A meta-analytic review of the correlation between peripheral oxytocin and cortisol concentrations

Accepted Manuscript A meta-analytic review of the correlation between peripheral oxytocin and cortisol concentrations Christopher A. Brown, Christopher Cardoso, Mark A. Ellenbogen PII: DOI: Reference:

S0091-3022(16)30052-8 http://dx.doi.org/10.1016/j.yfrne.2016.11.001 YFRNE 648

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Frontiers in Neuroendocrinology

Received Date: Revised Date: Accepted Date:

24 July 2016 3 November 2016 7 November 2016

Please cite this article as: C.A. Brown, C. Cardoso, M.A. Ellenbogen, A meta-analytic review of the correlation between peripheral oxytocin and cortisol concentrations, Frontiers in Neuroendocrinology (2016), doi: http:// dx.doi.org/10.1016/j.yfrne.2016.11.001

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A meta-analytic review of the correlation between peripheral oxytocin and cortisol concentrations Christopher A. Brown1, B.A., Christopher Cardoso2,3*, Ph.D., & Mark A. Ellenbogen1 Ph.D.

1

Centre for Research in Human Development, Department of Psychology, Concordia University,

Montreal, QC, Canada 2

Department of Neuroscience, Carleton University, Ottawa, ON, Canada

3

Institute of Mental Health Research, Royal Ottawa Mental Health Centre, Ottawa, ON, Canada

* Address correspondence to: Department of Neuroscience, Carleton University, 1125 Colonel By Dr, Ottawa, ON K1S 5B6, Canada. e-mail: [email protected]

2 Abstract The stress dampening effects of exogenous oxytocin in humans have been well documented. However, the relation between endogenous oxytocin and cortisol is poorly understood. We conducted a metaanalysis on the correlation between oxytocin and cortisol levels measured at baseline (k= 24, N = 739). The effect size for the baseline correlation statistic was small (Pearson r = .163, p = 0.008), with high heterogeneity (I2 = 67.88%). Moderation analysis revealed that studies where participants anticipated an experimental manipulation evidenced a greater positive correlation compared to those that did not (Pearson r = 0.318, p = .006). A supplementary analysis including additional studies indicated that oxytocin levels in unextracted samples were 60 times higher when using this questionable practice. The findings suggest that the interplay between oxytocin and cortisol is dynamic and sensitive to the anticipation of stress or novelty. Furthermore, extraction of oxytocin appears to be an essential methodological practice. Keywords: Oxytocin; Cortisol; Plasma; Meta-Analysis; Experimental Manipulation; Stress

3 1. Introduction In recent years, there has been increasing interest in the influence of the neuropeptide oxytocin in social behavior. Synthetic oxytocin, administered intranasally, has significant and widespread effects on different human behaviours, particularly those related to trust and social cognition (Meyer-Lindenberg et al., 2011). Oxytocin has also stimulated much interest for its potential role in stress regulation (Crespi, 2015). There is evidence that exogenous oxytocin dampens hypothalamic-pituitary-adrenal (HPA) activity in rodents (Neumann and Landgraf, 2012) and non-human primates (Parker et al., 2005). Consistent with what has been found in animal models, studies of intravenous oxytocin administration in humans have found corresponding decreases in levels of plasma cortisol in response to stress (Legros, 2001), and the administration of intranasal oxytocin is known to attenuate the salivary cortisol response in response to various stressors (Cardoso et al., 2013; Ditzen et al., 2009). In a recent meta-analytic review, intranasal oxytocin attenuated the cortisol response to a greater degree in human patient populations relative to healthy volunteer samples, and in studies with manipulations that more robustly activated the HPA axis relative to those that did not (Cardoso et al., 2014). These findings suggest that there may be a clinically-relevant interplay between these two hormonal systems during acute stress. Despite these advances using exogenous challenge studies and stress paradigms, less is known about the interaction between endogenous oxytocin and the HPA axis under more naturalistic conditions. In times of stress, there is evidence in human and animals that oxytocin and cortisol are coreleased and endogenous oxytocin was initially proposed as having anxiolytic effects. Jong and colleagues (2015) observed that oxytocin and cortisol are co-released in response to the Trier Social Stress Task (TSST), which parallels findings in non-human animal research which shows that oxytocin is released in response to stress, including physical immobilization and forced swimming (Lang et al., 1983). This release of oxytocin during stress may be necessary to down-regulate the cortisol response to stress (Taylor et al., 2000). For example, female mice who are oxytocin deficient due to genetic manipulation display more anxiety-related behaviour, release greater amounts of corticosterone after a psychogenic stressor and display greater stress-induced hypothermia compared to control mice (Amico et

4 al., 2004). In both male and female rats, central infusion of an oxytocin antagonist leads to increased activity of the HPA axis (Neumann et al., 2000). In humans, nursing mothers with higher levels of oxytocin in the periphery overall have improved mood and less anxiety and women who experienced childhood abuse displayed lower levels of CSF oxytocin and greater anxiety (Heim et al., 2009; Heinrichs et al., 2001). Despite evidence that oxytocin might attenuate stress reactivity and reduce anxiety, there are a number of findings showing opposite effects. For example, endogenous oxytocin in the periphery was associated with greater levels of interpersonal distress, greater relationship distress and greater attachment anxiety among females (Pierrehumbert et al., 2010; Tabak et al., 2011; Taylor et al., 2010; Weisman et al., 2013). In a recent study, high levels of plasma oxytocin were associated with fewer depressive symptoms and greater maternal sensitivity among postpartum mothers, but only for those women who reported high levels of psychosocial stress (Zelkowitz et al., 2014). In addition, oxytocin levels are higher in individuals with social anxiety disorder (Hoge et al., 2008). Thus, the anxiolytic and stress-dampening effects of oxytocin may be context-specific and may vary depending on the timing of the perceived stressor. Given the importance of these relations, examining key moderators of the relation between peripheral oxytocin and cortisol levels may help identify the factors that regulate these hormonal systems, which is crucial for a better understanding of how these systems interact. A concern related to drawing inferences from studies examining peripheral levels of oxytocin and cortisol relates to questions regarding the measurement of oxytocin in a given medium (e.g. blood, saliva or urine). While the measurement of cortisol in blood and saliva has been well established, many questions still remain concerning the measurement of oxytocin (McCullough et al., 2013). Earlier studies assessed oxytocin levels using a radioimmunoassay (RIA) in extracted blood samples, meaning that proteins and other molecules which are not oxytocin that might bind with the ligand and as a result be erroneously tagged as circulating oxytocin are removed prior to the assay. More recent studies have assessed oxytocin levels using an enzyme immunoassay (EIA), often in unextracted blood samples. The EIA method has yielded values for oxytocin that are 100 times larger than what was found using the RIA

5 assay with extracted samples (McCullough et al., 2013; Szeto et al., 2011). Such values are discrepant from those demonstrated in a study that used two-dimensional liquid chromatography separation with tandem mass spectrometry detection in samples of extracted oxytocin (Zhang et al., 2011). This method is highly sensitive and yielded oxytocin levels that are more consistent with those found in extracted oxytocin samples (McCullough et al., 2013; Zhang et al., 2011). Further research is required to fully understand the implications of these varying immunoassay techniques on stress research and this represents a critical gap in endogenous oxytocin research. For example Christensen et al. (2014) tested both EIA and RIA with and without extraction in a sample of 13 participants. In this sample, they observed that EIA without extraction lead to unacceptably poor coefficients of variation between duplicate samples. In addition, there were issues in the recovery of oxytocin levels in samples spiked with known levels of oxytocin for both EIA and RIA without extraction. These authors concluded that both RIA and EIA were valid measures, as long as extraction was performed. Another concern relates to the measurement of oxytocin in blood compared to saliva or urine. While measuring cortisol in saliva is well validated and has been a cornerstone of stress research for many years (Kirschbaum and Hellhammer, 1994), measuring oxytocin in saliva and urine is relatively new. Salivary estimates of oxytocin correlate modestly (r = .41 to .59) with measures of unextracted plasma oxytocin (Feldman et al., 2010; Grewen et al., 2010). This is in stark contrast to plasma and salivary cortisol measurements, which yield correlations greater than .90 (Kirschbaum and Hellhammer, 2000; McCullough et al., 2013). However, a recent study found detectable differences in salivary oxytocin levels in response to stimuli that are known stimulate peripheral and central release of the neuropeptide (Jong et al., 2015). The validity of measuring oxytocin in urine has also been questioned. There is little evidence that bio-available oxytocin is cleared in urine, and measures of urinary and plasma oxytocin are only weakly correlated (McCullough et al., 2013). Establishing greater methodological rigor and understanding how these differing oxytocin methodologies influence the empirical literature is a key concern to this field of research.

6 Using meta-analysis, the present study sought to determine whether peripheral oxytocin and cortisol levels, assessed at baseline during resting conditions, were related in studies measuring both hormones in blood and/or saliva. Given the wide range of findings in the literature (Alfven et al., 1994; Hew-Butler et al., 2008), it is important to determine whether there are reciprocal interactions between the systems, and more importantly, what types of contextual and methodological factors moderate this relationship. We put forth a number of predictions. First, we predicted that oxytocin and cortisol levels would show no relationship when examined at baseline during resting conditions. Next, we expected that a number of factors might moderate the relationship between oxytocin and cortisol levels at baseline. Based on studies showing that oxytocin and cortisol are co-released during stress (Neumann and Landgraf, 2012), and that oxytocin inhibits the rise of cortisol in response to challenge, (Cardoso et al., 2014), we expected that studies where participants anticipated a challenging or novel task would evidence a larger correlation, likely a negative one, between oxytocin and cortisol, signifying increased interplay between these systems. However, we could not rule out the possibility of a positive correlation between these two hormones at baseline in anticipation of a novel or stressful task, given that these hormones are initially positively coupled and that large scale meta-analytic observations have poor temporal precision. We tested this hypothesis by comparing studies where blood or saliva was collected immediately prior to a novel challenge (e.g. a laboratory challenge) with those studies where sampling did not occur in anticipation of a laboratory challenge. While there is strong evidence of oxytocinergic dampening of the HPA response to stress (Cardoso et al., 2014), indicating a negative relationship between systems over time in response to stress, we did not expect to observe this relationship in studies that did not employ an experimental manipulation. Finally, given the methodological concerns raised in McCullough et al. (2013), we predicted that studies without extraction would yield significantly higher (inflated) levels of oxytocin than those using extraction. Given concerns about the validity of this methodological practice, studies that did not employ extraction prior to assaying oxytocin were excluded from the analyses.

2. Method

7 2.1. Inclusion Criteria In order to be included in the present study, studies were required to meet the following criteria: (1) Measurement of endogenous oxytocin concentrations in blood or saliva 1 with prior extraction, (2) measurement of endogenous cortisol concentrations in blood or saliva, (3) samples taken at identical time points, (4) no manipulation of oxytocin or cortisol prior to baseline measurement, and (5) use of nonpregnant human participants. All participant populations and studies of all sample sizes were included in the analysis.

2.2. Study Variables To examine the correlation between oxytocin and cortisol at baseline, we computed a correlation statistic (Pearson r). Given that the baseline correlation was seldom reported in the manuscripts identified during the literature search, the present study necessarily required requesting original datasets from the corresponding authors of the published articles. Having access to the raw data afforded us the opportunity to apply statistical transformations to the data when required (described in the next section). Such transformations are of relevance given that it is known that oxytocin and cortisol values are often not normally distributed (described below). We included the following moderators in the analysis, which were coded by the first author (C.B.): average sample age, percentage of the sample that was female, percentage of the sample that had a diagnosis of a mental disorder, type of fluid collected for the assessment of oxytocin and cortisol (blood vs saliva), assay method used (RIA vs EIA), baseline levels of cortisol (standardized), and whether an experimental manipulation was employed subsequent to the collection of blood or saliva. Categorical variables were dummy coded (0,1) with 1 being the variable of interest. All moderators are listed in Table 1.

2.3. Search Strategies 1

Due the lack of a correlation between plasma and urinary oxytocin and the lack of evidence that oxytocin is cleared through urine (McCullough et al., 2013), we chose to exclude four studies of urinary oxytocin in the present meta-analysis

8 The search engines used were Web of Science and MEDLINE (Pubmed). The keywords used were “oxytocin” AND “cortisol”. There were no restrictions placed on publication date in either search, and the search included all articles published before December 15th, 2015. The second author (C.C.) completed the search. The search yielded k = 1027 articles. We eliminated articles for the following reasons: 259 were study duplicates, 366 used non-human subjects or were written in a foreign language, and 147 were not peer- reviewed original research articles. Of the remaining k = 255 articles, 193 were removed because they did not measure both oxytocin and cortisol simultaneously at baseline in either blood or saliva in a non-pregnant human sample. Of the remaining k = 63 articles, data for 34 studies could not be computed because authors could not be reached or denied a request for data. Of the remaining k = 29 studies, 5 did not use extraction prior to oxytocin assay and were removed from the analysis (although they were retained for descriptive purposes). The final number of studies included for analysis was k = 24 (N = 739). Of the 63 studies that were solicited for raw data, the pattern of missing and acquired data was determined to be completely random because no statistically significant relation between the observed and missing variables was detected χ 2(15) = 21.92, p = .11. Corresponding authors from all identified studies were contacted by email between 1 May 2014 and 7 January 2016. 2.4. Statistical Analysis We computed a Pearson r correlation to evaluate the relation between oxytocin and cortisol at baseline using the data obtained from the communications with the authors. In order to be consistent, these statistics were computed using the first available endocrine measurement since only three studies used multiple fluid samples to measure baseline values (Nissen et al., 1996; Strathearn, et al., 1999; Tabak et al., 2011). Given that oxytocin and cortisol values are generally not normally distributed, we examined the skewness and kurtosis values. Log transformation was applied to those sets of values that had skewness values +/- 3 and kurtosis values +/- 10. Variance for the correlation statistics was calculated using the compute.es package for R (R Core Team, 2013), which utilizes formulas found in Cooper et al. (2009). Effect size statistics were weighted by sample size using the metafor package for R (Viechtbauer,

9 2010). The 95% confidence intervals [lower bound, upper bound] were estimated using the metafor package, which uses formulas found in Cooper et al. (2009). We computed the test of heterogeneity with a Q-test to confirm that population effect sizes varied across samples, and we computed an I2 index to analyze the variance explained by this heterogeneity (Higgins and Thompson, 2002). Moderation analysis was also computed using metafor, and all studies were included in this analysis (k = 24). In addition, due to concerns about the low correlation between plasma and salivary oxytocin (McCullough et al., 2013), we conducted additional analyses with studies of salivary oxytocin removed (see Table 1). Finally, we generated a funnel plot and calculated Egger's Regression Test (Egger et al., 1997) to inspect publication bias in this research area (Sterne and Egger, 2001).

3. Results 3.1 Main effect: Correlation between oxytocin and cortisol The studies included in the meta-analysis (k = 24), along with listed descriptive and moderator variables, can be found in Table 1. The overall effect size estimate for the correlation between oxytocin and cortisol at baseline was statistically significant (Z = 2.670, p = 0.008, Pearson r = .164, 95% CI [0.044, 0.284]; See Fig 1). However, as expected, we found evidence to suggest significant heterogeneity in this effect across studies (χ2 (23) = 84.079, p < .0001, I2 = 67.88%). In this case, the I2 value suggests that 67.88% of the total variability in the effect size estimates can be attributed to variability in the correlation between oxytocin and cortisol across populations (i.e. different studies are capturing different population-level effects; Viechtbauer, 2010). This degree of heterogeneity suggests that there is a great deal of variability in baseline correlation statistics across studies, and that there is sufficient reason to probe for moderators within the data. Given potential concerns about the validity of measures of salivary oxytocin, we repeated the analysis with three studies removed (k = 21). The correlation was also found to be statistically significant (Z = 2.173, p = 0.030, Pearson r = .147, 95% CI [0.014, 0.279]) with high heterogeneity in this effect across studies (χ2 (20) = 82.514, p < 0.0001, I2 = 71.23%).

10 3.2 Moderation by context and individual differences Neither the sex composition of the sample (Z = -0.605, p = 0.545, Pearson r = -.124, 95% CI [0.527, 0.278]) nor the percentage of the sample with a mental disorder (Z = -0.756, p = 0.450, Pearson r = -.177, 95% CI [-0.636, 0.282]) moderated the relation between oxytocin and cortisol levels. However, we did observe a statistically significant moderating effect of studies that involved an experimental manipulation subsequent to sample collection (Z = 2.765, p= 0.006, Pearson r = .318, 95% CI [0.093, 0.543]; See Fig 1). After removing the three studies that measured oxytocin in saliva, the effect of moderation by experimental manipulation remained statistically significant (k = 21; Z = 2.488, p = 0.013, Pearson r = .309, 95% CI [0.066, 0.553]). Thus, both analyses are considered to be statistically comparable. We also assessed whether the moderation effect was due to the possibility of altered levels of cortisol in studies employing an experimental manipulation relative to those that did not. We found no evidence of this assertion: baseline cortisol levels (all standardized to ug/dl) did not moderate the correlation between oxytocin and cortisol levels in either the primary analysis (Z = -0.799, p = .424, Pearson r = -.051, 95% CI[-0.177, 0.744]) or the analysis excluding salivary oxytocin (Z = -1.024, p = .306, Pearson r = -.072, 95% CI[-0.208, 0.065]). We also found no evidence to suggest that this moderating effect was dependent on sex or lactation status, and it remained significant when controlling for these variables (data not shown). In sum, while the correlation between oxytocin and cortisol was not found to be sensitive to sex composition of the sample or the percentage of participants with a mental disorder. Studies that employed an experimental manipulation showed a large, statistically significant and positive correlation between oxytocin and cortisol in baseline samples.

3.3 Moderation by immunoassay methods Studies that did not employ extraction prior to oxytocin assay were excluded from analysis based on questions about the validity of this technique. The median values of the extracted samples of oxytocin (Mdn = 4.9 pg/ml, n = 739) were more than 60 times lower than the unextracted values (Mdn = 297.5

11 pg/ml; n = 372; See Fig. 2), which supports this contention. These numbers are consistent with, albeit more conservative than, those reported by McCullough et al. (2013), where extracted values were 100 times lower than unextracted values in a smaller sample (n=39). In addition, the fluid type sampled for oxytocin (Z = -.749, p = .454, Pearson r = -.148, 95% CI [-0.536, 0.240]) and cortisol (Z = -0.749, p = .454, Pearson r = -.148, 95% CI [-0.536, 0.240]) did not moderate the correlation between oxytocin and cortisol levels. In sum, these methodological differences did not influence the correlation between peripheral oxytocin and cortisol levels collected at baseline during resting conditions.

3.4 Publication bias Fig. 3 represents a funnel plot of the standard errors and correlations between oxytocin and cortisol. Egger’s Regression Test did not indicate evidence to suggest larger or smaller correlations between oxytocin and cortisol in larger samples (Z = -0.867, p = 0.386). In addition, conceptually, publication bias was unlikely in this sample of studies given that the outcome variable used in the present study, namely the correlation between oxytocin and cortisol at baseline, was not an outcome of interest for any of the studies included in the analysis.

4. Discussion In the present study, we predicted that there would be no main effect for the correlation between oxytocin and cortisol. We also predicted a negative correlation between endogenous oxytocin and cortisol in studies where participants anticipated a novel or stressful laboratory task. Contrary to this latter hypothesis, we observed a positive correlation between oxytocin and cortisol levels when participants were anticipating a novel or stressful laboratory procedure. We did not rule out this possibility at the outset—oxytocin and cortisol are co-released in response to stressful tasks in animal models (Neumann and Landgraf, 2012). It may be that this positive coupling represents an earlier temporal interplay between these systems, because while these two hormones are co-released initially, oxytocin lowers

12 cortisol over time in humans (Cardoso, Kingdon, & Ellenbogen, 2014). These results support the idea that the experience of anticipating a novel or challenging event in the environment modulates the relation between endogenous oxytocin and cortisol in the periphery. Studies with and without a laboratory manipulation may also be distinguished by the degree to which participants are acclimatized to the laboratory context. Specifically, the threat associated with a novel social context may have been minimized in studies with no experimental manipulation, as some of these studies were larger longitudinal studies with repeated visits, or studies taking place in a specialized clinics rather than research laboratories, where the participants may have been familiar with the staff and setting (Alfvén, 2004; Parker et al., 2010). The moderation effect is consistent with evidence that anticipating a novel or challenging event in the environment has important effects on neuroendocrine function. Balodis et al. (2010) examined various baseline indices of stress in order to establish an improved baseline for laboratory studies designed to elicit a cortisol response. The authors specifically examined the arrival index, which was defined as the area under the curve for cortisol between initial arrival in the lab and one hour later (Balodis et al., 2010). They observed that the change in cortisol from arrival to one hour later was highly correlated with the subsequent cortisol response to the TSST. The authors suggest that the arrival index may be a proxy for how individuals acclimate to their environment, which would be a laboratory setting in this case (Balodis et al., 2010). The present study further shows that the biological response to anticipating a novel or challenging event in the environment is not only a marker of HPA sensitivity to stress or habituation, but also that it may alter HPA coupling with the oxytocinergic system. In the present study, we predicted that studies which would be perceived as more stressful or novel to the participants (i.e. those involving an experimental manipulation) would evidence a more robust correlation between oxytocin and cortisol. The direction of the relationship was predicted to be negative, based on evidence that oxytocin attenuates the cortisol response to acute stress (Cardoso et al., 2014). In contrast, we observed a positive coupling of oxytocin and cortisol levels in studies where

13 participants anticipated an experimental manipulation. It is possible that novel and potentially threatening social environments simultaneously activate both systems. Consistent with this contention, these findings mirror earlier animal studies of oxytocin, where it was repeatedly shown in rats that oxytocin is released in response to stress alongside cortisol (Lang et al., 1983; Neumann et al., 1998; Wotjak et al., 1998). Given that exogenous oxytocin has been shown to have an inhibitory effect on peripheral cortisol levels (Cardoso et al., 2014), it may be that the relationship between oxytocin and cortisol at baseline captures an early interplay between the two systems where they are co-released. For example, adults experience both elevations in oxytocin and cortisol following a Trier Social Stressor Test (TSST) (Pierrehumbert et al., 2010). Another recent study of salivary oxytocin and cortisol found that the two hormones increased in tandem throughout a TSST procedure, depending on the use of oral contraceptives (Jong et al., 2015). Although oxytocin may attenuate HPA reactivity over time during an acute stress procedure, the initial oxytocin response to social threat (i.e. entering a laboratory to participate in an experiment) may occur in tandem with the HPA response. The present findings support the importance of a line of research investigating the coordination between stress markers both within the HPA axis and across biological systems rather than absolute or relative levels of a single marker (e.g. Laurent et al., 2016). Clearly, further research on the relation between cortisol and oxytocin levels at different stages of the cortisol response to stress, and across different contexts, is needed. The present findings, in conjunction with a previous meta-analysis (Cardoso et al., 2014), support the view that there is a greater interplay between oxytocin and cortisol during the anticipation of novelty or the experience of distress, relative to normative baseline conditions. This has also been observed in rodent models (Nishioka et al., 1998; Wotjak et al., 1998), and is consistent with the recent finding that the administration of exogenous oxytocin elicits greater attenuation of the cortisol response to stress during laboratory tasks that strongly activate the HPA-axis than those that only weakly stimulate it (Cardoso et al., 2014). The divergent findings between studies of baseline hormonal levels and acute stress highlight the fact that basal and stressful contexts likely activate HPA-oxytocinergic circuitry differentially, similar to the well-known distinctions in the HPA literature between mineralocorticoid

14 receptor-mediated basal regulatory functions and the glucocorticoid receptor-mediated stress adaptation functions (Sapolsky et al., 2000). However, the observed correlation between oxytocin and cortisol in this study does not explain the impact of these hormones on subjective mood state, which is often argued to be context-dependent (Bartz et al., 2011). Clearly, more experimental research is needed in order to tease apart the reciprocal interactions among hormones within the HPA axis, subjective mood state, and social context to uncover the nuanced relationship among these variables. Consistent with McCullough et al. (2013) and Christensen et al. (2014), we observed that oxytocin concentrations in unextracted samples are roughly 60 times larger than those in extracted samples. The values that we observed in the extracted samples of oxytocin are consistent with what Zhang et al. (2011) found using mass spectrometry, which further lends support to the use of oxytocin extraction. Using extraction prior to assaying oxytocin samples appears to be an essential methodological practice that measures biologically plausible levels of oxytocin in the periphery. We urge future researchers to consider this vital step when measuring plasma oxytocin. In the present study, we did not find support for sex moderating the correlation between oxytocin and cortisol. Due to the complex nature of hormone research in human men and women, given stark sex differences in a number of hormones that influence oxytocin and cortisol (i.e. estrogen, progesterone), the moderation analysis presented in this meta-analysis is no replacement for well-controlled experimental studies on the relation between oxytocin, cortisol, and sex. This is particularly true for the impact of lactation on the interplay between these hormones, which we could not evaluate because only two studies included in the analysis evaluated this subject. Thus, the relation between sex and the interplay between oxytocin and cortisol requires clarification in future studies. We also did not observe moderation of the relation between oxytocin and cortisol at baseline by the presence of a mental disorder in participants, in contrast to the acute stress literature (Cardoso et al., 2014). Yuen et al. (2014) found evidence that while concentrations of oxytocin are lower in depressed women, these concentrations are independent of cortisol (Yuen et al., 2014). It is important to note, however, that the proportion of mentally ill participants was rather small in this sample, and the impact of mental illness on the relation between

15 oxytocin and cortisol should be further investigated in larger studies with extensive sampling throughout the day. The present study is subject to a number of limitations, which exemplify many issues with this field of study. The most notable problem was that we were not able to use baseline concentrations of oxytocin as a moderator in the analyses because the variations between fluid medium and assay employed were too great. As a result, baseline samples were not necessarily comparable across studies and could not be entered into moderation analysis. Second, endogenous cortisol values are subject to wide variations throughout the day. While some studies reported the time of day that cortisol was sampled, the majority did not, so it was not possible to assess time of day effects systematically across studies. However, we examined whether cortisol levels at baseline, which is a proxy for time of day, moderated the correlation between oxytocin and cortisol, and found no evidence that it did. In addition, a previous study has demonstrated that diurnal cortisol and oxytocin are not correlated in animals and humans (Seckl & Lightman, 1987; Blagrove et al., 2012). Third, the present study conceptualized baseline samples as the first sample available in the dataset. These samples could have been taken at varying times following entry into the experimental setting. In addition, the estimated effect would be more reliable if the studies included in the present meta-analysis would have taken multiple baseline samples in order to ensure accurate baseline measurements, due to moment-to-moment changes in oxytocin and cortisol concentrations that are due to endogenous pulsatile release of these hormones (Amico et al., 1987; Balodis et al., 2010; Gimpl and Fahrenholz, 2001; Young et al., 2004). Fourth, with one exception (where the diagnosis was autism), major depressive disorder was the clinical diagnosis in all studies. A moderation analysis not presented here that omitted the single autism study revealed that percentage of depressed participants did not impact the correlation between oxytocin and cortisol. As an additional consideration, it is not known whether measures of oxytocin in blood provide any relevant information about central oxytocinergic function. While some studies show a positive correlation between measures of oxytocin in cerebrospinal fluid and plasma (Carson et al., 2014), others show no relation (Jokinen et al., 2012; Kagerbauer et al., 2013). The association between peripheral oxytocin concentrations and

16 psychological constructs is still a subject for debate and requires further investigation. Finally, 34 eligible studies were not included in the present meta-analysis because data for the studies could not be retrieved. Every effort to collect these data was made; however, some authors could not be contacted, some datasets were not available and a subset of authors refused the data request. In sum, a positive correlation between oxytocin and cortisol concentrations was found among studies where participants were anticipating the commencement of a novel laboratory task, indicating that anticipating a novel or challenging event in the environment may elicit coupling between these hormonal systems. While this meta-analysis lays the foundation to generate hypotheses on the interplay between endogenous oxytocin and cortisol in humans, it is important to note that meta-analytic studies are not a substitute for well-controlled experimental studies. Studies examining the impact of anticipating an experimental manipulation on the correlation between oxytocin and cortisol are warranted at this juncture. Future experimental research should also target neurobiological mechanisms underlying this possible relationship, and further examine how different contextual factors influence the interplay between these systems (Bartz et al., 2011).

17 Role of the Funding Source This research was supported by grants to Dr. Ellenbogen from the Canada Research Chair program (supported by the Social Sciences and Humanities Research Council of Canada; SSHRC). Christopher Cardoso is supported by a scholarship from the Canadian Institutes of Health Research (CIHR; Grant #148255). The funding agencies had no role in the production of the manuscript.

18 References *Articles with an asterisk were included in the primary study analyses †Articles with a cross were included only in the comparison of extracted and unextracted oxytocin values *Alfvén, G., 2004. Plasma oxytocin in children with recurrent abdominal pain. J. Pediatr. Gastroenterol. Nutr. 38, 513–7. *Alfven, G., Torre, B., Uvnäs‐ Moberg, K., 1994. Depressed concentrations of oxytocin and cortisol in children with recurrent abdominal pain of non‐ organic origin. Acta Paediatr. 1076–1080. Amico, J.A., Ulbrecht, J.S., Robinson, A.G., 1987. Clearance studies of oxytocin in humans using radioimmunoassay measurements of the hormone in plasma and urine. J. Clin. Endocrinol. Metab. 64, 340–345. doi:10.1210/jcem-64-2-340 Balodis, I.M., Wynne-Edwards, K.E., Olmstead, M.C., 2010. The other side of the curve: Examining the relationship between pre-stressor physiological responses and stress reactivity. Psychoneuroendocrinology 35, 1363–1373. doi:10.1016/j.psyneuen.2010.03.011 †Barraza, J.A., Zak, P.J., 2009. Empathy toward strangers triggers oxytocin release and subsequent generosity. Ann. N. Y. Acad. Sci. 1167, 182–9. doi:10.1111/j.1749-6632.2009.04504. 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, 301–9. doi:10.1016/j.tics.2011.05.002 *Blagrove, M., Fouquet, N.C., Baird, A.L., Pace-Schott, E.F., Davies, A.C., Neuschaffer, J.L., HenleyEinion, J.A., Weidemann, C.T., Thome, J., McNamara, P., Turnbull, O.H., 2012. Association of salivary-assessed oxytocin and cortisol levels with time of night and sleep stage. J. Neural Transm. 119, 1223–32. doi:10.1007/s00702-012-0880-1 *Bosch, O.G., Eisenegger, C., Gertsch, J., von Rotz, R., Dornbierer, D., Gachet, M.S., Heinrichs, M., Wetter, T.C., Seifritz, E., Quednow, B.B., 2015. Gamma-hydroxybutyrate enhances mood and prosocial behavior without affecting plasma oxytocin and testosterone. Psychoneuroendocrinology 62, 1–10. doi:10.1016/j.psyneuen.2015.07.167 Carson, D.S., Berquist, S.W., Trujillo, T.H., Garner, J.P., Hannah, S.L., Hyde, S.A., ... & Parker, K.J. (2014). Cerebrospinal fluid and plasma oxytocin concentrations are positively correlated and negatively predict anxiety in children. Mol Psychiatry. 20, 1085-90. Cardoso, C., Ellenbogen, M.A., Orlando, M.A., Bacon, S.L., Joober, R., 2013. Intranasal oxytocin attenuates the cortisol response to physical stress: a dose-response study. Psychoneuroendocrinology 38, 399–407. doi:10.1016/j.psyneuen.2012.07.013 Cardoso, C., Kingdon, D., Ellenbogen, M.A., 2014. A meta-analytic review of the impact of intranasal oxytocin administration on cortisol concentrations during laboratory tasks: Moderation by method and mental health. Psychoneuroendocrinology, 49, 161–170. doi:10.1016/j.psyneuen.2014.07.014

19 Christensen, J.C., Shiyanov, P.A., Estepp, J.R., Schlager, J.J., 2014. Lack of association between human plasma oxytocin and interpersonal trust in a prisoner’s dilemma paradigm. PLoS One 9, e116172. doi:10.1371/journal.pone.0116172 Cooper, H., Hedges, L. V, Valentine, J.C., 2009. The Handbook of Research Synthesis and MetaAnalysis, second ed. Russell Sage Foundation, New York, NY. Crespi, B.J., 2015. Oxytocin, testosterone, and human social cognition. Biol. Rev. doi:10.1111/brv.12175 Ditzen, B., Schaer, M., Gabriel, B., Bodenmann, G., Ehlert, U., Heinrichs, M., 2009. Intranasal oxytocin increases positive communication and reduces cortisol levels during couple conflict. Biol. Psychiatry 65, 728–731. doi:10.1016/j.biopsych.2008.10.011 Egger, M., Davey Smith, G., Schneider, M., Minder, C., 1997. Bias in meta-analysis detected by a simple, graphical test. BMJ 315, 629–634. Feldman, R., Gordon, I., Schneiderman, I., Weisman, O., Zagoory-Sharon, O., 2010. Natural variations in maternal and paternal care are associated with systematic changes in oxytocin following parentinfant contact. Psychoneuroendocrinology 35, 1133–1141. doi:10.1016/j.psyneuen.2010.01.013 †Feldman, R., Weller, A., Zagoory-Sharon, O., Levine, A., 2007. Evidence for a neuroendocrinological foundation of human affiliation: plasma oxytocin levels across pregnancy and the postpartum period predict mother-infant bonding. Psychol. Sci. 18, 965–70. doi:10.1111/j.1467-9280.2007.02010. *Ferreira, M.F., Sobrinho, L.G., Santos, M. a, Sousa, M.F., Uvnäs-Moberg, K., 1998. Rapid weight gain, at least in some women, is an expression of a neuroendocrine state characterized by reduced hypothalamic dopaminergic tone. Psychoneuroendocrinology 23, 1005–13. †Floyd, K., Pauley, P.M., Hesse, C., 2010. State and Trait Affectionate Communication Buffer Adults’ Stress Reactions. Commun. Monogr. 77, 618–636. doi:10.1080/03637751.2010.498792 Gimpl, G., Fahrenholz, F., 2001. The oxytocin receptor system: structure, function, and regulation. Physiol. Rev. 81, 629–83. †Gordon, I., Zagoory-Sharon, O., Schneiderman, I., Leckman, J.F., Weller, A., Feldman, R., 2008. Oxytocin and cortisol in romantically unattached young adults: associations with bonding and psychological distress. Psychophysiology 45, 349–52. doi:10.1111/j.1469-8986.2008.00649. †Grape, C., Sandgren, M., Hansson, L., 2002. Does singing promote well-being?: An empirical study of professional and amateur singers during a singing lesson. Behav. Sci. 38, 65–74. Grewen, K.M., Davenport, R.E., Light, K.C., 2010. An investigation of plasma and salivary oxytocin responses in breast- and formula-feeding mothers of infants. Psychophysiology. doi:10.1111/j.14698986.2009.00968. Heim, C., Young, L.J., Newport, D.J., Mletzko, T., Miller, A.H., Nemeroff, C.B., 2009. Lower CSF oxytocin concentrations in women with a history of childhood abuse. Mol. Psychiatry 14, 954–958. doi:10.1038/mp.2008.112

20 Heinrichs, M., Meinlschmidt, G., Neumann, I., Wagner, S., Kirschbaum, C., Ehlert, U., Hellhammer, D.H., 2001. Effects of suckling on hypothalamic-pituitary-adrenal axis responses to psychosocial stress in postpartum lactating women. J. Clin. Endocrinol. Metab. 86, 4798–4804. doi:10.1210/jcem.86.10.7919 *Hew-Butler, T., Noakes, T.D., Soldin, S.J., Verbalis, J.G., 2008. Acute changes in endocrine and fluid balance markers during high-intensity, steady-state, and prolonged endurance running: unexpected increases in oxytocin and brain natriuretic peptide during exercise. Eur. J. Endocrinol. 159, 729–37. doi:10.1530/EJE-08-0064 Higgins, J.P.T., Thompson, S.G., 2002. Quantifying heterogeneity in a meta-analysis. Stat. Med. 21, 1539–1558. doi:10.1002/sim.1186 Hoge, E.A., Pollack, M.H., Kaufman, R.E., Zak, P.J., Simon, N.M., 2008. Oxytocin levels in social anxiety disorder. CNS Neurosci. Ther. 14, 165–170. doi:10.1111/j.1755-5949.2008.00051.x *Hursti, T.J., Börjeson, S., Hellström, P.M., Avall-Lundqvist, E., Stock, S., Steineck, G., Peterson, C., 2005. Effect of chemotherapy on circulating gastrointestinal hormone levels in ovarian cancer patients: relationship to nausea and vomiting. Scand. J. Gastroenterol. 40, 654–61. doi:10.1080/0036552051001163 *Hysek, C.M., Domes, G., Liechti, M.E., 2012a. MDMA enhances “mind reading” of positive emotions and impairs “mind reading” of negative emotions. Psychopharmacology. 222, 293–302. doi:10.1007/s00213-012-2645-9 *Hysek, C.M., Schmid, Y., Rickli, A., Simmler, L.D., Donzelli, M., Grouzmann, E., Liechti, M.E., 2012b. Carvedilol inhibits the cardiostimulant and thermogenic effects of MDMA in humans. Br. J. Pharmacol. 166, 2277–2288. doi:10.1111/j.1476-5381.2012.01936.x *Hysek, C.M., Schmid, Y., Simmler, L.D., Domes, G., Heinrichs, M., Eisenegger, C., Preller, K.H., Quednow, B.B., Liechti, M.E., 2013. MDMA enhances emotional empathy and prosocial behavior. Soc. Cogn. Affect. Neurosci. doi:10.1093/scan/nst161 *Jansen, L.M.C., Gispen-de Wied, C.C., Wiegant, V.M., Westenberg, H.G.M., Lahuis, B.E., van Engeland, H., 2006. Autonomic and neuroendocrine responses to a psychosocial stressor in adults with autistic spectrum disorder. J. Autism Dev. Disord. 36, 891–9. doi:10.1007/s10803-006-0124-z Jokinen, J., Chatzittofis, A., Hellström, C., Nordström, P., Uvnäs-Moberg, K., Asberg, M., 2012. Low CSF oxytocin reflects high intent in suicide attempters. Psychoneuroendocrinology 37, 482-490. *Jong, T.R. de, Menon, R., Bludau, A., Grund, T., Biermeier, V., Klampfl, S.M., Jurek, B., Bosch, O.J., Hellhammer, J., Neumann, I.D., 2015. Salivary oxytocin concentrations in response to running, sexual self-stimulation, breastfeeding and the TSST: The Regensburg Oxytocin Challenge (ROC) study. Psychoneuroendocrinology 62, 381–388. doi:10.1016/j.psyneuen.2015.08.027 Kagerbauer, S.M., Martin, J., Schuster, T., Blobner, M., Kochs, E.F., Landgraf, R., 2013. Plasma oxytocin and vasopressin do not predict neuropeptide concentrations in human cerebrospinal fluid. J. Neuroendocrinol 25, 668-673.

21 *Keating, C., Dawood, T., Barton, D.A., Lambert, G.W., Tilbrook, A.J., 2013. Effects of selective serotonin reuptake inhibitor treatment on plasma oxytocin and cortisol in major depressive disorder. BMC Psychiatry 13, 124. doi:10.1186/1471-244X-13-124 Kirschbaum, C., Hellhammer, D.H., 2000. Salivary Cortisol, in: Fink, G. (Ed.), Encyclopedia of Stress. Academic Press, San Diego, pp. 379–383. Kirschbaum, C., Hellhammer, D.H., 1994. Salivary cortisol in psychoneuroendocrine research: recent developments and applications. Psychoneuroendocrinology 19, 313–333. Lang, R.E., Heil, J.W., Ganten, D., Hermann, K., Unger, T., Rascher, W., 1983. Oxytocin unlike vasopressin is a stress hormone in the rat. Neuroendocrinology 37, 314–316. Laurent, H.K., Lucas, T., Pierce, J., Goetz, S., Granger, D.A., 2016. Coordination of cortisol response to social evaluative threat with autonomic and inflammatory responses is moderated by stress appraisals and affect. Biol. Psychol. 118, 17–24. doi:10.1016/j.biopsycho.2016.04.066 Legros, J.J., 2001. Inhibitory effect of oxytocin on corticotrope function in humans: are vasopressin and oxytocin ying-yang neurohormones? Psychoneuroendocrinology 26, 649–55. Linnen, A.-M., Ellenbogen, M.A., Cardoso, C., Joober, R., 2012. Intranasal oxytocin and salivary cortisol concentrations during social rejection in university students. Stress 15, 393–402. doi:10.3109/10253890.2011.631154 McCullough, M.E., Churchland, P.S., Mendez, A.J., 2013. Problems with measuring peripheral oxytocin: can the data on oxytocin and human behavior be trusted? Neurosci. Biobehav. Rev. 37, 1485–92. doi:10.1016/j.neubiorev.2013.04.018 Meyer-Lindenberg, A., Domes, G., Kirsch, P., Heinrichs, M., 2011. Oxytocin and vasopressin in the human brain: social neuropeptides for translational medicine. Nat. Rev. Neurosci. 12, 524–38. doi:10.1038/nrn3044 Neumann, I.D., Johnstone, H.A., Hatzinger, M., Liebsch, G., Shipston, M., Russell, J.A., Landgraf, R., Douglas, A.J., 1998. Attenuated neuroendocrine responses to emotional and physical stressors in pregnant rats involve adenohypophysial changes. J. Physiol. 508, 289–300. Neumann, I.D., Wigger, A., Torner, L., Holsboer, F., Landgraf, R., 2000. Brain oxytocin inhibits basal and stress-induced activity of the hypothalamo-pituitary-adrenal axis in male and female rats: Partial action within the paraventricular nucleus. J. Neuroendocrinol. 12, 235–243. doi:10.1046/j.13652826.2000.00442.x Neumann, I.D., Landgraf, R., 2012. Balance of brain oxytocin and vasopressin: implications for anxiety, depression, and social behaviors. Trends Neurosci. 35, 649–59. doi:10.1016/j.tins.2012.08.004 Nishioka, T., Anselmo-Franci, J.A., Li, P., Callahan, M.F., Morris, M., 1998. Stress increases oxytocin release within the hypothalamic paraventricular nucleus. Brain Res. 781, 57–61.

22 *Nissen, E., Uvnäs-Moberg, K., Svensson, K., Stock, S., Widström, A.M., Winberg, J., 1996. Different patterns of oxytocin, prolactin but not cortisol release during breastfeeding in women delivered by caesarean section or by the vaginal route. Early Hum. Dev. 45, 103–18. Parker, K.J., Buckmaster, C.L., Schatzberg, A.F., Lyons, D.M., 2005. Intranasal oxytocin administration attenuates the ACTH stress response in monkeys. Psychoneuroendocrinology 30, 924–929. doi:10.1016/j.psyneuen.2005.04.002 *Parker, K.J., Kenna, H.A., Zeitzer, J.M., Keller, J., Blasey, C.M., Amico, J.A., Schatzberg, A.F., 2010. Preliminary evidence that plasma oxytocin levels are elevated in major depression. Psychiatry Res. 178, 359–62. doi:10.1016/j.psychres.2009.09.017 Pierrehumbert, B., Torrisi, R., Laufer, D., Halfon, O., Ansermet, F., Beck Popovic, M., 2010. Oxytocin response to an experimental psychosocial challenge in adults exposed to traumatic experiences during childhood or adolescence. Neuroscience 166, 168–77. doi:10.1016/j.neuroscience.2009.12.016 Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev. 21, 55–89. doi:10.1210/edrv.21.1.0389 R Core Team, 2013. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, http://www.R-project.org/. *Schmid, Y., Enzler, F., Gasser, P., Grouzmann, E., Preller, K.H., Vollenweider, F.X., Brenneisen, R., Müller, F., Borgwardt, S., Liechti, M.E., 2015a. Acute Effects of Lysergic Acid Diethylamide in Healthy Subjects. Biol. Psychiatry 78, 544–553. doi:10.1016/j.biopsych.2014.11.015 *Schmid, Y., Rickli, A., Schaffner, A., Duthaler, U., Grouzmann, E., Hysek, C.M., Liechti, M.E., 2015b. Interactions between Bupropion and 3,4-Methylenedioxymethamphetamine in Healthy Subjects. J. Pharmacol. Exp. Ther. 353, 102–111. doi:10.1124/jpet.114.222356 *Schmid, Y., Hysek, C.M., Simmler, L.D., Crockett, M.J., Quednow, B.B., Liechti, M.E., 2014. Differential effects of MDMA and methylphenidate on social cognition. J. Psychopharmacol. 28, 847–856. doi:10.1177/0269881114542454Seckl, J.R., Lightman, S.L., 1987. Diurnal rhythm of vasopressin but not of oxytocin in the cerebrospinal fluid of the goat: lack of association with plasma cortisol rhythm. J. Endocrinol. 114, 477–482. Seckl, J.R., Lightman, S.L., 1987. Diurnal rhythm of vasopressin but not of oxytocin in the cerebrospinal fluid of the goat: lack of association with plasma cortisol rhythm. J. Endocrinol. 114, 477–482. Sterne, J.A., Egger, M., 2001. Funnel plots for detecting bias in meta-analysis: guidelines on choice of axis. J. Clin. Epidemiol. 54, 1046–1055. *Strathearn, L., Fonagy, P., Amico, J., Montague, P.R., 2009. Adult attachment predicts maternal brain and oxytocin response to infant cues. Neuropsychopharmacology 34, 2655–66. doi:10.1038/npp.2009.103

23 Szeto, A., McCabe, P.M., Nation, D.A., Tabak, B.A., Rossetti, M.A., McCullough, M.E., Schneiderman, N., Mendez, A.J., 2011. Evaluation of enzyme immunoassay and radioimmunoassay methods for the measurement of plasma oxytocin. Psychosom. Med. 73, 393–400. doi:10.1097/PSY.0b013e31821df0c2 *Tabak, B.A., McCullough, M.E., Szeto, A., Mendez, A.J., McCabe, P.M., 2011. Oxytocin indexes relational distress following interpersonal harms in women. Psychoneuroendocrinology 36, 115–22. doi:10.1016/j.psyneuen.2010.07.004 Taylor, S.E., Klein, L.C., Lewis, B.P., Gruenewald, T.L., Gurung, R.A., Updegraff, J.A., 2000. Biobehavioral responses to stress in females: tend-and-befriend, not fight-or-flight. Psychol. Rev. 107, 411–429. Taylor, S.E., Saphire-Bernstein, S., Seeman, T.E., 2010. Are plasma oxytocin in women and plasma vasopressin in men biomarkers of distressed pair-bond relationships? Psychol. Sci. 21, 3–7. doi:10.1177/0956797609356507 *Tops, M., van Peer, J.M., Korf, J., Wijers, A. a, Tucker, D.M., 2007. Anxiety, cortisol, and attachment predict plasma oxytocin. Psychophysiology 44, 444–9. doi:10.1111/j.1469-8986.2007.00510. *Van Londen, L., Goekoop, J.G., Zwinderman, A.H., Lanser, J.B., Wiegant, V.M., De Wied, D., 1998. Neuropsychological performance and plasma cortisol, arginine vasopressin and oxytocin in patients with major depression. Psychol. Med. 28, 275–84. *Varga, K., Kekecs, Z., 2014. Oxytocin and cortisol in the hypnotic interaction. Int. J. Clin. Exp. Hypn. 62, 111–28. doi:10.1080/00207144.2013.841494 Viechtbauer, W., 2010. Conducting Meta-Analyses in R with the metafor Package. J. Stat. Softw. 36, 1– 48. Weisman, O., Zagoory-Sharon, O., Schneiderman, I., Gordon, I., Feldman, R., 2013. Plasma oxytocin distributions in a large cohort of women and men and their gender-specific associations with anxiety. Psychoneuroendocrinology 38, 694–701. doi:10.1016/j.psyneuen.2012.08.011 Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, F., Landgraf, R., Engelmann, M., 1998. Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience 85, 1209–1222. Young, E.A., Abelson, J., Lightman, S.L., 2004. Cortisol pulsatility and its role in stress regulation and health. Front. Neuroendocrinol. 25, 69–76. doi:10.1016/j.yfrne.2004.07.001 *Yuen, K.W., Garner, J.P., Carson, D.S., Keller, J., Lembke, A., Hyde, S.A., Kenna, H.A., Tennakoon, L., Schatzberg, A.F., Parker, K.J., 2014. Plasma oxytocin concentrations are lower in depressed vs. healthy control women and are independent of cortisol. J. Psychiatr. Res. 51, 30–6. doi:10.1016/j.jpsychires.2013.12.012 Zelkowitz, P., Gold, I., Feeley, N., Hayton, B., Carter, C.S., Tulandi, T., Abenhaim, H.A., Levin, P., 2014. Psychosocial stress moderates the relationships between oxytocin, perinatal depression, and maternal behavior. Horm. Behav. 66, 351–60. doi:10.1016/j.yhbeh.2014.06.014

24 Zhang, G., Zhang, Y., Fast, D.M., Lin, Z., Steenwyk, R., 2011. Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay. Anal. Biochem. 416, 45–52. doi:10.1016/j.ab.2011.04.041

25

Fig. 1 This figure is a forest plot for the average effect size (Pearson r) of the correlation between oxytocin and cortisol using a random-effects model. The first subgroup analysis in this figure, which is denoted with an asterisk, exclusively includes studies that employed an experimental manipulation (k = 17, n = 430). The overall effect size for all studies included in the metaanalysis is represented at the bottom of this figure (k = 24, N = 739). Studies are weighted by sample size, and larger filled circles for the effect size estimate represent larger samples. Error bars represent the 95% confidence interval.

26

Fig. 2 This figure represents raw case-level data for oxytocin concentrations in extracted and unextracted samples (pg/ml). Values higher than 1000 were included in the analyses, but are not presented in this figure (n = 25).

27

Fig. 3 This figure is a funnel plot of the standard errors and effect sizes (Pearson r) for the correlation between oxytocin and cortisol. Egger’s Regression Test did not indicate evidence to suggest larger or smaller correlations between oxytocin and cortisol in larger samples (Z = 0.867, p = 0.386).

28

Table 1. Descriptive statistics and moderator variables for the studies included in the meta-analysis (k = 29) Female Illness Pearson's (%) (%)a r

Transformationb

Cortisol Fluid

Oxytocin Fluid

Endurance Exercise Pharmacological Challengec Pharmacological Challenged

Blood

Blood

Yes

RIAo

Blood

Blood

Yes

RIA

Blood

Blood

Yes

RIA

Saliva

Saliva

Yes

EIAp

Blood

Blood

Yes

RIA

Blood

Blood

Yes

RIA

O

Sleep in Laboratory Pharmacological Treatmente Pharmacological Challengef Pharmacological Challengeg

Blood

Blood

Yes

RIA

0.23

OC

TSST

Saliva

Saliva

Yes

RIA

0

0.23

O

Relational Stress Task

Blood

Blood

Yes

RIA

0

0

0.21

O

Saliva

Saliva

Yes

EIA

25.2

50

0

0.10

O

Hypnosis Pharmacological Challengec h

Blood

Blood

Yes

RIA

31

21.3

8

42

0.10

C

Blood

Blood

Yes

RIA

Schmid et al. (2015b)

16

24.3

50

0

0.02

O

Blood

Blood

Yes

RIA

Hysek et al. (2012b)

16

24.2

50

0

-0.03

TSSTi Pharmacological Challengec j Pharmacological Challengec k

Blood

Blood

Yes

RIA

Strathearn et al. (2009)

57

28.8

100

0

-0.04

O

Bloodn

Bloodn

Yes

RIA

Bosch et al. (2015)

32

23.9

0

0

-0.08

O

Blood

Blood

Yes

RIA

Hursti et al. (2005)

14

56.3

100

0

-0.18

Interaction with Infant Pharmacological Challengel Pharmacological Treatmentm

Bloodn

Bloodn

Yes

RIA

Alfven et al. (2004)

42

9.5

71

0

0.31

None

Blood

Blood

Yes

RIA

Nissen et al. (1996)

37

29.0

100

0

-0.01

None

Blood

Blood

Yes

RIA

Study

N

Age

Hew Butler et al. (2008)

7

44.0

29

0

0.81

Hysek et al. (2012a)

48

25.8

50

0

0.63

Schmid et al. (2015a)

16

28.6

50

0

0.48

Blagrove et al. (2012)

20

20.5

50

0

0.42

C

Keating et al. (2013)

16

43.6

56

100

0.41

O

Tops et al. (2007)

18

41.0

100

0

0.40

Schmid et al. (2014)

30

24.0

50

0

0.36

De Jong et al. (2015)

30

23.5

50

0

Tabak et al. (2011)

35

19.3

100

Varga et al. (2014)

12

29.6

Hysek et al. (2013)

32

Jansen et al. (2006)

OC

Manipulation

OT Assay Extraction

29 Van Londen et al. (1998)

89

43.3

22

58

-0.08

Ferreira et al. (1998)

33

22.4

100

0

-0.08

Parker et al. (2010)

30

36.6

53

37

-0.10

Yuen et al. (2013)

44

39.4

61

61

-0.12

Alfven et al. (1994)

34

10.0

48

0

-0.26

O

None

Blood

Blood

Yes

RIA

None

Blood

Bloodn

Yes

RIA

O

None

Blood

Blood

Yes

RIA

OC

None

Blood

Blood

Yes

EIA

None

Blood

Blood

Yes

RIA

Note: Studies are ordered by effect size magnitude a

Besides Jansen et al. (2006) who studied autistic children, the rest of the samples were comprised of depressed participants O: Log transformation was applied to oxytocin values; C: Log transformation was applied to cortisol values c 3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') d Lysergic Acid Diethylamide e Selective serotonin reuptake inhibitor (SSRI) f Hydrocortisone g Methylphenidate h Ipsapirone i Trier Social Stress Task j Bupropion k Caverdilol l Gamma-hydroxybutyrate (GHB) m Chemotherapy n Denotes studies where hormones were measured in serum rather than plasma o Radioimmunoassay p Enzyme Immunoassay b

30

Highlights     

Peripheral oxytocin and cortisol samples were positively correlated on average across 24 studies. In samples anticipating an experimental manipulation, this correlation was greater Unextracted samples of peripheral oxytocin yield values 60x larger than extracted samples Individual differences in sample characteristics, including sex and clinical diagnosis, did not influence the correlation statistic. In summary, the interplay between oxytocin and cortisol in the periphery is dynamic and sensitive to contextual factors.