Participants

Participants were recruited through advertisements distributed via the Autism Society of Norway to user networks and specialist clinicians in the Oslo area. Eligible participants were male, aged 18 to 35 (inclusive), and had received a diagnosis of ASD from a specialized pediatric or psychiatric institution via multidisciplinary teams. Written confirmation of ICD-10 criteria ASD diagnosis was obtained from their treating clinicians and quality-controlled by a specialist study psychiatrist. Only males were selected because of their overrepresentation in ASD1 and to achieve a more homogenous study population. Exclusion criteria included psychiatric co-morbidity requiring acute intervention (for example, psychosis spectrum disorders) or IQ<75. A screening visit occurred before randomization at Oslo University Hospital. The Wechsler Abbreviated Scale of Intelligence43 and the Mini-International Neuropsychiatric Interview44 were administered by trained graduate students under the supervision of study physicians and clinical psychologists to index IQ and confirm the absence of psychiatric illnesses requiring intervention, respectively. A physical examination was performed by study physicians and nurses, including a 12-lead ECG and routine blood samples. Study physicians also confirmed normal nasal anatomy and patency in participants via physical examination under the supervision of an otolaryngologist, consistent with recent recommendations.32 Acoustic rhinometry data were collected by trained study staff under otolaryngologist supervision (SRE 2000; RhinoMetrics, Lynge, Denmark), yielding nasal valve dimensions (minimum cross-sectional area, summed left and right dimensions) and nasal cavity volume 2–5 cm from the nostrils. This trial was approved by the Regional Committee for Medical and Health Research Ethics (REC South East) and participants provided written informed consent before they participated. The study is registered at the EU Clinical Trials register (EudraCT no.: 2014-005452-26).

Study design

Participants received 8IU oxytocin intranasally, 24IU oxytocin intranasally, and an intranasal placebo treatment in a randomized, placebo-controlled, double-blind, three-period crossover design. Participants were randomized to one of six treatment sequences, using a three-period, three-treatment Latin square method (ABC—ACB—BAC—BCA—CAB—CBA in a 1:1:1 ratio; Supplementary Figure S1), with a minimum of 24 h between treatments to ensure adequate washout. Three participants, who were later randomized for treatment, took part in pilot tests of the social-cognitive and nasal spray administration procedures with open label placebo solution. An independent statistician (Smerud Medical Research International, Oslo, Norway) provided the randomization code, and both the participants and the research team were blinded to treatment. A pharmaceutical service provider (Farma Holding, Oslo, Norway) filled the oxytocin and placebo (matching liquid vehicle) formulations into the Breath Powered devices. All participants completed practice administration at every treatment session using an empty Breath Powered device under the supervision of study staff, before self-administering an intranasal treatment using another device. Participants began the social-cognitive tasks (see details below) 40 min after treatment administration in the following order during every visit: emotion sensitivity, Reading the Mind in the Eyes Test (RMET), emotional dot probe and emotional face-morphing (Figure 1). Each task took ~10 min to complete. Analysis of prior study data derived using the same emotion sensitivity primary outcome measure and Breath Powered device40 revealed a large effect size (partial η2=0.14). A power analysis using G*Power software45 indicated that a sample size of 18 would achieve 90% power for a repeated measures design, given a large effect size (partial η2=0.14) and α=0.05.

Figure 1 Task design. Participants were administered an intranasal solution with the social-cognitive tasks beginning 40 min after intranasal administration. Blood samples and the STAI responses (state-trait anxiety questionnaire) were also collected twice, before and after intranasal administration. Time is shown in minutes. Full size image

Breath Powered delivery device for nasal spray administration

The breath powered, closed-palate, bi-directional nasal spray (‘Breath Powered’) device (also known as the Exhalation Delivery System) capitalizes on two aspects of nasal anatomy to facilitate efficient posterior and superior delivery of medication in the nasal cavity46 (Supplementary Figure S2). As the user blows through the mouth against a resistance the soft palate automatically closes, creating an airtight seal isolating the nasal cavity from the oral cavity, preventing lung deposition and limiting gastrointestinal deposition.47 An optimized sealing nosepiece helps direct the exhaled breath and oxytocin aerosol into the upper-posterior nasal cavity. With a closed soft palate, airflow enters via one nostril and deposits the drug aerosol on target sites and then exits from the other nostril (that is, bi-directional delivery). These conditions create a positive variable pressure in the nasal cavity, which balances pressure across the soft palate, to prevent over-elevation and ensure a patent communication around and behind the nasal septum while also expanding the nasal valve and narrow slit-like nasal passages. This mechanism has been shown to produce improved delivery of drug beyond the nasal valve to target regions in the upper and posterior nasal cavity.46 As in a previous study, the device was further optimized for nose-to-brain deliver with an elongated nosepiece and sideways flexible tip to improve delivery to the most upper and posterior segments of the nasal cavity.40, 48

Co-primary outcome measures

We had two primary outcome measures: an overt emotion sensitivity task previously demonstrated to be modulated by oxytocin delivered via the Breath Powered device40 and a Norwegian translation of the RMET49 a commonly used task in oxytocin research17, 19, 26, 50, 51 to assess theory of mind performance. We hypothesized that oxytocin would increase emotion sensitivity and salience, and improve RMET performance. For the emotion sensitivity task, participants were presented with 20 male and female faces as used previously;40, 52 displaying angry, happy and emotionally ambiguous facial expressions derived from the Karolinska Directed Emotional Faces database.53 The task consisted of five blocks with 20 trials in each block. Each trial of ~6–8 s duration comprised the following sequence: fixation cross of 2 s duration→face presentation of 1 s duration→Q1 of 10 s duration (maximum response window, which terminated after participant response). Participants were asked either: ‘How angry is this person?’ (anchors: not angry—very angry) or ‘How happy is this person?’ (anchors: not happy—very happy). Participants were asked to rank their answer on a numerical rating scale from 1 to 5, with initial location of the cursor on the numerical rating scale randomized for each question. The primary outcome measures were the mean ratings for each category. The RMET is a 36-item battery indexing theory of mind ability,49 whereby participants are shown eye region images and asked which of four possible descriptions best describes what the person in the images is thinking or feeling. The percentage of correct responses was used as the outcome measure, as per prior research.19

Secondary outcome measures

Secondary outcomes included performance on an emotional dot probe task54 (covert emotional salience) and an emotional face-morphing task (speed of recognizing overt emotional stimuli). On the basis of prior reports, we hypothesized that oxytocin would increase covert emotional salience in the dot probe task55, 56 and increase the speed of recognizing overt emotional stimuli in the face-morphing task.57, 58 The dot probe task assesses attentional preference or bias between two stimuli that are presented for a short period of time. Each trial began with a fixation cross for 100 ms, followed by a face stimuli pair (using the same stimuli set from the emotion sensitivity task). One of three pairs of stimuli were presented: angry-ambiguous, happy-ambiguous and ambiguous-ambiguous. The stimuli (40 faces) were drawn from the same stimulus set as the emotion sensitivity task. Pairs were presented for 500 ms, with the probe appearing directly after the faces were present in the place of one of the faces. The probe either appeared behind the target (congruent trial), which was the emotional face for the angry-ambiguous and happy-ambiguous pairs, or non-target (incongruent trial). Participants were asked to indicate where the probe appeared as fast as possible by pressing one of two keys. A total of 160 trials were presented. To create a measure of attentional bias (ms), which was the outcome measure for this task for each stimuli pair (angry and happy), reaction times from congruent trials were subtracted from incongruent trials.

A face-morphing task was developed using the same stimuli to assess speed of emotion recognition. Faces morphed from ambiguous faces to either happy (50%) or angry faces (50%) over 10 s. Morphing videos were created using Fantamorph software (version 5.4.2, Abrosoft, USA). When selecting the starting frame as the ambiguous face and the end frame as the emotional face, each video displays a consistent 10-s morph between these two faces. Participants did not see the same face twice. Participants were instructed to indicate when they could recognize the emotion as either happy or angry. There were 38 trials in total.

The forty-item state-trait anxiety inventory STAI59 was also administered before intranasal administration to index state and trait anxiety. The twenty-item state anxiety portion of the STAI was administered again after the completion of the social cognition tasks to assess changes in state anxiety. After completing the STAI, participants were asked to guess which treatment they were randomized to for the present experimental session (oxytocin or placebo).

Pharmacokinetics

Blood samples were collected to assess peripheral levels of oxytocin, arginine vasopressin (AVP), and cortisol at baseline and 40 min after administration of study medication. Blood samples were centrifuged at 4 °C within 5 min of blood draw, after which plasma was frozen at −80 °C. Oxytocin concentrations were assessed with enzyme-linked immunosorbent assay (ELISA) using commercially available kits (Enzo Life Sciences, Farmingdale, NY, USA). AVP concentration was assessed with competitive radioimmunoassay using commercially available kits (BÜHLMANN Laboratories, Schönenbuch, Switzerland). Cortisol concentrations were measured by luminescence immunoassay (Siemens Immulite 2000XPi, Erlangen, Germany). The Oslo University Hospital hormone lab performed all assays using standard techniques (including sample extraction).

Statistical analysis

Statistical analysis was conducted using the R statistical package60 and JASP (https://jasp-stats.org) to examine the impact of treatment on social-cognitive and pharmacokinetic outcomes. A multilevel linear mixed-model (LMM) approach using the ‘nlme’ package (http://CRAN.R-project.org/package=nlme) was adopted to assess the effect of treatment on social-cognitive outcomes measures. This approach compares the fit of a null LMM model against a main effect LMM model, which yields a likelihood ratio and P-value. LMMs were chosen as repeated-measure analyses to assess main effects because they do not rely on complete data sets or the assumption of compound symmetry. For any significant main effects (P<0.05), post-hoc tests were performed to compare each treatment condition (with Tukey adjustment of critical P-values to correct for multiple comparisons). Cohen’s d was calculated as a measure of effect size with values of 0.2, 0.5 and 0.8 interpreted as small, medium and large effect sizes, respectively.61 For comparison and the calculation of effect sizes, repeated measures ANOVAs were also performed if there was <10% missing data, with Huynh-Feldt corrected statistics presented if sphericity was violated. Eta-squared (η2) was computed in JASP as a measure of ANOVA effect size, with values of 0.01, 0.06 and 0.14 interpreted as small, medium and large effect sizes, respectively.61 Figures illustrating main effects and interactions contain SE bars corrected for within-subjects data.62 This approach helps to ensure that non-overlapping SE bars better represent significant group mean differences, as uncorrected SE bars can be misleading when visualizing within-subjects data.63