https://doi.org/10.1177/01650254231208965 International Journal of Behavioral Development 1 –12 © The Author(s) 2023 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/01650254231208965 journals.sagepub.com/home/jbd Introduction Adolescence is an important transitional period, characterized by intensive social, emotional, and hormonal changes (Crone & Dahl, 2012). Optimal development involves fluent coordination between these domains, aiding adolescent adjustment to increas- ing social demands and stressors. The autonomic nervous system (ANS) consisting of the sympathetic (SNS) and parasympathetic (PNS) nervous systems and the hypothalamic–pituitary–adrenal (HPA) axis form the core bodily stress response system that establishes adaptive functioning and underlies mental health (Del Giudice et al., 2011). Production of both SNS-regulated alpha-amylase and HPA- regulated cortisol undergo considerable changes in adolescence (Susman et al., 2010). As this period is also characterized by heightened risk for mental health problems (Hazen et al., 2010), it is vital to understand the associations between mental health and alpha-amylase and cortisol. While the basal levels and responses to stress are agreed to be important for neuroendo- crinological functioning, less research has focused on the role of endocrinological symmetry between alpha-amylase and cortisol (Jones et al., 2020). This study analyzes how internalizing and externalizing symptoms in late adolescence are associated with dynamics between alpha-amylase and cortisol stress reactivity in a normative adolescent sample. Finally, previous research has mainly focused on either performance-related or social evalua- tive stress (Figueiredo et al., 2020; Wadsworth et al., 2019), whereas we focus on the more developmentally salient stress evoked by adolescent–mother conflict discussion. How is mental health associated with adolescent alpha-amylase and cortisol reactivity and coordination? Mervi Vänskä1, Samuli Kangaslampi1, Jallu Lindblom1,2, Raija-Leena Punamäki1, Mirva Heikkilä1, Lotta Heikkilä1, Aila Tiitinen3 and Marjo Flykt1,3 Abstract To better understand the role of neuroendocrinological regulation in adolescent mental health, stress reactivity needs to be analyzed through both the autonomic nervous system (ANS) and the hypothalamic–pituitary–adrenal (HPA) axis. Accordingly, this study examined how adolescents’ internalizing and externalizing mental health symptoms are associated with their salivary alpha-amylase and cortisol levels, responses, and coordination (symmetry versus asymmetry). We utilized a developmentally salient stress task of mother–adolescent conflict discussion. Eighty 18–20-year-old late adolescents (55% girls) participated in a home laboratory assessment involving a 10-min conflict discussion with their mothers. Five adolescent saliva samples were collected to measure alpha-amylase and cortisol levels before, immediately after, and in 10-min intervals following the conflict discussion, to indicate stress reactivity. Adolescents had reported their internalizing (depression, anxiety, somatization) and externalizing (inattention, hyperactivity, anger control problems) symptoms 1 year earlier as part of a prospective family study. Internalizing symptoms were associated with adolescents’ high baseline cortisol levels, but not with cortisol responses or alpha-amylase levels or responses. In contrast, externalizing symptoms were associated with blunted alpha-amylase responses. Neither internalizing nor externalizing symptoms were associated with asymmetry between alpha-amylase and cortisol reactivity. The mother–adolescent conflict discussion was relevant as a stress stimulus to induce neuroendocrinological stress responses in adolescents. The nature of mental health problems was important for stress reactivity, yet, we found no evidence about mental health problems being related to endocrinological asymmetry in adolescents. Keywords Stress reactivity, adolescence, HPA-axis, cortisol, alpha-amylase 1Tampere University, Finland 2University of Turku, Finland 3University of Helsinki, Finland Corresponding author: Mervi Vänskä, Faculty of Social Sciences/Psychology, Tampere University, FI-33014 Tampere, Finland. Email: mervi.vanska@tuni.fi 1208965 JBD0010.1177/01650254231208965International Journal of Behavioral DevelopmentVänskä et al. research-article2023 Empirical Paper 2 International Journal of Behavioral Development 00(0) Dual Stress Response System As the functioning of ANS and HPA are closely integrated and cross-regulated, they can be considered dual components of the human stress response system (Ellis et al., 2006). The Adaptive Calibration Model (ACM) by Del Giudice and colleagues (2011) informs about the integrated functioning of alpha-amylase and cortisol responses to stress and threat. Based on the evolutionary developmental framework (Boyce & Ellis, 2005), the ACM pos- tulates that multiple components of the stress response system, differing in timing and sensitivity to the type of stressor, work together to serve human growth, maturation, mental health, and social bonding in diversely demanding environments (Del Giudice et al., 2011). In line with that, the SNS activates an acute, “fight or flight” response by increasing heart rate and respiration and by releasing the catecholamines epinephrine and norepinephrine (Nater & Rohleder, 2009). Salivary alpha-amylase serves as a useful bio- marker of the rapid SNS reactivity (Granger et al., 2007). It con- centrates immediately, peaks at approximately 5–10 min and is attuned by 15 min after the stress trigger (Nater et al., 2013). The HPA axis, in turn, represents a delayed, long-term response to stressors that cannot be attuned by the ANS alone. Cortisol eleva- tion starts at around 5 min, peaks between 10 and 30 min and returns to baseline usually within 1 hr after the stress trigger (Allwood et al., 2011; Del Giudice et al., 2011). Considering the nature of stress, alpha-amylase has been sug- gested to be especially reactive to novelties, opportunities, and challenge (Schumacher et al., 2013; Van Stegeren et al., 2008), whereas unpredictable and uncontrollable events and threats to interpersonal security are known to trigger cortisol reactivity (Strahler et al., 2017). The potential stress-specific activation of alpha-amylase and cortisol may serve adaption by allowing a bet- ter goodness-of-fit in responding to demands that involve stress- ors of different nature (Van Stegeren et al., 2008). Mental Health Problems and Stress Response System Dysfunctional ways of regulating stress typically involve either neuroendocrinological hyper- or hypoactivation, evident in excessive or insufficient basal levels and increased or decreased reactivity to stress (Del Giudice et al., 2011; Guerry & Hastings, 2011). There is some evidence that these follow the well-estab- lished symptom dimensions (Kotov et al., 2017), in that hyperac- tivation associates with internalizing and hypoactivation with externalizing symptoms (Segal, 2016). It is noteworthy that there is some evidence also for the narrow symptom-specific dysregu- lation patterns, particularly regarding depression and anxiety (Fiksdal et al., 2019; Steudte-Schmiedgen et al., 2017; Yoon & Joormann, 2012). Yet, systematic reviews have also pointed out inconsistencies in the findings and criticized small samples and effect sizes (Figueiredo et al., 2020; Jones et al., 2020). Considering the high comorbidity between the narrow symptom categories, we focus on the broader dimensions of internalizing and externalizing. Concerning internalizing symptoms (e.g., depression and anxiety), systematic reviews generally suggest an association with increased cortisol levels and responses (Hartman et al., 2013; Wadsworth et al., 2019). The meta-analysis by Lopez-Duran and colleagues (2009) evidenced both higher cortisol basal levels and stronger responses to stress in depressed as compared to non-depressed adolescents. Similarly, adoles- cents with generalized or social anxiety showed increased corti- sol levels and responses (Funke et al., 2017; Segal, 2016). There is, however, also some evidence of depressive adolescents and adults showing low cortisol levels and responses (Burke et al., 2005; Harkness et al., 2011), particularly in chronic conditions (Booij et al., 2013). Internalizing problems have also been found to associate with dysregulated SNS (Segal, 2016), which is likely to represent the intensive distress and vigilance to threat inherent in the symp- toms. Along these lines, studies confirm that severe internalizing symptoms (Wadsworth et al., 2019) and anxiety disorders (Schumacher et al., 2013) in children and adolescents are associ- ated with high basal level and increased responses of alpha-amylase. Concerning externalizing symptoms (e.g., aggression and conduct disorders), systematic reviews suggest a general associa- tion with low cortisol basal levels and decreased responses to stress (Figueiredo et al., 2020; Guerry & Hastings, 2011). For instance, adolescents with externalizing (Hartman et al., 2013) and attention deficit hyperactivity disorder (ADHD) symptoms (Van West et al., 2009) showed lower cortisol levels and responses to stress than controls. Other studies found that aggression and conduct disorders were associated with adolescents’ lower corti- sol levels, but not with altered responses (Alink et al., 2008). Research further illustrates that externalizing symptoms are associated with hypoactivation of both HPA and SNS. For exam- ple, adolescents with externalizing problems showed decreased cortisol responses and low basal alpha-amylase levels (Bae et al., 2015), and those with severe aggression showed both decreased cortisol and alpha-amylase reactivity, indicated by levels and responses (Gordis et al., 2006). Wadsworth and colleagues (2019) further elaborated that externalizing symptoms were particularly evident among adolescents with both low cortisol and low alpha- amylase levels during the recovery from stress. However, there are also studies showing similar dysregulated stress reactivity, regardless of the nature (internalizing vs externalizing) of mental health problems (Lorber, 2004; Vigil et al., 2010). Cortisol and Alpha-Amylase Asymmetry The high interdependence between ANS and HPA makes it rea- sonable to anticipate some symmetry between the systems, meaning that marked activation in one is expected to be accom- panied by activity in the other. Instead, a failure in coordination, that is, asymmetry between these systems, can indicate dysregu- lation relevant for mental health problems (Bauer et al., 2002; Schumacher et al., 2013). Such asymmetry is indicated by a dis- proportionate ratio between alpha-amylase and cortisol levels and/or responses. Asymmetry is thus best apparent in interactive effects, for example, by symptoms associating with heightened cortisol reactivity only when alpha-amylase reactivity is blunted, or vice versa (Allwood et al., 2011; Bauer et al., 2002). Reviews provide some evidence that internalizing (but not externalizing) symptoms are associated with asymmetry between cortisol and alpha-amylase levels and responses (Jones et al., 2020; Wadsworth et al., 2019). As examples of asymmetric co- activation, clinically significant internalizing problems have Vänskä et al. 3 been found to associate with the combination of heightened alpha-amylase and blunted cortisol reactivity in children (Bae et al., 2015). However, other studies have reported internalizing symptoms to be associated with the opposite asymmetric pattern, that is, blunted alpha-amylase and heightened cortisol reactivity (Ali & Pruessner, 2012; Allwood et al., 2011). Further research is thus needed to clarify the role of asymmetry between cortisol and alpha-amylase reactivity in adolescent mental health. Interpersonal Stress While adolescents strive toward autonomy, their relationship with parents, especially the mother, remains an important socioe- motional context for their development and wellbeing (Rosenthal & Kobak, 2010). Hence, it is not surprising that the quality of the parent–adolescent relationship is important for adolescent mental health (Zhang et al., 2021). Researchers agree that the most stressful stimuli in childhood reflect threat to the primary source of attachment security, and in adolescence conflicts with parents can signify fear and threat of losing support and acceptance of the attachment figure (Gunnar et al., 2009). However, only a few studies regarding adolescent mental health and neuroendocrinological reactivity have used adoles- cent–parent conflict discussion as a stress stimulus. Both inter- nalizing and externalizing symptoms (Klimes-Dougan et al., 2001) as well as social withdrawal and social anxiety (Granger et al., 1994) were found to be associated with adolescent height- ened cortisol responses to conflict discussion. Despite the impor- tance of interpersonal conflict with parents as a cause of adolescent stress, previous studies have predominantly measured stress reactivity related to performance and social evaluative stress (such as public speaking in Trier’s Social Stress Task; Gunnar et al., 2009) and peer exclusion (Stroud et al., 2009). This study is one of the first to examine adolescent neuroendocrino- logical stress reactivity including both alpha-amylase and corti- sol in the context of adolescent–mother conflict discussion. Aims of the Study This study aims to describe associations between adolescent men- tal health problems and the levels, responses, and coordination between salivary alpha-amylase and cortisol in relation to the interpersonal stress of an adolescent–mother conflict discussion. First, we separately examine the associations between internaliz- ing (depression, anxiety, somatization) and externalizing (inatten- tion, hyperactivity, anger control problems) symptoms and the basal levels and responses of alpha-amylase and cortisol. Based on previous research (Figueiredo et al., 2020; Hartman et al., 2013), we hypothesize that adolescent internalizing symptoms are associated with heightened and externalizing symptoms with blunted levels and responses of alpha-amylase and cortisol across the stress task. Second, we examine how adolescent internalizing and externalizing symptoms are associated with the mutual coor- dination, that is, symmetry versus asymmetry, between alpha- amylase and cortisol reactivity. Based on previous research (Jones et al., 2020; Wadsworth et al., 2019), we assume internalizing symptoms to be associated with asymmetric responses, indicated either by blunted alpha-amylase and heightened cortisol, or by heightened alpha-amylase and blunted cortisol. Methods Participants A subsample of 80 adolescents at the age of 18–20 years (M = 19.27, SD = 0.48) were recruited during late 2018 to early 2020 from a prospective Finnish family study (Miracles of Development) to take part in a home laboratory assessment. The original sample, recruited in pregnancy, consisted of couples with infertility history and successful assisted reproductive treat- ment (ART; n = 484) and naturally conceiving couples (NC; n = 469). They further participated in questionnaire study waves during the child’s infancy, middle childhood, and late adoles- cence. The late adolescence questionnaire study involved 449 adolescents, 553 mothers, and 340 fathers, and took place about a year before the home laboratory assessment (late 2017 to early 2019), when the adolescents were 17−19 years old (M = 18.23, SD = 0.34). A more detailed description of the original sample is presented elsewhere (Tammilehto et al., 2021). A disproportionate stratified sampling procedure was used with the aim to collect a subsample of 100 late adolescents for the analysis of neuroendocrinological stress reactivity. However, due to the COVID-19 pandemic, sampling was interrupted in March 2020, and only 80 adolescents were examined. The sampling started in November 2018. Stratification was based on a risk index, focusing on multiple family risks during the pregnancy and infancy. The risk index was a sum of 21 variables that cover mothers’ and fathers’ mental health, interparental relationship, and parenting stress. Stratification was balanced using child sex and ART status. Thus, the subsample was representative of ado- lescents with high variability in the early life family environment (i.e., ranging from very low to very high risk). For details about the procedure, see the study by Ilomäki and colleagues (2022). The project complies with the Code of Ethics of the World Medical Association (WMA) Declaration of Helsinki 1964– 2014. The Ethical Board of Helsinki University Central Hospital approved the methods and data collection of the questionnaire study (HUS/1566/2017) and the home laboratory study (HUS/1477/2018). During both study phases, participants gave their written informed consent. In case the family indicated a need for psychological help, a psychotherapist in the research group was available for consultation. Furthermore, adolescents exhibiting clinically significant non-normality in neuroendo- crinological stress levels or responses were contacted and referred to further examination. Study Procedure Late adolescence questionnaire data were used in this study for the part of adolescent self-reported mental and somatic health and demographic factors as well as mother-reported socio-eco- nomic factors. For the home laboratory assessment, a researcher (who was also a psychologist) met the adolescent–mother dyads either at their home or at a family clinic, according to their prefer- ences. All visits took place in the late afternoon during the same hours and lasted for about 2 hr. Before the visit, the researcher sent instructions regarding the saliva sampling to the participating adolescents and mothers by e-mail or SMS. Adolescents were asked not to eat or drink any- thing, nor smoke for an hour before the session, and to refrain from 4 International Journal of Behavioral Development 00(0) alcohol use and heavy exercise from the previous night onwards. These requirements were aimed to prevent acute substance effects on HPA axis and ANS functioning (Strahler et al., 2017). Figure 1 presents the phases and time points of cortisol and alpha-amylase assessments. To sum it up, after arrival, the researcher introduced the tasks at hand and asked participants to fill in questionnaires about factors that may affect cortisol and alpha-amylase functioning. The participants were then asked to rinse their mouths (10–15 min before the first saliva sampling). The actual procedure involved: (a) a relaxation period when par- ticipants were asked to sit or lie down comfortably and listen to a 10-min relaxation tape that consisted of a progressive muscle relaxation and guided imagery; (b) the first (pre-conflict task) saliva sample collection; (c) defining the conflict discussion topic, that is, the adolescent recorded her or his topic together with the researcher; (d) the adolescent and the mother listened to the recording without the researcher’s presence and then dis- cussed about the conflict for 10 min, they were instructed to use the whole time and the discussion was video recorded; (e) the second (post-conflict task) saliva sample collection immediately after the conflict discussion; (f) evaluation of subjective stress during and satisfaction with the conflict discussion; (g) the third (early recovery phase) saliva sample collection at 10 min after the end of conflict discussion; (h) the fourth (mid-recovery phase) saliva sample collection after a free resting period and exactly 20 min after the end of conflict discussion; and (i) the fifth and final (late recovery phase) saliva sample was collected at 30 min after the end of conflict discussion. Importantly, from early recovery to late recovery phases, the participants were instructed to engage in calm activity, reading, or listening to music. Measures Mental Health Problems. Adolescent mental health problems were measured using the Self-Report of Personality─Adolescent (SRP─A) scales of the Behavior Assessment System for Chil- dren, Third Edition (BASC-3; Reynolds & Kamphaus, 2015). Six symptom scales were utilized: depression (12 items; e.g., “I just don’t care anymore”), anxiety (13 items; e.g., “I worry but I don’t know why”), somatization (7 items; e.g., “I have trouble breathing”), attention problems (8 items; e.g., “I am easily dis- tracted”), hyperactivity (8 items; e.g., “I have trouble sitting still”), and anger control problems (10 items; e.g., “When I get angry, I want to hurt someone”). The adolescents used dichoto- mous (1 = True or 0 = False) and 4-point Likert-type scale (from 0 = Never to 3 = Almost always) to answer whether or how often they had had the problem during the last month. Sum scores were constructed for internalizing (depression, anxiety, and somatization) and externalizing (attention problems, hyperactiv- ity, and anger control problems) symptoms, with higher scores indicating more severe symptoms. The reliabilities were α = .83 for internalizing and .71 for externalizing symptoms. Demographic and Health Information. Adolescents’ sex was determined from the original data collected right after birth. Ado- lescents self-reported on their age, education, and living arrange- ments. They further estimated their general somatic health, chronic illnesses, and psychiatric/neuropsychiatric disorders. Mothers reported about parental divorce and family’s level of monthly income. Right after the conflict discussion, adolescents self-evaluated the amount of subjective stress during the discus- sion (from 1 = not at all to 5 = extremely stressed). Factors Potentially Affecting Saliva Analyses. Factors that might affect saliva analyses were measured by 14 items, devel- oped particularly for this study and based on literature (Strahler et al., 2017). They covered chronic and transient illnesses, den- tistry, medications, contraceptives, nicotine, alcohol, and caf- feine use, heavy exercise, sleep quality, and major losses and stress during the past 6 months, as well as stressful events during the day of the saliva collection. Adolescent medication use was asked about generally, and also specifically concerning thyroxin and corticosteroids. Saliva Samples and Adolescent−Mother Conflict Discus- sion. ANS functioning was measured by alpha-amylase level in the first four collected saliva samples (pre-conflict task, post- conflict task, and recovery phases at 10 and 20 min), indicating rapid neuroendocrinological responses to the acute stressor. HPA axis functioning was measured by cortisol level in the five saliva Phases Duration Saliva sampling Preparation (20 min) and guided relaxation (10 min) Defining conflict (10 min) and conflict discussion (10 min) Resting Resting Resting End of the procedure 30 min 20 min 9 min 9 min 9 min ~40 min 1. 2. 3. 4. 5. Pre- conflict Post-conflict (+0 min) Recovery phases (+10min, +20min and +30min) Figure 1. Phases and Time Points of Saliva Assessments. Vänskä et al. 5 samples (pre-conflict task, post-conflict task, and recovery phases at 10, 20, and 30 min), indicating slower neuroendocrino- logical responses. Salivary samples were stored at −5°C in the laboratory of the Finnish Institute of Occupational Health. Alpha-amylase and cor- tisol were analyzed using a commercial expanded range high sen- sitivity Enzyme Immunoassays, EIA kit (Salimetrics; State College, PA). Inter- and intra-assay critical values were less than 5% and 10%, respectively. Statistical Analyses To answer the first research question about the associations between mental health symptoms and alpha-amylase and cortisol levels and responses, we used longitudinal linear mixed modeling (LMM) with restricted maximum likelihood estimation. In these models with alpha-amylase and cortisol values at each time point as outcomes, we included (a) the main effects of internalizing and externalizing symptoms on basal levels, (b) the linear and quad- ratic effects of time elapsed (in minutes since the pre-conflict task assessment), and (c) the interaction effects between internalizing and externalizing symptoms and linear and quadratic time. Random intercepts and random slopes for the effect of time on alpha-amylase and cortisol levels were included when they sig- nificantly improved model fit according to the log-likelihood test. Autoregressive covariance structures were included if models converged and they improved fit according to the log-likelihood test. Otherwise, variance component structures were used. Our second research question was about mental health prob- lems associating with asymmetry between alpha-amylase and cortisol. Similar to the study by Martinez-Torteya et al. (2017), we used areas under the curve (AUC) to analyze alpha-amylase and cortisol responses. However, in contrast to the study by Martinez-Torteya et al. (2017), who were interested in total response, we examined the change (increase or decrease) from pre-conflict task into the post-conflict task and recovery phases for both alpha-amylase and cortisol by using the AUC with respect to increase (AUCI; Pruessner et al., 2003). We calculated the AUCI using the summation of trapezoids method presented by Pruessner et al. (2003). Importantly, we tested the effects of asymmetry utilizing an interaction term of alpha-amylase AUCI and cortisol AUCI, computed using mean centered values. These interaction terms (as well as the corresponding main effect terms) were included in regression analysis as independent variables, and adolescent’s internalizing and externalizing symptoms as dependent variables. We used Spearman correlation analyses for examining associations between study variables, and analysis of variance (ANOVA) for associations between categorical demo- graphics, factors potentially affecting the saliva analyses, and alpha-amylase and cortisol reactivity. Data were missing for some mental health variables for 9 out of 80 participants and for the included confounding factors for 5 out of 80 participants. Data were excluded in a pairwise manner. Due to missing data, 67 participants contributed data to longitudinal LMM and regression models. Those excluded did not differ from those included in the LMM in terms of sex distribution, nor in basal levels or AUCI of cortisol and alpha-amylase. We carried out analyses of the two research questions by using R 4.0.5 (R Core Team, 2021) and the nlme 3.1-152 R package (Pinheiro et al., 2021) for LMM. Covariates and Outliers Adolescent sex was included as a covariate in all analyses as the literature suggests neuroendocrinological sex differences (Kudielka & Kirschbaum, 2005). Furthermore, contraception was used as a covariate in all analyses, as contraceptive medica- tion containing estrogen is known to affect stress hormone levels and responses (Gervasio et al., 2022). In LMM, the effects of the covariates were estimated for both the basal level and the linear and quadratic shapes of the responses. The data were screened for physiologically impossible or totally implausible values of alpha-amylase or cortisol, but none were identified. All cases with very high values (> 3 SD above mean) in (a) baseline values of alpha-amylase or cortisol, (b) alpha-amylase or cortisol responses (AUCI), or (c) the interaction of the AUCI of alpha-amylase and cortisol (a total of 6 out of 80 cases) were individually examined. One participant reported using glucocorticoid medication. As a sensitivity analysis, we ran all our statistical analyses excluding this participant, but this did not affect the results. We report the results with this participant included. To counter effects of extreme outliers, we winsorized alpha- amylase and cortisol values that were more than three standard deviations above the mean. Five values of four participants for alpha-amylase and 12 values of four participants for cortisol were winsorized due to high deviation from the mean. To account for non-normality, we further square-root transformed alpha-amyl- ase values and log-transformed cortisol values, in line with con- ventional practices in the field (Martinez-Torteya et al., 2017). Results Descriptive Statistics Table A1 (in Supplementary Appendix) presents demographic char- acteristics of the subsample. Approximately half (55%) of the ado- lescents were girls, and almost a half (49%) were conceived with ART. Three quarters (76%) lived with the nuclear family. About 17% had divorced parents. A majority (81%) studied in high school and about a tenth in vocational school. Almost half of the families had monthly income of more than 7,500 euros, close to average for Finnish families (Official Statistics of Finland, 2022). A majority (82%) of adolescents estimated their health as good or pretty good, and about a tenth reported a diagnosed psychiatric or neuropsychi- atric disorder (e.g., depression, eating disorder, or ADHD). Table A2 (in Supplementary Appendix) summarizes the pres- ence of factors potentially affecting the saliva analyses. About a third of the adolescents had had some transient illness in the past 6 months and 17% had chronic illness. More than a half had used painkillers during the two previous weeks and 18% were on regu- lar medication. Two participants used an asthma spray and one used thyroxine medication, but none used cortisone cream or pills. Nearly half of the girls indicated use of hormonal contraception. Regular smoking was rare (5%), but caffeine intake (80%) was common. Two-thirds (67%) reported moderate (i.e., one or two portions per week) alcohol use. About 13% of the adolescents reported major losses and stress during the past 6 months, and 13% reported stressful events during the saliva assessment day. The mean level of subjective stress during the conflict discussion was 1.97 (SD = 1.00), and did not differ significantly between boys (M = 1.82) and girls (M = 2.10), t(71.62) = 1.27, p = .208. 6 International Journal of Behavioral Development 00(0) To consider the need to include covariates in the main analyses in addition to sex and hormonal contraception, we examined whether demographics and factors potentially affecting the saliva analyses would be associated with the main study variables (i.e., mental health, alpha-amylase, and cortisol). ANOVAs showed that smoking was significantly associated with heightened mental health problems, for both internalizing, F(1,70) = 8.09, p = .006, η2 = .11, and externalizing symptoms, F(1,70) = 14.82, p = .0001, η2 = .18. As only a few (n = 5; 6%) adolescents smoked regularly and smoking was associated only with mental health (not with alpha-amylase or cortisol), we decided not to include it as an additional covariate. Table A3 (in Supplementary Appendix) shows the mean lev- els of alpha-amylase and cortisol at different assessment time points, separately for boys and girls. These mean levels are also visualized in Figures A1 and A2 (in Supplementary Appendix). Table 1 shows the correlations between the study variables, including maximum elevation (i.e., peak minus the pre-conflict task) values of alpha-amylase and cortisol. Female sex correlated with lower levels of maximum cortisol elevation (r = –.39) and lower AUCI of cortisol (r = –.38), and hormonal contraception correlated with lower maximum cortisol elevation (r = –.23). Externalizing symptoms (r = –.26) correlated with lower levels of maximum alpha-amylase elevation. Mental Health and Alpha-Amylase Levels and Responses Table 2 presents results on adolescents’ mental health problems associating with basal alpha-amylase levels (main effect) and responses to the conflict discussion (Time1 for linear and Time2 for quadratic shapes). Allowing for random intercepts and slopes improved model fit. Autoregressive covariance structure could not be tested due to model non-convergence. As hypothesized, externalizing symptoms were associated with blunted alpha- amylase responses, as indicated by significant interaction terms (i.e., Time1 × Externalizing for linear and Time2 × Internalizing for quadratic effects). Against our hypothesis, however, exter- nalizing symptoms did not significantly associate with low basal levels of alpha-amylase (non-significant externalizing main effect), and internalizing symptoms did not associate with either high basal levels or heightened responses of alpha-amylase. Concerning covariates, girls showed more blunted alpha-amyl- ase responses than boys (significant Time1 × Sex and Time2 × Sex effects). Mental Health and Cortisol Levels and Responses Table 3 presents results on adolescents’ mental health problems associating with basal cortisol levels (main effects) and responses to the conflict discussion (Time1 for linear and Time2 for quad- ratic shapes). Allowing for random intercepts and slopes improved model fit. Autoregressive covariance structure did not improve model fit, and was not included. As hypothesized, inter- nalizing symptoms were associated with high basal cortisol lev- els, indicated by significant main effect of these symptoms. Yet, against our hypothesis, internalizing symptoms were not associ- ated with heightened cortisol responses to the conflict discussion, as indicated by non-significant interaction effects with time (i.e., Time1 × Internalizing for linear and Time2 × Internalizing for Table 1. Correlations between Levels of Alpha-Amylase and Cortisol, Mental Health Variables, and Demographic Variables among Adolescents During the Family Conflict Discussion. Variable 1 2 3 4 5 6 7 8 9 1. AA T1 2. AA maximum elevation 0.23* [0.01, 0.43] 3. AUC AA 0.06 [−0.16, 0.28] 0.92** [0.88, 0.95] 4. Cortisol T1 0.20 [−0.02, 0.40] −0.03 [−0.25, 0.19] −0.11 [−0.32, 0.12] 5. Cortisol maximum elevation −0.08 [−0.30, 0.14] −0.12 [−0.33, 0.11] −0.07 [−0.29, 0.15] −0.15 [−0.36, 0.07] 6. AUC cortisol −0.11 [−0.33, 0.11] −0.04 [−0.25, 0.19] 0.06 [−0.17, 0.27] −0.15 [−0.36, 0.07] 0.82** [0.73, 0.88] 7. Female sex −0.07 [−0.29, 0.15] −0.09 [−0.30, 0.14] −0.16 [−0.37, 0.06] −0.11 [−0.32, 0.11] −0.39** [−0.56, −0.18] −0.38** [−0.56, −0.18] 8. Hormonal contraception 0.11 [−0.12, 0.33] 0.10 [−0.13, 0.32] 0.03 [−0.20, 0.25] 0.13 [−0.10, 0.35] −0.23* [−0.43, −0.00] −0.18 [−0.39, 0.05] 0.49** [0.30, 0.65] 9. Internalizing symptoms −0.06 [−0.29, 0.17] −0.16 [−0.38, 0.08] −0.12 [−0.34, 0.12] 0.11 [−0.13, 0.33] 0.06 [−0.17, 0.29] 0.01 [−0.22, 0.24] 0.24* [0.01, 0.45] 0.05 [−0.19, 0.28] 10. Externalizing symptoms −0.20 [−0.41, 0.03] −0.26* [−0.47, −0.03] −0.15 [−0.37, 0.09] −0.02 [−0.25, 0.21] 0.00 [−0.23, 0.24] −0.05 [−0.28, 0.19] 0.14 [−0.10, 0.36] −0.05 [−0.29, 0.19] 0.65** [0.49, 0.77] Note. AA: salivary alpha-amylase; cortisol: salivary cortisol; AUCI: areas under curve (with respect to increase). Log-transformed nmol/L values used for cortisol. Square-root-transformed nmol/L values used for alpha-amylase. Values in square brackets indicate the 95% confidence interval for each correlation. Total N = 80. Pairwise deletion used for missing data. *p < .05; **p < .01. Vänskä et al. 7 quadratic effects). Furthermore, against our hypothesis, external- izing symptoms did not significantly associate with either lower basal levels or blunted responses of cortisol. Concerning covari- ates, results show that sex predicted basal levels (main effect) and Table 2. Fixed Effects of Internalizing and Externalizing Mental Health Symptom on Levels of Salivary Alpha-Amylase at Different Time Points for Adolescents Experiencing Family Conflict Discussion. Predictor B SE 95% CI t Intercept 13.25 1.16 [10.97, 15.54] 11.44*** Time1 0.38 0.06 [0.26, 0.50] 6.23*** Time2 −0.009 0.001 [−0.01, −0.006] −6.75*** Main effects Female sex −0.77 1.27 [−3.14, 1.77] −0.61 Hormonal contraception 1.55 1.42 [−1.30, 4.40] 1.09 Internalizing symptoms 0.33 0.60 [−0.88, 1.53] 0.55 Externalizing symptoms −0.95 0.72 [−2.38, 0.48] −1.32 Interaction effects Time1 × Sex −0.13 0.07 [−0.27, −0.002] −2.00* Time2 × Sex 0.003 0.001 [0.0001, 0.006] 2.05* Time1 × Contraception 0.09 0.08 [−0.06, 0.24] 1.17 Time2 × Contraception −0.002 0.002 [−0.005, 0.001] −1.21 Time1 × Internalizing 0.02 0.03 [−0.05, 0.08] 0.57 Time2 × Internalizing −0.0006 0.0007 [−0.002, 0.0008] −0.80 Time1 × Externalizing −0.08 0.04 [−0.16, −0.005] −2.10* Time2 × Externalizing 0.002 0.0008 [0.0006, 0.004] 2.66** Note. CI: confidence interval. Based on 268 observations of 67 participants at four time points. Linear mixed model with random intercepts and slopes allowed. Dependent variable is square-root transformed level of salivary alpha-amylase measured in nmol/L. Main effects refer to baseline level of alpha- amylase. Time1 refers to linear effect of time elapsed since first measurement, measured in minutes. Time2 refers to quadratic effect of time elapsed since first measurement, measured in minutes. Unstandardized coefficients based on restricted maximum likelihood estimation presented. *p < .05; **p < .01; ***p < .001. Table 3. Fixed Effects of Internalizing and Externalizing Mental Health Symptoms Predicting Levels of Cortisol at Different Time Points for Adolescents Experiencing a Family Conflict Discussion. Predictor B SE 95% CI t Intercept 1.07 0.13 [0.81, 1.33] 8.05*** Time1 0.01 0.006 [0.002, 0.02] 2.38* Time2 −0.0003 0.00009 [−0.0004, −0.0001] −3.63*** Main effects Female sex −0.30 0.15 [−0.59, −0.01] −2.07* Hormonal contraception 0.26 0.16 [−0.07, 0.59] 1.58 Internalizing symptoms 0.15 0.07 [0.01, 0.29] 2.17* Externalizing symptoms −0.13 0.08 [−0.29, 0.04] 1.54 Interaction effects Time1 × Sex −0.02 0.006 [−0.03, −0.006] −2.94** Time2 × Sex 0.0002 0.0001 [0.00004, 0.0004] 2.42* Time1 × Contraception −0.003 0.007 [−0.02, 0.01] −0.46 Time2 × Contraception 0.00009 0.0001 [−0.0001, 0.0003] 0.78 Time1 × Internalizing 0.003 0.003 [−0.003, 0.009] 1.12 Time2 × Internalizing −0.00004 0.00005 [−0.0001, 0.00005] −0.96 Time1 × Externalizing −0.004 0.004 [−0.01, 0.002] −1.26 Time2 × Externalizing 0.00008 0.00005 [−0.00002, 0.0002] 1.53 Note. CI: Confidence interval. Based on 335 observations of 67 participants at five time points. Linear mixed model with random intercepts and slopes allowed. Dependent variable is log transformed level of salivary cortisol measured in nmol/L. Main effects refer to baseline level of cortisol. Time1 refers to linear effect of time elapsed since first measurement, measured in minutes. Time2 refers to quadratic effect of time elapsed since first measurement, measured in minutes. Unstandardized coefficients based on restricted maximum likelihood estimation presented. *p < .05; **p < .01; ***p < .001. cortisol responses in terms of both the linear (Time1 × Sex) and quadratic (Time2 × Sex) effect of time. Controlling for other covariates, girls showed lower basal levels of cortisol and more blunted cortisol responses than boys. 8 International Journal of Behavioral Development 00(0) Asymmetry between Alpha-Amylase and Cortisol Reactivity Against our hypotheses, the results showed no associations between adolescent mental health and the asymmetry of alpha- amylase and cortisol reactivity. More precisely, in regression analyses, neither internalizing nor externalizing symptoms were associated with the total amount of alpha-amylase and cortisol responses (i.e., AUCI) nor their interaction (i.e., AUCI cortisol × AUCI alpha-amylase), all p > .05. Thus, the results did not pro- vide support our hypothesis that high levels of internalizing symptoms would be associated with alpha-amylase−cortisol asymmetry. See Table A4 (in Supplementary Appendix) for details. Discussion Interpersonal conflicts are stressful in general and also for ado- lescents who seek autonomy from their parents and build novel bonds outside the home. We analyzed how mental health prob- lems in late adolescence were associated with the levels, responses, and coordination (symmetry vs asymmetry) between ANS-regulated alpha-amylase and HPA-regulated cortisol in a conflict discussion with the mother. Based on previous research, we hypothesized internalizing symptoms to associate with neu- roendocrinological hyperactivation and externalizing symptoms with hypoactivation (Figueiredo et al., 2020; Hartman et al., 2013). In line with our hypotheses, the nature of symptoms turned out to be decisive: internalizing symptoms associated with heightened baseline cortisol levels, whereas externalizing symp- toms associated with blunted alpha-amylase responses to the adolescent–mother conflict discussion. Against our hypothesis, however, adolescent internalizing symptoms did not to associate with the asymmetry between alpha-amylase and cortisol reactivity. The main function of alpha-amylase and cortisol production is stress adaptation and homeostatic maintenance of optimal neu- roendocrinological activity (Del Giudice et al., 2011). People suf- fering from mental health problems typically show either excessive or insufficient basal levels and altered responses to stress, reflecting regulatory dysfunction (Guerry & Hastings, 2011; Jones et al., 2020). Our findings concur with studies of depressive or anxious adolescents showing neuroendocrine hyperactivity, especially heightened cortisol levels (Hartman et al., 2013; Lopez-Duran et al., 2009), and aggressive or antiso- cial adolescents showing hypoactivity, indicated by blunted reac- tivity (Figueiredo et al., 2020). However, the associations between mental health and alpha-amylase and cortisol in our study were not as comprehensive as expected. Internalizing symptoms were associated only with high basal cortisol levels, but not with cortisol responses or alpha-amylase levels or responses during the conflict discussion. Externalizing symp- toms in turn were associated only with blunted alpha-amylase responses, but neither with low basal alpha-amylase levels nor with cortisol levels or responses. Dynamic biological, psychological, and behavioral processes may explain our findings about the specific associations between internalizing and externalizing symptoms and neuroendocrino- logical levels and responses to interpersonal stress. Internalizing symptoms are characterized by maladaptive regulation of negative emotions, which can be exemplified in rumination (Olatunji et al., 2013). Although the focusing on one’s negative emotions and thoughts reflects a mental attempt to solve prob- lems, the judgmental and repetitive nature of rumination tends to maintain dysphoric mood and distress (Michl et al., 2013). High cortisol basal levels and increased responses to stress reflect dys- functional maintenance of physiological homeostasis (Allwood et al., 2011), which was found to be associated with rumination (Zoccola & Dickerson, 2012). Other emotion regulation and per- sonality factors may similarly play important roles in explaining the association. For example, neuroticism and introversion as personality traits have been found to be linked with less-than- optimal cortisol regulation patterns (Hauner et al., 2008). Considering links between behavior and biological processes can help explain why adolescents’ externalizing symptoms were associated with blunted alpha-amylase responses. According to the sensation seeking model (Zuckerman et al., 1980), low neu- roendocrinological activity creates an unpleasant physiological state that evokes a need for stronger stimulation, which in turn is characteristic to externalizing behaviors, such as conduct disor- ders and aggression. This “unresponsivity” characteristic of externalizing problems also concur with findings that low neu- roendocrinological activity is associated with fearlessness in the face of danger and insensitivity to other people’s emotions (Blair, 2001). This may, in turn, hinder anticipation and learning of con- sequences that typically inhibit aggression and deceit in interper- sonal relations (Figueiredo et al., 2020). Yet, previous research has observed these processes in relation to more extreme forms of antisociality (Wright et al., 2019). Our findings are important as they reveal a similar link between externalizing symptoms and blunted alpha-amylase responses in a normative late adolescent group. Coordination between alpha-amylase and cortisol reactivity has been suggested as important for mental health, with asym- metry indicating dysregulation and thus mental health problems (Bauer et al., 2002; Wadsworth et al., 2019). Surprisingly, how- ever, our results provided no indication about the role of asym- metry in adolescents’ mental health. Hence, we found no support for the hypothesis that internalizing symptoms would associate with the pattern of low alpha-amylase and high cortisol levels and responses or vice versa. Reviews emphasize that results con- cerning coordination between alpha-amylase and cortisol are still mixed and based on small samples or specific groups, such as male participants only (Jones et al., 2020). It is possible that the links between mental health and asymmetry are more complex than currently understood. It is noteworthy that in modeling asymmetry, we relied on a method introduced by Martinez-Torteya et al. (2017), where coordination is reflected as the interaction of alpha-amylase and cortisol AUC. This method emphasizes asymmetry in the overall neuroendocrinological responses. Alternatively, for example, the interaction could have been analyzed in separate sequences or focusing on individual points of measurement. Yet, as the rates of reaction, peak, and recovery differ both between alpha-amylase and cortisol and between individuals, such approaches would not have been straightforward to accomplish. However, caution is needed before making definitive conclusions about the role of asymmetry between ANS and HPA. For example, it is possible that asymmetry between ANS and HPA may not always indicate regulation problems but can rather reflect a sequential nature of Vänskä et al. 9 the stress response system: a stressful situation may not trigger a cortisol response if the stress can be efficiently managed by the faster-responding mechanisms of the ANS (Del Giudice et al., 2011). Instead, HPA may only be engaged to challenges that require substantial, longer-lasting mobilization of resources. More research with varying approaches to the asymmetry and interactions are needed. Adolescent sex was important in endocrinological reactivity, as girls, compared to boys, showed lower basal levels and corti- sol and alpha-amylase responses in the conflict discussion with mother. The result concurs with research reporting more blunted cortisol and alpha-amylase reactivity to psychological stressors in women compared to men, who typically show increased reac- tivity (Kudielka & Kirschbaum, 2005). Different from earlier performance-related stress tasks, our task involved interpersonal stress with the mother, which may represent gender-specific stressor to adolescents. However, boys and girls did not differ in their subjective evaluation of the stress. Our result is an impor- tant contribution to the literature, as findings on sex differences in adolescence are inconsistent and research on alpha-amylase is scarce (Panagiotakopoulos & Neigh, 2014; Strahler et al., 2017). It is noteworthy that besides adolescent sex, other demographic factors were not significant to the cortisol and alpha-amylase lev- els or responses in our study. Finally, our sample represented a unique reproductive group, as half of the participating adolescents were conceived with ART. Some research shows higher medical risks in offspring after ART (Bergh & Wennerholm, 2020). These risks can suggest vulnera- bility to neuroendocrinological dysfunction in ART children and adolescents, but there are still very few studies (Belva et al., 2013). Our previous research, based on the same prospective study, found only minor differences in the daily cortisol levels between ART and NC children in middle childhood (Vänskä et al., 2016), and the current findings did not show any differ- ences in cortisol or alpha-amylase reactivity between ART and NC adolescents. Therefore, our results can provide some reassur- ance that infertility and ART do not place children’s neuroendo- crinological development at a long-term risk. Strengths and Limits of the Study Our results confirmed some general observations regarding the biobehavioral associations between internalizing and externaliz- ing problems and alpha-amylase and cortisol. The salivary prod- ucts of the ANS and HPA showed distinct response profiles for alpha-amylase as a rapid mobilizing response to stress and for cortisol as a later response important for recovery (Jones et al., 2020; Nater & Rohleder, 2009). The alpha-amylase response reached a peak and recovered more quickly than the cortisol response. Furthermore, sex differences in girls’ blunted and boys’ heightened cortisol and alpha-amylase reactivity support earlier findings (Kudielka & Kirschbaum, 2005; Panagiotakopoulos & Neigh, 2014). Yet, our study also has limitations regarding the research set- ting and sample size. Instead of a task-performance or social- evaluative situation, the adolescent stress responsivity in our study was analyzed in the context of an adolescent–mother con- flict discussion. Although our research setting can be considered as relatively ecologically valid, it can as well be criticized for not including fathers. Furthermore, our results are based on five time points for cortisol and four for alpha-amylase assessments. In an ideal setting, there would have been two baseline assessments, instead of our one, before the stress task to account for anticipa- tory stress (Gordis et al., 2006; Gunnar et al., 2009). Although the data collection was carried out mainly in the adolescents’ homes, the novel situation and the psychologist conducting the assess- ment may have initiated endocrinological responses. We did, however, include a relaxation task at the beginning of the experi- ment to reduce anticipatory stress. Finally, our analyses were based on one-time measurements of mental health and neuroendocrinological stress reactivity. As such, our results are correlational in nature and do not allow for conclusions about causal influences (Hamaker, 2023). Future studies could analyze directional effects using repeated meas- ures panel designs. Furthermore, our sample size was relatively small (80 adolescents in total), which led to only moderate sta- tistical power that may have prevented some effects from reach- ing statistical significance in our sample. Due to the small sample, we could only study the associations between adoles- cent mental health problems and neuroendocrinological reactiv- ity within the whole sample. Yet, the associations may have differed between girls and boys, and thus using sex as a mod- erator rather than covariate might have been informative. 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