Introduction

Oral submucous fibrosis (OSMF) is an insidious chronic disease and one of the most common potentially malignant conditions seen in the Indian population.[1,2] It affects most of the soft tissues of the oral cavity including buccal mucosa, labial mucosa, tongue, soft palate and hard palate. The prevalence of OSMF in India has been estimated with a broad age range from 11-60 years, male to female ratio of 2:1 and with a malignant transformation rate of 1.5-15% of all cases.[3]

Though the aetiology of OSMF is multi-factorial, areca-nut chewing has been reported to be the most common cause. The other causative agents include intake of spicy food, genetic predisposition and nutritional deficiency.[4,5] OSMF arises due to disturbances in the homeostatic equilibrium between the degradation and synthesis of the collagen leading to fibrosis and other changes.[6]

The diagnosis of OSMF is usually based on the clinical findings in patients with a habit of arecanut chewing. Osmf is clinically characterized by presence of fibrous bands, altered oral mucosa texture leading to blanching of mucosa and burning sensation mostly while having spicy foods. As a result of the fibrosis of the oral tissues, reduction in mouth opening and cheek flexibility along with difficulty in tongue movements, difficulty in swallowing, decreased salivary flow, and shrunken uvula will be present in later Stages.[7]

However clinical examination alone may be subjective and the severity may be overestimated or underestimated depending on the clinical experience of the practitioner. For this reason, severity is assessed through both clinical and histopathological staging. Histologically as the disease progresses, there will be increased submucosal thickness and hypertrophy of muscles along with decreased vascularity and epithelial atrophy due to obliteration of blood vessels.[8] Increased submucosal thickness is said to be due to fibrosis which is caused by increased collagen activity or decreased collagen degradation and increased activity of proliferation of fibroblasts. These changes in submucosal thickness progresses through stages where submucosa cannot be distinguished from underlying muscle layer.[9]

As it is a diffuse condition, wrong site for biopsy of the disease is possible which leads to an improper histopathological grading. In severe Stages where the mouth opening is minimal, it is difficult to take a biopsy and it is not feasible to take biopsies repeatedly to determine the progression of the disease and also to check for the improvement after the treatment is employed. All these disadvantages can be overcome by using USG as an adjuvant investigation modality with an extra-oral approach.

Ultrasonography (USG) is a non-invasive, non-ionizing, safe, readily available and cost effective modality for imaging superficial structures of the head and neck region.[10] Because of its non-invasive nature and safety, it has better patient acceptance.[11,12] In addition, because of the wider area that can be imaged, USG may be used to determine the extent and severity of the involvement of the tissues in OSMF, thus supplementing clinical and histological details. Some studies reported that USG can delineate feeble fibrotic bands before they can clinically be detected.

The hypothesis studied in this study is that there is a significant positive correlation between submucosal thickness measured by ultrasonography and the clinical staging of Oral Submucous Fibrosis. Specifically, it is anticipated that as the clinical Stage of OSMF progresses from Stage I to Stage III, the submucosal thickness will increase correspondingly, reflecting the extent of fibrosis and disease severity.

So the study aimed to measure submucosal thickness in OSMF patients using ultrasonography by quantifying submucosal thickness at various oral sites in patients diagnosed with OSMF and comparing these measurements with the clinical Stages focusing on analyzing the relationship between the submucosal thickness values and the clinical Stages of OSMF. The goal was to determine if there is a statistically significant correlation between increasing submucosal thickness and advancing clinical Stages of the disease.

By establishing this correlation, the study aimed to determine the utility of USG as a non-invasive tool for evaluating the severity of OSMF, thereby enhancing disease assessment and management.

Material and Methods

The total sample size for the study was 44. It was a case-control study including 33 patients clinically diagnosed with OSMF, categorized into three Stages (Stage I, II, and III) and 11 healthy controls. This study was conducted in Department of Oral Medicine and Radiology, Yenepoya Dental College, Yenepoya (Deemed to be University), Mangalore, Karnataka and Department of Radiology, Yenepoya Medical College, Mangalore, Karnataka, after getting ethical clearance from the institutional ethical committee (Protocol no. YEC2/1071). Patients who were clinically diagnosed with various stages of OSMF were taken as cases. Healthy individuals with no other oral lesions and no habit history were taken as controls. Exclusion Criteria included patients with known history of restricted mouth opening owing to causes other than OSMF, previously treated cases of OSMF, any systemic and skin diseases which included scleroderma, amyloidosis, diabetes mellitus and hypertension and radiation therapy done in the head and neck region. In this study, the independent variable was the clinical stages of Oral Submucous Fibrosis (OSMF), while the dependent variable was the submucosal thickness measured through ultrasonography. The analysis also considered age and sex as covariates.

The USG images were obtained from the Department of Radiology, Yenepoya Medical College and Hospital, Yenepoya deemed to be University, Mangalore using Philips Clear VUE 850 and a multi-frequency linear transducer probe with the frequency range from 3-12 Mhz. Each sample of the selected group was informed about the study in patient’s known language and informed consent was obtained. Detailed history was elicited from each subject and the data was entered into a structured proforma. The clinical examination was carried out and patients were divided into Stage I-Early OSMF, Stage II-Moderate OSMF and Stage III-Severe OSMF. Healthy individuals with no habit history or any systemic illnesses were taken as controls. So, in total the study group consisted of 44 patients (11 cases in each stage of OSMF and 11 controls) divided into 4 groups (Group 1-Stage 1, Group 2 –Stage 2, Group 3-Stage 3 and Group 4-Controls). As a part of clinical examination, the maximum mouth opening was measured using a vernier caliper (normal distance between central incisor tips) and cheek flexibility was checked (CF = V1-V2, two points measured between; V2 = marked at 1/3rd the distance from the angle of the mouth on a line joining the tragus of the ear and the angle of the mouth and V1 = the subject was then asked to blow his cheeks fully, and the distance measured between the two points marked on the cheek). After this extra oral USG Scanning was performed by a trained radiologist with the patient in supine position using a medical sonographic unit Philips Clear VUE 850 and a multi-frequency linear transducer probe with the frequency range from 3-12 Mhz.(Fig.1)

Fig.1: Ultrasonic imaging of submucosal thickness of a healthy control

Prior to commencing imaging, participants were instructed to retain water in their mouth to outline the empty space within the oral cavity. The transducer probe was positioned to avoid excessive compression of soft tissues, as heightened contact pressure during imaging could impact measurements. Therefore, to achieve precise measurements of submucosal thickness, the probe was gently brought into contact with the surface. The probe was positioned along imaginary planes on the buccal mucosa as follows: longitudinal (outer canthus of the eye to 1cm above the lower border of the mandible) and transverse (commissure of the lip to the tragus of the ear). These reference planes were considered for ultrasonographic imaging of buccal mucosa. For imaging upper and lower labial mucosa, the transducer probe will be placed in the mid-region of philtrum and mentalis region, respectively. The real-time imaging of submucosa of buccal and labial mucosa was done(Fig.2a and b)

Fig.2a: USG image of measurement of submucosal thickness of buccal mucosa	Fig.2b: USG image of measurement of submucosal thickness of labial mucosa

Ultrasonographic measurements were taken from six different locations for each patient i.e right anterior buccal mucosa, right posterior buccal mucosa, left anterior buccal mucosa, left posterior buccal mucosa, upper labial mucosa and lower labial mucosa. The measurements were taken in centimetres. The maximum measurement among these six values for each patient was taken as the representative value of submucosal thickness for that patient

Statistical Analyses:

Descriptive Statistics: The distribution of participants across different Stages of OSMF was summarized using counts and percentages for categorical variables like sex and age groups.

Mean and Standard Deviation Calculation: For continuous variables such as submucosal thickness, the mean and standard deviation were calculated to assess the central tendency and variability across different OSMF Stages.

Confidence Intervals: 95% confidence intervals were calculated for the mean submucosal thickness in each Stage to estimate the range within which the true mean is likely to fall.

Post Hoc Analysis: Differences in mean submucosal thickness between the Stages were evaluated using post hoc comparisons, with corresponding p-values to determine statistical significance.

Significance Testing: The study used p-values to assess the statistical significance of the differences in submucosal thickness between various OSMF Stages, with lower p-values indicating more significant differences.

Results

Table 1 presents the distribution of sex and age across different Stages of the OSMF (Stage I, Stage II, and Stage III). The data is categorized by sex (female and male) and age groups (<30, 30-50, and ≥50 years), with each category showing the number of cases (n) and the corresponding percentage for each Stage.

• Sex: There was one female participant in Stage II (100.0%). The male participants were evenly distributed across the Stages, with 11 males in Stage I (25.6%), 10 in Stage II (23.3%), and 11 in Stage III (25.6%).
• Age:
• <30 years (n=8): The youngest age group had 2 participants (25.0%) in Stage I, 1 participant (12.5%) in Stage II, and 1 participant (12.5%) in Stage III.
• 30-50 years (n=28): The majority of participants fell within this age range. with 8 in Stage I (28.6%), 6 in Stage II (21.4%), and 9 in Stage III (32.1%).
• ≥50 years (n=8): The oldest age group showed a distribution where 1 participant (12.5%) was in Stage I, 4 participants (50.0%) were in Stage II, and 1 participant (12.5%) was in Stage III.

Table 2 shows the relationship between different covariates and the mean submucosal thickness (with standard deviation) across control and various disease Stages. The analysis is divided by sex and age:

• Sex:
• There was one female participant, who was in Stage II, with a mean thickness of 0.130 mm
• The mean submucosal thickness in males increased progressively across the stages: Control (0.049 mm), Stage I (0.080 mm), Stage II (0.118 mm), and Stage III (0.196 mm). The standard deviation also increased with disease severity, reflecting greater variability in thickness in more advanced Stages.
• Age:
• <30 years (n=8): The mean thickness increased with disease stage: Control (0.050 mm), Stage I (0.080 mm), Stage II (0.110 mm), and Stage III (0.164 mm).
• 30-40 years (n=8): A similar trend was observed with increasing thickness from Control (0.050 mm) to Stage III (0.207 mm), and increasing variability in thickness as indicated by the rising standard deviation.
• ≥50 years (n=28): In this age group, the mean thickness also increased with the disease stage. (Table 2)

Table 3 focuses on the relationship between the mean submucosal thickness and disease staging, including the 95% confidence interval for the mean, standard deviation, and test statistics:

• Control: The mean thickness was 0.049 mm with a narrow confidence interval (0.047 mm to 0.051 mm) and a very low standard deviation (0.003 mm). The test statistics showed a significant difference in thickness compared to the other Stages (p < 0.0001).
• Stage I: Mean thickness increased to 0.08 mm with a slightly wider confidence interval (0.07 mm to 0.09 mm) and a higher standard deviation (0.014 mm).
• Stage II: The mean thickness further increased to 0.119 mm, with a wider confidence interval (0.097 mm to 0.14 mm) and a standard deviation of 0.032 mm.
• Stage III: The most advanced stage had the highest mean thickness of 0.196 mm, the widest confidence interval (0.156 mm to 0.235 mm), and the largest standard deviation (0.059 mm), indicating greater variability in submucosal thickness. (Table 3).

Table 4 provides a post hoc analysis of the differences in mean submucosal thickness between different stages, along with the corresponding p-values to determine statistical significance:

• Control vs. Stage I: The mean difference in thickness was -0.031 mm, with a significant p-value (0.00013).
• Control vs. Stage II: A larger mean difference of -0.07 mm was observed, with a similarly significant p-value (0.00013).
• Control vs. Stage III: The greatest mean difference was -0.146 mm, with an even more significant p-value (0.00005).
• Stage I vs. Stage II: The mean difference was -0.039 mm, with a p-value of 0.01213.
• Stage I vs. Stage III: The mean difference increased to -0.116 mm, with a highly significant p-value (0.00027).
• Stage II vs. Stage III: The difference in mean thickness was -0.077 mm, with a significant p-value (0.0084).

Each comparison showed statistically significant differences in submucosal thickness between the different stages of the disease, suggesting that as the disease progresses, submucosal thickness increases significantly (Table 4).

Discussion

Oral submucous fibrosis (OSMF) represents a significant health concern in populations where areca nut chewing is prevalent, particularly in regions like India. Its insidious nature and potential for malignant transformation underscore the importance of early diagnosis, monitoring the progression and improvement following treatment.

Pre-malignant lesions and conditions like OSMF are said to predominantly affect middle-aged to older individuals with a noticeable male predominance.[13] In our study, the age range was found to be between 20-60 years with male predominance. Out of 33 cases of OSMF included in the study there was only one female patient confirming the male predominance. The higher prevalence of OSMF in males compared to females can be attributed to various factors such as higher rates of areca nut and alcohol use among males in many cultures, which are significant risk factors for OSMF.[14]

The study found that the distribution of male participants was relatively even across all Stages of OSMF, while there was only one female participant, who was in Stage II (Table 1). This suggests that while the progression of OSMF may not differ significantly between sexes, the limited number of female participants makes it difficult to draw definitive conclusions about the impact of sex on disease progression.

The analysis of age groups revealed that participants aged 30-50 years were the most affected, with the majority falling within this age range across all Stages. This pattern indicates that OSMF predominantly affects individuals in their middle ages. The younger (<30 years) and older (≥50 years) age groups had fewer participants, but it is notable that the oldest group showed a higher proportion in Stage II, suggesting that age might influence the Stage at which the disease is diagnosed, with older individuals possibly experiencing more advanced Stages.

Traditionally, clinical examination has been the mainstay for assessing Oral Submucous Fibrosis (OSMF), relying on subjective observations and measurements. However, this may not be effective for early diagnosis before visible signs manifest. Tiwari et.al[9] and Manjunath K et.al.,[15] in their studies found that USG can delineate feeble fibrotic bands before they can clinically be detected confirming that USG can also be used for screening of this condition at an early stage.

Biopsy, particularly incisional biopsy, is often employed to confirm, know the severity of the disease and Stage them accordingly or in cases where malignancy is suspected. This approach may be also limited by factors such as inter-observer variability and difficulty in evaluating patients with advanced disease manifestations, such as severe trismus. The introduction of ultrasonography as an adjuvant investigative tool for OSMF offers a promising alternative, addressing some of the limitations associated with traditional clinical and histopathological examination. Ultrasonography (USG) emerges as a promising non-invasive technique for assessing OSMF with advantages such as real-time imaging, non-ionizing radiation, and the ability to visualize changes in superficial structures like the buccal and labial mucosa.

The diagnostic potential of USG in OSMF has been reported by C. Krithika et al.,[16] and Thapasum AF et al.[1] where the characteristics of the buccal mucosa was evaluated using USG. In studies conducted by Manjunath K et al.,[15], Rashmi Kewal et al.,[10] and Lakshmi Kavitha et al.,[11] clinical and histopathological examinations of OSMF were compared with findings from ultrasonographic techniques. All of these studies showed that ultrasonography is an effective adjuvant tool for OSMF, providing qualitative and quantitative information without causing discomfort to patients. However, despite these advantages, the utility of USG in OSMF patients is still not in routine use.

Hence the study aimed to assess and stage OSMF patients based on clinical signs and symptoms and subsequently measure the submucosal thickness of buccal and labial mucosa using ultrasonography thereby providing quantitative data to supplement clinical assessments. By comparing these measurements with those of normal healthy individuals, the study sought to identify any significant differences in the submucosal thickness indicative of OSMF. Furthermore, the research intended to establish a correlation between the severity of OSMF, as determined clinically, and ultrasonographic measurements of submucosal thickness.

By providing objective measurements of submucosal thickness, ultrasonography offers a more standardized approach to assessing disease severity. The results of this study also revealed submucosal thickness increased significantly with disease progression. In males, as well as across various age groups, the mean thickness rose from 0.049 mm in the control group to 0.196 mm in Stage III. This increase was accompanied by a rise in standard deviation, indicating greater variability in thickness at more advanced stage. The results suggest that submucosal thickness serves as a reliable indicator of disease severity in OSMF and were consistent with the results of the studies conducted by Devathambi et.al.,[2]and Lakshmi Kavitha et.al., [11] which also showed an increase in submucosal thickness as OSMF stages advanced compared to controls. Importantly, age did not significantly affect the pattern of submucosal thickening. The progression of submucosal thickness was primarily driven by the disease stage rather than by age. Due to the uneven distribution of samples between sexes, broad conclusions about the impact of sex on submucosal thickness could not be drawn.

It was also found in the current study that, two patients clinically staged as Stage I had markedly increased submucosal thickness compared with the other Stage I patients which is a finding not reported in any of the past studies. This indicates the possibility that USG can show us the precise severity level of the disease through the layers of the mucosa even when the progression and severity of the disease is not visible clinically.

The post hoc analysis also confirmed that the differences in submucosal thickness between stages were statistically significant. Each stage showed a marked increase in thickness compared to the previous one, with the most significant differences observed between the control group and Stage III. These findings reinforce the utility of submucosal thickness as a quantitative measure for assessing the progression of OSMF.

The current study aligns partially with the findings of Rangaiah et al.,[17], who observed elevated submucosal thickness at the anterior and posterior buccal mucosa as well as the upper and lower labial mucosa in OSMF cases compared to controls. However, they found no significant relationship between ultrasonography (USG) findings and clinical assessments, largely due to a higher proportion of subjects in clinical Stages III (50%) and IVa (20%). Kumar et al.[8] reported similar study results, noting a notable rise in submucosal thickness among OSMF cases compared to controls. However, they encountered challenges in establishing statistical significance across stages and ultrasonographic findings due to a heterogeneous distribution of cases across the stages. In contrast, our study observed a significant correlation between OSMF stages and ultrasonographic findings, which can be attributed to a more uniform distribution of cases across all OSMF groups.

Additionally, it was also observed that some patients had difference in submucosal thickness between different sides and sites. This difference was attributed to the reason that some of these patients had the habit of retention and chewing arecanut more on one side compared to the other side, more anteriorly than posteriorly and vice versa, leading to comparatively increased thickness on the side and site of maximum chewing and retention. The site of retention and chewing influences the extent of fibrosis and hence justifies the difference in thickness at different sites in the same patient. This is the reason why in our study, the maximum measurement of submucosal thickness was taken as the representative value for all the patients.

From all these above mentioned findings, the current study aligns with existing literature that demonstrates the potential of ultrasonography as a valuable adjunctive tool in assessing and monitoring OSMF progression. The observed increase in submucosal thickness with advancing disease stages is consistent with prior findings, highlighting the progressive nature of OSMF.

Moreover, the non-invasive nature of ultrasonography makes it a safe and well-tolerated imaging modality, suitable for repeated evaluations over time. This allows for longitudinal monitoring of disease progression and treatment response, facilitating early intervention and personalized patient care. Additionally, ultrasonography may offer advantages in cases where traditional biopsy techniques are challenging or contraindicated. Further research and clinical validation are warranted to fully establish the utility of USG in OSMF effectively.

The findings of this study underscore the clinical significance of utilizing ultrasonography as an adjuvant tool in OSMF. Traditional clinical examination alone may not provide sufficient information to accurately characterize disease severity. By incorporating ultrasonographic measurements of submucosal thickness, clinicians can obtain quantitative data to correlate with clinical staging. The ability to correlate ultrasonographic measurements with clinical staging can facilitate a more comprehensive understanding of disease progression and treatment response.

Acknowledgments And Disclosure Statements

The authors would like to extend our sincere thanks to Mrs. Yashaswini for her tremendous guidance for the statistical analyses. The authors report no conflicts of interest related to this study.

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