Oxytocin’s social and stress-regulatory effects in children with autism and intellectual disability: a protocol for a randomized placebo-controlled trial

Abstract

Background

Oxytocin is increasingly considered as a new pharmacological option for mitigating social difficulties and regulating stress in autism spectrum disorder. However, in prior trials, autistic individuals with co-occurring intellectual disability (ID) have largely been overlooked, despite their high prevalence, poorer outcome, and the enhanced need but reduced availability of therapeutic interventions. Prior studies have also overlooked the importance of standardizing the context in which oxytocin is administered, rendering outcomes from prior trials inconclusive.

Methods

To meet these limitations, we propose a double-blind, randomized, placebo-controlled trial investigating the effects of intermittent multiple-dose intranasal oxytocin administration (4 weeks, 24 IU 3x/week), administered within a standardized psychosocial stimulating context at special need schools, in 80 children with autism and co-occurring ID (4–13 years old). Clinical-behavioral as well as stress-regulatory effects of oxytocin will be evaluated using the Autism Treatment Evaluation Checklist (ATEC), the Brief Observation of Social Communication Change (BOSCC) expert rating scale, and measurements of high-frequency heart rate variability (HF-HRV), a validated index of parasympathetic autonomic nervous system activity. To assess the possibility of retention and/or late-emerging effects, outcomes will be assessed immediately after the administration regime, and at two follow-up sessions, four-weeks and six months after administration.

Discussion

Significant clinical-behavioral improvements on the ATEC and BOSCC, and significantly higher parasympathetic HF-HRV power in the oxytocin compared to the placebo group would confirm beneficial clinical-behavioral and stress-regulatory effects of oxytocin in autistic children with co-occurring ID. This would also corroborate the use of intermittent dosing schemes in combination with concomitant psychosocial stimulation.

Trial registration

The trial was registered on the 9th of December 2022 at the European Clinical Trial Registry (EudraCT 2022-002423-36) and on 20th of September the trial was transferred to the EU Clinical Trial Register (EU CT 2024-513436-14).

Supplementary Information

The online version contains supplementary material available at 10.1186/s12888-025-06617-6.

Background

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition marked by challenges in social communication and interaction, along with restrictive and repetitive behaviors and interests (RRBI) [1]. These difficulties often result in considerable distress, reduced quality of life, and limited social integration for individuals with autism and their families [2]. Traditionally, interventions have focused on behavioral approaches aimed at enhancing social communication and mitigating the impact of RRBIs, as biomedical and pharmacological interventions have yet to demonstrate consistent efficacy [3].

Recent research points to disrupted oxytocinergic signaling as a potential factor in autism [4, 5], leading to increased interest in the intranasal administration of oxytocin as a pharmacological option aimed at improving stress regulation and alleviating the core social difficulties characterizing autism [6, 7]. However, results from previous studies using both single and multiple doses of oxytocin have been inconsistent, with some showing improvements in social and repetitive behaviors, while others found no significant difference compared to placebo [7]. Several factors may account for these discrepancies in efficacy and have been put forward in literature [8].

A first critical factor is the dosing regimen. Optimal regimens have not been conclusively determined, though moderate intervention periods (4–6 weeks) with intermittent dosing (24–32 international units (IU) every other day) have shown promise [9–14]. Second, the social environment in which oxytocin is administered is also vital, with better outcomes observed when administration occurs in socially supportive settings [12, 15, 16]. This underscores the context-dependent nature of oxytocin’s effects, as the hormone may modulate neural processes that increase the salience of social stimuli rather than directly causing social behavioral improvements [15]. Third, there is a need for more precise and sensitive outcome measures. Traditional diagnostic tools, such as the Social Responsiveness Scale (SRS) [17] and the Autism Diagnostic Observation Schedule (ADOS) [18], though reliable for diagnosis, may not effectively capture changes over time. Hence, incorporating behavioral scales designed to detect intervention-induced changes, along with objective biomarkers related to stress and neurophysiological regulation, is essential. Fourth, individual differences have also been put forward as a determining factor of oxytocin’s effect. For instance, sex-related differences of oxytocin’s effect remain an understudied field within autism research [19]. Likewise, despite the high prevalence of co-occurring intellectual disability (ID) in 30% of individuals with autism [20], nearly all study have systematically excluded participants with lower cognitive abilities, which may limit the generalizability of their findings [21]. Emerging evidence also suggests that the developmental stage of participants is a critical determinant, with younger children potentially deriving greater benefit from oxytocin administration [4, 22].

In this paper, we present the protocol for a randomized, double-blind, placebo-controlled clinical trial with parallel design. This trial aims to test the tolerability and efficacy of multiple-dose, chronic oxytocin administration (four weeks of intranasal administration of 24 IU, intermittently administered 3x/week) on social functioning and cardiac parasympathetic activity in a representative sample of 4-to-13-year-old autistic children with co-occurring ID (40 oxytocin / 40 placebo). Considering increasing insights that the context in which oxytocin is administered may impact outcome and potentially explain response heterogeneity within and across studies [15, 23–26], our study will adopt a consistent pairing of each oxytocin administration with a standardized psychosocially stimulating context. Importantly, the oxytocin administration will be administered directly in the special needs schools of the participating children, ensuring a broader peer-group interaction within a trusted, positive environment, as well as a highly standardized context. As detailed in the methods section, to provide a psychosocially stimulating context, children will engage in interactive group and imitation games, as well as sensory stimulating and relaxing activities which have been shown to stimulate the endogenous oxytocinergic system [27, 28]. The Brief Observation of Social Communication Change (BOSCC) will be used as a validated observational measure to assess oxytocin-induced changes in reciprocal interactions and RRBI. The BOSCC is an abbreviated, researcher-assessed standardized observation scale developed for detecting subtle changes in social communication behaviors over brief periods [29]. Additionally, the Autism Treatment Evaluation Checklist (ATEC), a caregiver-rated scale specifically designed to measure changes in autism characteristics over time and validated for children with co-occurring ID, will also serve as a primary outcome measure [30]. The ATEC will be assessed by both parents and teachers of the participating children, providing a multi-informant evaluation of oxytocin’s impact from caregivers closely connected to the child. Furthermore, we will measure cardiac parasympathetic activity through high-frequency heart rate variability (HF-HRV) as a biophysiological marker of oxytocin’s stress-regulating effects [31–33]. To assess potential long-term effects, outcome measures will be taken immediately after the intervention and at follow-up assessments four weeks and six months later.

Methods

Design

General trial design

The study encompasses a double-blind, placebo-controlled trial with between-subject design examining the effect of intranasal oxytocin on clinical-behavioral outcomes and cardiac parasympathetic activity in autistic children with co-occurring ID. Children will, depending on randomization, receive a dose of 24 IU of intranasal oxytocin three days per week (i.e., Monday, Wednesday, Friday), for a period of four weeks or an identical-looking placebo nasal spray at the same frequency for the same period. Nasal sprays will be administered in the morning at school and children will engage in a play session upon each nasal spray administration. This particular approach ensures that the psychosocial context of the placebo and oxytocin group will be highly standardized and accurately matched. Oxytocin nasal spray effects on clinical-behavioral outcomes and cardiac parasympathetic activity will be examined during three assessment sessions; (i) at baseline (T0), which takes place at least one day before the first administration; (ii) immediately after the four-week administration period (T1), which takes place at least one day after the last administration; (iii) at a follow-up session four-weeks after the last nasal spray administration (T2). Additionally, a long-term follow-up assessment will be obtained for a subset of clinical-behavioral outcomes, through remote collection six months after cessation of the nasal spray administration period (T3). Please see Fig. 1A, for a visualization of the trial design and Fig. 1B for the planned CONSORT Flow diagram, visualizing the number of participants randomized and analyzed.

Fig. 1.

Visualization of the trial design. (A) Trial Design Chart. Outcome measurements will be performed at baseline (T0), immediately after the oxytocin administration period (N = 40) or placebo (N = 40) administration (T1), and at two follow-up sessions, i.e. four weeks (T2) and six months (T3) after administration. (B) CONSORT flow diagram. Autism Treatment Evaluation Checklist (ATEC); Brief Observation of Social Communication Change (BOSCC)

Participants

Recruitment

Participants will be recruited through special needs schools and Autism Reference Centers across Flanders between September 2023 and November 2025.

Inclusion/exclusion criteria

Principal inclusion criteria comprise having both clinical diagnoses of ASD and ID and being enrolled in a special needs school, specifically for children with ID. The diagnoses have to be established by a multidisciplinary team based on the criteria of the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-5) [1]. Children within a chronological age range of 4–13 years will be included. Female participants need to be premenarchal, as endogenous oxytocin concentrations may fluctuate during the menstrual cycle [34]. Principal criteria for exclusion comprise active intake of anti-epileptic medication; active medical problem (e.g., cardiac pathology or serious liver, renal problems which influence the metabolism of oxytocin); known genetic syndrome that interacts with the hormonal reproductive system (e.g., Prader-Willi or Angelman syndrome); significant hearing or vision impairments; known hypersensitivity to active substance or ingredients of the nasal spray; previous chronic oxytocin administration; and simultaneous participation in another pharmacological clinical trial.

Background phenotypic information

Estimates of cognitive functioning in terms of intelligence quotient (IQ) will be assessed via the Raven’s 2 Progressive Matrices [35], a nonverbal test for assessing general cognitive ability in individuals aged 4 through 90 years, hence, suitable for nonverbally autistic children. The test examines cognitive functions such as perception and attention to visual detail, inductive reasoning, broad visual intelligence, fluid intelligence, simultaneous processing, classification and spatial ability, and working memory. Estimates of adaptive functioning will be assessed via the parent-rated Adaptive Behavior Assessment System-3 (ABAS-3). The ABAS-3 [36] is a 241-item questionnaire that assesses adaptive behaviors in three domains: ‘Social’, ‘Conceptual’, and ‘Practical’. It covers subscales like ‘Communication’, ‘Societal Skills’, ‘Self-Care’, and more. For each item, the parent indicates on a four-point Likert scale the extent to which the participant spontaneously exhibits a specific behavior in a context in which it is needed or expected. Lower scores indicate lower levels of adaptive behavior.

Sample size

As outlined in more detail in the sampling plan section, a total of 80 children (40 in each intervention arm) will participate in the trial.

Co-occurring conditions, medication use and participation in behavioral therapies/trainings

The presence of co-occurring psychiatric conditions (e.g., attention deficit/hyperactivity disorder), concurrent psychoactive medication use (defined as use within four weeks before study enrollment) and participation in ongoing behavioral therapies/trainings will be established through parent reports.

Intervention

Study medication

Participants will be randomized to receive either oxytocin (Oxytocin, CD Pharma) or placebo nasal sprays, administered in identical blinded amber 10 ml glass bottles with metered pump. The placebo spray will consist of physiological water, sodium chloride (NaCl 0.9%) solution, with added preservatives (aqua conservans, chlorbutanol, glycerinmethocel). Nasal spray preparation, packaging, blinding and randomization (permuted-block randomization) will be performed by pharmacy A15 (The Netherlands). Participants will be randomly assigned in a 1:1 ratio, balanced for chronological age and biological sex. All research staff conducting the trial, participants, their parents and teachers will be blinded to nasal spray allocation. After the last assessment of the last participant, trial staff will be unblinded regarding nasal spray allocation.

Dosing

Children will receive a dose of 24 IU nasal spray or placebo in the morning (administered as three puffs of 4 IU in each nostril), similar to dosing schemes adopted in young autistic children [10, 12]. The nasal spray will be administered three times weekly at school (i.e., intermittent dosing: on Monday, Wednesday and Friday), for a period of four weeks (12 doses in total). The four-week duration aligns with previous trials involving autistic children [11] and adults [37] and reflects recent insights into optimal dosing regimens for similar trials [8]. To standardize the context during the expected time window of actively circulating oxytocin in the central nervous system, children will engage in a play session starting 30 min after the nasal spray administration. The play session will last approximately 15–40 min, thereby overlapping with the optimal time window at which central peak oxytocin concentrations have been identified in cerebrospinal fluid [38–40].

Psychosocially stimulating context

During every play session, children will be stimulated to engage in four short, semi-standardized social activities of approximately 3–10 min each, at school, in a classroom context. Components of the play session were chosen to encompass tasks and behaviors known to stimulate the endogenous oxytocinergic system [27, 28]. Specifically, the play session will encompass the following four activities: (i) an initial warming-up part, involving singing and dancing together; (ii) an imitation game, in which children are stimulated to imitate animal or bodily movements of one another/the activity leader; (iii) an interactive group game, for instance a ballgame; and (iv) the session will close with a relaxation part, during which children will massage one another/themselves or play with sensory toys.

Compliance and adverse event monitoring

In this study, rigorous compliance monitoring will be implemented for both nasal spray administration and play session. Notably, these procedures will be executed directly by the investigators, distinguishing this approach from prior trials where oxytocin administration and contextual factors relied upon parental involvement. Additionally, a medication diary will be employed to document the precise date and time of nasal spray and play session administration. Further, the total amount of administered nasal spray fluid will be monitored. During the nasal spray administration period, participants will be screened tightly for reports of potential adverse events through a side-effect report form made available to parents and teachers.

Parent reported beliefs about allocated nasal spray

At the post administration and first follow-up sessions (i.e. T1, T2), parents and teachers will be asked to report their belief about nasal spray allocation. This includes reporting (i) whether they believe the child received either the oxytocin or placebo nasal spray; (ii) how certain they are of their report (scored on a five-point Likert scale); (iii) whether the administration of the nasal spray has improved the child’s behavior (scored on a five-point Likert scale); and (iv) whether the administration of the nasal spray has reduced the child’s stress (scored on a five-point Likert scale).

Primary outcome measures

The following behavioral-clinical outcomes and stress physiological outcome will be included as primary endpoints of the trial:

Autism treatment evaluation checklist (ATEC)

The ATEC questionnaire was specifically developed as an instrument for parents and caregivers to describe children’s current functioning and behaviors, as a way of monitoring the impact of interventions [42, 43]. It is a highly sensitive instrument for evaluating autism-related behaviors in autistic children with co-occurring ID in educational settings [30]. It comprises the following subscales: ‘Speech/Language Communication’ (14 items), ‘Sociability’ (20 items), ‘Sensory/Cognitive Awareness’ (18 items), and ‘Health/Physical/Behavior’ (25 items), which are scored on a three- to four-point Likert scale. The scores from each subscale are combined to calculate a Total Score, which ranges from 0 to 179 points. A higher score indicates a higher presence of ASD characteristics. The ATEC will be completed by the child’s parents and teacher at each assessment session (T0, T1, T2 and T3).

Brief observation of social communication change (BOSCC)

The BOSCC is an abbreviated observation scale specifically designed for the evaluation of treatment responses in ASD, assessing subtle changes in social communication behaviors over brief periods of time [29]. The BOSCC coding scheme consists of 15 items scored using a six-point Likert scale. The scheme comprises three subscales: ‘Social Communication Behavior’ (8 items), ‘Restricted and Repetitive Behavior’ (4 items) and ‘Other Abnormal Behavior’ (3 items). Only the domains ‘Social Communication Behavior’ and ‘Restricted and Repetitive Behavior’ will be taken into account for calculating the BOSCC Total Score. A higher score indicates a higher severity of ASD characteristics. The BOSCC will be administered and video-rated by trained researchers based on semi-structured naturalistic social interaction/play sessions held with the child at assessment sessions T0, T1 and T2.

Cardiac parasympathetic activity - High frequency heart rate variability (HF-HRV)

Electrocardiography (ECG) recordings will be performed with a bipolar lead set up (sampling rate: 512 Hz). One ECG electrode will be placed under the right collarbone and the other one on the left ribcage. A ground electrode consisting of a CMS-electrode and a DRL-electrode will be used as third electrode to make up Einthoven’s Triangle. The CMS-electrode will be placed under the left collarbone (opposite to the first electrode) and the DRL-electrode will be placed on the left arm or ankle. ECG measurement will be recorded during a 7-minute ‘resting-state’ condition, while children are presented with the movie paradigm ‘Inscapes’ [44]. This movie displays abstract shapes without a narrative or scene-cuts, thereby providing sufficient stimulation to ensure participant compliance in terms of wakefulness and motion reduction while minimizing cognitive load.

To assess HF-HRV, time intervals between R-peaks will be assessed using Kubios HRV Premium software (version 3.5.0) [45] for deriving continuous inter-beat-intervals (IBI). All IBI time series will be manually inspected prior to analysis and automatic artifact removal, as implemented in Kubios, will be applied. Participants will be removed from the primary analyses if less than 3 min of noise-free data are available and/or > 5% ectopic beats are identified [32, 46]. For each of the remaining subjects, HRV frequency domain analyses will be performed using Fast Fourier Transformation (FFT) based on Welch’s periodogram. Percentage power values will be computed for the high-frequency component of the HRV signal, which in children is defined between 0.24 and 1.04 Hz [47]. Rest-ECG recordings will be collected at assessment session T0, T1 and T2.

Secondary and exploratory outcome measures

Clinical-behavioral secondary outcomes

The study will also include the following clinical-behavioral scales as secondary endpoints: (i) the Repetitive Behavior Scale-Revised (RBS-R) [48, 49] rated by the parents and teacher about the child at T0, T1, T2 and T3; (ii) the Developmental Behavior Checklist (DBC) [50, 51], rated by the parents about their child at T0 and T3; and (iii) the Perceived Stress Scale (PSS) [52], rated by the parent as a self-report index of perceived (family) stress at T0, T1, T2 and T3.

• i.The RBS-R assesses severity of repetitive behaviors and has been reported effective at measuring intervention outcomes for ASD with co-occurring ID [30]. The questionnaire consists of 43 items covering six subscales: ‘Sameness Behavior’ (11 items), ‘Self-Injurious Behavior’ (8 items), ‘Stereotypical Behavior’ (6 items), ‘Compulsive Behavior’ (8 items), ‘Ritualistic Behavior’ (6 items) and ‘Restricted Behavior’’ (4 items). Items are scored on a four-point Likert scale. Higher scores indicate more severe repetitive behaviors.
• ii.The DBC assesses behavioral and emotional problems of children with developmental and intellectual disabilities. The questionnaire consists of 95 items and covers five subscales: ‘Disruptive & Antisocial Behavior’ (27 items), ‘Introverted Behavior’ (31 items), ‘Communication Disturbances’ (13 items), ‘Anxiety’ (9 items), and ‘Social Deficits’ (10 items), scored on three-point Likert scales. A higher score indicates more emotional and behavioral problems.
• iii.The PSS assesses the parent’s own perceived stress over a defined period (e.g., the past month). It does not focus on specific stressors but instead assesses the parent’s overall perception of stress in their life. The 10-item Dutch version will be used, in which items are scored on a four-point Likert scale, with higher scores indicating more perceived stress.

Stress physiological secondary outcomes

Alongside ECG measurements, the 7-minute ‘resting-state’ condition will simultaneously assess other physiological parameters: (i) electroencephalography (EEG); (ii) skin conductance; and (iii) respiration. Resting state EEG, skin conductance and respiration recordings will be collected at assessment sessions T0, T1 and T2.

• i.EEG will be recorded using a 16-channel EEG headcap system (BioSemi ActiveTwo, The Netherlands). Prior to recording, the participant’s head is measured to locate the center. A cap with embedded electrodes, secured with a chinstrap, is then placed over the head. Electrodes (BioSemi Pin-Type Active-electrodes) are attached using easy snap-on connectors after application of electrode gel onto the participant’s scalp.
• ii.Two silver chloride (Ag-AgCl) electrodes (BioSemi ActiveTwo Galvanic Skin Response electrodes) will be attached on the instep of the foot [53]. By applying a tiny bit of current (undetectable by the participant) on those electrodes attached to the skin, the resistance (or conductance) of the skin will be measured.
• iii. To measure breathing, a belt with respiration sensor will be placed on the chest (BioSemi ActiveTwo Respiration Belt), which measures the relative expansion and contraction of the chest. The belt can be applied on top of the participant’s clothing.

Exploratory hormonal outcomes

Previous research highlighted the role of person-dependent factors in mediating response variability. Particularly, in a prior oxytocin trial with autistic children, significant improvements in social functioning (SRS-2 Total Score) were only observed when baseline variations in plasma oxytocin levels were included as a covariate into the analyses [11]. To enable similar sensitivity analyses in the current study, assessment of variations in salivary oxytocin levels will be performed using Salivette cotton swabs (Sarstedt AG & Co., Germany) at baseline (T0). Furthermore, to explore the biological effects of chronic intranasal oxytocin administration on endogenous oxytocinergic function, salivary oxytocin levels will also be measured at each post administration study session (T1, T2, T3) [4, 54]. In addition to oxytocin levels, salivary cortisol levels will be assessed from the same Salivette cotton swabs. Salivary sampling will be performed in the morning, acquired at home, within 30 min after awakening and before breakfast. Oxytocin salivary levels will be analyzed using enzyme immunoassay oxytocin ELISA kits from Enzo Life Sciences, Inc., USA, in accordance with the manufacturer’s instructions (similar to prior studies [55, 56]). Cortisol salivary levels will be analyzed using the Salivary Cortisol Enzyme Immunoassay Kits by Salimetrics. Sample concentrations (100 µl/well) will be calculated conform plate-specific standard curves.

Exploratory epigenetic outcome

To explore the epigenetic effects of chronic intranasal oxytocin administration, another saliva sample measuring the oxytocin receptor gene [OXTR, Online Mendelian Inheritance in Man (OMIM) entry 167055] will be collected. Saliva samples for DNA methylation (DNAm) will be collected at each assessment session (T0, T1, T2, T3) using the Oragene DNA (OG-500) collection kit (DNA Genotek, Canada). In line with previous studies, variations in OXTR DNAm will be assessed at three CpG sites of the OXTR gene, previously shown to be impacted in autism (i.e., -934, − 924 and − 914) [4, 54].

Sampling plan

Sample size

As this is the first study examining chronic oxytocin administration effects in autistic children with co-occurring ID, power calculations could not be based on prior studies with similar outcomes and designs. To obtain an approximation, a pooled (meta-analytic) effect size was calculated based on prior chronic oxytocin administration studies with young autistic children (without ID), adopting the SRS-2 as primary outcome. These include studies by Yatawara et al. 2016 [10] (5 weeks, 24 IU 1x/day); Parker et al. 2017 [11] (4 weeks, 24 IU 2x/day); Le et al. 2022 [12] (5 weeks, 24 IU 1x/ every other day), and Guastella et al. 2022 [13] (12 weeks, 16 IU 2x/day). Using G*Power software (independent t-test, α = 0.05; two-tailed; power = 0.95), it was estimated that a minimum sample size of 76 (38 oxytocin/38 placebo) would have 95% power to detect an effect size as large as the identified meta-analytic effect size, Cohen’s d = 0.84.

Note however, that in the majority of these prior studies, oxytocin was administered without standardizing the context of the daily administration, which is planned in the current trial, i.e. through implementation of the playsession at each oxytocin administration. Calculation of the estimated sample size based on effect sizes from the only study who did include this standardization of (social) context, i.e., Le et al., 2022 [12], rendered an estimated sample size of n = 26 (13 oxytocin/13 placebo) and n = 36 (18 oxytocin/18 placebo), respectively for primary outcomes SRS-2 and ADOS-2 Total Score. While the current trial will include different outcomes compared to these prior studies, that are tailored more specifically to the targeted populations of children with co-occurring ID, the ADOS-2 can be considered similar to the planned inclusion of the BOSCC, since both consist of semi-structured standardized assessments evaluating social communication and repetitive behavior in ASD [29, 57, 58]. Nevertheless, the BOSCC is considered to be more sensitive than the ADOS-2, as it has been specifically designed to pinpoint subtle intervention effects [29]. The planned total sample size of 80 children (40 oxytocin/40 placebo) is therefore expected to yield sufficient statistical power for detecting medium-to-large clinical effects if present.

Analysis plan

Statistical analysis plan

First, mixed-effects models will be constructed separately for each assessment time point (T1, T2, T3), i.e., allowing a direct examination of whether oxytocin can induce beneficial changes as evident at the immediate post-intervention assessment T1. Exploratory, the assessments at T2 and T3 will allow examining whether any oxytocin administration effect shows a retention after cessation of the nasal spray administration period, or whether possible oxytocin administration effects emerge with a delay, e.g., only discernible four weeks after cessation of the administrations. Cohen’s d effect sizes of between-group differences will be reported, where 0.2 is indicative of a small effect, 0.5 a medium effect and 0.8 a large effect.

Next, to directly explore evolution in administration responses among the different assessment time points, outcome measure scores will be subjected to linear mixed-effects models with the between-subject factor ‘nasal spray’ (oxytocin, placebo) and within-factor ‘assessment time point’ (T1, T2, T3) as well as their interaction. Here, baseline T0 scores assessed pre-treatment will be included as covariate.

A modified intention-to-treat format will be adopted including all randomized participants who completed the baseline session and at least one post or follow-up session. Accordingly, only participants with missing data on all assessment time points will be considered a drop-out of that particular outcome measure. Also, participants with a missing baseline score for a particular outcome will be excluded list-wise for that particular outcome measure.

Regarding the handling of missing data, the planned mixed models will be able to handle moderate amounts of missing data provided they are missing at random. To examine whether data were missing at random, we will compare dropout rate between treatment arms, examine whether baseline scores are similar between dropouts and completers, as well as differences in response variables up to the point of premature withdrawal.

All analyses will be reported with a statistical significance threshold of α = 0.05, two-tailed. Statistical results will be focused on the reports of the primary outcomes, and in analogy to prior clinical trial reports (e.g [12, 13])., these results will be reported in full, without explicit corrections for multiple comparisons.

Electronic supplementary material

Below is the link to the electronic supplementary material.

List of Abbreviations

ASD

Autism Spectrum Disorder

RRBI

Restricted and Repetitive Behaviors and Interests

IU

International Units

SRS

Social Responsiveness Scale

ADOS

Autism Diagnostic Observation Schedule

ID

Intellectual Disability

BOSCC

Brief Observation of Social Communication Change

ATEC

Autism Treatment Evaluation Checklist

HF-HRV

High-Frequency Heart Rate Variability

DSM-5

Diagnostic and Statistical Manual of Mental Disorders, fifth edition

IQ

Intelligence Quotient

ABAS-3

Adaptive Behavior Assessment System-3

ECG

Electrocardiography

FFT

Fast Fourier Transformation

RBS-R

Repetitive Behavior Scale-Revised

DBC

Developmental Behavior Checklist

PSS

Perceived Stress Scale

EEG

Electroencephalography

OXTR

Oxytocin Receptor Gene

DNAm

DNA methylation

Author contributions

GR performed investigation, project administration, formal analysis- and writing-original draft. AT and ET performed investigation, project administration, formal analysis and writing-review & editing. JP contributed to conceptualization, supervision, writing-review & editing. ED did investigation, resources, writing-review & editing. JS and BB have done conceptualization, resources, funding acquisition, supervision and writing-review & editing. KA performed conceptualization, project administration, formal analysis, writing-original draft, funding acquisition, supervision. All authors read and approved the final manuscript.

Funding

This work is supported by a TBM grant of the Flanders Fund for Scientific Research (FWO-TBM T001821N) granted to K.A., B.B. and J.S. J.S. also obtained a Research Council KU Leuven C2-Project Grant (C24M/21/045) to support the planned research activities of the TBM project. As part of the C2-project Grant and the FWO-TBM project grant the protocol has undergone peer review. Further, J.P. is supported by a postdoctoral fellowship of the Flanders Fund for Scientific Research (FWO; 1257621 N) and AT by an aspirant fellowship of the Flanders Fund for Scientific Research (FWO; 11PW324N). The funding sources had no further role in study design; and will have no role on the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Data availability

According to the FAIR data principle (Findable, Accessible, Interoperable, and Reusable), all the datasets generated and/or analyzed during the current study will be made available in the University of Leuven’s Research Data Repository (RDR KU Leuven), accessible and citable through a digital object identifier, DOI.

Declarations

Ethics approval and consent to participate

Written informed consent will be obtained from the child’s parents or legal guardians prior to study enrollment. Consent forms and study design were approved by the Ethics Committee for Biomedical Research at the University of Leuven, KU Leuven (S66832) in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki and its later amendments). The trial will be monitored by the Clinical Trial Center at the University Hospital of Leuven. All trial staff obtained Good Clinical Practice certification and will be trained in the study protocol.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.