We here report the detection of statistically significant and positive correlations between left hand 2D:4D ratios, of phalanges and metacarpals plus phalanges, measured in boys and girls diagnosed with ASD and chronologic age, skeletal age and BMI. No statistically differences were detected in 2D:4D ratios by sex, however rating of skeletal maturity using the TW3 method suggested that relative digit development was not complete in our sample of children. This might have caused misinterpretation of the 2D:4D ratio in attempting to determine exposure to androgens in utero. The latter is underscored by the absence of associations between chronologic age, skeletal age and BMI with the 2D:4D ratio of metacarpals but the detection of significant positive correlations with the 2D:4D ratio of phalanges (i.e., may reflect differential bone development ‘within-digit’ at these ages). Left hand 2D:4D ratios among ASD cases may thus not be fixed throughout childhood and, therefore, during this life phase might not accurately reflect prenatal androgen exposure.
Studies of terminated fetuses, at various stages of gestation, have lead to the conviction that sex-specific differences in relative digit length begin as early as three months gestation and become greater over time [18
]. Sex differences in 2D:4D from as early as one year of age have been reported by several investigators. For example, McIntyre et al. [20
] reported sex differences between males and females from data obtained in a longitudinal serial study of 124 subjects, aged one to 17 years of age. Likewise, a mean 2D:4D for the phalanges equal to 0.98 for 400 males aged two to 25, and 1.00 for 400 females of similar age was reported from a cross-sectional study [9
]. No such difference was found in the current study using 2D:4D ratios for the phalanges, the metacarpals, or the sum of phalanges and metacarpals, and, in fact, we observed moderate sized effects [16
] in the direction opposite of that which we anticipated (i.e., boys demonstrated greater average 2D:4D than girls). We suspect that our observations are a reflection of problems with interpreting 2D:4D data because of the high degree of variability in children whose digits are still maturing. However, the limited sample of girls available for this study (n=6) does not provide sufficient statistical power for the detection of 2D:4D sex differences and thus point estimates for sex-differences are likely to be substantially influenced by sampling error. It is worth noting that our finding of statistically similar metacarpal 2D:4D ratios for boys and girls has been recently reported for metacarpal ratios in African-American and European-American adults [21
In addition to comprising participants diagnosed with ASD, our study sample was generally younger than those for which statistically significant associations were reported for digit ratio and sex. If a trend towards a sexually dimorphic relative digit length was not in fact in effect within the age range of the majority of our subjects, then variability within-subject may have introduced exposure misclassification into the study and potentially biased our results towards the null hypothesis. The possibility that post-natal factors may influence relative finger bone length has been previously raised [18
]. Investigators have speculated that relative finger growth may not be complete until some time during adolescence [19
]. In a longitudinal sample of two to 10 year olds, McIntyre and colleagues [22
] reported a declining 2D:4D ratio from subjects six to eight years of age and found that the ratio was an unreliable marker for sex. Thus, while sex-specific differences in relative digit length may be initiated in utero,
they might vary during childhood growth in keeping with the differential maturation of the digits, to appear fixed after puberty when hand maturation is complete.
Prior to the completion of puberty, the non-uniform pattern of vertebrate digit development (i.e., sequential progression from the lateral to the medial aspect of the hand when in anatomical position) might introduce substantial within-subject variability into 2D:4D ratios [23
]. Our findings, though cross-sectional in nature, suggest that digit ratio remains a dynamic quantity until later in development among children with ASD, and may thus not be a useful indicator of prenatal androgen exposure among ASD cases during active phases of growth. This was corroborated by bone maturity estimates employing the 3rd
digits, which indicated that a substantial proportion of study subjects had medial digits that were less mature than those more lateral and could therefore presumed to be shorter relative to their expected size at maturation. This would tend to bias the 2D:4D ratio upward. The statistically significant correlations between 2D:4D and measures for age (i.e., chronologic age) and growth (i.e., skeletal age) further suggest ongoing development of the ratio of digits among subjects in this study. However, the cross-sectional nature of this study precludes assessment of temporality with regard to digit immaturity. While the positive correlations detected between 2D:4D and chronologic age [22
] and BMI [24
] are not novel, to our knowledge, this report constitutes the first report of a positive correlation between 2D:4D ratio and skeletal age.
There are several limitations to the current study which necessitate caution in the interpretation of the study results. A fundamental condition to our interpretation of the study results is that left-hand 2D:4D ratios are valid markers of androgen exposure in utero
, a reasonable presumption [25
]. However, right hand 2D:4D ratios are known to be more strongly sexually differentiated than left hand 2D:4D ratios and frequently show more pronounced effects with target traits [26
]; these measures were unfortunately not available for this study. The limited sample size available for this study, with regard to girls and non-Caucasian races/ethnicities in particular, undermined our ability to detect previously reported differences in digit ratios by these characteristics [28
]. Moreover, the absence of control group radiographs precluded comparison of digit ratio between ASD cases and controls, an unfortunate consequence of the nature of the parent study from which this secondary analysis was completed.
Our study employed a minority method, hand radiographs, to ascertain 2D:4D [29
] and thus limits our ability to evaluate our results in the context of prior studies. Comparative studies employing hand radiographs are limited [30
]. Differences, in particular between radiographs, photocopies, and direct measures of 2D:4D ratios may be due in part to hand position and incorporation, or lack thereof, of soft tissue, basal and tip fat pads in particular, the development of which may also be responsive to androgens [32
]. Thus, comparisons using the results from this study must be made with caution.
This study of boys and girls with ASD raises several questions with regard to the nature of 2D:4D ratios throughout growth and development. We present several indicators of inconsistent digit maturation among our subjects raising the possibility of ongoing relative digit length development among children aged four to eight years and diagnosed with ASD. This study showed no significant difference in 2D:4D ratios between the sexes and point estimates for boys were higher than those for girls, which we suspect is a consequence of dynamic 2D:4D ratios at these ages, in spite of the small number of girls in this study. In conclusion, our study suggests that 2D:4D ratios should be used cautiously as a marker for prenatal androgen exposure among young children when investigating the ASD hyper-androgenic hypothesis as ongoing 2D:4D development may lead to erroneous interpretation.