ADHD Atten Def Hyp Disord (2014) 6:231–239 DOI 10.1007/s12402-014-0145-y
ORIGINAL ARTICLE
Oxytocin plasma concentrations in children and adolescents with autism spectrum disorder: correlation with autistic symptomatology Regina Taurines • Christina Schwenck • Benjamin Lyttwin • Martin Schecklmann • Thomas Jans • Lennart Reefschla¨ger • Julia Geissler • Manfred Gerlach • Marcel Romanos
Received: 12 January 2014 / Accepted: 16 June 2014 / Published online: 3 July 2014 Ó Springer-Verlag Wien 2014
Abstract Findings from research in animal models and humans have shown a clear role for the neuropeptide oxytocin (OT) on complex social behaviors. This is also true in the context of autism spectrum disorder (ASD). Previous studies on peripheral OT concentrations in children and young adults have reported conflicting results with the initial studies presenting mainly decreased OT plasma levels in ASD compared to healthy controls. Our study therefore aimed to further investigate changes in peripheral OT concentrations as a potential surrogate for the effects observed in the central nervous system (CNS) in ASD. OT plasma concentrations were assessed in 19 male children and adolescents with ASD, all with an IQ [ 70 (age 10.7 ± 3.8 years), 17 healthy male children (age 13.6 ± 2.1 years) and 19 young male patients with attention deficit hyperactivity disorder (ADHD) as a clinical control group (age 10.4 ± 1.9 years) using a validated radioimmunoassay. Analysis of covariance revealed significant group differences in OT plasma concentrations (F(2, 48) = 9.574, p \ 0.001, g2 = 0.285; plasma concentrations ASD 19.61 ± 7.12 pg/ml, ADHD 8.05 ± 5.49 pg/ml, healthy controls 14.43 ± 9.64 pg/ml). Post hoc R. Taurines (&) B. Lyttwin T. Jans L. Reefschla¨ger J. Geissler M. Gerlach M. Romanos Department of Child and Adolescent Psychiatry and Psychotherapy, University of Wu¨rzburg, Fu¨chsleinstraße 15, 97080 Wu¨rzburg, Germany e-mail: [email protected] C. Schwenck Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Goethe-University, Frankfurt/M., Germany M. Schecklmann Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany
analyses showed significantly higher concentrations in children with ASD compared to ADHD (p \ 0.001). After Bonferroni correction, there was no significant difference in ASD in comparison with healthy controls (p = 0.132). A significant strong correlation between plasma OT and autistic symptomatology, assessed by the Autism Diagnostic Observation Schedule, was observed in the ASD group (p = 0.013, r = 0.603). Patients with ADHD differed from healthy control children by significantly decreased OT concentrations (p = 0.014). No significant influences of the covariates age, IQ, medication and comorbidity could be seen. Our preliminary results point to a correlation of OT plasma concentrations with autistic symptom load in children with ASD and a modulation of the OT system also in the etiologically and phenotypically overlapping disorder ADHD. Further studies in humans and animal models are warranted to clarify the complex association of the OT system with social impairments as well as stress-related and depressive behavior and whether peripheral findings reflect primary changes of OT synthesis and/or release in relevant areas of the CNS. Keywords Autism spectrum disorder (ASD) High functioning Oxytocin Plasma Attention deficit hyperactivity disorder (ADHD)
Introduction Autism spectrum disorder (ASD) is characterized by deficits in social communication and interaction as well as stereotypic, rigid behaviors (DSM-5, American Psychiatric Association 2013). As the highly conserved neuropeptide oxytocin (OT) is a key modulator of diverse aspects of affiliation in mammals, including maternal behavior,
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sexual reproduction, pair bonding, social communication and social memory (de Wied et al. 1993; Insel 1997), it was suggested that the core symptoms of social impairments in ASD might be associated with a primary dysfunction of the central OT system (Insel et al. 1999; Lukas and Neumann 2013). OT is produced in supraoptic and paraventricular hypothalamic neurons and exerts effects in central brain regions and peripheral tissue. The target regions of the centrally projecting neuropeptidergic neurons are mainly involved in the modulation of emotions, stress coping and social behaviors. OT is synaptically released for a rapid information processing as well as non-synaptically also for long-term effects (Lukas and Neumann 2013). To act at distance, e.g., during labor or lactation, OT is send to and released by the posterior pituitary gland into the blood stream (Insel 1997). Findings from (epi-)genetic studies point to a possible involvement of the OT system in ASD pathophysiology, such as associations of variants of the OT receptor gene, OXTR, with autistic symptoms (e.g., Wu et al. 2005; Jacob et al. 2007; Campbell et al. 2011) and abnormalities in OXTR methylation in patients with ASD (Gregory et al. 2009; Kumsta et al. 2013). Knocking out both OXTR alleles resulted in impaired sociability and preference for social novelty, impaired cognitive flexibility, increased aggression and seizure susceptibility in mice, thus representing a putative neurobehavioral model for ASD (Sala et al. 2011). Results from research in diverse rodent models support a crucial regulatory capacity of OT concerning social cognition (Lukas and Neumann 2013). Due to the ethically and methodologically limited means to investigate central OT in humans, in this study, peripheral neuropeptide concentrations were measured as a potential surrogate for dysregulation of the central OT system. Initial reports and a 2010 study assessing plasma OT concentrations of patients with ASD found reduced OT levels in ASD compared with normally developing subjects (Modahl et al. 1998; Green et al. 2001; Al-Ayadhi 2005; Andari et al. 2010). These findings resulted in the hypothesis that low peripheral OT concentrations might be etiologically relevant and associated with ASD core symptoms. However, high OT plasma concentrations in humans and animal models were repeatedly found following negative moods, such as anxiety, depression and stress, with OT exerting anxiolytic effects and positive ways of stress coping (Insel 2010; Neumann and Landgraf 2012; Lukas and Neumann 2013). Therefore, results of elevated OT levels in young adolescent persons with ASD (Jansen et al. 2006) can be interpreted as a physiological reaction on negative moods and stress—frequently seen associated with
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ASD phenotype (Corbett et al. 2009; Spratt et al. 2012; Lai et al. 2013). In a recent study, no differences in OT plasma concentrations were observed in well-characterized collectives of autistic boys and girls compared to healthy controls; however, an association of higher OT levels with elevated anxiety scores and female sex (Miller et al. 2013). Varying results on OT dysregulation in plasma of autistic patients might also be due to abnormalities in OT peptide processing in ASD (Green et al. 2001), differences in study design, including patients of different age, gender, comorbidity and intellectual functioning as well as diverse analytical methods to quantify OT (McCullough et al. 2013). Furthermore, in some previous studies, ASD patients were not diagnosed according to the current diagnostic gold standard (Lord et al. 1989, 1994). Trying to translate the preliminary preclinical evidence of a potential pro-social effect of OT into treatment options, synthetic OT was administered intranasally, per inhalation or infusion. Results of such studies in healthy persons showed that OT administrations increased components of trust and the ability to perceive others in ways that facilitate affiliation (Heinrichs et al. 2003; Green and Hollander 2010; Bartz et al. 2011). In individuals with ASD, OT administration reduced the severity and frequency of repetitive behaviors (Hollander et al. 2003), improved emotion recognition (Guastella et al. 2010) and enhanced development of social memories (Hollander et al. 2007). However, recent randomized, placebo-controlled studies in patients with ASD did not reveal significant effects of intranasal OT on social cognition or general behavioral adjustment (Anagnostou et al. 2012; Dadds et al. 2013). In conclusion, there is evidence from animal research as well as (epi-)genetic and plasma studies in humans that dysregulation of the OT system is associated with social impairment, also in the context of ASD. The aim of our study was to further investigate the role of OT in ASD by means of peripheral quantification in well-characterized high-functioning autistic male children as a potential surrogate for effects in the CNS. Furthermore, association of plasma OT values and autistic symptom load should be explored. ASD is highly comorbid with attention deficit hyperactivity disorder, ADHD (Noterdaeme and Wriedt 2010). Both disorders share genetic factors and phenotypic features (Rommelse et al. 2010; Williams et al. 2010; Taurines et al. 2012), such as difficulties in social interactions and communication (Santosh and Mijovic 2004; Reiersen et al. 2007). Therefore, we investigated male children with ADHD as a clinical control group in order to estimate the categorical specificity of our results.
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anxiety disorder diagnosis, and five had an oppositional defiant disorder or conduct disorder. Patients with ASD or ADHD were excluded in case of a known severe somatic or neurological disorder, schizophrenia or an intelligence level below IQ 70. Healthy control subjects were screened for behavioral problems by the Child Behavior Check List (CBCL) (Achenbach and Edelbrock 1981; Do¨pfner et al. 1994) and excluded if they exhibited T-scores of [64 in the CBCL internal, external or total score, suffered from a somatic or neurological disease or were taking any medication. IQ was assessed with German assessment tools based on the following intelligence tests: Wechsler Intelligence Scale for Children (Wechsler 1949; Tewes et al. 1999; Petermann and Petermann 2007), Kaufman Assessment Battery for Children (Kaufman and Kaufman 2004; Melchers and Preuß 2009) or Culture Fair Intelligence Test (Cattell 1949; Cattell et al. 1997; Weiß 1998). The choice of different measures was due to individual clinical necessities.
Materials and methods Subjects After approval by the local ethics committee (Wuerzburg study numbers 8/06 and 227/09), this study was done in accordance with the Declaration of Helsinki. Patients were recruited at the Department for Child and Adolescent Psychiatry and Psychotherapy of the University Hospital of Wuerzburg, and written informed consent was gained. In our study, 19 male children and adolescents with ASD (age range 6.0–17.6 years), 17 normally developing male controls (age range 10.9–17.6 years) and 19 male children with ADHD (age range 6.5–14.0 years) as a clinical control group were investigated. One-way ANOVA revealed significant differences of age and IQ: ASD patients: age 10.7 ± 3.8 years, IQ 97 ± 14; ADHD patients: age 10.4 ± 1.9 years, IQ 98 ± 10; healthy controls: age 13.6 ± 2.1 years, IQ 114 ± 13 (see also Table 1 for demographics and group differences). ASD patients were diagnosed by an experienced child psychiatrist according to ICD-10 criteria. They were included, if at least two of the three behavioral subscales of the Autism Diagnostic Interview-Revised (ADI-R) passed the cutoff or if the sum of communication and social interaction cutoff for ‘‘autism spectrum’’ of the Autism Diagnostic Observation Schedule (ADOS) was reached (Lord et al. 1989, 1994; Ruehl et al. 2004; Boelte et al. 2006). The ASD group consisted of patients with highfunctioning autism, atypical autism and Asperger Syndrome. Most frequent clinical comorbidity of autistic patients was ADHD (68 %), and three individuals had an ICD-10 mood or anxiety disorder diagnosis. ADHD patients with predominantly inattentive, predominantly impulsive and combined symptomatology according to DSM-IV criteria were included. Four individuals with ADHD had a comorbid ICD-10 mood or Table 1 Demographics and group comparisons
ADI-R Autism Diagnostic Interview-Revised, ADOS Autism Diagnostic Observation Schedule, ANCOVA analysis of covariance, ANOVA analysis of variance, CBCL Child Behavior Check List, DSM-IV Diagnostic and Statistical Manual of Mental Disorders, fourth Edition
Plasma sample collection and radioimmunoassay Fasting blood samples were collected between 7.30 and 10.00 a.m. by standard phlebotomy in pre-chilled EDTA tubes. They were immediately stored on ice and centrifuged at 4 °C for 15 min at 1,6009g. Blood plasma was separated, and 300 ll aliquots were stored at -80 °C until analysis. A standardized and validated radioimmunoassay (RIA) was performed according to the protocol of Neumann et al. (2013), Kagerbauer et al. (2013). Data analysis and statistics Statistical analyses were performed with the software SPSS 18.0.0 (SPSS Inc., USA). OT group differences were analyzed by analyses of variance (ANOVA) post hoc tests
ASD
ADHD
Controls
Group comparisons
Sample size (n)
19
19
17
–
Plasma oxytocin (pg/ml)
19.6 ± 7.1
8.0 ± 5.5
14.4 ± 9.6
F = 10; df = 2,48; p \ 0.001 (ANCOVA)
Age (years)
10.7 ± 3.8
10.4 ± 1.9
13.6 ± 2.1
F = 7; df = 2,52; p = 0.001 (ANOVA)
IQ
97 ± 14
98 ± 10
114 ± 13
F = 10; df = 2,50; p \ 0.001 (ANOVA)
Comorbidity (n)
17
11
None
–
Comorbid mood or anxiety disorder (n) On medication (n)
3
4
None
–
13
7
None
–
Diagnosis
ADI-R, ADOS
DSM-IV
CBCL
–
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with the between-subjects factor group (ASD, ADHD, control children). Since groups differed significantly in these variables, age and IQ were included as covariates to control for possible confounding effects (ANCOVA) in separate analyses. A potential influence of the covariates medication status, a comorbid diagnosis of ICD-10 mood or anxiety disorder and comorbidity in general, was tested by subgroup analyses (patients with vs. without medication/comorbidity) using Student’s t test (after normal distribution was confirmed by Kolmogorov–Smirnoff test). Considering a comorbid ADHD diagnosis in autistic patients, OT plasma concentrations in ASD ? ADHD and ASD–ADHD patients were compared by Student’s t test. As data were normally distributed, the Pearson rank correlation coefficient was computed. Statistical significance was predefined as p \ 0.05, two sided. Bonferroni tests were conducted for post hoc tests where indicated with corrections for multiple testing. Above-mentioned multiple testing of subgroups (medication, anxiety/mood, ASD ? ADHD, comorbidity in general) resulted in an adjusted p \ 0.0125.
Results A significant strong correlation between plasma OT concentrations and ASD symptomatology, assessed by the relative ADOS sum of communication and social interaction scores, was observed in autistic patients (p = 0.012, r = 0.609). In patients with ADHD, a medium negative correlation of oxytocin plasma concentrations with IQ (p = 0.021, r = -0.555), but not age (p = 0.972, r = 0.009) was found. Such correlations of OT levels could not be found in patients with ASD concerning age (p = 0.274, r = 0.264) or IQ (p = 0.921, r = -0.025), neither in healthy control children (age: p = 0.617, r = 0.131; IQ: p = 0.692, r = -0.104). Neither age nor IQ confounded group differences in OT plasma concentrations between patients with ASD, ADHD and healthy controls, as indicated by ANCOVA (F(2, 48) = 9.574, p \ 0.001, g2 = 0.285). Subgroup analyses of patients with versus without medication/comorbidity did not show significant effects of the covariates medication, comorbid mood or anxiety disorder diagnosis and comorbidity in general (results not shown). Mean OT plasma concentrations of 19.61 ± 7.12 pg/ml in the ASD group, 8.05 ± 5.49 pg/ml in male children with ADHD and 14.43 ± 9.64 pg/ml in male normally developing children were measured (see Table 1). As ANCOVA can produce or hide effects in the case of differences between group means (Miller and Chapman 2001), additionally one-factorial ANOVA was performed
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Fig. 1 Oxytocin plasma concentrations. Oxytocin (OT) plasma concentrations in 19 male children and adolescents with autism spectrum disorder (ASD), 19 male children with attention deficit hyperactivity disorder (ADHD) and 17 healthy male control subjects (mean concentration ± SD)
and showed consistently a significant group difference of OT plasma concentrations, as indicated by a significant, large group main effect (F(2, 52) = 11.279, p \ 0.001, g2 = 0.303). Post hoc Bonferroni test revealed significantly increased OT concentrations in children and adolescents with ASD compared to children with ADHD (p \ 0.001). After corrections for multiple testing with the Bonferroni test, there was no significant difference between ASD and healthy controls (p = 0.132). OT plasma concentrations differed significantly in ADHD patients and healthy controls with lower levels in the ADHD group (p = 0.042) (see Fig. 1). Considering a diagnosis of comorbid ADHD in patients with ASD, there was no significant difference in OT plasma concentrations in ASD ? ADHD patients (n = 13) compared to ASD– ADHD subjects (n = 5; t = 0.345, df = 16, p = 0.734).
Discussion Findings from research in animal models and humans emphasize the role of the neuropeptide OT and the associated vasopressin system on complex social behaviors (Insel 1997; Lukas and Neumann 2013). In our study, we aimed to further clarify the impact of the OT system on ASD pathophysiology by measuring its basal plasma
Oxytocin plasma concentrations
concentrations and possible correlations with ASD symptomatology in well-characterized male autistic children and adolescents with high-functioning intellectual ability to identify potential surrogates of effects in the CNS. Due to an etiological and phenotypic overlap of the disorders, male children with ADHD were included as clinical control group to estimate the categorical specificity of our results. In autistic patients, we found increased plasma OT levels compared to normally developing boys and young male patients with ADHD. After adjustment for multiple testing with the conservative Bonferroni test, there was only a significant difference between patients with ASD and ADHD. In the ASD group, a significant strong correlation of OT plasma concentrations and autistic symptomatology was observed. Boys with ADHD differed from the healthy control group by significantly reduced OT values. The OT neuropeptide is released from the posterior pituitary gland in response to diverse stimuli, such as sexual stimulation, birth, nursing and stress. Relevant to social neuroscience, OT expressing neurons in the hypothalamus also project centrally. As a characteristic of the OT and associated vasopressin system, many actions seem to be exerted in a gender-specific way (Insel 2010; Neumann and Landgraf 2012). Due to the high heritability of ASD, it was hypothesized that genetic factors such as polymorphic variation of the OXTR and epigenetic effects result in a dysregulated OT and vasopressin system with etiological impact on ASD cores symptoms (for a review see, e.g., Insel 2010). Considering the initial studies on peripheral OT plasma measurements, it was suggested that low plasma levels are associated with autistic symptomatology (Modahl et al. 1998; Green et al. 2001; Al-Ayadhi 2005). The finding in Modahl’s initial study that OT levels correlated positively with some facets of autistic symptomatology in patients, such as deficits in social awareness and language development, already made clear, that a simple OT deficit or excess model for ASD seems to be inadequate to integrate findings. Adding to the hypothesis of a potential pro-social effect of OT, initial studies on synthetic OT administration in healthy humans resulted in an increase of trust and facilitated affiliation (Heinrichs et al. 2003; Green and Hollander 2010; Bartz et al. 2011), although a minor part of the studies even revealed anti-social effects under particular circumstances (Bartz et al. 2011). Initial promising pro-social actions of OT in persons with ASD (Hollander et al. 2003, 2007; Guastella et al. 2010) were followed by randomized, placebo-controlled studies that did not reveal significant effects of intranasal OT on social cognition or general behavioral adjustment in ASD (Anagnostou et al. 2012; Dadds et al. 2013).
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High peripheral levels of OT in humans were observed in reaction to negative emotions involving social distress, anxiety and depression (Turner et al. 1999; Gimpl and Fahrenholz 2001; Taylor et al. 2006, 2010; Tops et al. 2007; Cyranowski et al. 2008; Holt-Lunstad et al. 2011; Miller et al. 2013; Weisman et al. 2013). In rodent models, diverse forms of stress resulted in elevated OT plasma levels (Nishioka et al. 1998; Wotjak et al. 1998; Grippo et al. 2007), amongst it the exposure to novel environment and novel conspecific, stimuli that are known to be stressful for individuals with ASD (Gimpl and Fahrenholz 2001). Because the stress-induced central OT release ameliorates symptoms of anxiety and depression (Gimpl and Fahrenholz 2001), as a unifying principal of OT action, the facilitation of social encounters—by reducing associated anxiety and stress—was suggested (McCarthy 1995; Gimpl and Fahrenholz 2001; Neumann and Landgraf 2012), whereby the anxiolytic effects and modulation of physiological stress responses are mainly exerted at the level of the paraventricular nuclei and the amygdala (Kirsch et al. 2005; Domes et al. 2007, for a review, see Neumann and Landgraf 2012). The pro-social effect of OT discussed above can be interpreted as an anxiolytic action in a social context, the reduction of social anxiety as a prerequisite for social interaction (Neumann and Landgraf 2012). The anxiolytic and pro-social effects of OT are probably exerted in modulation with monoaminergic systems (such as the for ASD etiology discussed serotonin system) and steroid hormones, such as estradiol (Neumann and Landgraf 2012). Therefore initial findings of ‘‘low OT’’ in ASD might be interpreted to reflect a genetically caused etiopathologically relevant lack of this pro-social neuropeptide with the consequences of impairments in social communication and interaction as well as stereotypic behavior. ‘‘High OT’’ in ASD might, however, might be understood as a secondary phenomenon, a physiological response on negative moods and stressors—often apparent in ASD (Corbett et al. 2009; Spratt et al. 2012; Lai et al. 2013)—with the aim to ameliorate anxiety. Both explanatory models might be of validity, perhaps in different patient subgroups. Therefore our observation of elevated, however, not significantly increased OT plasma levels in male autistic children and adolescents with high-functioning cognitive ability in comparison with healthy children—that differ from initial findings on this subject—might be influenced by aspects of the autistic phenotype, respectively, comorbid conditions and elevated levels of stress. Other work groups observed—also in contrast to initial reports—elevated (Jansen et al. 2006) or unchanged (Miller et al. 2013) OT plasma levels in ASD compared to matched healthy developing controls. As Miller et al. (2013) reported of an
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association of high OT concentration with elevated anxiety scores, in future studies a detailed, dimensional assessment of comorbid anxiety and depression symptoms by specific diagnostic instruments might be desirable. To account for the varying results on OT plasma concentrations, furthermore it has been suggested that OT is processed differently in the brains of autistic patients, rather than simply at a different level: In young patients with ASD, OT pro-hormone seems to be incompletely converted into the fully processed OT, the bioactive amidated form of the neuropeptide (Green et al. 2001). As the OT processing system underlies a physiological maturation process (Whitnall et al. 1985; Morris et al. 1992), a failure of developmental progression in peptide processing, impacting on ASD phenotype, was suggested (Green et al. 2001). In our project, we found a positive correlation of OT plasma concentrations with autistic symptomatology, assessed by the relative ADOS sum of communication and social interaction score. These findings are in accordance with Modahl et al.’s initial study, who described that autistic individuals with higher OT levels were more socially and developmentally impaired (Modahl et al. 1998): They showed a greater lack of imitation, lack of modesty and less appropriate personal barriers on the Wing’s autism symptom checklist. A lack of association of OT concentrations with autistic symptomatology in some studies (Jansen et al. 2006) might result from using the ADI-R for ASD diagnostics that describes autistic symptomatology in childhood, but not current symptom load in adulthood. To our knowledge for the first time, peripheral OT values were investigated in the context of ADHD and significantly reduced OT concentrations were observed in boys with this disorder. In the group of autistic children, comorbidity with ADHD did not significantly modify OT values. Children with ADHD clinically present with difficulties in social interactions and communication (Santosh and Mijovic 2004; Reiersen et al. 2007) that exceed those, which are found in healthy control subjects (Hattori et al. 2006). Some impairments of social cognition domains resemble in ASD and ADHD, such as difficulties in emotion recognition, but there are also distinct patterns: Deficits in theory of mind, e.g., are characteristic for autistic patients and not associated with ADHD symptomatology (Taurines et al. 2012). In a first molecular genetic study on the relevance of OXTR gene variants in ADHD, there was no general association of OXTR single nucleotide polymorphisms (SNPs) with the global phenotype ADHD, but one SNP was associated with social cognitive impairments in a subgroup of patients (Park et al. 2010). Our preliminary results give first hints to dysregulation in this neuropeptide system in ADHD or in the context of associated comorbidity, such as oppositional defiant or conduct
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disorder. The finding of reduced OT levels in children with ADHD—in contrast to possibly expected increased concentrations due to high levels of stress and negative mood in this disorder (Taurines et al. 2010)—supports a potential pathophysiological impact of this finding. In future approaches, OT challenging studies in patients with ADHD might be of interest, considering the above-mentioned deficits in emotion recognition in ADHD, our results of decreased OT plasma levels and potential improvements of synthetic OT on recognition of basic facial expressions in healthy persons (Shahrestani et al. 2013). Such approaches might be useful to gain more insights into a potential link between ADHD symptomatology, respectively, its comorbidity and the OT system and to test potential pharmacological interventions. The diversity of hitherto existing study results on peripheral OT concentrations in the field of ASD research may be influenced by differences in study designs concerning diagnostic process, as the current gold standard instruments were used just in a few studies and just partly. Differing results might also be due to inclusion, e.g., of patients of different age and pubertal status, gender, comorbid conditions and level of intellectual functioning. High variability might also be due to different OT assays used, such as different RIAs or Enzyme Linked Immunosorbent Assays, and differences in preceding sample preparation. Evaluation of commercially available OT assays revealed a lack of reliability when used on unextracted samples of human fluids (McCullough et al. 2013). These assays yielded OT estimates that were wildly discrepant with an extensive body of earlier findings in validated, but more laborious assays, as they tag other molecules additionally to OT (McCullough et al. 2013). Samples in our study were extracted and quantified using a highly sensitive and specific radioimmunoassay (RIAgnosis, Munich, Germany) that was strictly standardized and validated in many animal and human studies using a wide variety of stimuli (hypertonicity, parturition, lactation, stress, etc.) to reliably detect the bioavailable neuropeptide in plasma and in central compartments, such as CSF (Neumann et al. 2013; Kagerbauer et al. 2013). Limitations of our study are relatively small sample sizes, age differences, co-medication, mostly psychostimulants, but also antidepressants and antipsychotics in the patient groups. Data on effects of methylphenidate on OT synthesis and release are missing. Results on possible effects of antidepressants (Uvna¨s-Moberg et al. 1999; Ozsoy et al. 2009; Keating et al. 2013) and antipsychotics (Uvna¨s-Moberg et al. 1992; Glovinsky et al. 1994) are inconsistent. Patients with ADHD were diagnosed by an experienced child psychiatrist according to DSM-IV criteria categorically, but unfortunately, diagnoses were not accompanied by a dimensional diagnostic instrument.
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As a further limitation of the study design in general, a recent study found no correlation between OT concentrations in plasma and CSF in 41 non-neurological and nonpsychiatric patients (Kagerbauer et al. 2013). Nevertheless, it could be clearly demonstrated that social stimuli impact also on peripheral concentrations of OT in humans (e.g., Wismer Fries et al. 2005; Seltzer et al. 2010; Schneiderman et al. 2012). Studies in rat models added to findings in humans, indicating that peripheral neurohypophysial secretion into the blood and central release of OT may occur simultaneously, but also independently of each other (Lukas and Neumann 2013). Therefore, results from neurohormone analyses in peripheral blood in general have to be interpreted cautiously and complemented by further studies on OT release in humans and studies on animal models to examine whether abnormal peripheral concentrations reflect primary changes in for the disorder relevant structures of the CNS. Strengths of our study can be seen in ASD diagnostics that included the current ‘‘gold standard’’ measures ADOS and ADI-R (Lord et al. 1989, 1994). In several published studies on plasma OT concentrations in humans, ASD diagnosis was given according to DSM-IV criteria, but was not confirmed by these internationally valid, currently state of the art instruments. Exceptions are the following studies: Jansen et al. (2006), Andari et al. (2010) who used the ADI-R (Jansen et al. 2006; Andari et al. 2010) and Miller et al. (2013), who used the ADOS (Miller et al. 2013) for autism diagnosis. Recruiting groups of pure male subjects in our study aimed to rule out gender effects on OT release (Insel 1997). Further strengths of our project include the above-mentioned strictly standardized and validated OT assay (Kagerbauer et al. 2013) and a standardized protocol with time of blood withdrawal, fasting in the morning, to rule out potential circadian effects on plasma oxytocin concentrations, as suggested in the literature (Landgraf et al. 1982; Devarajan and Rusak 2004). For the first time, still in an exploratory approach, peripheral OT concentrations were assessed also in male children with an etiologically and phenotypically overlapping disorder, ADHD. In summary, we found significant group differences, assessing plasma OT in male children and adolescents with ASD, ADHD and healthy controls and a correlation of peripheral OT levels with autistic symptomatology in patients with ASD. However, a great individual variability throughout all subjects was obvious. Preliminary results also point to peripheral OT modulations in children with ADHD. Our findings add to animal research, fMRI and (epi-)genetic studies demonstrating crucial effects of the neuropeptide OT on human affiliative behavior and suggesting a potential pathophysiologic role in ASD. A simple OT deficit or excess model for ASD seems to be inadequate to integrate complex relationships of peripheral
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OT measurements with autistic phenotype, comorbid symptom facets and other modulating factors. In the future prospective studies, following autistic children until adulthood are needed to elucidate in more detail influences of gender, age, phenotype, development, intellectual functioning, etc., on central and peripheral OT concentrations. Acknowledgments We wish to thank the patients and families who participated in this study as well as Dr. Alex C. Conner for his support with manuscript preparation.
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ORIGINAL ARTICLE
Oxytocin plasma concentrations in children and adolescents with autism spectrum disorder: correlation with autistic symptomatology Regina Taurines • Christina Schwenck • Benjamin Lyttwin • Martin Schecklmann • Thomas Jans • Lennart Reefschla¨ger • Julia Geissler • Manfred Gerlach • Marcel Romanos
Received: 12 January 2014 / Accepted: 16 June 2014 / Published online: 3 July 2014 Ó Springer-Verlag Wien 2014
Abstract Findings from research in animal models and humans have shown a clear role for the neuropeptide oxytocin (OT) on complex social behaviors. This is also true in the context of autism spectrum disorder (ASD). Previous studies on peripheral OT concentrations in children and young adults have reported conflicting results with the initial studies presenting mainly decreased OT plasma levels in ASD compared to healthy controls. Our study therefore aimed to further investigate changes in peripheral OT concentrations as a potential surrogate for the effects observed in the central nervous system (CNS) in ASD. OT plasma concentrations were assessed in 19 male children and adolescents with ASD, all with an IQ [ 70 (age 10.7 ± 3.8 years), 17 healthy male children (age 13.6 ± 2.1 years) and 19 young male patients with attention deficit hyperactivity disorder (ADHD) as a clinical control group (age 10.4 ± 1.9 years) using a validated radioimmunoassay. Analysis of covariance revealed significant group differences in OT plasma concentrations (F(2, 48) = 9.574, p \ 0.001, g2 = 0.285; plasma concentrations ASD 19.61 ± 7.12 pg/ml, ADHD 8.05 ± 5.49 pg/ml, healthy controls 14.43 ± 9.64 pg/ml). Post hoc R. Taurines (&) B. Lyttwin T. Jans L. Reefschla¨ger J. Geissler M. Gerlach M. Romanos Department of Child and Adolescent Psychiatry and Psychotherapy, University of Wu¨rzburg, Fu¨chsleinstraße 15, 97080 Wu¨rzburg, Germany e-mail: [email protected] C. Schwenck Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Goethe-University, Frankfurt/M., Germany M. Schecklmann Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany
analyses showed significantly higher concentrations in children with ASD compared to ADHD (p \ 0.001). After Bonferroni correction, there was no significant difference in ASD in comparison with healthy controls (p = 0.132). A significant strong correlation between plasma OT and autistic symptomatology, assessed by the Autism Diagnostic Observation Schedule, was observed in the ASD group (p = 0.013, r = 0.603). Patients with ADHD differed from healthy control children by significantly decreased OT concentrations (p = 0.014). No significant influences of the covariates age, IQ, medication and comorbidity could be seen. Our preliminary results point to a correlation of OT plasma concentrations with autistic symptom load in children with ASD and a modulation of the OT system also in the etiologically and phenotypically overlapping disorder ADHD. Further studies in humans and animal models are warranted to clarify the complex association of the OT system with social impairments as well as stress-related and depressive behavior and whether peripheral findings reflect primary changes of OT synthesis and/or release in relevant areas of the CNS. Keywords Autism spectrum disorder (ASD) High functioning Oxytocin Plasma Attention deficit hyperactivity disorder (ADHD)
Introduction Autism spectrum disorder (ASD) is characterized by deficits in social communication and interaction as well as stereotypic, rigid behaviors (DSM-5, American Psychiatric Association 2013). As the highly conserved neuropeptide oxytocin (OT) is a key modulator of diverse aspects of affiliation in mammals, including maternal behavior,
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sexual reproduction, pair bonding, social communication and social memory (de Wied et al. 1993; Insel 1997), it was suggested that the core symptoms of social impairments in ASD might be associated with a primary dysfunction of the central OT system (Insel et al. 1999; Lukas and Neumann 2013). OT is produced in supraoptic and paraventricular hypothalamic neurons and exerts effects in central brain regions and peripheral tissue. The target regions of the centrally projecting neuropeptidergic neurons are mainly involved in the modulation of emotions, stress coping and social behaviors. OT is synaptically released for a rapid information processing as well as non-synaptically also for long-term effects (Lukas and Neumann 2013). To act at distance, e.g., during labor or lactation, OT is send to and released by the posterior pituitary gland into the blood stream (Insel 1997). Findings from (epi-)genetic studies point to a possible involvement of the OT system in ASD pathophysiology, such as associations of variants of the OT receptor gene, OXTR, with autistic symptoms (e.g., Wu et al. 2005; Jacob et al. 2007; Campbell et al. 2011) and abnormalities in OXTR methylation in patients with ASD (Gregory et al. 2009; Kumsta et al. 2013). Knocking out both OXTR alleles resulted in impaired sociability and preference for social novelty, impaired cognitive flexibility, increased aggression and seizure susceptibility in mice, thus representing a putative neurobehavioral model for ASD (Sala et al. 2011). Results from research in diverse rodent models support a crucial regulatory capacity of OT concerning social cognition (Lukas and Neumann 2013). Due to the ethically and methodologically limited means to investigate central OT in humans, in this study, peripheral neuropeptide concentrations were measured as a potential surrogate for dysregulation of the central OT system. Initial reports and a 2010 study assessing plasma OT concentrations of patients with ASD found reduced OT levels in ASD compared with normally developing subjects (Modahl et al. 1998; Green et al. 2001; Al-Ayadhi 2005; Andari et al. 2010). These findings resulted in the hypothesis that low peripheral OT concentrations might be etiologically relevant and associated with ASD core symptoms. However, high OT plasma concentrations in humans and animal models were repeatedly found following negative moods, such as anxiety, depression and stress, with OT exerting anxiolytic effects and positive ways of stress coping (Insel 2010; Neumann and Landgraf 2012; Lukas and Neumann 2013). Therefore, results of elevated OT levels in young adolescent persons with ASD (Jansen et al. 2006) can be interpreted as a physiological reaction on negative moods and stress—frequently seen associated with
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ASD phenotype (Corbett et al. 2009; Spratt et al. 2012; Lai et al. 2013). In a recent study, no differences in OT plasma concentrations were observed in well-characterized collectives of autistic boys and girls compared to healthy controls; however, an association of higher OT levels with elevated anxiety scores and female sex (Miller et al. 2013). Varying results on OT dysregulation in plasma of autistic patients might also be due to abnormalities in OT peptide processing in ASD (Green et al. 2001), differences in study design, including patients of different age, gender, comorbidity and intellectual functioning as well as diverse analytical methods to quantify OT (McCullough et al. 2013). Furthermore, in some previous studies, ASD patients were not diagnosed according to the current diagnostic gold standard (Lord et al. 1989, 1994). Trying to translate the preliminary preclinical evidence of a potential pro-social effect of OT into treatment options, synthetic OT was administered intranasally, per inhalation or infusion. Results of such studies in healthy persons showed that OT administrations increased components of trust and the ability to perceive others in ways that facilitate affiliation (Heinrichs et al. 2003; Green and Hollander 2010; Bartz et al. 2011). In individuals with ASD, OT administration reduced the severity and frequency of repetitive behaviors (Hollander et al. 2003), improved emotion recognition (Guastella et al. 2010) and enhanced development of social memories (Hollander et al. 2007). However, recent randomized, placebo-controlled studies in patients with ASD did not reveal significant effects of intranasal OT on social cognition or general behavioral adjustment (Anagnostou et al. 2012; Dadds et al. 2013). In conclusion, there is evidence from animal research as well as (epi-)genetic and plasma studies in humans that dysregulation of the OT system is associated with social impairment, also in the context of ASD. The aim of our study was to further investigate the role of OT in ASD by means of peripheral quantification in well-characterized high-functioning autistic male children as a potential surrogate for effects in the CNS. Furthermore, association of plasma OT values and autistic symptom load should be explored. ASD is highly comorbid with attention deficit hyperactivity disorder, ADHD (Noterdaeme and Wriedt 2010). Both disorders share genetic factors and phenotypic features (Rommelse et al. 2010; Williams et al. 2010; Taurines et al. 2012), such as difficulties in social interactions and communication (Santosh and Mijovic 2004; Reiersen et al. 2007). Therefore, we investigated male children with ADHD as a clinical control group in order to estimate the categorical specificity of our results.
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anxiety disorder diagnosis, and five had an oppositional defiant disorder or conduct disorder. Patients with ASD or ADHD were excluded in case of a known severe somatic or neurological disorder, schizophrenia or an intelligence level below IQ 70. Healthy control subjects were screened for behavioral problems by the Child Behavior Check List (CBCL) (Achenbach and Edelbrock 1981; Do¨pfner et al. 1994) and excluded if they exhibited T-scores of [64 in the CBCL internal, external or total score, suffered from a somatic or neurological disease or were taking any medication. IQ was assessed with German assessment tools based on the following intelligence tests: Wechsler Intelligence Scale for Children (Wechsler 1949; Tewes et al. 1999; Petermann and Petermann 2007), Kaufman Assessment Battery for Children (Kaufman and Kaufman 2004; Melchers and Preuß 2009) or Culture Fair Intelligence Test (Cattell 1949; Cattell et al. 1997; Weiß 1998). The choice of different measures was due to individual clinical necessities.
Materials and methods Subjects After approval by the local ethics committee (Wuerzburg study numbers 8/06 and 227/09), this study was done in accordance with the Declaration of Helsinki. Patients were recruited at the Department for Child and Adolescent Psychiatry and Psychotherapy of the University Hospital of Wuerzburg, and written informed consent was gained. In our study, 19 male children and adolescents with ASD (age range 6.0–17.6 years), 17 normally developing male controls (age range 10.9–17.6 years) and 19 male children with ADHD (age range 6.5–14.0 years) as a clinical control group were investigated. One-way ANOVA revealed significant differences of age and IQ: ASD patients: age 10.7 ± 3.8 years, IQ 97 ± 14; ADHD patients: age 10.4 ± 1.9 years, IQ 98 ± 10; healthy controls: age 13.6 ± 2.1 years, IQ 114 ± 13 (see also Table 1 for demographics and group differences). ASD patients were diagnosed by an experienced child psychiatrist according to ICD-10 criteria. They were included, if at least two of the three behavioral subscales of the Autism Diagnostic Interview-Revised (ADI-R) passed the cutoff or if the sum of communication and social interaction cutoff for ‘‘autism spectrum’’ of the Autism Diagnostic Observation Schedule (ADOS) was reached (Lord et al. 1989, 1994; Ruehl et al. 2004; Boelte et al. 2006). The ASD group consisted of patients with highfunctioning autism, atypical autism and Asperger Syndrome. Most frequent clinical comorbidity of autistic patients was ADHD (68 %), and three individuals had an ICD-10 mood or anxiety disorder diagnosis. ADHD patients with predominantly inattentive, predominantly impulsive and combined symptomatology according to DSM-IV criteria were included. Four individuals with ADHD had a comorbid ICD-10 mood or Table 1 Demographics and group comparisons
ADI-R Autism Diagnostic Interview-Revised, ADOS Autism Diagnostic Observation Schedule, ANCOVA analysis of covariance, ANOVA analysis of variance, CBCL Child Behavior Check List, DSM-IV Diagnostic and Statistical Manual of Mental Disorders, fourth Edition
Plasma sample collection and radioimmunoassay Fasting blood samples were collected between 7.30 and 10.00 a.m. by standard phlebotomy in pre-chilled EDTA tubes. They were immediately stored on ice and centrifuged at 4 °C for 15 min at 1,6009g. Blood plasma was separated, and 300 ll aliquots were stored at -80 °C until analysis. A standardized and validated radioimmunoassay (RIA) was performed according to the protocol of Neumann et al. (2013), Kagerbauer et al. (2013). Data analysis and statistics Statistical analyses were performed with the software SPSS 18.0.0 (SPSS Inc., USA). OT group differences were analyzed by analyses of variance (ANOVA) post hoc tests
ASD
ADHD
Controls
Group comparisons
Sample size (n)
19
19
17
–
Plasma oxytocin (pg/ml)
19.6 ± 7.1
8.0 ± 5.5
14.4 ± 9.6
F = 10; df = 2,48; p \ 0.001 (ANCOVA)
Age (years)
10.7 ± 3.8
10.4 ± 1.9
13.6 ± 2.1
F = 7; df = 2,52; p = 0.001 (ANOVA)
IQ
97 ± 14
98 ± 10
114 ± 13
F = 10; df = 2,50; p \ 0.001 (ANOVA)
Comorbidity (n)
17
11
None
–
Comorbid mood or anxiety disorder (n) On medication (n)
3
4
None
–
13
7
None
–
Diagnosis
ADI-R, ADOS
DSM-IV
CBCL
–
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with the between-subjects factor group (ASD, ADHD, control children). Since groups differed significantly in these variables, age and IQ were included as covariates to control for possible confounding effects (ANCOVA) in separate analyses. A potential influence of the covariates medication status, a comorbid diagnosis of ICD-10 mood or anxiety disorder and comorbidity in general, was tested by subgroup analyses (patients with vs. without medication/comorbidity) using Student’s t test (after normal distribution was confirmed by Kolmogorov–Smirnoff test). Considering a comorbid ADHD diagnosis in autistic patients, OT plasma concentrations in ASD ? ADHD and ASD–ADHD patients were compared by Student’s t test. As data were normally distributed, the Pearson rank correlation coefficient was computed. Statistical significance was predefined as p \ 0.05, two sided. Bonferroni tests were conducted for post hoc tests where indicated with corrections for multiple testing. Above-mentioned multiple testing of subgroups (medication, anxiety/mood, ASD ? ADHD, comorbidity in general) resulted in an adjusted p \ 0.0125.
Results A significant strong correlation between plasma OT concentrations and ASD symptomatology, assessed by the relative ADOS sum of communication and social interaction scores, was observed in autistic patients (p = 0.012, r = 0.609). In patients with ADHD, a medium negative correlation of oxytocin plasma concentrations with IQ (p = 0.021, r = -0.555), but not age (p = 0.972, r = 0.009) was found. Such correlations of OT levels could not be found in patients with ASD concerning age (p = 0.274, r = 0.264) or IQ (p = 0.921, r = -0.025), neither in healthy control children (age: p = 0.617, r = 0.131; IQ: p = 0.692, r = -0.104). Neither age nor IQ confounded group differences in OT plasma concentrations between patients with ASD, ADHD and healthy controls, as indicated by ANCOVA (F(2, 48) = 9.574, p \ 0.001, g2 = 0.285). Subgroup analyses of patients with versus without medication/comorbidity did not show significant effects of the covariates medication, comorbid mood or anxiety disorder diagnosis and comorbidity in general (results not shown). Mean OT plasma concentrations of 19.61 ± 7.12 pg/ml in the ASD group, 8.05 ± 5.49 pg/ml in male children with ADHD and 14.43 ± 9.64 pg/ml in male normally developing children were measured (see Table 1). As ANCOVA can produce or hide effects in the case of differences between group means (Miller and Chapman 2001), additionally one-factorial ANOVA was performed
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Fig. 1 Oxytocin plasma concentrations. Oxytocin (OT) plasma concentrations in 19 male children and adolescents with autism spectrum disorder (ASD), 19 male children with attention deficit hyperactivity disorder (ADHD) and 17 healthy male control subjects (mean concentration ± SD)
and showed consistently a significant group difference of OT plasma concentrations, as indicated by a significant, large group main effect (F(2, 52) = 11.279, p \ 0.001, g2 = 0.303). Post hoc Bonferroni test revealed significantly increased OT concentrations in children and adolescents with ASD compared to children with ADHD (p \ 0.001). After corrections for multiple testing with the Bonferroni test, there was no significant difference between ASD and healthy controls (p = 0.132). OT plasma concentrations differed significantly in ADHD patients and healthy controls with lower levels in the ADHD group (p = 0.042) (see Fig. 1). Considering a diagnosis of comorbid ADHD in patients with ASD, there was no significant difference in OT plasma concentrations in ASD ? ADHD patients (n = 13) compared to ASD– ADHD subjects (n = 5; t = 0.345, df = 16, p = 0.734).
Discussion Findings from research in animal models and humans emphasize the role of the neuropeptide OT and the associated vasopressin system on complex social behaviors (Insel 1997; Lukas and Neumann 2013). In our study, we aimed to further clarify the impact of the OT system on ASD pathophysiology by measuring its basal plasma
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concentrations and possible correlations with ASD symptomatology in well-characterized male autistic children and adolescents with high-functioning intellectual ability to identify potential surrogates of effects in the CNS. Due to an etiological and phenotypic overlap of the disorders, male children with ADHD were included as clinical control group to estimate the categorical specificity of our results. In autistic patients, we found increased plasma OT levels compared to normally developing boys and young male patients with ADHD. After adjustment for multiple testing with the conservative Bonferroni test, there was only a significant difference between patients with ASD and ADHD. In the ASD group, a significant strong correlation of OT plasma concentrations and autistic symptomatology was observed. Boys with ADHD differed from the healthy control group by significantly reduced OT values. The OT neuropeptide is released from the posterior pituitary gland in response to diverse stimuli, such as sexual stimulation, birth, nursing and stress. Relevant to social neuroscience, OT expressing neurons in the hypothalamus also project centrally. As a characteristic of the OT and associated vasopressin system, many actions seem to be exerted in a gender-specific way (Insel 2010; Neumann and Landgraf 2012). Due to the high heritability of ASD, it was hypothesized that genetic factors such as polymorphic variation of the OXTR and epigenetic effects result in a dysregulated OT and vasopressin system with etiological impact on ASD cores symptoms (for a review see, e.g., Insel 2010). Considering the initial studies on peripheral OT plasma measurements, it was suggested that low plasma levels are associated with autistic symptomatology (Modahl et al. 1998; Green et al. 2001; Al-Ayadhi 2005). The finding in Modahl’s initial study that OT levels correlated positively with some facets of autistic symptomatology in patients, such as deficits in social awareness and language development, already made clear, that a simple OT deficit or excess model for ASD seems to be inadequate to integrate findings. Adding to the hypothesis of a potential pro-social effect of OT, initial studies on synthetic OT administration in healthy humans resulted in an increase of trust and facilitated affiliation (Heinrichs et al. 2003; Green and Hollander 2010; Bartz et al. 2011), although a minor part of the studies even revealed anti-social effects under particular circumstances (Bartz et al. 2011). Initial promising pro-social actions of OT in persons with ASD (Hollander et al. 2003, 2007; Guastella et al. 2010) were followed by randomized, placebo-controlled studies that did not reveal significant effects of intranasal OT on social cognition or general behavioral adjustment in ASD (Anagnostou et al. 2012; Dadds et al. 2013).
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High peripheral levels of OT in humans were observed in reaction to negative emotions involving social distress, anxiety and depression (Turner et al. 1999; Gimpl and Fahrenholz 2001; Taylor et al. 2006, 2010; Tops et al. 2007; Cyranowski et al. 2008; Holt-Lunstad et al. 2011; Miller et al. 2013; Weisman et al. 2013). In rodent models, diverse forms of stress resulted in elevated OT plasma levels (Nishioka et al. 1998; Wotjak et al. 1998; Grippo et al. 2007), amongst it the exposure to novel environment and novel conspecific, stimuli that are known to be stressful for individuals with ASD (Gimpl and Fahrenholz 2001). Because the stress-induced central OT release ameliorates symptoms of anxiety and depression (Gimpl and Fahrenholz 2001), as a unifying principal of OT action, the facilitation of social encounters—by reducing associated anxiety and stress—was suggested (McCarthy 1995; Gimpl and Fahrenholz 2001; Neumann and Landgraf 2012), whereby the anxiolytic effects and modulation of physiological stress responses are mainly exerted at the level of the paraventricular nuclei and the amygdala (Kirsch et al. 2005; Domes et al. 2007, for a review, see Neumann and Landgraf 2012). The pro-social effect of OT discussed above can be interpreted as an anxiolytic action in a social context, the reduction of social anxiety as a prerequisite for social interaction (Neumann and Landgraf 2012). The anxiolytic and pro-social effects of OT are probably exerted in modulation with monoaminergic systems (such as the for ASD etiology discussed serotonin system) and steroid hormones, such as estradiol (Neumann and Landgraf 2012). Therefore initial findings of ‘‘low OT’’ in ASD might be interpreted to reflect a genetically caused etiopathologically relevant lack of this pro-social neuropeptide with the consequences of impairments in social communication and interaction as well as stereotypic behavior. ‘‘High OT’’ in ASD might, however, might be understood as a secondary phenomenon, a physiological response on negative moods and stressors—often apparent in ASD (Corbett et al. 2009; Spratt et al. 2012; Lai et al. 2013)—with the aim to ameliorate anxiety. Both explanatory models might be of validity, perhaps in different patient subgroups. Therefore our observation of elevated, however, not significantly increased OT plasma levels in male autistic children and adolescents with high-functioning cognitive ability in comparison with healthy children—that differ from initial findings on this subject—might be influenced by aspects of the autistic phenotype, respectively, comorbid conditions and elevated levels of stress. Other work groups observed—also in contrast to initial reports—elevated (Jansen et al. 2006) or unchanged (Miller et al. 2013) OT plasma levels in ASD compared to matched healthy developing controls. As Miller et al. (2013) reported of an
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association of high OT concentration with elevated anxiety scores, in future studies a detailed, dimensional assessment of comorbid anxiety and depression symptoms by specific diagnostic instruments might be desirable. To account for the varying results on OT plasma concentrations, furthermore it has been suggested that OT is processed differently in the brains of autistic patients, rather than simply at a different level: In young patients with ASD, OT pro-hormone seems to be incompletely converted into the fully processed OT, the bioactive amidated form of the neuropeptide (Green et al. 2001). As the OT processing system underlies a physiological maturation process (Whitnall et al. 1985; Morris et al. 1992), a failure of developmental progression in peptide processing, impacting on ASD phenotype, was suggested (Green et al. 2001). In our project, we found a positive correlation of OT plasma concentrations with autistic symptomatology, assessed by the relative ADOS sum of communication and social interaction score. These findings are in accordance with Modahl et al.’s initial study, who described that autistic individuals with higher OT levels were more socially and developmentally impaired (Modahl et al. 1998): They showed a greater lack of imitation, lack of modesty and less appropriate personal barriers on the Wing’s autism symptom checklist. A lack of association of OT concentrations with autistic symptomatology in some studies (Jansen et al. 2006) might result from using the ADI-R for ASD diagnostics that describes autistic symptomatology in childhood, but not current symptom load in adulthood. To our knowledge for the first time, peripheral OT values were investigated in the context of ADHD and significantly reduced OT concentrations were observed in boys with this disorder. In the group of autistic children, comorbidity with ADHD did not significantly modify OT values. Children with ADHD clinically present with difficulties in social interactions and communication (Santosh and Mijovic 2004; Reiersen et al. 2007) that exceed those, which are found in healthy control subjects (Hattori et al. 2006). Some impairments of social cognition domains resemble in ASD and ADHD, such as difficulties in emotion recognition, but there are also distinct patterns: Deficits in theory of mind, e.g., are characteristic for autistic patients and not associated with ADHD symptomatology (Taurines et al. 2012). In a first molecular genetic study on the relevance of OXTR gene variants in ADHD, there was no general association of OXTR single nucleotide polymorphisms (SNPs) with the global phenotype ADHD, but one SNP was associated with social cognitive impairments in a subgroup of patients (Park et al. 2010). Our preliminary results give first hints to dysregulation in this neuropeptide system in ADHD or in the context of associated comorbidity, such as oppositional defiant or conduct
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disorder. The finding of reduced OT levels in children with ADHD—in contrast to possibly expected increased concentrations due to high levels of stress and negative mood in this disorder (Taurines et al. 2010)—supports a potential pathophysiological impact of this finding. In future approaches, OT challenging studies in patients with ADHD might be of interest, considering the above-mentioned deficits in emotion recognition in ADHD, our results of decreased OT plasma levels and potential improvements of synthetic OT on recognition of basic facial expressions in healthy persons (Shahrestani et al. 2013). Such approaches might be useful to gain more insights into a potential link between ADHD symptomatology, respectively, its comorbidity and the OT system and to test potential pharmacological interventions. The diversity of hitherto existing study results on peripheral OT concentrations in the field of ASD research may be influenced by differences in study designs concerning diagnostic process, as the current gold standard instruments were used just in a few studies and just partly. Differing results might also be due to inclusion, e.g., of patients of different age and pubertal status, gender, comorbid conditions and level of intellectual functioning. High variability might also be due to different OT assays used, such as different RIAs or Enzyme Linked Immunosorbent Assays, and differences in preceding sample preparation. Evaluation of commercially available OT assays revealed a lack of reliability when used on unextracted samples of human fluids (McCullough et al. 2013). These assays yielded OT estimates that were wildly discrepant with an extensive body of earlier findings in validated, but more laborious assays, as they tag other molecules additionally to OT (McCullough et al. 2013). Samples in our study were extracted and quantified using a highly sensitive and specific radioimmunoassay (RIAgnosis, Munich, Germany) that was strictly standardized and validated in many animal and human studies using a wide variety of stimuli (hypertonicity, parturition, lactation, stress, etc.) to reliably detect the bioavailable neuropeptide in plasma and in central compartments, such as CSF (Neumann et al. 2013; Kagerbauer et al. 2013). Limitations of our study are relatively small sample sizes, age differences, co-medication, mostly psychostimulants, but also antidepressants and antipsychotics in the patient groups. Data on effects of methylphenidate on OT synthesis and release are missing. Results on possible effects of antidepressants (Uvna¨s-Moberg et al. 1999; Ozsoy et al. 2009; Keating et al. 2013) and antipsychotics (Uvna¨s-Moberg et al. 1992; Glovinsky et al. 1994) are inconsistent. Patients with ADHD were diagnosed by an experienced child psychiatrist according to DSM-IV criteria categorically, but unfortunately, diagnoses were not accompanied by a dimensional diagnostic instrument.
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As a further limitation of the study design in general, a recent study found no correlation between OT concentrations in plasma and CSF in 41 non-neurological and nonpsychiatric patients (Kagerbauer et al. 2013). Nevertheless, it could be clearly demonstrated that social stimuli impact also on peripheral concentrations of OT in humans (e.g., Wismer Fries et al. 2005; Seltzer et al. 2010; Schneiderman et al. 2012). Studies in rat models added to findings in humans, indicating that peripheral neurohypophysial secretion into the blood and central release of OT may occur simultaneously, but also independently of each other (Lukas and Neumann 2013). Therefore, results from neurohormone analyses in peripheral blood in general have to be interpreted cautiously and complemented by further studies on OT release in humans and studies on animal models to examine whether abnormal peripheral concentrations reflect primary changes in for the disorder relevant structures of the CNS. Strengths of our study can be seen in ASD diagnostics that included the current ‘‘gold standard’’ measures ADOS and ADI-R (Lord et al. 1989, 1994). In several published studies on plasma OT concentrations in humans, ASD diagnosis was given according to DSM-IV criteria, but was not confirmed by these internationally valid, currently state of the art instruments. Exceptions are the following studies: Jansen et al. (2006), Andari et al. (2010) who used the ADI-R (Jansen et al. 2006; Andari et al. 2010) and Miller et al. (2013), who used the ADOS (Miller et al. 2013) for autism diagnosis. Recruiting groups of pure male subjects in our study aimed to rule out gender effects on OT release (Insel 1997). Further strengths of our project include the above-mentioned strictly standardized and validated OT assay (Kagerbauer et al. 2013) and a standardized protocol with time of blood withdrawal, fasting in the morning, to rule out potential circadian effects on plasma oxytocin concentrations, as suggested in the literature (Landgraf et al. 1982; Devarajan and Rusak 2004). For the first time, still in an exploratory approach, peripheral OT concentrations were assessed also in male children with an etiologically and phenotypically overlapping disorder, ADHD. In summary, we found significant group differences, assessing plasma OT in male children and adolescents with ASD, ADHD and healthy controls and a correlation of peripheral OT levels with autistic symptomatology in patients with ASD. However, a great individual variability throughout all subjects was obvious. Preliminary results also point to peripheral OT modulations in children with ADHD. Our findings add to animal research, fMRI and (epi-)genetic studies demonstrating crucial effects of the neuropeptide OT on human affiliative behavior and suggesting a potential pathophysiologic role in ASD. A simple OT deficit or excess model for ASD seems to be inadequate to integrate complex relationships of peripheral
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OT measurements with autistic phenotype, comorbid symptom facets and other modulating factors. In the future prospective studies, following autistic children until adulthood are needed to elucidate in more detail influences of gender, age, phenotype, development, intellectual functioning, etc., on central and peripheral OT concentrations. Acknowledgments We wish to thank the patients and families who participated in this study as well as Dr. Alex C. Conner for his support with manuscript preparation.
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