000129

Abstract

The Correlation of the Lifestyle and Medical Conditions with the Incidence of Micronuclei in the Vaginal Epithelial Cells

The objective of this study was determination of the causative factors of the cytogenetic damage (expressed as incidence of micronuclei) of the vaginal epithelial cells in the patients with/without the symptoms of the genital infections. 197 women in the age range from 18 to 65 years were included in the study. After the completion of questionnaire all the patients were subjected to Pap test, cervical swabs for the presence of aerobic bacteria, yeasts, Ureaplasma urealyticum, Chlamydia trachomatis, Mycoplasma and hrHPV DNA. Separate samples of vaginal secretion were taken for micronucleus assay.

Keywords: Lifestyle; Environmental and Medical Exposure; Micronucleus assay; Vaginal epithelial cells, Bacterial infection; Viral infection; Yeast infection

Keywords

Lifestyle; Environmental and Medical Exposure; Micronucleus assay; Vaginal epithelial cells, Bacterial infection; Viral infection; Yeast infection

Introduction

Scaled epithelial cells of the cervix are of the great importance in cytology [1]. Staining of these cells according to Papanicolaou can detect abnormal changes. This can range from premalignant to malignant transformations. Cervical cancer is the third most common form of the cancer in women. This form of cancer occurs as the result of changes in the progression of low grade squamous intraepithelial lesions (LGSIL) in the high grade lesions (HGSIL). The transition from low to high grade lesions is characterized by a high degree of nuclear atypia, and the inability of cell differentiation in the superficial layers of the epithelium. It is believed that these changes are accompanied by genetic instability and mutations, such as loss or gain of chromosome fragments, which would explain the occurrence of specific chromosomal aberrations [2].
Chromosomes or parts of the chromosomes that fail to incorporate into the cell nucleus during the cell division forming micronuclei (MN). MN are the indicators of chromosomal damage [3]. Various endogenous factors contribute to the spontaneous occurrence of micronuclei. Besides, the substances normally present in the environment, habits and lifestyle could be also significant promoting factors. Knowledge of the mechanisms and causes of spontaneous micronuclei formation is very important for the proper interpretation of micronucleus assay. All processes that directly promote DNA, chromosomes and spindle damage will contribute to an increase in the number of micronuclei [4]. Oxidative processes that take place within the cell and organism are particularly significant [5] as well as gene polymorphism, and different gene mutations that can lead to genome instability [6].
The aim of this research was determination of the frequency of micronuclei occurred in the vaginal epithelial cells of 197 Croatian women as well as the correlation of these results with the lifestyle and general health status of the woman. The presence of the symptoms of infection and the presence of genital infections were also correlated with the total number of MN.

Materials and Methods

Study design
Detailed description of the study design was described in our previous work [7]. 200 patients (80 patients without the symptoms of vaginosis serving as a control group and 120 patients with the symptoms of vaginosis serving as an experimental group) in the age range from 18 to 65 years were included in the study. All of them agreed to participate in the investigation and allowed data publication, confirming that by signing the written informed consent form. Three participants were withdrawn from the study due to pregnancy discovered at their first checkup (among them two with the symptoms of infection). After the completion of questionnaire containing the questions about the health status and lifestyle, all 197 patients were subjected to Pap test, cervical swabs for the presence of aerobic bacteria, yeasts, Ureaplasma urealyticum, Chlamydia trachomatis, Mycoplasma sp., and hrHPV DNA as well as vaginal secretion the for detection of the presence of micronucleated cells. Following the analysis of Pap test and cervical swabs, it was discovered that most of the asymptomatic participants were positive to either fungal, bacterial or viral infections or all the three, and the opposite, some patients from the experimental group who exhibited symptoms (secretion, etching) were found negative to all the conducted tests. Due to the above mentioned reasons, the entire population (all the 197 participants) was considered as one group while performing statistical evaluation.
For Ureaplasma urealyticum, Chlamydia trachomatis, Mycoplasma sp. Aptima swab kits (containing sterile swab, transport tube with transport medium) were used (Gen-Probe Incorporated). ThinPrep kit with liquid-based medium was used for the collection and transport of the samples for Pap test and HPV detection (Hologic). For aerobic bacteria Gemini kit (Gemini) was used for sampling and transport. Yeasts were collected with dry swabs and put in transport tube. The swabs for analysis of metabolites were put into transport tube and kept at -400C until analysis. All samples were transported into laboratory within two hours from sampling [7].
Microbiological examination of uterine cervix swabs was performed at the Department of Microbiology of Dr. Andrija Štampar Institute of Public Health [7]. Two separate swab samples were used to perform bacterial and mycotic examinations. One swab sample was used for Gram stain so as to determine the number of polymorfonuclear leukocytes and the presence of multicellular yeast forms. The other swab sample was immersed into Amies transport medium and used for cultivation of possible causative agents.
Yeasts were grown on Sabouraud Glucose Agar and Candida ID 2 chromogenic agar medium (BioMerieux). Nutrient agar plates were incubated at 35°C for 24 hours and at room temperature for the next 48 hours [7]. Candida species were identified by a germination test, the colony appearance on chromogenic and sweet corn agar, and using a standard yeast identification system (API-Candida). C. dubliniensis was identified at the Croatian Ministry of Health and Social Welfare Reference Centre for Mycological Diagnosis of Systemic and Disseminated Infections within the Croatian National Institute of Public Health.
All microorganisms cultured from the cervical smears were identified by routine laboratory methods, including the API test (Bio- Merieux). Diagnosis of genital Mycoplasmas was confirmed using semi quantitative tests for cultivation and Mycofast and MacoIST (Bio-Merieux), while molecular detection is performed using real time polymerase chain reaction (PCR) (Applied Biosystem) [7].
Swabs for Pap test were fixed with alcohol and have been stained according to Papanicolaou protocol [7].
HPV DNA detection and typing was done by polymerase chain reaction (PCR) according to the protocol developed by Fujinaga et al. [8].
Analysis of micronuclei was conducted in Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb. 197 samples of vaginal smears after slides staining according to the modified method [4] were analyzed under a light microscope (Olympus CX41, Tokyo, Japan) with a 40 × 10 magnification. At each slide a 1000 cells were examined in order to determine the total number of micronuclei.
Statistical analysis
For statistical evaluation Statistica 6.0 and MedCalc software packages were employed. Original values without any data transformations were used in the study. In order to determine the influence of lifestyle and medical condition of the tested population on the formation of micronuclei multiple regression analysis and advanced regression model (expressed in the form of Pareto Charts of t values) were used. Results of all 197 samples were used in the model. The following predictor variables were used for that purpose: (1) age, (2) occupation, (3) smoking habits, (4) alcohol intake, (5) marital status, (6) number of partners in the last 12 months, (7) type of contraception if any, (8) number of births, (9) normal birth (Y, N), (10) number of induced miscarriages, (11) number of spontaneous miscarriages, (12) professional exposure (dust, organic/inorganic chemicals, irradiation), (13) sporting, (14) type of nutrition, (15) coffee intake, (16) intake of minerals, (17) intake of vitamins (18) intake of antioxidants, (19) tumor illness, (20) virus illness, (21) Chronic illness (22) use of antibiotics-orally in the last 30, (23) use of antibiotics-locally in the last 30 days, (24) use of antimycotics-orally in the last 30 days, (25) use of antimycotics-locally in the last 30 days, (26) intake of other medications in the last 30 days, (27) presence of symptoms of vaginal infections (itching, burning, vaginal secretion, incontinence), (28) exposure to hard physical conditions, (29) exposure X-rays, (30) exposure to ultrasound, (31) exposure to CT, (32) drug abuse. These two statistical methods were also used for the assessment of the influence of the predictor variables (simptoms, Pap test, hrHPV, aerobic bacteria, Ureaplasma urealyticum, Chlamydia trachomatis, fungi) on the formation of micronuclei. Statistical significance was set to p<0.05 in all conducted tests.

Results

Description of the tested population
The frequency and the percentage of the variables: occupational exposure, marital status, number of partners, number of births, type of birth, spontaneous miscarriage, induced miscarriage are shown in Table 1. 174 of 197 (85.71%) of the women were not in the course of its life been occupationally exposed to some of the toxic agents. As regards to marital status 101 (49.75%) of the women at the time of testing were married. The number of partners in the last year ranged from 0 to 5. Women with one partner (74.88% of the population) are predominant, followed by 2 partners (7.88% of the population). The number of births also varied from 0 to 5. Women who have not given birth (45.81% of the population) are prevalent. Among the women who gave birth most of them had two births (26.60% of the population), followed by women with one birth (16.26% of the population). Most of the women had normal delivery (87.68% of the population). 85.71% of the population had no miscarriages while the rest of the population had 1-5 spontaneous miscarriages with the highest incidence one miscarriage (5.42%). 86.70% of the population had no induced miscarriages while 7.88% of the women had one miscarriage, and 0.99% had two miscarriages.
Table 1: The frequency (N) and the percentage of predictor variables: occupational exposure, marital status, number of sexual partners in the last year, the total number of births, type of birth, the total number of spontaneous miscarriages, the total number of induced miscarriages.
The frequency and the percentage of the independent variables: the use of contraception, the type of diet, vitamins intake, antioxidants intake, minerals intake, herbal preparations intake, smoking habit, alcohol consumption habit, coffee consumption, narcotics abuse, exposure to chemical agents are presented in Table 2. From the tested population 27.59% of the women used some form of contraception. As regards to diet cooked food is prevalent (53.20% of the population), followed by the combination of cooked and baked foods (28.08% of the population). Vitamins were taking 18.22% of the population. 7.39% of them used antioxidants, 8.37% taking minerals regularly, while herbal preparations used 5.42% of the population. At the time of completion of the questionnaire 27.57% of the women were smokers, 32.02% had consumed some form of alcohol while 80.79% of the population consumed coffee. Narcotics have used 3.94% of the population while only one woman (0.49% of the population) was exposed to the chemical agents.
Table 2: The frequency (N) and the percentage of independent variables: the use of contraception, the type of diet, taking vitamins, taking antioxidants, taking minerals, taking herbal preparations, smoking habits, alcohol consumption, coffee consumption, narcotics use, exposure to chemical agents.
The frequency and the percentage of predictor variables: sporting, exposure to heavy physical work, viral diseases, chronic diseases, tumor disease, HPV, medical exposure to X-rays, medical exposure to CT irradiation, medical irradiation exposure to ultrasound, taking antibiotic-locally, taking antibiotic-orally, taking antimycotics locally, taking antimycotics orally, taking other medications are shown in Table 3. From the tested population 29.06% of the women at the time of completing of the questionnaire were engaged in some type of sport activity. To heavy physical work 6.89% of the women were exposed. Viral diseases were present in 24.14% of the population while the same percentage of the population had some of the chronic diseases. Tumor diseases were present in 4.43% of the population and HPV in 13.30% of the population. 15.27% of the population was medically exposed to the X-rays, 3.45% to the CT-irradiation, and 30.54% to the ultrasound irradiation. Local application of antibiotics was characteristic of 7.39% of the population, and oral intake of 54.68% of the population. Local antimycotics were used by 18.72% of the population, and oral by the 1.97% of the population while the various symptoms were present in 57.67% of the population (including different types of secretion, itching and incontinence).
Table 3: The frequency (N) and the percentage of each predictor variables describing sport activity, general health status, medical exposure to irradiation, various medication intake in the last month and presence of symptoms.
The frequency and the percentage of each parameter obtained by the Pap smears and cervical swabs were presented in our previous work [5]. Briefly, in 36.3% of the total population low grade lesions were obtained by Pap test. Aerobic bacteria were isolated in 72.1% of the patients, yeasts in 18.3% of the samples, Ureaplasma urealyticum in 31% of the samples, Chlamydia trachomatis and Mycoplasma sp. were isolated in 2.5% and 1% of the samples, respectively while hrHPV DNA was isolated from 25.9% of the tested samples.
The incidence of micronuclei
The frequency of micronuclei in 197 of the analyzed samples showed log-normal distribution (Figure 1). The number of micronuclei ranged from 0 (in 7 analyzed samples) to 22 (three samples analyzed). Most of the samples (33 of them) had 6 micronuclei, while the five and nine micronuclei were found in 20 samples. The mean value of micronuclei per 1000 cells examined were 7.17, while the median value was 6 micronuclei. The results are characterized by a large standard deviation (4.47), while the relative standard deviation was 62%.
Figure 1: The distribution of micronuclei in vaginal epithelial cells in 197 of the analyzed samples of vaginal secretion.
In this work the frequency of micronuclei was correlated with the variables describing the patients’ lifestyle and various stress factors, as well as the exposure to irradiation as part of a medical examination. Multiple regression analysis revealed a good (R=0.69), statistically significant (p=0.0441) correlation between the predictor variables and the frequency of micronuclei. A statistically significant influence was found for the following variables: medical exposure to X-rays (p=0.0115), coffee consumption (p=0.0471) and alcohol consumption (p=0.0497). Radiation exposure and alcohol consumption have a positive impact, while coffee intake has a negative impact on the frequency of micronuclei.
The results of multiple regression analysis and Pareto charts showed that the exposure to X-rays had the highest influence on the frequency of micronuclei (Figure 2). Indeed, the mean value of micronuclei in the patients exposed to X-rays was 10.9 (range 5-19) compared to 7.17 for the total tested population. Regular alcohol consumers also showed somewhat higher frequency of micronuclei (8.23) in comparison with the total population, while coffee consumers had a slightly lower mean value (6.82).
Figure 2: Pareto charts of the dependence of predictor variables (living and working habits and health status of the women) on the frequency of micronuclei.
The symptoms, the results of the cervical smears on aerobic bacteria, yeasts, Ureaplasma urealyticum, Chlamydia trachomatis, hrHPV and Pap smears, were also correlated with the frequency of micronuclei. The results presented by Pareto diagram (Figure 3) showed that scaled epithelial cells in the patients with cervical lesions have a higher incidence of micronuclei. In our study, there was no significant difference in the frequency of micronuclei between low and high grade cervical lesions. Higher frequency of micronuclei was also observed in the patients positive to hrHPV (mean value 9.52).
Figure 3: Pareto charts of the dependence of the predictor variables (the symptoms, the results of Pap smears and cervical swabs) on the frequency of micronuclei.
Although the multiple regression analysis showed a certain degree of the correlation (R=0.41) between the predictor variables and the frequency of micronuclei, none of those variables showed a statistically significant influence.

Discussion

Basic statistical evaluation of our data revealed the median value of 6 MN per 1,000 cells. In the majority of the tested samples the number of micronuclei ranged from 6 to 9. Those results were very similar and in the range of the previously published data. Bonassi et al. [9] after evaluation of nearly 5000 individual data from 23 international databases reported median value of 6.5 micronuclei per 1000 cells with the range between 3 and 12 micronuclei.
Previous research has shown that high-risk HPV infections are the primary etiologic factor of cervical cancer. It has been proven that the HPV was the expresser of oncogenic proteins p53 and Rb, that interfere with the cell growth control [10-12]. This may result in the occurrence of genetic instability, which leads to the cell transformations. We also found that patients positive to C. trachomatis had higher frequency of micronuclei compared to the total population.
The comparison of our results with the results of other researchers is somewhat difficult due to the differences in the study design and differences in the populations tested. For these reasons, the total number of micronuclei from available literature data (ranging between 7 and 10 micronuclei per 1000 cells) [13-17] differ slightly compared to our results.
In some studies there was a positive correlation between the frequency of micronuclei with age [9,13]. Consequently, with increasing age of the patient a series of the processes at the level of the cells and organisms occurred that may lead to increased levels of the primary DNA damage, increased frequency of chromosomal aberrations as well as spontaneous generation of the aneuploidy [6,18,19].
Literature data considering the medical exposure to irradiation during diagnostic examinations (ultrasound, X-rays) and its contribution to the formation of the micronuclei are contradictory. Modern X-ray machines are technically advanced, so the lower doses of irradiation will be received during the diagnostic examination resulted in lower genetic damage. After a short exposure to X-ray irradiation it is not likely to expect a significant genetic damage. However, it is well known that a certain part of the population may be more genetically susceptible compared to the total population. Consequently, the data on the medical exposure to X-rays in the last month is important parameter for interpretation of the results of micronucleus test. Diagnostic examinations in which contrast media is used could be also attributed with the increased number of micronuclei [20-26].
On the basis of available literature data, it could be concluded that micronucleus assay has a significant advantage over other cytogenetic techniques because it enables the assessment of the damage at the level of function and integrity of the cell’s spindle [6]. Consequently, determination of the total number of micronuclei could be used as a reliable indicator of cytogenetic damage.
Furthermore, micronucleus assay is useful analytical tool for detecting the instabilities of the genome that are associated with an increased risk of cancer [27-30]. The causal link between the increased number of micronuclei and genitourinary cancers was confirmed [23]. It has been shown that regardless of the cancer localization the patients before starting with the therapy have elevated levels of micronuclei in comparison to the general population [24,31-35].

Conclusion

The results of the micronucleus test conducted on the samples of vaginal secretion revealed that in 190 of 197 tested samples micronucleated cells were present. The number of micronuclei per 1000 analyzed vaginal epithelial cells ranged from 1 to 22 (median=6 MN). This median value was within the range obtained from the available literature data. Multivariate analysis showed good (R=0.69), statistically significant correlations (p=0.0441) between the frequency of micronuclei and predictor variables describing the lifestyle. Among them, the exposure to X-rays irradiation during medical examination, alcohol consumption and coffee consumption are the variables with the highest, statistically significant influence. The patients with cervical lesions, presence of hrHPV DNA and/or Chlamydia trachomatis also exhibited increased values of micronucleated cells. In conclusion, a micronucleus assay could be used as an additional analytical tool in the prediction of genetic instability that may lead to cervical cancer.

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