Hormonal changes in exfoliated normal buccal mucosal cells

Abstract

Aims:

The present study was undertaken as a baseline study to evaluate the nuclear diameter (ND), cell diameter (CD) and nuclear-cytoplasmic ratio (N:C) and their variation with age in normal buccal smears of female subjects.

Materials and Methods:

Buccal smears were collected from 400 apparently healthy individuals. After fixation in 95% alcohol, the smears were stained using the standard Papanicolaou laboratory procedure. The CD and ND were measured using a stage micrometer and an ocular micrometer. Statistical analysis of the data was performed using the one-way analysis of variance to evaluate the difference in the ND, CD and N:C with age in females. Multiple range tests by Tukey-honestly significant difference procedure were performed to evaluate the significance between various age groups.

Results:

The results showed an increase in ND and CD from the 5-10 age groups to 15-35 age groups. In the 15-35 age groups, there was a decline in ND and CD when comparing the first and the second half of the menstrual cycle, but the difference was not significant. There was further decline in ND and CD in the 45-60 age groups. Variations were also seen in ratio N: C. Pattern of exfoliation also varied in each age group.

Conclusion:

Age-related alterations are observed in buccal smears.

Introduction

Oral mucosa appeared to be the true reflector of the systemic status of an individual. Alterations in the various systems are attributed to hormones. Hormones play a wide role in human tissues, which may bring about a physiological or a pathological change.

The diagnostic value of intraoral cytology has already been established particularly in screening for malignant lesions, owing much to its technical simplicity, cost-effectiveness and lack of pain induced to the patient. The initial studies of normal epithelial smears were established by Montgomery in 1951,[1] which was later used as baseline information for many other studies carried out on oral epithelial cells. However, very few recent studies are reported on physiological alterations in the buccal mucosa.

In the context of hormonal changes, the normal oral cytological pattern must be established for both men and women, in the latter especially in relation to the menstrual cycle.[2]

Sex steroids play a major role in the life of a woman. It is known that from the time of childhood, through menstrual age to menopausal stage, a woman undergoes cyclic fluctuations in her hormone levels.[3] Abnormal levels of sex hormones have been shown to influence the maturation of mucosa, but the presence of physiological cyclic patterns in the oral mucosa has not been satisfactorily demonstrated.

The present study uses exfoliative cytology and micrometry for scrutinizing clinically normal appearing buccal mucosal smears of female subjects through all ages and were evaluated quantitatively to assess the effect of hormonal changes by estimating the cell diameter (CD) and nuclear diameter (ND) and to quantify the age-related changes in the above variables along with a qualitative analysis.

Materials and Methods

A total of 400 apparently healthy female subjects with no known history of any systemic disease or therapeutic medication were selected for the study. All the subjects were free of any deleterious habits such as smoking, alcohol consumption or use of tobacco in other forms. No detailed biochemical, radiological or other laboratory investigations were carried out to establish their normal status.

The subjects were divided into four groups, with 100 patients per group, based on their age. No control groups are included [Table 1].

Table 1.

Grouping of subjects

The microscopic slides were cleaned with water, dried and numbered. Using a wet wooden spatula, exerting gentle pressure, cells were scraped from the clinically normal appearing buccal mucosa. Scrapings were smeared on to the slide. Slides were fixed in 95% alcohol for ½ h. Air drying of the smears was avoided as it leads to alterations in the cellular morphology. Smears were stained using the Rapid-Pap kit manufactured by Biolab Diagnostics Pvt. Ltd.

The slides were then subjected to cytomorphometric assessment with the aid of an ocular micrometer and a stage micrometer. The Papanicolaou stained cells were examined under ×40 compound microscope (Magnus BX 40). Unfolded cells with a clear outline were only selected for the study. CD and ND of the exfoliated cells were measured using a calibrated ×40 objective for all groups. The sampling was done in a stepwise manner, moving the slide from left upper corner to the right and then down in order to avoid measuring the same cells again. The cells were identified based upon the morphology and staining characteristics. Only intermediate cells were selected for the study. Dimensions of 20 cells were taken for each slide.

The average of the values was calculated and recorded as the mean ND and the mean CD for each subject. The ratio of the ND to CD was calculated and recorded as the nuclear-cytoplasmic ratio (N:C) ratio. All these values are tabulated and subjected to statistical analysis to compare if there is any change in the cellular and nuclear dimensions with respect to age.

One-way analysis of variance was performed to evaluate the difference in the ND, CD and N: C with age in females. Multiple range tests by Tukey's-honest significant difference procedure were performed to evaluate the significance between various age groups at 5% level.

Results

ND

There are significant differences in ND in different age groups except for Group II and Group III. The mean ND was found to increase from 10.35 μ in subjects in 5-10 years of age (Group I) to a maximum of 11.02 μ in 15-35 years of age (Group II).

In Group III age of 15-35 years, the mean value progressively decreases to 10.86 μ, but no significant change was seen in the nucleus diameter in the cells exfoliated in Group II with Group III (P = 0.057).

In Group IV, age group of 45-60 years, the mean ND declined to 9.29 μ [Table 2].

Table 2.

Comparison of nuclear diameter of buccal mucosal cells in different age groups in females using one-way ANOVA and Tukey-HSD procedure

CD

The difference in CD in various age groups is significant. The mean CD was found to increase from 65.84 μ in subjects in 5-10 age groups (Group I) to a maximum of 77.10 μ in 15-35 age group (Group II) showing significant difference.

There is a decrease in CD from 77.10 μ to 74.57 μ in Group III with a significant difference. CD value decreases further to 54.45 μ in Group IV (45-60 years) and statistical differences exist between this group and the remaining groups [Table 3].

Table 3.

Comparison of cell diameter of buccal mucosal cells in different age groups in females using one-way ANOVA and Tukey-HSD procedure

N:C

There is a significant difference in N:C in various age groups except for Group II and III. The mean NC was found to decrease from 0.37 μ in subjects in 5-10 age groups to 0.34 μ in 15-35 age group (Group II) showing a significant difference.

In Group III the mean N:C shows a slight increase from 0.34 μ to 0.35 μ, but there is no significant difference in N:C between Group II and Group III. In Group IV, the mean N:C further increased to 0.41 μ [Table 4].

Table 4.

Comparison of nuclear-cytoplasmic ratio of buccal mucosal cells in different age groups in females using one-way ANOVA and Tukey-HSD procedure

Pattern of maturation

The pattern of exfoliation was also observed in each group. Group I shows a predominance of parabasal cells followed by intermediate cells. No superficial cells were observed. In Group II, during the first phase of the menstrual cycle, superficial cells were predominant followed by intermediate cells, which indicate a complete maturation has taken place [Figures 1-2].

Figure 1.

Buccal mucosal cells from different layers (Pap, 100)

Figure 2.

Buccal mucosal cells showing variation in nuclear and cellular diameter (Pap, x400)

In Group III, during the second phase of the menstrual cycle numerous intermediate cells were found and very few superficial cells. Parabasal cells were not observed in both Group II and Group III in most of the smears taken. In Group IV, intermediate cells were predominant and absence of superficial cells and parabasal cells were observed [Table 5].

Table 5.

Pattern of maturation of buccal mucosal cells in females of different age groups

Discussion

The results of this study showed a significant difference in the ND, CD and N:C in females of various age groups. There was an increase in the ND and CD from the 5-10 years age group to 15-35 years age group [Tables 2 and 3]. This may be attributed to the rise in estrogen and progesterone levels in their blood. The hike in their levels leads to a direct impact causing the acceleration of cell metabolism and growth. This is reflected as an increase in the dimensions. This was followed by a decline in the ND and CD from the 15-35 years age group to the 45-60 years age group [Table 3]. The decline in their dimensions could be due to the decrease in the serum estrogen levels in the post-menopausal females.

Estrogen influences the cytodifferentiation of stratified squamous epithelium. The effects of oestrogen hormone are regulated through cellular receptors. These receptors were identified in many cells of the body including vaginal epithelium.

Ziskin and Moulton[4] disclosed well-expressed rhythmic alterations in cells of the oral cavity coinciding with changes found in vaginal smears, thus reflecting the hormonal state of the menstrual cycle.

Erler et al.[5] detected weak estrogenic activity in buccal epithelial tissue. Bercovici et al.[6] compared the hormonal content of the vagina and oral mucosa of menopausal women and they concluded that the maturation values were high in buccal smears of the menopausal women when compared with vaginal smears and it may not be the result of hormonal effect, but that of local mechanisms or irritating factors.

In the study by Nayar and Sivapathasundharam,[7] the ND was reported to increase with age and CD to decrease with age. Our results are in conformity with their study.

In a study conducted by Laufer et al.[8] in 1982, there is a gradual increase in estrogen during the first half of the menstrual cycle followed by a decline. In the second half of the menstrual cycle, there is a progressive increase in progesterone level. During the menstrual period, there is a decline in the level of the two hormones. Our results also showed an initial rise in the ND and CD during the first half of the menstrual cycle followed by a decline in the second half in the 15-35 years age group, but the differences were not significant.

Montgomery[1] concluded that there is no relationship between the cell colour and sex, age or menstrual cycle of the individual. This was contradictory to our study. There was variation in the type of cells observed in each group.

Peters and Rijsinghani[9] in 1956 concluded that epithelial lining of the oral cavity does not respond rhythmically to hormonal fluctuations.

Main and Ritchie[2] in 1967 observed presence of intermediate cells followed by superficial cells during the first half of the cycle and curled, folded spinous cells during the second half.

Studies of Leimola-Virtanen et al.[10] in 1997 revealed cell patterns in both samples were nearly identical, oestrogen may not be the only factor causing maturational changes in buccal epithelial cells. In our study, Group I showed less signs of maturation. The cells in Group II (first half of the menstrual cycle) showed complete maturation has taken place and a decline in Group III (second half of the menstrual cycle). Complete absence of parabasal cells were observed. The cells in Group IV (post-menopausal group) showed only parabasal cells indicating lack of maturation. This can also be attributed to other factors underlying the aging process.

Recent researches show that cytomorphometric analysis of buccal mucosal cells as an aid in diagnosis of type 2 diabetes mellitus. However, the age and sex of the patient, hormonal status and habit history of the patient should be also considered as these factors influence the morphometric values of epithelial cells.[11,12,13] Considerable reduction in ND and CD of buccal mucosal cells was observed in another study conducted on tobacco smokers.[14,15] Analysis of cytomorphometric values of exfoliated mucosal cells in malignant and premalignant lesions also has shown variation from the normal values.[16]

In the present study, morphometric measurements were done with the help of an eyepiece micrometer and stage micrometer, which permits only linear measurements of cell and nucleus. As the dimensions are measured manually, the results may not be exactly representing the hormonal status of an individual. To improve the accuracy, it would be better to use image analysis software using the automated computer as it measures the area of nucleus and cell.

Secondly, a study of fewer subjects with samples taken on a daily basis repeating for two or three menstrual cycles for the same subject would have achieved results displaying details in depth and accuracy. However, to confirm one's hormonal status, it is mandatory to know the serum estrogen and progesterone level. Finally, each study also differs in the number of cells being counted per slide, type of fixatives used, time lapse of staining following smear preparation and staining techniques. Due to the absence of established guidelines for cytological studies, it might lead to discrepancies in these reports.

Conclusion

The present study shows age-related variations in ND, CD and N:C ratio and variations in the pattern of exfoliated normal buccal mucosal cells in female subjects and these probably reflect fluctuations in the hormonal levels. Although considering cytomorphometric methods as a diagnostic aid in detection of systemic diseases, factors which influence the morphology of cells should also be given importance as they can affect the results.