Experimental empathy induction promotes oxytocin increases and testosterone decreases

Hormones and Behavior 117 (2020) 104607

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Experimental empathy induction promotes oxytocin increases and testosterone decreases

T

Tanya L. Procyshyna,b, Neil V. Watsonc, Bernard J. Crespia,

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a

Department of Biological Sciences, 8888 University Drive, Simon Fraser University, Burnaby V5A 1S6, Canada Autism Research Centre, Department of Psychiatry, University of Cambridge, Cambridge CB2 8AH, UK c Department of Psychology, 8888 University Drive, Simon Fraser University, Burnaby V5A 1S6, Canada b

ARTICLE INFO

ABSTRACT

Keywords: Oxytocin Testosterone Empathy Systemizing

Oxytocin and testosterone coordinate adaptive social behaviors with stimuli in the environment. Administration of oxytocin and testosterone is associated with increased and reduced indicators of empathy, respectively, but how levels of these hormones are jointly affected by naturalistic empathy-inducing stimuli remains unclear. In this study, salivary oxytocin and testosterone levels were measured in 173 healthy adults before and after watching a video involving a gravely ill child. Participants also completed questionnaires to assess psychological variables predicted to affect oxytocin reactivity (Autism-Spectrum Quotient, Interpersonal Reactivity Index, Empathy and Systemizing Quotients). On average, there was a 14% increase in oxytocin (p = 0.003) and 4% decrease in testosterone (p = 0.001) pre- to post-video. Opposite directional changes in hormone levels occurred together, as supported by a chi-square test (p < 0.001) and a circular statistics test (p < 0.05). Considered separately, psychological traits did not predict hormone levels or changes to any appreciable degree. However, oxytocin and testosterone changes were linked with empathy relative to systemizing such that: (1) ‘Empathy Bias’ was associated with a large oxytocin increase but little change in testosterone, while (2) ‘Systemizing Bias’ and ‘Balance’ between empathy and systemizing were associated with a decrease in testosterone but little change in oxytocin. These findings suggest that participants were divisible into ‘high oxytocin responders’ (relatively empathetic) and ‘high testosterone responders’ (balanced or systemizing-biased). These findings support a model of joint, opposite changes in oxytocin and testosterone under experimental empathy induction, with high, somewhat predictable, diversity in individual responses.

1. Introduction Hormones are evolutionarily ancient regulators of social behavior, playing central roles in parental care, affiliation, anxiety, and aggression across vertebrate species (Goodson, 2005). Alterations to hormone levels or hormone receptors in the central nervous system can exert powerful effects on social behavior (Donaldson and Young, 2008), and stimuli from the environment—including social encounters—can in turn influence hormone levels (Bosch and Neumann, 2012; Eisenegger et al., 2011). Through such bidirectional interactions, hormone systems serve as mechanisms for coordinating adaptive social cognition and behavior with the changing environments that social species inhabit. Research into the extent to which hormonal factors influence human social behavior has focused mainly on two hormones with apparently opposite roles: the neuropeptide oxytocin and the sex steroid testosterone (Table 2 in Crespi, 2016). According to a social-

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evolutionary framework, these opposite effects can be explained by oxytocin promoting activation of neural regions that subserve mentalizing—described as “engaging in social cognition and making sense of each other, and ourselves, in terms of subjective empathic and cognitive states”, while testosterone favors “self-oriented, ego-centric, and nonsocial attention and information processing” (Crespi, 2016). In line with this framework, endogenous oxytocin levels are positively associated with empathy, attachment, and sensitive parenting (Daughters et al., 2017; Feldman et al., 2012; Gordon et al., 2008), while testosterone levels are positively associated with intrasexual competition and aggression (Zilioli and Bird, 2017) but negatively associated with parenting in both sexes (Grebe et al., 2019; Kuzawa et al., 2010). Variation in oxytocin and testosterone levels has also been reported in neurodevelopmental conditions. Some studies report elevated testosterone and reduced oxytocin being linked with autism spectrum conditions (Auyeung et al., 2009; Bakker-Huvenaars et al., 2018; Xu

Corresponding author. E-mail address: [email protected] (B.J. Crespi).

https://doi.org/10.1016/j.yhbeh.2019.104607 Received 17 May 2019; Received in revised form 27 September 2019; Accepted 28 September 2019 0018-506X/ © 2019 Elsevier Inc. All rights reserved.

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et al., 2013), but elevated oxytocin and reduced testosterone among individuals subject to psychotic-affective conditions, including schizophrenia (see Crespi, 2016). Dysregulated neurohormonal systems may thus be related to the alterations in social behavior that characterize some psychiatric conditions. Considered together, such findings from experimental, correlational, and clinical research support a broadly social role for oxytocin and an asocial or antisocial role for testosterone. Most studies to date have considered and analyzed oxytocin and testosterone separately, such that there have been few direct tests of a framework for joint oxytocin-testosterone effects. The primary goal of the current study was thus to test for joint, opposite changes in oxytocin and testosterone in response to a naturalistic, empathy-inducing stimulus. To do so, we quantified changes in salivary oxytocin and testosterone in typical young adults who watched a validated experimental video of a father talking about his gravely ill child (Barraza and Zak, 2009) to induce feelings of empathy. Participants reported their subjective emotional responses to the video and completed a set of questionnaires to assess variation in relevant psychological traits: the Autism-Spectrum Quotient (AQ), Interpersonal Reactivity Index (IRI), Empathy Quotient (EQ), and Systemizing Quotient (SQ), as described in more detail below. These data were used to determine if and how psychological factors influenced basal hormone levels or hormonal responses to the video. Several specific predictions follow below, from theory and previous relevant studies. First, the experimental video was predicted to mediate an increase in oxytocin and a decrease in testosterone relative to baseline. Moreover, the oxytocin increase and testosterone decrease were expected to occur together, as predicted from studies suggesting prosocial and antisocial/asocial roles for oxytocin and testosterone, respectively. This prediction is predicated on previous studies showing inverse and opposite associations of oxytocin and testosterone with one another across diverse contexts (reviewed in Crespi, 2016; see also Dai et al., 2017; Khan et al., 2018; Nicholson and Jenkin, 1995; Dzirbíková et al., 2018), including parenting (reviews in Grebe et al., 2019; BakermansKranenburg and van IJzendoorn, 2018), the context of the experimental video used in the present study. Second, basal oxytocin and oxytocin increase were predicted to correlate positively with EQ and IRI scores (and negatively with SQ and AQ scores), while basal testosterone and any testosterone increases (if observed) were predicted to correlate positively with SQ and AQ scores (and negatively with EQ and IRI scores). Moreover, the presence and magnitude of testosterone decreases due to the emotional video might be expected, all else being equal, to be associated with higher EQ and IRI scores and lower SQ and AQ scores. Hormone levels were also, specifically, predicted to be associated with Baron-Cohen's (2002) metric of empathizing (the drive to share and understand the feelings of other people) relative to systemizing (the drive to construct or analyze rule-based inanimate systems). Empathizing and systemizing are quantified using the self-report Empathy Quotient and Systemizing Quotient questionnaires (Baron-Cohen et al., 2003; Baron-Cohen and Wheelwright, 2004; Wakabayashi et al., 2006), and their relative magnitudes provide a metric of autism-associated cognition and affect (Baron-Cohen, 2002). In this framework, individuals can be classified as having an ‘Empathizing Bias’ (high EQ relative to SQ), a ‘Balance’ between the two, or a ‘Systemizing Bias’ (high SQ relative to EQ), where the biases are quantified using standardization to avoid the statistical problems of ratios (Goldenfeld et al., 2005). This EQ- and SQ-based classification scheme has been used to predict autism spectrum traits (as involving high systemizing relative to empathizing) and associated psychological and hormonal phenotypes (e.g., Auyeung et al., 2006; Greenberg et al., 2018; Kidron et al., 2018). In our study, we thus predict that an empathizing bias should be associated with a greater increase in oxytocin and/or a greater decrease in testosterone in response to the video, and a systemizing bias may be associated with a smaller increase in oxytocin and/or a smaller decrease in testosterone. This second set of predictions is predicated on numerous previous

findings, including: (1) positive associations of oxytocin with measures of empathy, including EQ (Bartz et al., 2010; De Dreu and Kret, 2016; Feeser et al., 2015); (2) negative associations of testosterone with measures of empathy (e. g., Hermans et al., 2006; Chapman et al., 2006; van Honk et al., 2011), and a positive (for prenatal testosterone) association with systemizing (Auyeung et al., 2006); and (3) associations of low oxytocin and high testosterone with autism and autism-associated traits (reviews in Baron-Cohen et al., 2011; Auyeung et al., 2013; Crespi, 2016). 2. Materials and methods 2.1. Experimental design After giving informed consent, participants provided a saliva sample for baseline hormone measurement and completed a demographic questionnaire. Next, they watched a short video about a young child with terminal cancer narrated by the child's father. This video has been shown to increase oxytocin levels measured in blood (Barraza and Zak, 2009). After watching the video, participants reported their empathyrelated and distress-related responses to the video on the Emotion Ratings questionnaire described below, and provided a second saliva sample. Collection of a third saliva sample began 20 min after the video. Between saliva samples, participants completed the psychological questionnaires. This experiment was approved by the Simon Fraser University Office of Research Ethics (study number 2015s0228). 2.2. Participants In total, 173 participants (94 women) were recruited from a Canadian university, receiving either course credit or $10 for participation. Participants were screened for medical conditions, use of drugs and hormones with endocrine effects (hormonal contraceptive use in women was allowed), and avoided food and drink for a minimum of 1 h before participation. The mean and standard deviation (SD) participant age was 20.4 ± 2.5 years. Participants self-reported diverse ethnic backgrounds: 41% were East Asian, 27% Caucasian, 22% South Asian, and 10% other or multiple ethnicities. No participants reported having children or being pregnant. In women, hormone levels were not significantly associated with stage of menstrual cycle or use of hormonal contraception (Supplementary Table 1). 2.3. Salivary collection and hormone analysis Collection of saliva samples took place between 1:00 pm and 5:00 pm to minimize effects of diurnal variation. Saliva was collected by passive drool into pre-chilled polypropylene 15 ml tubes and kept on ice until a sufficient volume (> 2 ml) was collected, then immediately frozen at −20 °C. Three saliva samples were collected per participant: (A) a baseline sample, (B) an immediately post-video sample, and (C) a 20-minute post-video sample. Due to the volume of saliva required, participants took upwards of 10 min to provide each sample. Oxytocin was measured at all three time points, and testosterone was measured only at timepoints A and C. The timings of the saliva samples were selected based on our interest in quantifying multiple hormones. In previous studies, a time period of 15–20 min between stimulus and sampling has been sufficient to observe an endogenous change in salivary testosterone levels (e.g., Roney et al., 2007; van Anders et al., 2012) as well as salivary oxytocin levels (e.g. de Jong et al., 2015; Brondino et al., 2017; Schladt et al., 2017). Oxytocin was also quantified at time point B (immediately post-video) as this was the sampling time used by Barraza and Zak (2009) in the original study that used the emotional experimental video. Percent change in hormone levels in the present study is reported as the change from baseline to 20 min postvideo (timepoint A–C), unless otherwise stated. Prior to assay, samples were thawed at 4 °C and centrifuged at 4 °C 2

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at 1600 ×g for 15 min. All samples were run in duplicate and samples from the same individual were always analyzed together on the same plate. Oxytocin was assayed on the first freeze-thaw cycle using Enzo Life Sciences enzyme-linked immunosorbent assay kit ADI-901-153A. Consistent with protocols described in the literature (e.g., Daughters et al., 2015; Nishizato et al., 2017), samples were concentrated by lyophilization, reconstituted, incubated overnight as per kit instructions, and analyzed on a Biotek plate reader at 405 nm. Oxytocin concentrations were then calculated from standard curves. The intra- and inter-assay coefficients of variability were < 10% and < 18%, respectively, for n = 17 plates; these coefficients fall within the tolerance ranges of 12.6–13.3% and 11.9–20.9%, respectively, reported in the kit instructions. The validity of oxytocin measured in extracted versus unextracted body fluid, as well as the relationship between peripheral oxytocin and central oxytocin, has been questioned (McCullough et al., 2013). However, the ELISA kit used in this study has undergone rigorous testing since the publication of such critiques and is highly specific to oxytocin (i.e., it does not detect vasopressin). Furthermore, any methodological issues should affect samples in a similar manner and are not expected to bias the results in any particular way. Salivary testosterone was quantified using Salimetrics enzymelinked immunosorbent assay kit 1-2312-5. This assay kit is designed specifically for use with saliva and no concentration step is necessary. After centrifugation, 20 μl of saliva supernatant was incubated as per kit instructions and read at 450 nm. Testosterone concentrations were calculated from standard curves. The intra-assay coefficient of variability was < 5% and the inter-assay coefficients of variability were < 10% and < 15% for the high and low controls, respectively, for n = 10 plates.

assessed by repeated measures analysis of variance (ANOVA) in SPSS, with time point as a within-subjects variable and sex as a betweensubjects variable. Circular statistics tests were performed using the ‘circular’ package (version 0.4-7) for R. Associations between hormonal and psychological variables were assessed using Pearson product-moment correlations controlled for sex, where appropriate, and with adjustments for multiple comparisons as described. These associations were also evaluated in a multivariate context using best subsets regression, an exploratory model building method that, in this case, considers the ability of all subsets of the independent variables (gender and questionnaire scores) to predict changes in oxytocin or testosterone, as optimized by adjusted R-squared using the ‘leaps’ package in R (version 3.0; Lumley, 2009). Individuals with missing or invalid data were excluded from analyses involving that particular variable, but retained for other analyses; thus, sample sizes differ among tests to a small degree. Two individuals were unable to provide sufficient saliva for the quantification of both hormones. Due to the lower volume of saliva required for the testosterone assay, samples from these individuals were analyzed for testosterone only. One individual was excluded from all analyses due to atypical results on genetic analyses and extremely high testosterone levels (> 3 SD above sex-specific mean). 3. Results 3.1. Questionnaire scores As found in previous work (Baron-Cohen, 2010; Davis, 1983; Rong et al., 2010), women scored higher than men on the IRI and its subscales and, marginally non-significantly, on the EQ, while men scores higher on the AQ and SQ (Table 1). Women reported a greater empathyrelated response to the video, while there was no sex difference in distress-related response. The empathy- and distress-related response questionnaires showed good internal consistency, with Cronbach's alpha values of 0.840 and 0.763, respectively.

2.4. Psychological questionnaires Participants completed several questionnaires to assess variation in socio-cognitive traits postulated to be relevant to oxytocin and testosterone levels. The Autism-Spectrum Quotient (AQ) (Baron-Cohen et al., 2001) quantifies autistic-like traits in individuals of normal intelligence. The Interpersonal Reactivity Index (IRI) (Davis, 1980) assesses four distinct components of empathy: empathic concern, perspective taking, personal distress, and fantasy scale. The short-form Empathy Quotient (EQ) and Systemizing Quotient (SQ) assess social (empathizing) and non-social (systemizing) aspects of cognition (Wakabayashi et al., 2006); ‘Empathy Bias’ or ‘Systemizing Bias’ was then calculated by subtracting the standardized EQ score from the standardized SQ score. Due to significant sex differences in EQ and SQ scores, an individual's category by this metric was determined here by using the mean and SD for their respective sex; thus, the proportion of individuals in each of the three categories within each sex in this study is comparable. Three categories are used: ‘Systemizing-Biased’ (difference scores ≥ 1), ‘Balanced’ (difference scores −1 to 1), and ‘EmpathyBiased’ (difference scores ≤ 1). Participants also completed an Emotion Response questionnaire created for this study (Supplementary material), modeled on that used by Barraza and Zak (2009), after watching the video. Participants were instructed to indicate, on a scale from 1 to 5, how strongly they experienced 12 emotions. “Empathy Response” was determined by summing the ratings of six emotions (sympathetic, warm, compassionate, tender, soft-hearted, and moved), and “Distress Response” was determined by summing the ratings of the other six emotions (anxious, annoyed, sad, distressed, frightened, and disturbed). For all questionnaires employed here, higher scores indicate higher endorsement of the emotion or phenotype.

3.2. Changes in salivary oxytocin and testosterone Salivary oxytocin levels varied significantly across the three time points for the overall population (repeated measures ANOVA, F2,166 = 8.9, p = 0.003, partial eta squared = 0.051; Table 2). Oxytocin levels did not differ significantly between men and women (F = 1.4, p = 0.24, partial eta squared = 0.008), and there was no Table 1 Comparison of questionnaire scores between women and men participants. Questionnaire

Women Mean ± SD N = 94

Men Mean ± SD N = 78

Difference (p value)

Autism-Spectrum Quotient (AQ) Interpersonal Reactivity Index (IRI) Empathic concern Fantasy scale Personal distress Perspective taking Empathy Quotient (EQ)

17.3 ± 5.4 N = 89 72.9 ± 13.4 N = 91 21.0 ± 4.9 19.3 ± 5.2 13.7 ± 4.6 19.1 ± 4.5 25.6 ± 7.5 N = 92 14.7 ± 7.9 N = 91 23.4 ± 4.1 N = 93 9.4 ± 3.8 N = 94

19.6 ± 5.8 N = 76 65.6 ± 10.6 N = 74 17.9 ± 4.9 17.7 ± 5.1 12.4 ± 4.3 17.6 ± 4.2 23.45 ± 6.6 N = 74 23.0 ± 8.9 N = 77 20.1 ± 5.1 N = 78 8.7 ± 3.5 N = 78

0.008**

Systemizing Quotient SQ Empathy Response

2.5. Statistical analyses

Distress Response

Statistical tests were performed in SPSS (version 24) or R (version 3.5.1). Pre to post-video changes in salivary hormone levels were

* p < 0.05 ** p < 0.01 3

< 0.001** < 0.001** 0.04* 0.06 0.03* 0.05 < 0.001** < 0.001** 0.20

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Table 2 Salivary hormone levels across time points.

Both sexesa Women Men Women Men

Oxytocin Mean (SD) Testosterone Mean (SD)

N

A Baseline (pg/ml) Mean ± SD N

B Immediately post-video (pg/ml) Mean ± SD

C 20-min post-video (pg/ml) Mean ± SD

170 94 76 94 78

106.5 ± 71.0 98.6 ± 62.3 116.2 ± 79.7 82.5 ± 28.2 159.6 ± 47.8

118.0 ± 93.2 116.2 ± 92.6 120.2 ± 94.5 n.a. n.a.

117.0 ± 86.6 108.1 ± 70.0 127.9 ± 102.8 76.7 ± 26.0 154.2 ± 48.6

a Repeated measures ANOVA indicated no significant gender difference in oxytocin levels, consistent with previous studies of salivary oxytocin (e.g., Nishizato et al., 2017; Koven and Max, 2014). Welch's t-tests (not shown) were performed to confirm that the difference in oxytocin levels between women and men was not significant at any of the three time points.

Table 3 Paired comparisons of hormone levels across time points.

Oxytocin Testosterone

Time

Mean difference (pg)

Std. error

p-Value

95% confidence interval of difference

A–B A–C B–C A–C

−10.8 −11.3 −0.53 5.6

4.1 3.8 4.7 1.7

0.009* (0.027 with Bonferroni correction) 0.003* (0.01 with Bonferroni correction) 0.91 (1.0 with Bonferroni correction) 0.001**

(−18.8, −2.7) (−18.8, −3.8) (−9.8, 8.7) (2.3, 8.9)

Time points: A = baseline; B = immediately post-video; C = 20 min post-video. * p < 0.05 ** p < 0.01

significant Time × Gender interaction (F = 0.15, p = 0.70, partial eta squared = 0.001). Post-hoc paired comparisons (Table 3) revealed that oxytocin increased from baseline to the immediately post-video sample and 20-minute post video sample (average percent change of +14.9% and +13.9% from baseline, respectively), but did not differ between the two post-video samples. Testosterone levels decreased significantly from the pre-video sample to the 20-minute post-video saliva sample (repeated measures ANOVA, F1,169 = 10.9, p = 0.001, partial eta squared = 0.060) for the overall population. As expected, men had higher testosterone levels than women (F = 189.9, p < 0.001, partial eta squared = 0.053), but there was not a significant interaction between Time × Gender (F = 0.11, p = 0.92, partial eta squared = 0.00). Relative to baseline, the average testosterone change for the overall population was −4%.

and the red arrow indicating the mean direction of joint hormone change. The quadrants of the circle in Fig. 2 correspond with the quadrants in Fig. 1 for the chi-square test. For the overall population, Rao's Spacing Test rejects the null hypothesis of uniformity (test statistic = 147.6, p < 0.05), supporting the presence of directionality in the dataset. The mean direction is 2.03 rad, which falls in quadrant II, i.e., an oxytocin increase and testosterone decrease. In the separate tests by gender, Rao's Spacing Test rejects the null hypothesis of uniformity for women (test statistic = 159.7, p < 0.001) but not men (test statistic = 144.1, p = 0.14). Nevertheless, men and women show a similar mean directions of change falling in quadrant II (1.93 rad for women, 2.21 rad for men), indicating increased oxytocin paired with decreased testosterone. The gender differences in hormone responses thus appear to be smaller than the individual differences.

3.3. Relationship between oxytocin change and testosterone change

3.4. Relationships among hormonal and psychological variables

Depending on the direction of hormone change (increase or decrease), each participant was assigned to one of four categories, as shown in Fig. 1: (I) oxytocin increase, testosterone increase; (II) oxytocin increase, testosterone decrease; (III) oxytocin decrease, testosterone decrease; (IV) oxytocin decrease, testosterone increase. The results of a χ2 test for the overall population (χ2 = 26.7, p < 0.001) indicate that the observations are not equally distributed between the four quadrants, with the largest proportion (39%) of individuals in quadrant II and the smallest proportion of individuals (11%) in the opposite quadrant, IV. As an additional test of joint hormonal change, circular statistical tests were employed. By this test, percentage changes in oxytocin and testosterone are represented as two sides of a triangle, which allows the angle of change from the origin (0,0) to be calculated trigonometrically. Rao's Spacing Test of Uniformity is then used to test for the presence or absence of underlying directionality. This test is based on the idea that, in the absence of underlying directionality, observations should be evenly spaced around the circle; clustered observations or unusually large spaces between observations thus constitute evidence for directionality (Levitin and Russell, 2014). Fig. 2 presents the circular data for the overall population and separately by gender, with each black arrow representing one individual

Correlations between questionnaire scores and hormonal variables are presented in Table 4; additional correlations, including separate analyses for men and women and correlations between questionnaires, are presented in Supplementary Tables S2–S4. Empathy Response to the video was not related to any hormonal variable, although it was positively correlated (nominally) with IRI and EQ (r = 0.47 and 0.24, respectively, p < 0.001 and < 0.01), and negatively correlated (nominally) with AQ and SQ (r = −0.17 and − 0.19, respectively, p < 0.05 both tests) in the overall population. Distress Response was positively correlated with IRI (r = 0.16, p < 0.05) and negatively correlated, nominally, with testosterone change (r = −0.16, p < 0.05), suggesting that individuals who found the video more emotionally distressing showed larger decreases in salivary testosterone. Oxytocin change was not related to total IRI, EQ, or AQ scores; however, there was a nominal, positive correlation between oxytocin change and IRI-Perspective Taking (r = 0.18, p < 0.05). Baseline oxytocin and testosterone levels were not associated with psychological variables in the overall population. None of the correlations of psychological with endocrine variables survived statistical adjustment for multiple comparisons (Table 4). Best subsets regression identified a model comprising SQ, IRIPersonal Distress, IRI-Perspective Taking, and Distress response as the 4

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Fig. 1. Scatterplot of pre- to post-empathy induction percent change in oxytocin and testosterone for all participants (N = 167). Each dot indicates one individual; red dots indicate women, blue dots indicate men. Quadrant I indicates increased oxytocin, increased testosterone; Quadrant II indicates increased oxytocin, decreased testosterone; Quadrant III indicates decreased oxytocin, decreased testosterone; and Quadrant 4 indicates deceased oxytocin, increased testosterone. Oxytocin change is the average of the percent change from baseline to time points B and C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

F2,158 = 0.073, p = 0.93, partial eta squared = 0.01; testosterone F2,159 = 2.3, p = 0.10, partial eta squared = 0.028). By contrast, individuals in the three categories differed in percent change oxytocin (F2,158 = 3.87, p = 0.023, partial eta squared = 0.047), with three- to fourfold higher increases for Empathy-Biased individuals (mean change 30.9% versus 8.8% for Balanced and 6.9% for Systemizing-Biased) (Fig. 3; see Supplementary Figs. S1 and S2 for results separated by sex). By Tukey's HSD post-hoc test, the mean oxytocin change for the Empathy-Biased individuals differed significantly (p = 0.025) from that for the Balanced individuals and nearly significantly (p = 0.059) for the Systemizing-Biased individuals. Individuals in the three empathizing/systemizing categories also differed in percent change testosterone (F2,159 = 4.80, p = 0.0094, partial eta squared = 0.057), with mean changes of +3.6% for Empathy-Biased individuals, −5.3% for Balanced individuals, and − 7.9% for Systemizing-Biased individuals (Fig. 4; see Supplementary Figs. S1 and S2 for results separated by gender). By Tukey's HSD posthoc test, the mean testosterone change for Empathy-Biased individuals differed significantly from that for Balanced (p = 0.021) and Systemizing-Biased (p = 0.014) individuals.

best predictor of oxytocin response (% change), which yielded an overall adjusted R-squared of 0.051 (p = 0.016), with significant coefficients for SQ (t = −2.02, p = 0.029) and IRI-Perspective Taking (t = 2.12, p = 0.036) (Supplementary Fig. S3). For testosterone response (% change), the best model comprised gender, EQ, SQ, and Distress response, which was marginally non-significant (p = 0.059) with an adjusted R-squared of 0.056; none of the predictor variables was individually significant at 0.05 or below (Supplementary Fig. S4). These findings suggest that higher SQ scores were associated with lower increases in oxytocin, while higher IRI-Perspective Taking scores were associated with greater oxytocin increases. 3.5. Oxytocin and testosterone measures in relation to empathizing and systemizing Participants were categorized as ‘Empathy-Biased’, ‘Balanced’, or ‘Systemizing-Biased’ based on their EQ scores relative to SQ scores, as described above. ANOVAs comparing baseline oxytocin and testosterone levels showed no differences between the three categories (oxytocin

Fig. 2. Circular plots of angles derived from joint percent change in salivary oxytocin and testosterone. Plot a) is for the overall population, b) is women only, and c) is men only. Each black arrow indicates the joint change in oxytocin and testosterone for one individual. The red arrow indicates the mean change. Quadrant I indicates increased oxytocin, increased testosterone; Quadrant II indicates increased oxytocin, decreased testosterone; Quadrant III indicates decreased oxytocin, decreased testosterone; and Quadrant 4 indicates deceased oxytocin, increased testosterone. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

5

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decrease empathy, respectively (reviewed in Bos et al., 2012; Crespi, 2016). As the subject of the video was a child, who may have been perceived as especially vulnerable due to illness, the results can additionally be interpreted as consistent with the literature on hormones and parental care, which indicates positive associations between parenting and oxytocin and negative associations between parenting and testosterone (Feldman and Bakermans-Kranenburg, 2017; Holtfrerich et al., 2018). The similarities in the roles of oxytocin and testosterone across administration studies, compared to our naturalistic experimental study, highlight the interactive relationship between hormones and sociality, where hormone levels may both cause changes in social behavior and change in response to social stimuli. We also demonstrated that increases in oxytocin and decreases in testosterone tended to occur together, and we used circular statistical techniques in a novel way to demonstrate the presence of underlying directionality in our dataset on simultaneous changes in two hormones. This finding of a joint change supports the social-evolutionary framework for the roles of these two hormones (Crespi, 2016), which states that oxytocin promotes social cognition, testosterone promotes antisocial or asocial cognition, and increases in both hormones should thus usually be incompatible. Despite the overall pattern of oxytocin increase and testosterone decrease found here, only 39% of the participants showed paired hormone changes that were in the predicted directions (Figs. 1 and 2). How can this variation be explained? Our analyses show that it cannot be explained by scores on any of the questionnaires considered individually to any appreciable degree, since all relevant correlation coefficients were small, as well as being non-significant after statistical adjustment. Indeed, the only moderately strong association of hormonal change with psychological traits was the positive correlation of IRI Perspective-Taking with oxytocin increase (found in both the bivariate and best subsets regression analyses). This finding is consistent with selective increases in the IRI-Perspective Taking subscale after oxytocin administration to patients with schizophrenia reported in two previous studies (Gibson et al., 2014; Halverson et al., 2019), and with the effects of this hormone in facilitating externally directed social attention (Yao et al., 2018), it but provides little explanatory power or insight. In

Table 4 Correlations of hormonal variables with psychological variables for the overall population.

AQ IRI total IRI - Empathic concern IRI - Fantasy scale IRI - Personal distress IRI - Perspective taking EQ SQ Empathy Response Distress Response

Baseline Ta

Baseline OT

% change T

% change OTb (A–C)

−0.03 0.13 0.13

−0.06 −0.04 −0.11

−0.06 0.03 −0.02

−0.07 0.04 0.05

0.09 0.00 0.12

−0.03 −0.07 0.09

0.03 0.03 0.05

−0.08 0.03 −0.04 −0.04

0.03 0.00 −0.04 −0.12

0.10 −0.05 −0.08 −0.16⁎

0.00 −0.10 0.16⁎ 0.09 −0.06 −0.06 −0.12

Due to significant sex differences in testosterone levels, correlations between testosterone and psychological variables for the overall population were calculated in SPSS as partial correlations controlling for sex. b Correlations separated by gender are presented in Supplementary Tables S3 and S4. ⁎ p < 0.05, unadjusted for multiple comparisons; not significant after10fold Bonferroni adjustment. a

4. Discussion The current study investigated if and how endogenous levels of the hormones oxytocin and testosterone jointly changed in response to an experimental video intended to induce empathy, a socio-cognitive state or trait previously linked to these hormones. Additionally, we tested if hormonal variables (baseline hormone levels or pre- to post-video percentage changes in hormone levels) were associated with psychological variables, as predicted from theory and previous studies. On average, viewing the experimental video mediated a 15% increase in salivary oxytocin and a 4% decrease in salivary testosterone among participants. The directions of these observed changes were consistent with predictions from the hormone administration literature, namely that oxytocin and testosterone are expected to increase and

Fig. 3. Changes in oxytocin in relation to Empathy and Systemizing scores. Each red dot represents one individual. The black dots indicate the means and the whiskers indicate the standard deviations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 6

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Fig. 4. Changes in testosterone in relation to Empathy and Systemizing scores. Each blue dot represents one individual. The black dots indicate the means and the whiskers indicate the standard deviations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

administration) in the former. Strathearn et al. (2018) recently described a direct connection of oxytocin with systemizing, showing that oxytocin administration abolished preference for highly-systemized images among individuals with autism, which implicates low oxytocin in high systemizing as well as low empathy. In our study, AQ scores were, as expected, positively correlated with Empathizing relative to Systemizing (r = 0.26, p < 0.01); AQ did not, however, show an association with either hormone, suggesting that Empathizing relative to Systemizing is more directly related to hormonal systems than is AQ itself. Most generally, these results suggest that a notable degree of the individual variation in naturalistic and experimental changes and responses to levels of oxytocin, and likely testosterone, is associated with individual variation in empathy relative to systemizing. This study is subject to several limitations. First, both hormones were measured in saliva. Although oxytocin measured in saliva was recently shown to correlate with central oxytocin better than oxytocin measured in plasma (Martin et al., 2018), salivary oxytocin measures are known to show high levels of variability between individuals, which may underlie the weak nature of the relationships between some of the hormonal and psychological variables in this study. MacLean et al. (2019) provide a comprehensive discussion of the diverse causes of variability in oxytocin quantification. The baseline oxytocin values here are comparable to those reported in other studies using lyophilized saliva samples (Daughters et al., 2015; Nishizato et al., 2017) with similarly high variability between individuals. The baseline testosterone values for men and women in our sample are comparable to the ranges reported by the kit manufacturer; the somewhat higher than expected testosterone concentration for women may reflect the relatively young age of our sample or seasonal variation (Keevil, 2017; Stanton et al., 2011). Al-Dujaili and Sharp (2012) provide important insights into complications salient to measuring testosterone in women. Due to the limitations of salivary hormone analysis, we make no attempt to compare our hormone values with other samples. Notably, the salivary hormone measures across timepoints are highly correlated within individuals (time A to time C, r = 0.92 for testosterone, r = 0.83 for oxytocin). Second, our data on individual responses to the video itself were

addition, the Distress Response to the video was negatively correlated with testosterone change in our study, meaning that individuals who rated the video as more distressing showed greater decreases in testosterone. Kuo et al. (2016) reported that fathers' testosterone levels decreased on average after watching infants in distress, although, as in our results, there was high variability in individual testosterone responses. Finally, in the best subsets regression analysis, higher SQ scores were associated with reduced OT increase (or a reduction in OT); this finding supports an inverse association of SQ with empathic response. In contrast to the bivariate and multiple regression tests, analyses using Baron-Cohen's (2002) empathizing/systemizing theory, which are based on Empathy Quotient relative to Systemizing Quotient scores, show significant associations with both oxytocin and testosterone changes (Fig. 3) such that average increases in oxytocin were much higher (30.9%) among Empathy-Biased individuals than among Balanced (8.8%) and Systemizing-Biased (6.9%) individuals. By contrast, average decreases in testosterone were found among individuals with Balanced (−5.3%) and Systemizing-Biased (−7.9%) individuals, but not among individuals with Empathy-Biased individuals (who actually showed a slight average testosterone increase of 3.6%). These results suggest that individuals exhibit differential oxytocin, compared to testosterone, reactivity as a function of their Empathizing/Systemizing balance, with Empathy-Biased individuals showing reactivity to the experimental stimulus mainly via oxytocin changes (‘high oxytocin responders’), while Systemizing-Biased and Balanced individuals reacted mainly via large testosterone decreases coupled with much smaller elevations in oxytocin (‘high testosterone responders’). This is an unexpected and intriguing result, which may help to explain the relatively low oxytocin system reactivity found among individuals with autism in some studies (reviewed in Crespi, 2016), given that autism is linked with high systemizing and low empathy (Baron-Cohen, 2010). Oxytocin has previously been associated with individual variation in EQ and empathizing, in that administration of this hormone increases measures of empathic performance for typical subjects with low EQ scores, but not for those with high EQ scores (Feeser et al., 2015; Hirosawa et al., 2012), suggesting higher oxytocin reactivity (to 7

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limited to the Emotion Ratings questionnaire, which showed only weak associations with hormone level changes. Third, the participants were providing questionnaire data after the empathy induction, so it was possible that their self-report replies were affected by hormone changes that it mediated. Although intranasal oxytocin administration can affect self-report measures of, for example, personality (Cardoso et al., 2012), whether such effects can occur under naturalistic induction is unknown. The low magnitude of the correlations (Table 4) of hormone levels with self-report measures suggests, in any case, that any such effects would be weak. An important strength of this study is its use of naturally-induced responses, for multiple hormones, with relatively large samples and multiple predictor variables. Naturally-induced responses avoid potential experimental artifacts due to hormone administration, which is commonly outside of natural contexts and can supersede physiological levels. Measurement of multiple hormones is likewise important, as most hormonal responses probably involve trajectory changes in multiple hormone systems, such as the steroids and peptides as shown here. Finally, simple endogenous responses to induction correspond more closely to what happens in nature than do most experiments; as such, they may give more realistic read-outs for analyzing the connections of psychological and behavioral traits with endocrine traits. Further research is needed to understand mechanisms that connect oxytocin with testosterone responses and effects in the central nervous system. A suite of studies in rodents and humans has documented direct molecular and physiological interactions between components of the oxytocin and testosterone systems that give rise to inverse, antagonistic effects (Dai et al., 2017; Khan et al., 2018; Nicholson and Jenkin, 1995; Dzirbíková et al., 2018); however, none of these studies focused on the human brain or social behavior. To the extent that these systems typically interact in ways that vary across individuals depending upon their psychological makeups, future studies of neuropeptide and steroid effects in human behavior could usefully consider a selected suite of hormones simultaneously and determine what psychological traits most directly mediate their effects. Based on our results, it would be especially interesting to determine if and how levels of oxytocin, vasopressin, testosterone, and other neuroactive hormones such as allopregnanolone (which mediates aspects of mood) (Bäckström et al., 2014) change during simulations of other naturalistic situations, such as social bonding, social competition, or interactions involving high levels of complex mentalizing or negative mood.

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