Digit ratio (2D:4D) and congenital adrenal hyperplasia (CAH): Systematic literature review and meta-analysis

Highlights

• Congenital adrenal hyperplasia (CAH) involves elevated prenatal testosterone.

• There is a sex difference in the digit ratio (2D:4D) that may involve prenatal androgen exposure.

• Article presents a systematic review and meta-analysis of CAH and 2D:4D.

• Low (male-typical) 2D:4D is associated with CAH and the effects are small-to-medium in size.

• Effect sizes observed here are ~50% smaller than those of an earlier meta-analysis.

Abstract

The ratio of length between the second and fourth fingers (2D:4D) is commonly used as an indicator of prenatal sex hormone exposure. Several approaches have been used to try to validate the measure, including examining 2D:4D in people with congenital adrenal hyperplasia (CAH), a suite of conditions characterised by elevated adrenal androgen production secondary to defective steroidogenesis. We present a systematic review and meta-analysis that examines the relationship between these two variables. Twelve articles relating to nine CAH cohorts were identified, and 2D:4D comparisons have been made between cases and controls in eight of these cohorts. Altogether, at least one 2D:4D variable has been compared between n = 251 females with CAH and n = 358 unaffected females, and between n = 108 males with CAH and n = 204 unaffected males. A previous meta-analysis (Hönekopp and Watson, 2010) reported lower right hand (R2D:4D) and left hand (L2D:4D) digit ratios in patients with CAH relative to sex-matched controls. Our meta-analysis showed the same pattern, with medium effect sizes for R2D:4D and small effect sizes for L2D:4D. Differences of small magnitude were also observed for M2D:4D, and no significant effects were observed for D_[R-L]. Notably, the only effects that remained statistically significant when stratified by sex were R2D:4D in males and L2D:4D in females, and the average effect size had reduced by 46.70% since the meta-analysis of Hönekopp and Watson (2010). We also found that individual comparisons in this literature were considerably underpowered, and that patterns of sexual dimorphism in 2D:4D were similar in CAH samples as in typically developing populations. Findings are discussed in relation to the prenatal androgen hypothesis as well as alternative explanations.

Introduction

Digit ratio (2D:4D) is typically lower in males than females, with a slightly larger sex difference present for the right hand (Hönekopp and Watson, 2010). The measure has been suggested to index the level of exposure to foetal testosterone (Brown et al., 2002; Manning et al., 1998) or the ratio of foetal testosterone to foetal oestradiol (Lutchmaya et al., 2004; Manning, 2011; Zheng and Cohn, 2011). Nevertheless, relatively few studies have validated the measure in human populations. Some research has directly manipulated foetal hormones in animal models (Abbott et al., 2012; Auger et al., 2013; Huber et al., 2017; Romano et al., 2005; Saino et al., 2007; Talarovičová et al., 2009; Zheng and Cohn, 2011), though the effects reported have not always been consistent. For instance, although Zheng and Cohn (2011) and Huber et al. (2017) both examined the effects of prenatal hormone exposure in CD-1 mice, the studies reported effects in opposing directions. Early manipulation of hormones is unethical in human studies, meaning that researchers have had to rely on other methods, such as correlating 2D:4D with hormone concentrations in amniotic fluid (Lutchmaya et al., 2004; Richards et al., 2020a; Richards et al., 2019; Ventura et al., 2013), umbilical cord blood (Çetin et al., 2016; Hickey et al., 2010; Hollier et al., 2015; Mitsui et al., 2015, Mitsui et al., 2016; Whitehouse et al., 2015), or the maternal circulation (Barona et al., 2015; Hickey et al., 2010; Richards et al., 2019; Ventura et al., 2013). The results of studies in humans broadly point towards a negative correlation between foetal testosterone exposure and 2D:4D, although statistically significant effects are accompanied by many null findings (Richards, 2017), and publication bias may be an issue.

Another approach for determining the efficacy of 2D:4D has been to examine whether it is associated with medical conditions characterised by atypical androgen activity. Two studies (Berenbaum et al., 2009; van Hemmen et al., 2017) have reported evidence of feminised 2D:4D ratios in phenotypically female (46XY) individuals with complete androgen insensitivity syndrome (CAIS), although it should be noted that the variance for 2D:4D in this population appears to be comparable to that of controls despite the complete lack of androgen sensitivity (Berenbaum et al., 2009; see also commentary by Wallen, 2009). Manning et al. (2013) showed that digit ratios were higher (i.e. more female-typical) in males with Klinefelter syndrome (47XXY) than in their unaffected relatives. However, this effect is difficult to interpret considering that prenatal testosterone levels in males with Klinefelter syndrome do not appear to differ from those of typically developing males (Ratcliffe et al., 1994).

A promising area of research has examined individuals with congenital adrenal hyperplasia (CAH). CAH is a family of autosomal recessive conditions characterised by impairment of one of five enzymes required to synthesise cortisol from cholesterol. This causes an accumulation of adrenocorticotrophic hormone (ACTH) secondary to negative feedback, which results in overstimulation of the adrenal cortex and increased adrenal androgen production (New, 2006). Most cases (90–95%) of CAH are caused by 21–hydroxylase (21–OH) deficiency, with three main phenotypes being distinguishable (for a comparison of symptom profiles, see New, 2006). The most severe form, classical salt-wasting (SW) CAH, involves impairment of aldosterone synthesis, a symptom that is absent overall in classical simple-virilizing (SV) CAH; both SW and SV are characterised by genital ambiguity in female (46XX) patients. Pharmacological treatment for classical CAH due to 21-OH deficiency typically begins soon after birth, and the condition has been found to occur in approximately 1 in 14,000 live births (Pang et al., 1988). Non-classical CAH due to 21-OH deficiency does not present with aldosterone impairment nor typically with genital ambiguity, and can go undetected (Levine et al., 1980) particularly in males. The non-classical or late-onset form is diagnosed when symptoms present later in life (Kisch et al., 1987; New et al., 1981), and is more common than classical CAH, with reported prevalence ranging from 1 in 27 to 1 in 300, depending on the ethnic group studied (Hannah-Shmouni et al., 2017; New, 2006).

CAH provides an opportunity for researchers to examine the organisational effects of elevated androgen exposure during gestation. There is some evidence for behavioural masculinisation and defeminisation in CAH, with such observations being particularly pertinent in 46XX female-assigned cases because prenatal androgen concentrations may not only affect external somatic sex structures, but also bipotential areas in the brain, leading to modification of behavioural/psychological outcomes (see Cohen-Bendahan et al., 2005; Hines, 2004; Hines et al., 2015; Jordan-Young, 2012). The early androgenic effects of CAH in males however are less clear, as feedback mechanisms may lead to normalisation of androgen levels via reduced production by the testes (Pang et al., 1979; for a discussion, see Mathews et al., 2004). Evidence for this is provided by the observation that amniotic testosterone levels for 46XY CAH foetuses tend not to be clearly distinguishable from those of typically developing 46XY foetuses (Pang et al., 1980; Wudy et al., 1999), though such observations have relied on very small samples. However, it does appear possible that following an initial elevation, testosterone concentrations may be relatively typical in males with CAH.

Hines et al. (2003) reported that females with CAH outperformed their unaffected female relatives on two tasks assessing targeting performance. The tasks employed included measures of visuomotor spatial ability that have been found to demonstrate a large male advantage in the typically-developing population (Watson, 2001). In a study by Collaer et al. (2009), females with CAH also outperformed unaffected female relatives on motor and visuomotor tasks (grip strength and targeting), which have shown a male advantage in previous research (e.g. Miller et al., 1993), even after controlling for weight and height. The enhanced targeting performance in females with CAH was still present after adjusting for grip strength, leading the researchers to point towards an organisational influence of prenatal androgens on the neural regions dedicated to targeting (Collaer et al., 2009).

Behavioural masculinisation in females with CAH may only occur in traits which show a particularly large sex difference. Alternatively, as studies of CAH populations typically utilise small samples due to the rarity of the condition, they may lack the statistical power required to reliably detect effects of small or medium size. A way to overcome this limitation is to pool the findings of individual studies into a meta-analysis, which provides an indication of the presence (or absence) of an effect as well as its size. Using this technique, Puts et al. (2008) reported that females with CAH display an advantage on spatial tasks, whereas males with CAH display a disadvantage. However, although a more recent meta-analysis (Collaer and Hines, 2020) including a larger number of samples replicated the finding of reduced overall spatial ability in males with CAH relative to male controls, it did not find any difference between females with CAH and female controls. A possible interpretation of these contradictory findings is that early studies (and hence meta-analyses of those early studies) are more likely to report statistically significant effects in small samples whereas later studies often report smaller (or null) effects when attempting to replicate them in larger samples.

As CAH (at least in females) is associated with elevated prenatal androgen levels, and 2D:4D is hypothesised to indicate individual differences in foetal testosterone exposure, it follows that they should be related. A meta-analysis of early studies of CAH case-control studies (Hönekopp and Watson, 2010) showed that digit ratio for the right hand (R2D:4D) (d = −0.91, p < 0.001) and left hand (L2D:4D) (d = −0.75, p = 0.007) were significantly lower (i.e. more male-typical) in females with CAH relative to female controls; R2D:4D (d = −0.94, p = 0.061) and L2D:4D (d = −0.63, p = 0.013) were also lower in males with CAH relative to male controls, albeit the effect for R2D:4D was not statistically significant at the p < 0.050 level. This pattern of results is consistent with prenatal androgen exposure being elevated in both males and females with CAH, and so runs contrary to the idea that feedback mechanisms can normalise testosterone levels in males with CAH via downregulation of testicular androgen production.

Although behavioural effects associated with CAH may be explainable by environmental influences (Hines et al., 2015; Jordan-Young, 2012) such as the presence and extent of genital virilisation, and alterations in the way that parents, teachers and others interact with children with CAH, it seems unlikely that these could affect a person's digit ratio. However, it should be acknowledged that although CAH research may indicate that elevated prenatal testosterone exposure causes physical differences, such as a masculinised 2D:4D ratio, these findings cannot necessarily be extrapolated to indicate a similar influence on the developing brain.

The current study aims to build on the earlier meta-analysis of Hönekopp and Watson (2010) by updating their analysis to include new studies and incorporating a full systematic review of the relevant literature. Hönekopp and Watson (2010) included their analysis of the relationship between 2D:4D and CAH within an article with a much broader remit. Therefore, this literature has yet to be comprehensively reviewed. We also extend their analysis in other ways. As it has been suggested that the right-left difference in digit ratio (D_[R-L]) can provide a further marker of prenatal sex hormone activity, with low R2D:4D relative to L2D:4D hypothesised to indicate high androgen exposure (Manning, 2002; Manning et al., 2014), this variable is also considered in the current study. Furthermore, some studies report on the average 2D:4D across the right and left hands (M2D:4D). Because studies comparing digit ratios between patients with CAH and controls have not so far investigated D_[R-L] or M2D:4D, we contacted the authors of relevant papers to request the necessary data. We hypothesised that R2D:4D, L2D:4D, and D_[R-L] would each be significantly lower in males and females with CAH relative to male and female controls, respectively. Although not initially considered in our pre-registration (see next section), we also hypothesised that M2D:4D would be significantly lower in males and females with CAH relative to male and female controls, respectively, and, additionally, examined whether 2D:4D variables exhibit similar sexual dimorphism in CAH samples as they do in general population studies.