Mandibular prognathism caused by the anterior protrusion of the lower jaw is considered one of the most severe dento-maxillo-facial abnormalities. Apart from the unesthetic facial profile, mandibular prognathism(class III skeletal base) produces impaired mastication and speech problems. Though various factors contribute to mandibular prognathism, the effect of certain hormones like growth hormone, adrenal androgens and parathormone on skeletal structures has interested many researchers and orthodontists in recent years. The role of sex hormones on bone growth has received considerable importance in research studies.(1,2) Testosterone and oestrogen have a direct and sex-specific stimulatory activity on skeletal growth.(3) Testosterone has anabolic effects which include growth of muscle mass and strength, increased bone density, stimulation of linear growth and bone maturation. On the other hand, oestrogen plays a protective role in bone growth rather than bone production. Among these two sex hormones, studies have shown that testosterone contributes more to accelerated mandibular growth.(4,5) Likewise, the digit growth and patterning in the human hand are determined by the sex hormones.(6) The 2nd and 4th digit present a pattern of length symmetry with the 3rd digit in the centre. The ratio of the length of two fingers namely the index finger and the ring finger is affected by exposure to androgens and the ratio is sexually dimorphic. This 2D:4D ratio has variations among males and females. Females present greater 2nd digit length and higher 2D:4D ratio while males present with a smaller 2D:4D ratio with the 4th digit being longer than the 2nd digit.(7) The variation in the ratio of 2nd to 4th digit length is explained by oestrogen and testosterone concentrations. This ratio has been suggested to serve as a pointer to the prenatal hormonal environment of a foetus, particularly testosterone.(8) The second-to-fourth digit ratio has been linked to numerous human traits associated with sex hormones and is considered an early morphological marker for many human behaviours that persist into adult life. This ratio is considered to be determined as early as in utero or at least before two years of age and remains constant throughout life.(9,10) The shared factor influencing both mandibular development and digit ratio appears to be testosterone concentration. One prior study reported that the 2D:4D ratio can be used as an early marker for mandibular prognathism in subjects with anteroposterior dysplasia.(11) To appreciate and gain deeper insights, the present study was undertaken to investigate the relationship between testosterone levels and the efficacy of the 2D:4D digit ratio as a predictive marker for mandibular prognathism.

This was a hospital-based cross-sectional clinical study conducted in the Department of Orthodontics, Tamil Nadu Government Dental College, India, between 2013 and 2015 with the patients who reported for correction of malocclusions. This study received approval from the institutional ethics committee. The sample size was calculated using the F test in G power version 3.1.9.2. The study subjects were 180 mesomorphic patients in the age group of 18-25 years. They were divided into three groups based on Angle’s classification of malocclusion. The skeletal base of Class I(normal) patients had orthognathic maxilla and mandible, Class II (retrognathic)patients had orthognathic maxilla with retrognathic mandible and Class III(prognathic) patients had orthognathic maxilla with prognathic mandible. Sixty patients(30 males and 30 females)from each class were taken for study. Subjects were detailed about the study and written consent was obtained before participation. Patients who were medically compromised, patients with gonadal disorders, patients on hormonal therapy, patients with a history of trauma to teeth, jaws and fingers, obese and left-handed individuals, ectomorphic, endomorphic individuals, and patients with a previous history of orthodontic treatment were excluded for the study. All patients had the normal orientation of the maxilla to the cranial base with an SNA of 82±3. Classification of the patients as class I was based on ANB angle 0–4 degrees and a Wits appraisal ±1 mm, as class II with ANB >5 and Wits appraisal >4 mm and as class III when ANB angle less than 1 and a Wits appraisal of <−2 mm. Lateral cephalometric radiographs of all patients were taken using the standardised procedure for evaluating the cranial structure. All radiographs were of good quality and showed the craniofacial complex. The images were calibrated and all linear measurements were corrected for magnification difference prior to statistical analysis. 2.1 Assessment of 2D:4D digit ratio: Lengths of the second digit and fourth digit of the right hand were measured on the ventral surface of the palm from the tip of the finger to the bottom crease of the finger.(8,12) All measurements were made with a digital vernier calliper. Second digit to fourth digit ratio (2D:4D) was calibrated and noted for each subject. 2.2 Assessment of Testosterone Intravenous blood (2ml) was collected from the forearm of all the patients between 9.00 a.m. and 12.00 noon. This was done to avoid diurnal variation of hormones. Estimation of total serum testosterone level was done by the ECLIA (Electrochemiluminescence immunoassay) method. The assay was carried out with an Elecsys Testosterone II kit (Cayman Chemical’s EIA kits, USA) with a cobas e immunoassay analyser. The total duration of the assay was 18 minutes. The analyser automatically calculated the analyte concentration of each sample (either in ng/ml, ng/dL or nmol/L). The normal reference for testosterone levels in males is 2.8-8ng/ml and, in females it is-0.06-0.82ng/ml. 2.3 Statistical Analysis: The initial statistical analysis of the collected data was done with the normality tests Kolmogorov-Smirnov test and Shapiro-Wilk test. Parametric methods were applied to analyse the data. Pearson and Spearman's Rank correlations were calculated to assess the linear relationship between the 2D:4D ratio and testosterone concentrations for each gender and overall samples. Independent samples t-test was carried out to compare mean values between genders for 2D:4D ratio and testosterone concentrations. To compare the mean values between the three classes one-way ANOVA was applied followed by Tukey’s HSD post hoc tests for multiple pairwise comparisons. To analyse the data SPSS (IBM SPSS Statistics for Windows, Version 26.0, Armonk, NY: IBM Corp. Released 2019) was used.

3.1 The 2D:4D: The overall(180 subjects) 2D:4D ratio was statistically significant with p <0.001 at 95% CI. Males had a mean 2D:4D ratio of 0.89134 and females had a mean of 0.93757. The mean ratio was significantly higher for females. (Table I). TABLE: I Independent samples t-test to compare mean values between Genders (Overall) Variable Gender N Mean Std Dev 95% CI for Mean p-value LL UL 2D/4D Ratio Male 90 .89134 .060036 .87877 .90392 <0.001 Female 90 .93757 .080893 .92062 .95451 Testosterone levels Male 90 5.63274 1.208873 5.37955 5.88594 <0.001 Female 90 .40079 .173384 .36447 .43710 The interclass comparisons of the mean value of the 2D:4D ratio for males and females were tabulated. (Table II). Males lacked significance while females had significant mean values at 95% CI with p <0.001. For females, multiple interclass comparisons were performed using Turkey HSD post hoc tests using the 2D:4D mean ratio. Statistically significant mean differences were obtained when class III(prognathic) was compared with class I(normal) and class II(retrognathic). This showed that females with class III skeletal base had an increased fourth digit when compared to class I as previously described.(11) Comparing retrognathic subjects with prognathic individuals in terms of the 2D:4D ratio did not yield significant results.(11) TABLE: II One-way ANOVA to compare mean values between Classes in each Gender Gender Variable Group N Mean Std Dev 95%CI for Mean p-value LL UL Male 2D/4D Ratio Class -I 30 .89893 .053517 .87895 .91892 0.593 Class -II 30 .89210 .071386 .86544 .91876 Class -III 30 .88300 .054491 .86265 .90335 Testosterone levels Class -I 30 5.58557 1.436357 5.04922 6.12191 0.333 Class -II 30 5.42733 1.286913 4.94679 5.90787 Class -III 30 5.88533 .808511 5.58343 6.18724 Female 2D/4D Ratio Class -I 30 .98060 .051342 .96143 .99977 <0.001 Class -II 30 .95670 .064937 .93245 .98095 Class -III 30 .87540 .083288 .84430 .90650 Testosterone levels Class -I 30 .26963 .131191 .22065 .31862 <0.001 Class -II 30 .43377 .187522 .36374 .50379 Class -III 30 .49897 .104845 .45982 .53812 3.2 Testosterone concentrations: The total testosterone values estimated for both genders were normally distributed with a P value of 0.001 at a confidence interval of 95%(Table I). Overall males had a mean testosterone value of 5.63274 and females had 0.40079. Particularly females had mean values that were statistically significant. Interclass comparison among females showed significant (p<0.001) values when Class I(normal) was compared with Class II(retrognathic) and Class III(prognathic). (Table III) TABLE: III Tukey HSD Post Hoc Tests for Multiple Pairwise Comparisons Gender Variable Group Mean Difference 95% CI for Mean diff p-value LL UL Female 2D/4D Ratio Class -I Class -II .023900 -.01784 .06564 0.364 Class -III .105200 .06346 .14694 <0.001 Class -II Class -III .081300 .03956 .12304 <0.001 Testosterone levels Class -I Class -II -.164133 -.25361 -.07465 <0.001 Class -III -.229333 -.31881 -.13985 <0.001 Class -II Class -III -.065200 -.15468 .02428 0.197 3.3 The 2D:4D ratio and testosterone levels: Testosterone levels were negatively correlated with 2D:4D ratio (r = -0.413, p<0.001)overall. (Table IV) Male testosterone levels were correlated with 2D:4D ratio as r = -0.544 (p<0.001) and females as r = -0.669 (p<0.001). (Table V) TABLE: IV Correlations between 2D/4D Ratio and Testosterone levels (Overall) 2D/4D Ratio Testosterone levels Correlation -0.413 P-value <0.001 N 180 TABLE: V Correlations between 2D/4D Ratio and Testosterone levels in each Gender Gender 2D/4D Ratio Male Testosterone levels Correlation -0.544 P-value <0.001 N 90 Female Testosterone levels Correlation -0.669 P-value <0.001 N 90 3.4 The 2D:4D ratio, testosterone and mandibular prognathism: The relationship between 2D:4D ratio, testosterone concentrations and mandibular base is particularly significant in females in the current study. Class III tendency females showed a low 2D:4D ratio and higher testosterone concentrations. (Graph 1,2)

Hormones act as a general epigenetic factor, particularly the sex hormones that have stimulatory effects on skeletal growth.(4) Sex hormones also determine the digit dermatoglyphics in humans by length and dimensions which are laid as early as seven weeks in-utero.(10,12) High 2D:4D ratios are characteristics of females and lower values are characteristics of males. (13,14) The 2D:4D ratio is shown to be stable throughout life and negatively related to adult testosterone levels.(15) The ratio has been considered a predictor of the degree of sexual dimorphism and other sex hormone-mediated traits that persist throughout adult life.(7,9,16) Mandibular growth can be one such trait influenced by sex hormones. Regarding sagittal jaw growth, the impact of sex hormones is markedly evident during the pubertal period in both males and females.(17) Hence young adult groups were studied for testosterone concentrations and mandibular growth in the present study.(18) Premkumar et al in 2013(11) conducted a study on the 2D:4D ratio’s association with mandibular dysplasia. They compared the 2D:4D ratio among subjects with normal mandible, retrognathic mandible and prognathic mandible. Significant results were obtained in comparing retrognathic and prognathic mandibles in males. Among females, they got significant results when they compared normal and prognathic mandibles. They have stated that the testosterone alterations might be a causative factor to the difference in the 2D:4D ratio in the three classes of mandible. They also reported that this trait which is set in utero under the influence of sex hormones like testosterone would provide an alternative way to determine the anteroposterior growth of the mandible. However, the study did not measure the testosterone levels to validate this claim. Hence the present study was conducted to analyse this aspect. In the present study, 2D:4D values revealed consistent findings in the interclass comparison similar to the previous study. Overall prognathic subjects displayed a lower 2D:4D ratio, with increased length of the fourth finger. Particularly the females with mandibular prognathism exhibited a significant value when compared with other classes. The present study was investigated with a minimal required sample size, to explore the relationship and identify any potential influence of testosterone on the relationship of digit ratio to mandibular excess. However, when testosterone levels were investigated, we could appreciate notable findings like in 2D:4D values. Class I (normal) showed significant differences with retrognathic and prognathic subjects which can be attributed to testosterone levels that play a role in influencing mandibular growth. Surprisingly, no significant difference was observed in testosterone levels between retrognathic and prognathic individuals, and this phenomenon remains unexplained. Testosterone levels are subjected to variation among genders and across the three classes of mandibular growth patterns making it challenging to generalize the findings. Though there were some findings favouring the previous study results, that prognathic females with lower 2D:4D ratios and increased testosterone levels, the same cannot be generalised across all classes and conditions. This study suggests that testosterone may be one of the influencing factors for the 2D:4D ratio as a predictor of mandibular prognathism. Even genetic predisposition plays a role in jaw growth, partly through hormonal mechanisms, making genetic influence undeniable. More studies in future are needed in larger populations and varied ethnic groups to find a consistent correlation between 2D:4D and prognathic mandible. The influence of both sex hormones on skeletal growth at different ages from in utero to adult life (9) can be studied and correlated with that of the digit ratio to have a more conclusive understanding.

Assessing the concentration of testosterone showed a relationship between 2D:4D and mandibular prognathism which is consistent with the previous study. But it also suggests that testosterone may not be the exclusive factor contributing to the 2D:4D ratio’s role as a predictor for mandibular prognathism. Conflict of interest: None declared. Financial aid: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgement: We acknowledge all the patients and their parents who actively participated in the study.

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