Lower digit ratio and higher endogenous testosterone are associated with lower empathic accuracy

Hormones and Behavior 119 (2020) 104648

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Lower digit ratio and higher endogenous testosterone are associated with lower empathic accuracy ⁎

Jonas P. Nitschke , Jennifer A. Bartz

T

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Department of Psychology, McGill University, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: Testosterone Digit ratio Empathic accuracy Social cognition Person perception

Empathy is a cornerstone of human sociality. It has important consequences for our interpersonal relationships and for navigating our social world more generally. Although research has identified numerous psychological factors that can influence empathy, evidence suggests that empathy may also be rooted in our biology and, in particular, the gonadal steroid hormone testosterone. To date, much of the research linking testosterone and empathy has focused on the 2D:4D ratio (i.e., the ratio of the lengths of the index and ring fingers), and the results have been mixed. These mixed results, however, may be due to reliance on self-report measures to assess empathy, which can be vulnerable to self-presentation, as well as social-cultural norms about gender/sex differences in empathy. Moreover, although some have argued that digit ratio is an indicator of prenatal androgen exposure, the evidence for this to date is weak. Here, we aimed to follow up on this prior work, using a naturalistic empathic accuracy task in which participants dynamically track, in real-time, the emotional state of targets. We show that lower digit ratio (Study 1; N = 107) and higher circulating testosterone (Study 2; N = 76) are associated with poorer empathic accuracy performance; critically, these effects hold when controlling for sex/gender. In neither study, however, did we find effects on self-reported empathy. Our results highlight the limitations of self-report measures and support the notion that endogenous testosterone levels as well as 2D:4D ratio are related to key social-cognitive competencies like empathic accuracy.

1. Introduction It is well-known that social connections are important for physical and mental well-being (Holt-Lunstad et al., 2010). For this reason, understanding the factors that facilitate, or hinder, the social skills that help us to connect with others is crucial. In this regard, the ability to understand the emotions and intentions of others, or cognitive empathy (Ickes, 1993), is an important component of human sociality (De Waal, 2010). Although numerous psychological factors have been shown to predict abilities in cognitive empathy (Zaki and Cikara, 2015), research suggests that variation in cognitive empathy is also rooted in our biological make-up and, in particular, the androgenic steroid hormone testosterone, a major sex hormone in both men and women (e.g., Knickmeyer and Baron-Cohen, 2006). Careful inspection of the literature, however, reveals that research supporting the link between testosterone and empathy is mixed. This, however, may be due, at least in part, to methodological issues related to the assessment of empathy. Much of the research to date relies on self-report measures, which can be vulnerable to self-presentation and other sources of influence. Moreover, when researchers have assessed empathy behaviorally, they

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typically rely on relatively simplistic task-based measures, which can be vulnerable to floor/ceiling effects, and restricted range; moreover, such simplistic tasks may not completely capture the kinds of complex social cognitive processes used in everyday social interactions. Here we aimed to address these gaps in the literature by investigating the association between testosterone and empathy using a naturalistic empathic accuracy task. In addition, we sought to extend prior research by investigating both digit ratio as well as endogenous testosterone levels. Most of the research implicating testosterone in empathy (and in social skills more generally) has focused on prenatal androgen exposure in utero and, in lieu of the former, digit ratio, whereas research looking at available endogenous testosterone is lacking. This is problematic, however, as it is unclear whether testosterone impairs the development of social skills like empathy, or whether testosterone also influences social cognitive processes in real time. Moreover, although some have argued that digit ratio is an indicator of prenatal androgen exposure, to date the evidence for this association is weak (Berenbaum et al., 2009; Hönekopp and Watson, 2010; Manning and Fink, 2017; Richards, 2017a, 2017b; Richards et al., 2019; Wallen, 2009). Testosterone plays a key role in the development of secondary

Corresponding authors at: Department of Psychology, McGill University, 2001 Avenue McGill College, Montreal H3A 1G1, Canada. E-mail address: [email protected] (J.P. Nitschke).

https://doi.org/10.1016/j.yhbeh.2019.104648 Received 25 March 2019; Received in revised form 22 November 2019; Accepted 23 November 2019 0018-506X/ © 2019 Elsevier Inc. All rights reserved.

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game. Other researchers, however, have not found reliable associations between the 2D:4D ratio and empathy. Voracek and Dressler (2006) found no association between digit ratios and either the EQ or SQ (N = 423); nor was 2D:4D associated with performance on the RMET (cf. Valla et al., 2010). Similarly, in a sample (N = 95) of secondary students, Kempe (2009) found no association between 2D:4D ratios and empathy assessed with the EQ. Finally, in what is probably the largest study to date (N = 201,895), Manning et al. (2010) found no association between 2D:4D and the EQ (although 2D:4D ratio was associated with the SQ). Of note, however, this was an online study and digit ratio was assessed by the participants with a ruler, a methodological factor that could have introduced error (e.g., Hönekopp and Watson, 2010); moreover, this study used brief 10-item versions of the EQ and SQ. How do we reconcile the (relatively consistent) findings from the studies that assess androgen exposure directly with the more inconsistent findings from the 2D:4D studies? One possibility is that digit ratio is not a sensitive measure of prenatal androgen exposure. As noted, evidence supporting the association between digit-ratio and prenatal androgen exposure is weak. Although studies have shown associations between digit ratio and prenatal androgen exposure in rodents (e.g., Auger et al., 2013; Talarovicová et al., 2009; Zheng and Cohn, 2011) and humans (McIntyre, 2006; Mitsui et al., 2016; Ventura et al., 2013), other studies have found no association between digitratio and prenatal androgen exposure (e.g., Hickey et al., 2010; Hollier et al., 2015; Richards et al., 2019). Moreover, it has been argued that digit ratio is likely influenced by factors other than prenatal testosterone including, for example, gonadal hormone ratios (Wallen, 2009) and/or genetic variations that influence gonadal hormones (Berenbaum et al., 2009; Warrington et al., 2018). Thus the imprecision of 2D:4D ratio as an indicator of prenatal androgen exposure could undermine the ability to detect an association between testosterone and empathy (Leslie, 2019). Sample size and/or gender composition may also play a role in the mixed findings, given that some studies show gender specific effects of digit ratio. Another factor that may contribute to the mixed research findings is that the vast majority of the 2D:4D studies relied on self-report measures of empathy, which can be problematic. First, people may lack insight to their actual empathic abilities. Indeed, self-reported empathy does not track tightly with performance on task based empathy measures (Zaki et al., 2008), or with neurophysiological measures of empathic arousal (Michalska et al., 2013). Second, even if people have insight into their empathic abilities, it is well known that self-report measures are vulnerable to self-presentation motives and other sources of influence. Indeed, in the case of empathy, there are strong socialcultural expectations that women are, in general, more empathic than men (Baez et al., 2017; Christov-Moore et al., 2014; Eagly and Wood, 1991; Eisenberg and Lennon, 1983; Ickes et al., 2000), and these cultural expectations may bias people's self-assessments. In line with this idea, meta analytic studies (Ashmore et al., 1986; Thompson and Voyer, 2014) show fairly large effects sizes for the association between gender/ sex and self-reported empathy (d = −0.99), but only small to moderate effect sizes for the association between gender/sex and task based measures like the Picture Story Exercise (d = −0.10) (see: Ashmore, 1990). These findings suggest that expectations about gender/sex roles may be guiding self-perceptions of empathy since self-report is more vulnerable to self-presentation motives compared to task-based measures. Thus, the first aim of the present investigation was to investigate the association between digit ratio and cognitive empathy. To this end, we used a naturalistic empathic accuracy task that requires perceivers to dynamically track, in real time, the emotions of real targets discussing autobiographical experiences (Zaki et al., 2008). We predicted that lower digit ratio would be associated with poorer empathic accuracy performance (controlling for sex/gender). We also took this opportunity

sexual characteristics early on in maturation, as well as in the development of the central nervous system (Bale, 2015). Research indicates that the organizational effects of androgen exposure on the brain during the sensitive period of gestation have been linked to more male-stereotypical cognition and behavior (Auyeung et al., 2013; Baron-Cohen et al., 2015, 2005; Hines, 1982), including poorer interpersonal skills and empathy (for a review, Knickmeyer and Baron-Cohen, 2006). In this regard, high levels of testosterone exposure in utero have been proposed to lead to an “extreme male brain,” characterized by increased tendencies to systemize, and difficulties understanding others and making accurate predictions about the social world (Baron-Cohen et al., 2005, 2003). The most direct, and consistent, evidence linking prenatal androgen exposure and empathy is based on research assessing testosterone exposure directly via amniotic fluid collected during gestation. For example, Knickmeyer et al. found that higher androgen exposure was associated with poorer social relationship quality, as well as less frequent use of intentional propositions, an index of mentalizing, in fouryear olds (Knickmeyer et al., 2006, 2005). Chapman et al. (2006) found that higher androgen exposure was associated with poorer performance on the Reading the Mind in the Eyes Test (RMET; Baron-Cohen et al., 2001), a measure of cognitive empathy, in six- to eight-year olds. Chapman et al. also assessed the mother's perceptions of her child's empathic abilities (with the child version of the Empathy Quotient); here, prenatal androgen exposure was associated with the mother's perceptions, but only for boys. More recently, Lombardo et al. (2018) found that prenatal androgen exposure was associated with decreased resting-state connectivity in the “social brain” default mode network that is, brain areas implicated in both mentalizing and self-referential cognition - in adolescents between the ages of 13 and 17. Finally, studies of individuals with Congenital Adrenal Hyperplasia (CAH), a sexual development disorder associated with increased androgen exposure pre-birth, corroborate the link between prenatal androgen exposure and empathy. For example, Mathews et al. (2009) found that women with CAH endorsed more male-stereotypical traits (e.g., dominance) compared to a control group, and Khorashad et al. (2018) found that young adult women with CAH performed more poorly on the RMET as compared to matched healthy control women. Taken together, there is consistent evidence linking prenatal androgen exposure, when assessed directly, to social skills and empathy more broadly. Often, however, it is not feasible to obtain direct measures of prenatal androgen exposure in utero; consequently, researchers have relied on measures like the 2D:4D ratio (i.e., the ratio of the index finger to the ring finger) - a putative indirect measure of prenatal androgen exposure. In contrast to fetal androgen studies, results from the 2D:4D ratio studies have been mixed, with some studies finding that lower digit ratio is associated with reduced empathy, but others finding no relationship. As examples for the former, in a large mixed sex sample (N = 120), Kempe and Heffernan (2011) found that lower 2D:4D ratios were associated with lower self-reported empathy, an effect that was specific to women. In a study of Swedish male adults, von Horn et al. (2010) found that lower 2D:4D ratios were associated with lower scores on the Empathy Quotient (EQ; Chapman et al., 2006) and higher scores on the Systemizing Quotient (SQ; Baron-Cohen et al., 2003). Replicating these findings, Wakabayashi and Nakazawa (2010) found that lower 2D:4D ratios were associated with lower scores on the EQ and higher scores on the SQ in a large mixed gender sample (N = 348) of Japanese university students. In the same vein, Barona et al. (2015) found that children with lower digit ratios performed more poorly on a facial emotion recognition task, and parents reported worse social communication skills (N = 7159), although these latter effects were only significant in boys. Finally, although not empathy per se, Moskowitz et al. (2015) found that men with lower 2D:4D ratios engaged in less agreeable behavior towards a partner during real-life social interactions, and Millet and Dewitte (2009, 2006) found that those with lower digit ratios engaged in less prosocial behavior in the dictator 2

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J.P. Nitschke and J.A. Bartz

monophasic oral contraceptives.

to test for the association between 2D:4D ratio and self-reported empathic abilities using a more typical self-report measure of empathy. The second aim of this research was to investigate the association between endogenous testosterone levels and empathic accuracy. Although there are fewer studies looking at circulating testosterone than 2D:4D ratio, preliminary evidence indicates that circulating testosterone may also be linked with empathy. Harris et al. (1996) found that higher levels of circulating testosterone were associated with lower levels of self-reported prosociality, including empathy and prosocial care (as well as higher levels of antisocial traits, like aggression, anger, and hostility). Ronay and Carney (2013) found that endogenous testosterone was negatively associated with a behavioral measure of cognitive empathy. In this research, Ronay and Carney (2013) measured endogenous testosterone and then had participants engage in an economic role-playing task in which they had to make sell-pitches to another participant. Afterwards, participants rated their own thoughts and feelings, and the performance of their interaction partner. These participant-partner ratings were then correlated to create an index of accurate empathic understanding, or “empathic accuracy”. Consistent with Harris et al. (1996), results showed that higher endogenous testosterone levels were associated with lower empathic abilities. Notably, in a second study, Ronay and Carney (2013) had peers rate participants' empathic abilities. Corroborating the findings from their first study, participants with higher testosterone levels were rated as less empathic by their peers. Finally, the aforementioned study by Lombardo et al. (2018), which reported a negative relationship between prenatal androgen exposure and default brain network, found a similar relationship between circulating levels of testosterone and resting state connectivity. Based on these studies, we predicted that higher endogenous testosterone levels would be associated with poorer empathic accuracy performance (again, controlling for sex/gender). As in Study 1, we also took this opportunity to test for the association between endogenous testosterone levels and self-reported empathic abilities.

2.3. Measures 2.3.1. Interpersonal reactivity index (IRI; Davis, 1983) The IRI is a 28-item self-report scale designed to measure individual differences in trait empathy. Specifically, it consists of 4-subscales measuring: Perspective Taking (“I try to look at everybody's side of a disagreement before I make a decision”); Empathic Concern (“I often have tender, concerned feelings for people less fortunate than me”); Personal Distress (“In an emergency situation, I feel apprehensive and ill-at-ease”); and Fantasy (“I really get involved with the feelings of the characters in a novel”). Participants rate each item on a 5-point Likert scale ranging from 1 (“does not describe me well”) to 5 (“describes me very well”). 2.3.2. Empathic accuracy task (EA; Zaki et al., 2008) In this task, participants watch six videos of individuals discussing negative personal life events (e.g., losing one's job, death of parents, rejection). While watching each video, participants used key presses on a computer keyboard to continuously rate how positive or negative they thought the target felt at each moment during the narrative. This was done using a 9-point Likert-scale (ranging from ‘very negative’ to ‘very positive’) displayed on the computer screen (for further descriptions see: Zaki et al., 2008). Importantly, the individuals in the video (targets) also rated their own emotional experience while discussing the events on the same 9-item Likert scale. Empathic accuracy is operationalized as the correlation between the continuous ratings made by perceivers and targets. Participants watched a total of 6 videos with negatively valenced life stories. This resulted in a total of 635 accuracy ratings: 107 participants times 6 videos (a total of 7 video clip accuracy scores were excluded because the participants did not generate enough responses to calculate a score). Participants were randomly assigned to watch one of two possible sets of videos, each with an equal number of male and female targets.

2. Study 1

2.3.3. Digit ratio To assess digit ratio, we took a high-resolution scan (1200 dpi × 1200 dpi) of participants' right hands so that we could assess the ratio of the length of the 2nd digit (index finger) and 4th digit (ring finger). These scans were then evaluated by two coders to assess digit ratio. Specifically, coders followed the procedure described by Voracek and Offenmüller (2007) and Moskowitz et al. (2015) in which each digit was measured from the midpoint of the ventral basal crease to the fingertip on the right hand (following suggestions by Hönekopp and Watson, 2010). Inter-rater reliability was calculated using Intraclass Correlation Coefficients (ICCs; Bartko, 1966; Shrout and Fleiss, 1979) in which raters are assumed to be a random sample, and reliability is provided for the average of the raters. The ICCs for the average of the two raters exceeded 0.88, indicating excellent inter-rater reliability (Cicchetti, 1994). Scores from the two raters were averaged. Of note, although digit ratio can be measured with both hands, or by the Dr – l (i.e., subtracting the left hand ratio from that of the right; e.g., Manning, 2002), we chose to measure the right hand in the present investigation to be consistent with prior studies on digit ratio and empathy, which typically measured digit ratio with the right hand. Moreover, as Hönekopp and Watson (2010) summarize, right hand 2D:4D might be a better indicator of prenatal androgenization than the left hand 2D:4D. Finally, there is some evidence for stronger effects for right hand 2D:4D (e.g., Moskowitz et al., 2015).

2.1. Overview Participants first provided informed consent and then completed a series of self-report questionnaires, including a measure of trait empathy (see below). Afterwards, participants completed a computerbased task assessing empathic accuracy as well as some other tasks not reported here1; digit ratio was assessed at the end of the study. Study procedures took approximately 1 h, and participants were compensated $10 for their time. The study was approved by the McGill University Institutional Review Board and conducted in accordance with the Declaration of Helsinki. 2.2. Participants One-hundred and seven healthy participants, 29 men (mean age = 23.2, SD ± 3.95) and 78 women (mean age = 21.8, SD ± 2.90), with no current history of medical or psychiatric illness were recruited. Recruitment criteria included: no recreational drug use, consuming less than ten alcoholic beverages a week, smoking less than 7 cigarettes a day.2 Women were either regularly menstruating or on 1 These data, as well as the data from Study 2, were collected as part of a larger program of research designed to test multiple, unrelated, hypotheses about hormones and social cognition and behavior. Participants in “Study 1” were taken from the baseline testing day from one of the studies in that research program. 2 These criteria were required primarily for some of the other procedures that were part of the larger program of research but are also relevant to this study as there is evidence that these factors can influence gonadal hormonal functioning (Peugh and Belenko, 2001).

2.4. Statistical analyses We first conducted a MANOVA to investigate the effects of gender on digit ratio and on age. As noted, previous research has reported gender/sex differences for digit ratio (e.g., Hönekopp and Watson, 3

Hormones and Behavior 119 (2020) 104648

J.P. Nitschke and J.A. Bartz

Table 1 Multilevel models predicting empathic accuracy with gender/sex and digit ratio or testosterone.a Study 1 b

Intercept Gender/sex DR/testosteronea

Study 2

b

SE (b)

df

t

bb

SE (b)

df

t

0.23 −0.01 0.03

0.06 0.02 0.01

13.02 103.94 102.88

3.53 −0.49 2.77⁎⁎

0.22 −0.06 −0.04

0.06 0.04 0.02

16.03 69.85 69.84

3.73 −1.47 −2.42⁎

Notes: Analyses were based on: Study 1 = 635 observations; Study 2 = 448 observations. a Study 1 = 2D:4D ratio. Study 2 = endogenous salivary testosterone. b Unstandardized. ⁎ p < 0.05. ⁎⁎ p < 0.005.

and beyond any effects due to gender/sex differences in digit ratio, suggesting that it is digit ratio per se that is implicated in empathic accuracy performance. Regarding the self-report measures of empathy, the multiple regression analyses with the subscales of the IRI as DVs, and gender/sex and digit ratio as IVs did not yield any significant results for digit ratio, or the interaction between digit ratio and gender/sex (all ps > 0.1). Thus, digit ratio did not significantly predict self-reported empathic abilities.4

2010). Age was included to ensure that men and women did not differ in this regard. To test our main hypothesis, i.e., whether digit ratio is associated with empathic accuracy, we conducted a mixed-linear model analysis (MLM; Baguley, 2012; Holmes Finch et al., 2014) in which empathic accuracy scores were nested within participants. Empathic accuracy scores were entered as the dependent variable, with gender/sex (0 = men; 1 = women) and digit ratio as fixed-effects, and subject-ID and video-name as random-effects. As noted, in addition to investigating the association between digit ratio and empathic performance, we took this opportunity to investigate whether digit ratio is associated with self-reported empathy. To this end, we conducted a series of multiple regression analyses for all subscales of the IRI as the DVs, and gender/sex (0 = men; 1 = women) and digit ratio as IVs. For all analyses digit ratio values were z-standardized. Reported confidence intervals were bootstrapped. All statistical analyses were conducted using R (R Core Team, 2018) and the lme4-package for MLM (Bates et al., 2015).

4. Study 2 4.1. Overview Participants came to the lab and first completed an informed consent document and then a series of self-report questionnaires, including the IRI; participants then provided a saliva sample for the testosterone assay (note: as the questionnaires took approximately 40-min to complete, we could attenuate the influence of external factors - e.g., a stressful or competitive experience (Lennartsson et al., 2012; Zilioli and Bird, 2017) - on basal testosterone levels prior to taking the saliva sample). Following procedures unrelated to the present investigation,5 participants completed the same empathic accuracy task as in Study 1. Study procedures took approximately 2 h and participants were compensated $20 for their time. The study was approved by the McGill University Institutional Review Board and conducted in accordance with the Declaration of Helsinki.

3. Results As expected, and consistent with prior research, the results of the MANOVA testing for gender/sex differences in digit ratio revealed a significant difference between men and women, F(1,105) = 11.017, p = 0.001, η2 = 0.095. Men's ratios (M = 0.956, SD ± 0.032) were smaller than women's ratios (M = 0.977, SD ± 0.028). The results of the MANOVA testing for gender/sex differences for age revealed no significant differences between the two groups for age (F (1,105) = 3.901, p = 0.051, η2 = 0.036). The MLM predicting empathic accuracy scores with gender/sex and digit ratio as fixed effects, and subject-ID and video-name as random effects, revealed a significant effect for digit ratio, b = 0.0298 (SE ± 0.0108; 95% - CI[0.006, 0.052]), t(102.88) = 2.765, p < 0.007, but no effect of gender/sex, b = −0.012 (SE ± 0.02416, 95% - CI[−0.065, 0.036]), t(103.94) = −0.49, p = 0.626 (see Table 1). Including the set of videos participants viewed as a covariate did not change the results, suggesting that the effects were not specific to specific targets or video topics. In a subsequent analysis, we included the digit ratio × gender/sex interaction term; the digit ratio × gender/ sex was not significant, t(102.17) = 0.64, p = 0.52, and including the interaction term did not result in a more parsimonious model fit (noninteraction model, AIC = −91.37; interaction Model, AIC = −89.79, p = .51).3 Thus, as predicted, lower digit ratio was associated with poorer empathic-accuracy performance; critically, this effect is above

4.2. Participants The sample consisted of 76 healthy participants, with no current history of medical or psychiatric illness; there were 30 men (mean age = 22.1 m SD ± 3.00) and 46 women (mean age = 21.6, SD ± 2.60). As in Study 1, exclusion criteria included recreational drug use, consuming more than ten alcoholic beverages a week, and smoking > 7 cigarettes a day. Women were either regularly 4

Although the IRI subscales were unrelated to digit ratio (and empathic accuracy), we re-ran our main analysis with IRI subscales as covariates. Including the IRI subscales did not change the reported results (i.e., the effect of digit ratio on empathic accuracy remained significant after accounting for the IRI and none of the IRI subscales predicted empathic accuracy; see Supplemental Materials for details). This suggests that the effect of digit ratio on empathic accuracy was not driven by individual differences in trait empathy, nor did trait empathy mediate the effect. 5 As noted, participants were taken from a larger program of research. Study 2 participants were from a study in which we randomly assigned participants to either a stress condition, in which they underwent the Trier Social Stress Test (TSST; Kirschbaum et al., 1993), or a no stress conditions (the “placebo” TSST; Het et al., 2009); we focused only on the control condition participants as our primary goal was to replicate and extend prior work on the effects of testosterone on empathy. Study 2 data did not overlap with that from Study 1.

3 Of note, although the digit ratio × gender/sex interaction was not significant, we ran the model separately for men and women for the interested reader. Results showed that lower digit ratios significantly predicted poorer empathic accuracy for women, whereas the effect was not statistically significant for men (although the direction of the effect was the same as it was for women; see Supplementary Materials).

4

Hormones and Behavior 119 (2020) 104648

J.P. Nitschke and J.A. Bartz

(see Table 1). Including the set of videos participants viewed as a covariate did not change the results. In a subsequent analysis, we included the testosterone × gender/sex interaction term; the testosterone × gender/sex was not significant, t(69.17) = 1.37, p = 0.17, and including this interaction term did not result in a more parsimonious model fit (non-interaction model, AIC = 64.56; interaction model, AIC = 64.62, p = 0.164).6 Thus, as predicted, higher levels of endogenous testosterone were associated with lower empathic-accuracy scores. Critically, as in Study 1, the effect of testosterone is above and beyond that related to gender/sex differences.7 See Fig. 1. Finally, the multiple regression analyses with the subscales of the IRI as DVs, and gender/sex and testosterone as IVs did not yield any significant results for testosterone, or the interaction between testosterone and gender/ sex. Hence, endogenous levels of testosterone did not significantly predict self-reported empathic abilities.8

menstruating or were on monophasic oral contraceptives. 4.3. Measures 4.3.1. Testosterone assay Participants drooled approximately 1.5 mL of saliva through a straw into a passive drool device (Eppendorf AG, Hamburg, Germany). Saliva samples were immediately put into a laboratory freezer at e20C. Testosterone was assayed using the DRG salivary Testosterone ELISA kit with monoclonal (mouse) as anti-testosterone antibody (DRG Instruments GmbH, Marburg, Germany). All samples were assayed in duplicates, subsequent analyses used the mean value. Intra-assay coefficient of variation (CV) for each sample was < 12.5% (mean CV = 3.01, SD ± 2.36). Testosterone levels were in the normal ranges (cf. Andersson et al., 2017; Mehta and Josephs, 2010). One participant's testosterone value was higher than 3 SD from the mean, to minimize leverage the value was windsorized to 3 SD for all further analyses.

6. General discussion

4.3.2. Empathic accuracy task (EA; Zaki et al., 2008) We used the same EA task described in Study 1. Participants watched a total of 6 videos with negatively valenced life stories. This resulted in a total of 448 accuracy ratings: 76 participants times 6 videos (a total of 8 video clip accuracy scores were excluded because the participants did not generate enough responses to calculate a score). Participants were randomly assigned to watch one of two possible sets of videos, each set had an equal number of male and female targets. The sets were different from the ones used study 1, though some videos overlapped.

In Study 1, we showed that digit ratio, a putative indirect measure of prenatal androgen exposure, was associated with performance on a naturalistic empathic accuracy task (Zaki et al., 2008). In Study 2, we extended this finding by showing that circulating testosterone was also associated with empathic accuracy performance. Of note, and consistent with prior work, we found that sex/gender was associated with both digit ratio and circulating testosterone, with men showing smaller ratios, and higher levels of circulating testosterone, than women. Critically, though, the effects of digit ratio, and testosterone, on empathic accuracy remained significant when controlling for gender/sex, indicating that the empathic accuracy effects we observed were specifically due to digit ratio (Study 1) and testosterone (Study 2) and not gender/sex differences. Interestingly, we did not find an association between 2D:4D or circulating testosterone on the self-report measure of empathy. Although these null effects could be due to lack of statistical power, they are consistent with the mixed findings in the 2D:4D literature - many of which relied on self-report measures of empathy. Our results suggest that the mixed findings in the 2D:4D-empathy literature may be partly due to the use of self-report measures to assess empathy. As noted, cultural expectations about gender/sex roles (Baez et al., 2017; Christov-Moore et al., 2014; Eagly and Wood, 1991; Eisenberg and Lennon, 1983; Ickes et al., 2000) may influence peoples self-

4.4. Statistical analyses As in Study 1, we first conducted a MANOVA to assess gender/sex differences in age to ensure that men and women did not differ on these variables. We also included levels of testosterone as an additional dependent variable, as previous research has reported gender/sex differences for endogenous testosterone (e.g., Hönekopp and Watson, 2010). To test our main hypothesis, we again conducted a MLM (MLM; Baguley, 2012; Holmes Finch et al., 2014) for our empathic-accuracy scores, with gender/sex (0 = men; 1 = women) and testosterone-values as fixed-effects, and subject-ID and video-name as random-effects. In addition to investigating the association between testosterone and empathic performance, we investigated whether testosterone is associated with self-reported empathy. To this end, we conducted a series of multiple regression analyses for all subscales of the IRI as the DVs, and gender/sex (0 = men; 1 = women) and endogenous testosterone as IVs. For all analyses, testosterone values were z-standardized. Reported confidence intervals were bootstrapped. All statistical analyses were conducted using R (R Core Team, 2018) and the lme4-package for MLM (Bates et al., 2015).

6 As with Study 1, although the testosterone × gender/sex interaction was not significant, we ran the model separately for men and women for the interested reader. Results show that higher levels of testosterone significantly predicted poorer empathic accuracy for men, whereas the effect was not statistically significant for women (although the direction of the effect was the same as it was for men; see Supplementary Materials). 7 As noted, our primary goal in Study 2 was to conceptually replicate and extend prior research on testosterone and empathy. That said, research has shown that stress can moderate the effect of gonadal hormone status on social behavior (Mehta and Prasad, 2015). As the larger program of research included a stress manipulation condition, we conducted additional analyses that included participants in the stress condition for the interested reader. Results showed that the stress manipulation significantly moderated the effect of testosterone on empathic accuracy, such that the simple effect of testosterone on empathic accuracy was significant in the control condition (z = −2.8, p = 0.01) but not in the stress condition (z = 0.8, p = 0.4). These results suggest that there might be boundary conditions for the effects of testosterone on empathic accuracy and, specifically, that contextual factors such as stress might negate these effects. 8 Although the IRI subscales were unrelated to testosterone (and empathic accuracy), we re-ran our main analysis with the IRI subscales as covariates. Including the IRI subscales did not change the reported results (i.e., the effect of testosterone on empathic accuracy remained significant even after accounting for the IRI; see Supplemental Materials for details). This suggests that the effect of testosterone on empathic accuracy was not driven by individual differences in trait empathy, nor did trait empathy mediate the effect.

5. Results The results of the MANOVA testing for gender/sex differences in age, revealed no significant differences between the two groups for age (F(1,74) = 0.82, p = 0.37). As with digit ratio in Study 1, there was a significant gender/sex difference for testosterone-values (F (1,74) = 68.32, p > 0.00, η2 = 0.48), with men having significantly higher levels of endogenous testosterone than women. Testosterone in men: mean = 145.22 pg/mL, SD ± 60.52; Testosterone in women: mean = 53.43 pg/mL, SD ± 36.54. The results of the MLM explaining EA-scores, with gender/sex and testosterone-values as fixed-effects, and subject-ID and video-name as random factors, revealed a significant effect of testosterone, b = −0.043 (SE ± 0.018; 95% - CI[−0.077, −0.010]), t (69.84) = −2.42, p = 0.018, and no effect of gender/sex, b = −0.056, (SE ± 0.038; 95% - CI[−0.129, 0.021], t(69.85) = −1.47, p = 0.146 5

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Fig. 1. The association between Digit-Ratio (Study 1; panel A) and Testosterone (Study 2; panel B) and the Empathic Accuracy task (EA). A) Higher digit ratios are associated with better performance on the EA task. B) Higher levels of endogenous testosterone are associated with poorer performance on the EA task. Empathic accuracy was operationalized as the correlation between participants' continuous ratings of what the target was feeling and the targets' continuous self-ratings of what he or she was feeling; in this way, a score of 1 would indicate perfect correspondence, or accuracy.

Hermans et al., 2006; Wright et al., 2012; for a review: Bos et al., 2012; however: Nadler et al., 2019). Social cognitive abilities are often conceptualized as relatively fixed, trait-like characteristics, but these data suggest that may not always be the case, and that interpersonal processes, like empathy, may fluctuate considerably even within individuals across situations. Indeed, as with other hormonal effects (e.g., Bartz et al., 2011; Nitschke et al., 2018), contextual and person specific factors might have to be considered when studying the effects of testosterone on behavior and cognition (Carré and Archer, 2018). Relatedly, some have argued that the effects of testosterone on behavior are dependent on other hormones. For example, according to the “Dual Hormone Hypothesis” the effect of testosterone on dominance depends on circulating levels of cortisol (Mehta and Prasad, 2015; van Honk et al., 2010; however see: Dekkers et al., 2019). This hypothesis stems from the fact that both the hypothalamus-pituitary-gonadal axis (HPG) and the hypothalamus-pituitary-adrenal (HPA) are activated in response to challenging situations (Dickerson and Kemeny, 2004; Turan et al., 2015; Wingfield and Sapolsky, 2003), and it is hypothesized that the HPG and HPA, once activated, interact in an antagonistic, inhibitory way (Viau, 2002) to jointly influence behavior. Although not the primary focus of the present investigation, consistent with the Dual Hormone hypothesis, our supplementary analyses from Study 2 (footnote 7) showed that acute psychosocial stress negated the effects of testosterone on empathic accuracy, suggesting important boundary conditions for this effect. Finally, the current findings should be considered alongside their limitations. First, as noted elsewhere, using digit ratio as an indirect measure of fetal androgen exposure remains contentious (e.g., Berenbaum et al., 2009; Hönekopp and Watson, 2010; Manning and Fink, 2017; Richards, 2017a, 2017b); thus, the reported digit ratio findings must be seen in this context. Second, although our inter-rater reliability for the 2D:4D ratio was high, we did not test intra-rater reliability for the 2D:4D ratios. Finally, although gender/sex did not moderate the effects of testosterone (or digit ratio) on empathic accuracy, we may have been underpowered to detect such an effect. Future research is needed to more systematically assess moderation by gender/ sex. In sum, we show that lower 2D:4D ratios and higher endogenous levels of the sex hormone testosterone are associated with poorer empathic accuracy, in this case dynamically tracking another's emotional state. These findings support the notion that key social-cognitive competencies related to empathy are not only rooted in our psychological make-up and experience, but also in our biology.

assessments and, in this way, distort any associations between 2D:4D and empathy. One novel contribution of this research is that we observed similar effects for digit ratio and circulating testosterone. Given that digit ratio and circulating testosterone levels are typically unrelated (for a review: Hönekopp et al., 2007), our findings suggest two pathways by which testosterone could impair empathy. Acknowledging the controversy regarding digit ratio, this index is thought to capture organizational effects of testosterone exposure during gestation (indeed, evidence suggests that digit ratio is not related to testosterone levels during later development; Muller et al., 2011). By contrast, circulating testosterone levels are activational, and reflect current functioning. Our 2D:4D findings suggest that high prenatal androgen exposure may impair children's abilities to learn empathic skills (cf. Chapman et al., 2006) related to identifying and tracking others emotions and/or mental states. Our testosterone findings, however, suggest that variation in circulating testosterone could disrupt empathy even if the skill exists. It is also possible that prenatal androgen exposure and circulating testosterone could have interactive effects. For example, some have argued that the organizational effects of prenatal androgen exposure may moderate the sensitivity of circulating testosterone levels (Berenbaum et al., 2009; Knickmeyer and Baron-Cohen, 2006). In this way, circulating testosterone levels could be disruptive for abilities that are relatively intact, especially if the circuitry is vulnerable, or sensitive to, the effects of testosterone, as might be the case with prenatal androgen exposure. Supporting this idea, van Honk et al. (2011) showed that women with lower (vs. higher) digit ratios were more susceptible to the detrimental effects of testosterone administration on RMET performance. Similarly, Carré et al. (2015) showed that only men with a combination of high psychopathic traits and lower digit ratios showed impaired RMET performance following testosterone administration. Notably, digit ratio has also been shown to moderate the effects of testosterone administration on other social behaviors like cooperation (Montoya et al., 2013; van Honk et al., 2012). That said, as noted, evidence that digit ratio is an indicator of prenatal androgen exposure is weak; there are likely many other factors that influence digit ratio and future work is needed to probe whether these factors account for the effects of digit ratio on empathic accuracy that we observed. It is well-established that various situational factors can influence circulating testosterone levels, including competitions for status and/or dominance (Archer, 2006; Mazur and Booth, 1998; Wingfield et al., 1990). Our observation that circulating testosterone was associated with empathic abilities raises the question of whether even very acute, situationally induced changes in testosterone could undermine empathy in specific situations. Indeed, this is consistent with work showing that acute testosterone administration can impair empathy (Bos et al., 2010; 6

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Funding

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This study was funded by an Natural Sciences and Engineering Research Council (NSERC) Discovery grant awarded to JAB (#RGPIN04241). JPN holds a doctoral scholarship from Fonds de Recherche du Québec-Société et Culture (FRQSC). The authors have no financial or other conflicts of interest to declare. Author contributions JPN and JAB developed the study concept and study design. JPN conducted the data analyses, the ELISA analyses, and drafted the manuscript. JAB provided critical revisions. All authors approved the final version of the manuscript for submission. We thank all the students that assissted with the data collection, and especially Emma Galarneau and Ajar Diushekeeva for their considerable work on this project. We would also like to thank Dr. Jeff Mogil and Dr. Sarah Rosen for assisting us with the ELISA assays. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yhbeh.2019.104648. References Andersson, C.R., Bergquist, J., Theodorsson, E., Ström, J.O., 2017. Comparisons between commercial salivary testosterone enzyme-linked immunosorbent assay kits. Scand. J. Clin. Lab. Invest. 77, 582–586. Archer, J., 2006. Testosterone and human aggression: an evaluation of the challenge hypothesis. Neurosci. Biobehav. Rev. 30, 319–345. Ashmore, R.D., 1990. Sex, gender, and the individual 486–526. Ashmore, R.D., Del Boca, F.K., Wohlers, A.J., 1986. Gender stereotypes, in: The Social Psychology of Female–Male Relations. Elsevier, pp. 69–119. Auger, J., Le Denmat, D., Berges, R., Doridot, L., Salmon, B., Canivenc-Lavier, M.C., Eustache, F., 2013. Environmental levels of oestrogenic and antiandrogenic compounds feminize digit ratios in male rats and their unexposed male progeny. Proc. Biol. Sci. 280, 20131532. Auyeung, B., Lombardo, M.V., Baron-Cohen, S., 2013. Prenatal and postnatal hormone effects on the human brain and cognition. Pflugers Arch. 465, 557–571. Baez, S., Flichtentrei, D., Prats, M., Mastandueno, R., Garcia, A.M., Cetkovich, M., Ibáñez, A., 2017. Men, women… who cares? A population-based study on sex differences and gender roles in empathy and moral cognition. PLoS One 12, e0179336. Baguley, T., 2012. Serious Stats: a guide to advanced statistics for the behavioral sciences. Macmillan International Higher Education. Bale, T.L., 2015. Epigenetic and transgenerational reprogramming of brain development. Nat. Rev. Neurosci. 16, 332–344. Barona, M., Kothari, R., Skuse, D., Micali, N., 2015. Social communication and emotion difficulties and second to fourth digit ratio in a large community-based sample. Mol. Autism 6, 68. Baron-Cohen, S., Wheelwright, S., Hill, J., Raste, Y., Plumb, I., 2001. The “Reading the Mind in the Eyes” test revised version: a study with normal adults, and adults with asperger syndrome or high-functioning autism. J. Child Psychol. Psychiatry 42, 241–251. Baron-Cohen, S., Richler, J., Bisarya, D., Gurunathan, N., Wheelwright, S., 2003. The systemizing quotient: an investigation of adults with Asperger syndrome or highfunctioning autism, and normal sex differences. Philos. Trans. R. Soc. Lond. B Biol. Sci. 358, 361–374. Baron-Cohen, S., Knickmeyer, R.C., Belmonte, M.K., 2005. Sex differences in the brain: implications for explaining autism. Science 310, 819–823. Baron-Cohen, S., Auyeung, B., Nørgaard-Pedersen, B., Hougaard, D.M., Abdallah, M.W., Melgaard, L., Cohen, A.S., Chakrabarti, B., Ruta, L., Lombardo, M.V., 2015. Elevated fetal steroidogenic activity in autism. Mol. Psychiatry 20, 369–376. Bartko, J.J., 1966. The intraclass correlation coefficient as a measure of reliability. Psychol. Rep. 19, 3–11. Bartz, J.A., Zaki, J., Bolger, N., Ochsner, K.N., 2011. Social effects of oxytocin in humans: context and person matter. Trends Cogn. Sci. 15, 301–309. Bates, D., Mächler, M., Bolker, B., Walker, S., 2015. Fitting linear mixed-effects models using lme4. Journal of Statistical Software, Articles 67, 1–48. Berenbaum, S.A., Bryk, K.K., Nowak, N., Quigley, C.A., Moffat, S., 2009. Fingers as a marker of prenatal androgen exposure. Endocrinology 150, 5119–5124. Bos, P.A., Terburg, D., van Honk, J., 2010. Testosterone decreases trust in socially naive humans. Proc. Natl. Acad. Sci. U. S. A. 107, 9991–9995. Bos, P.A., Panksepp, J., Bluthé, R.-M., van Honk, J., 2012. Acute effects of steroid hormones and neuropeptides on human social-emotional behavior: a review of single administration studies. Front. Neuroendocrinol. 33, 17–35. Carré, J.M., Archer, J., 2018. Testosterone and human behavior: the role of individual and contextual variables. Curr Opin Psychol 19, 149–153.

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