Assessment of skeletal maturity using salivary dehydroepiandrosterone sulfate as a biomarker: A clinical study

INTRODUCTION

Timing of early orthodontic treatment has been based on stages of skeletal maturation as they relate to peak height velocity or to maximal treatment response.^[1] Literature shows that the skeletal effect of the appliances was more pronounced during the peak height velocity periods than during the prepeak period.^[2-4] Craniofacial development has been compared mainly with general body height and in relation to skeletal maturation, evaluated with hand-wrist radiographs. Skeletal maturation assessment based on cervical vertebral examination is the most widely used indicator. The cervical vertebral maturation method relies on inter-stage comparisons of the shape and depth of the inferior concavity and the height and shape of the cervical vertebrae bodies in different growth phases. However, the disadvantage of this method is the challenge of visualizing the subtle changes in the vertebrae, due to an improper neck posture while taking the radiograph, or blocking out of the cervical vertebrae structures by the thyroid collar.^[5] Therefore, detecting skeletal maturation using biochemical markers has recently become the focus of an increasing number of studies, especially since they overcome the previously mentioned limitations.^[6] Biomarkers have been associated with bone metabolism and cartilage development and production of signals that stimulate growth and development of craniofacial structures and mandibular condyles. The 19-carbon steroids dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S) are the most abundant steroid hormones in the body. They are produced and secreted from the adrenal cortex into the circulation. These sex steroids play major roles in stimulating reproductive glands to produce the sex steroid hormones: Testosterone and estrogen. In growing individuals, the progressively increasing level of DHEA-S can lead to an increase in linear growth velocity and advanced bone age.^[7] Therefore, the aim of the study is to evaluate skeletal maturation using salivary DHEA-S levels and their correlation with existing skeletal maturity indicators represented by 4 cervical vertebrae growth stages.

MATERIALS AND METHODS

The present study was done on 48 participants (26 male and 22 female) under the age group of 9–16 years who visited the department seeking orthodontic treatment. A general and oral examination was done to exclude: Patients with growth abnormalities, subjects with systemic diseases, subjects under medications, presence of micro-injury to the oral mucosa, and subjects with deciduous tooth exfoliation within 2 days. Selected patients were asked to sign a written consent form by their parents upon explanation of the entire procedure. Radiographic examination of each subject included the pre-existing lateral cephalogram which was analyzed using Baccetti and McNamara^[8] stagings [Figure 1]. The subjects belonging to each of the 4 stages were segregated and then divided into 4 groups consisting of 12 subjects each. Using a radiographic view and 0.003-inch lacquered polyester matte acetate tracing paper with a 0.3 mm lead pencil, a single examiner evaluated, analyzed, and measured every record to eliminate inter-examiner variability.^[9,10] Following that, the patients were told to rinse their mouths well to get rid of any food particles and to just drink water. Following a 60-min duration, the patients were instructed to gather their saliva in their mouths and drool into the sterile plastic tube until it reached the 5-mm threshold. The samples were then collected by the examiner and stored immediately at −4℃ in a domestic freezer. All samples were collected at 10 am–11 am, keeping in mind the diurnal variation of DHEA-S^[11] and centrifuged at 5000 rpm for 5 min using the Remi centrifuge precision refrigerated (CPR) plus centrifuge [Figure 2a and b]. 3 mL of supernatant was transferred to storage containers [Figure 2c]. These vials were transferred to the laboratory freezer to be stored at −80℃ within 4 h^[12] [Figure 2d]. On the day of assay, the saliva samples as well as the reagents were brought to room temperature and an enzyme-linked immunosorbent assay (ELISA)-based DHEA-S Estimation Kit (KRISHGEN Biosystems) that has been approved by the Food and Drug Administration was used to estimate the concentration of salivary DHEA-S [Figure 3a]. The competitive binding concept explains the solid phase ELISA, which was used in the test [Figure 3b and c]

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Figure 1: Lateral cephalogram showing C2, C3, and C4 vertebrae.

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Figure 2: (a) Centrifuging machine. (b) Centrifuged saliva. (c) Micropipetted supernatant saliva. (d) −80° laboratory freezer.

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Figure 3: (a) Dehydroepiandrosterone-sulfate kit with reagents. (b) Adding reagents. (c) Color-changing indicating presence of antibodies.

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Statistical analysis: Descriptive statistics of the explanatory and outcome variables were calculated by mean, standard deviation for quantitative variables, frequency, and proportions for qualitative variables. Inferential statistics like Chi-square test was applied for qualitative variables to find the association. Independent sample t-test was applied to compare the DHEA-S levels between the groups (male vs. female). Analysis of variance (ANOVA) test was applied to compare the mean DHEA-S levels among the groups (based on CVMI stage) with post hoc Bonferroni for intergroup comparison.

Pearson’s correlation was applied to correlate age and DHEA-S levels based on CVMI stage. The level of significance for the study is set at 5%.

RESULTS

The study consists of subjects from age 9–16 years who were classified into 4 groups based on CVMI stagings. Mean age was highest in CVMI stage 5 (14.83 ± 0.577), followed by stage 4 (13.33 ± 0.492), stage 3 (12.25 ± 0.965), and stage 2 (10.83 ± 0.835) [Table 1].

While comparing the DHEA-S levels between the gender within the groups, females showed higher mean DHEA-S levels in stage 2 (12.70 ± 0.70), stage 3 (16.23 ± 0.32), stage 4 (17.60 ± 0.96), and Stage 5 (22.66 ± 3.54). Independent sample t-test showed no statistically significant difference between the groups (p > 0.05), as shown in [Table 2].

The DHEA-S level when analyzed in sample size of 12 per group was found to be 12.03 ± 0.87 nmol/mL for CVMI 2, 15.12 ± 1.33 nmol/mL for CVMI 3, 18.9 ± 0.81 nmol/mL for CVMI 4, and CVMI5 to be 22.11 ± 3.17 nmol/mL. Mean DHEA-S levels were highest in CVMI stage 5. ANOVA test was applied which showed statistically significant difference among the groups with respect to DHEA-S levels (p = 0.001) as shown in [Table 3]. On conducting inter-CVMI comparison using post-hoc Bonferroni test, there was statistically significant difference seen between all CVMI stages (p < 0.05) as shown in [Table 4].

Pearson’s correlation test which was applied to correlate age and DHEA-S levels based on CVMI stage showed positive, strong, and significant correlation in stage 2 (r = 0.644; p = 0.024) and stage 3 (r = 0.649; p = 0.022). Positive, very weak correlation was found in stage 4 (r = 0.023; p = 0.944) and stage 5 (r = 0.026; p = 0.935), as shown in [Table 5].

DISCUSSION

Skeletal maturation assessment plays an important role during optimal orthodontic diagnosis and treatment planning.^[13] According to Baccetti et al., the ideal biologic indicator for an individual’s skeletal maturity should be measured by the following characteristics: it should be efficient in identifying the peak of mandibular growth, simple, and non-requisite of additional radiation exposure.^[14] Previous studies done to check the assessment of skeletal maturation using biomarkers, where DHEA and DHEA-S biomarkers have been reported to play a major role in stimulating growth and proliferation of epiphyseal cartilage and initiation of growth hormone by mitogen-activated protein kinase signaling pathways which are necessary for the expression of osteoblast-specific genes.^[15]

The present study evaluated the skeletal maturation using salivary DHEA-S levels and their correlation with existing skeletal maturity indicators represented by 4 cervical vertebrae maturation stages. DHEA-S was chosen in this study as the serum content of DHEA-S is 300–500 times higher than DHEA. While DHEA is more active at the tissue level, DHEA-S is albumin-bound and forms a circulating reservoir, hence the increased concentration.^[16] Srinivasan and Premkumar^[17] have conducted a study taking advantage of increased serum DHEA-S concentration correlating with the hand-wrist radiograph to confirm its usefulness as a skeletal maturity indicator. Saliva was selected over blood as saliva carries many advantages:^[18]

1. It is easy to collect

2. Non-invasive

3. Handling of samples is safer

4. Convenient to store

5. Affordable.

Also, using saliva as a biomarker over gingival crevicular fluid (GCF) sampling can reduce the long waiting period during GCF collecting which can be upsetting for both children and adults.

Despite the relatively modest sample size of 48, standardized sample collection, strict inclusion criteria, and uniform analytical procedures were employed to enhance internal validity. Statistically significant correlations observed in the study suggest that the sample size was adequate to address the primary objective.

DHEA-S shows diurnal variation; hence, the collection of samples was standardized within 10 am–11 am, as this time window corresponds to relatively stable salivary hormone levels.^[11] To further reduce potential confounding, participants were instructed to refrain from eating, drinking (except water), strenuous physical activity, and stressful activities for at least 1 h before sample collection. However, complete control of all potential confounders such as individual stress levels and dietary habits was not feasible, which is recognized as a limitation of the study. Hucklebridge et al.^[11] investigated the secretory pattern of DHEA at 3-h intervals over 12 h following awakening and found salivary DHEA levels remained stable after falling from high levels upon awakening to noticeably lower levels 3 h later. Furthermore, Matchock et al.^[19] found a diurnal rhythm in boys and girls during puberty. Although gender differences were reported descriptively, the primary objective of the study was not to evaluate sex-specific effects but to assess the overall association between salivary DHEA-S levels and CVMI stages. The sample size was calculated to address this primary outcome and may therefore be underpowered to detect subtle gender-based differences. Consequently, gender-wise findings were interpreted cautiously and are presented as exploratory observations. This has been acknowledged as a limitation of the study. Among different ways of collecting saliva, the best method is by passive drooling into a tube without any stimulation, as stated by Hofman.^[20] Absorbent pads or cotton rolls if used may be subjected to bacterial degradation over time. Furthermore, chemical substances added to the absorbing pads or cotton rolls may interfere with the saliva.

According to Thomadaki et al.,^[21] decreasing the incubation temperature slows the rate at which the salivary protein degrades. To stop bacterial development and additional salivary molecule breakdown, specimens were immediately kept at 4°C domestic freezer and then transferred to −80°C laboratory freezer post centrifugation with little to no deterioration. Existing evidence suggests that sample stored at −4°C is stable up to 6 h without undergoing significant metabolic changes.^[12] Results of the present study showed a progressive increase in DHEA-S levels as the skeletal maturation progresses, following the normal release pattern displayed by DHEA-S which peaks 20–30 years later. The DHEA-S levels were low during CVMI-1 stage and sharply increased at CVMI Stage 3, followed by a gradual increase as skeletal maturation progressed, almost reaching the highest value in the completion stage at CVMI stage 5 with the mean value of being 17.28 ng/mL for females and 16.09 ng/mL for males which corresponds with the findings of a related study by Sultana et al.,^[22] Matchock et al.,^[19] Srinivasan and Premkumar.^[17] These investigations have demonstrated a considerable rise before puberty, a peak around 25 years, a reduction throughout the reproductive stage, and a rise before menopause, indicating a potential involvement of the hypothalamo-pituitary axis.

Among all the CVMI stages, there was a statistically significant difference in the means of salivary DHEA-S levels, except between CVMI-S 1 and 2 and between CVMI-S 4 and 5. Srinivasan and Premkumar assessed the DHEA-S levels in serum as a skeletal maturation indicator where they found a sharp increase just before puberty, followed by a progressive increase until the early twenties.^[17] Venkatagiriappa et al.^[23] used the blood spot collection technique to evaluate the level of DHEA-S based on the hand and wrist maturity index and they observed a similar increase pattern, but this increase was statistically insignificant. They explained that the difference in their results could be attributed to the extensive variation of the DHEA-S values found in all their study groups. Anusuya et al.^[24] has gone beyond previous reports and showed a different pattern of DHEA-S levels in serum. They found that the highest mean values of DHEA-S were observed in pubertal phases at CVMI-4 and CVMI-3 in males and females with a statistically significant difference among each CVMI stage except between CVMI-5 and 6. The outcome of this study revealed that the maturation of cervical vertebrae elevates with increasing age and could be used for the evaluation of skeletal maturity.

CONCLUSION

Based on the results obtained from the current study:

• CVMI stage 2 = 9–13.9 nmol/mL

• CVMI stage 3 = 14–16.4 nmol/mL

• CVMI stage 4 = 16.5–19.3 nmol/mL

• CVMI stage 5 = 19.4–29.5 nmol/mL.

In conclusion, the relationship between salivary DHEA-S levels and cervical maturational stages demonstrates the potential utility of salivary DHEA-S as a prospective marker of skeletal maturation to pinpoint the pubertal growth spurt.