Role of the Gut Vascular Barrier and Microbiota in Autism Spectrum Disorders

The presence of gastrointestinal symptoms will be assessed at recruitment (T0) using the GI Severity Index.

Subjects recruited will be treated with the postbiotic-based dietary supplement, PostbiotiX Comfort®, once daily for 1 month.

The GI Severity Index will be repeated within one week after the end of the treatment (T1) to quantify changes in bowel regulation and one month after the end of the treatment (T2) to assess the stability of the observed changes.

Standardized behavioral assessments will be conducted through parents/caregivers reported questionnaires to evaluate the effects of postbiotic supplementation on sleep regulation. Sleep disturbances will be assessed through validated instruments designed to investigate difficulties related to sleep onset and maintenance, respiratory sleep disturbances, disorder of arousal, wake-sleep transition, excessive daytime sleepiness, and nocturnal hyperhidrosis.

Sleep regulation will be assessed using the italian version of the Sleep Disturbance Scale for Children. The scale consists of 26 items rated on a 5-point Likert scale (1 = never; 2 = occasionally; 3 = sometimes; 4 = often; 5 = always). The total raw score ranges from 26 to 130, with higher scores indicating greater severity of sleep disturbances.

Assessments will be conducted at three time points: baseline (T0), at enrollment in the interventional study; post-treatment (T1), within one week after the end of treatment; and follow-up (T

Standardized behavioral assessments will be conducted through parents/caregivers reported questionnaires to evaluate the effects of postbiotic supplementation on behavioral regulation. Emotional and behavioral regulation will be assessed through validated instruments designed to investigate the presence and severity of emotional and behavioral problems, including internalizing and externalizing symptoms.

Behavioral and emotional problems will be assessed using the Italian versions of the Child Behavior Checklist (CBCL) according to age. The instrument consists of items rated on a 3-point Likert scale (0 = not true; 1 = somewhat or sometimes true; 2 = very true or often true). Higher scores indicate greater severity of emotional and behavioral problems. Raw scores will be converted into standardized T-scores (mean = 50, standard deviation = 10).

Assessments will be performed at three time points: baseline (T0), at enrollment in the interventional study; post-treatment (T1)

Standardized behavioral assessments will be conducted through parents/caregivers reported questionnaires to evaluate the effects of postbiotic supplementation on sensory profile.

The sensory profile will be assessed using the Sensory Profile-2 (Italian version), completed by parents/caregivers for children aged 3-8 years. The questionnaire evaluates sensory functioning, self-regulation behavioral responses and the frequency of behavioral responses to environmental sensory stimuli. Raw scores are calculated for each sensory domain; higher scores indicate greater deviation from typical sensory processing patterns.

Assessments will be performed at three time points: baseline (T0), at enrollment in the interventional study; post-treatment (T1), within one week after the end of treatment; and follow-up (T2), within one month after the end of treatment to assess the stability of the observed changes. All questionnaires completed by the parents/caregivers will subsequently be discussed with

Fecal samples obtained from control subjects and children with ASD with and without gastrointestinal symptoms. For the latter group, samples will be collected both prior to the initiation of supplement therapy (T0) and after treatment (T1).

Taxonomic and functional profiling of the fecal microbiota will be performed using shallow shotgun metagenomic sequencing. Total bacterial DNA will be extracted from fecal samples and sequenced using Illumina platforms. Taxonomic analyses will be performed at Species-level Genome Bin (SGB) resolution (8th taxonomic rank). Functional analyses will be based on destratified gene family and metabolic pathway abundances, with pathway annotation performed against the MetaCyc database. Differential abundance analyses (DAAs) will be performed on taxonomic profiles expressed as relative abundances and on gene family and pathway profiles normalized to copies per million (CPM) Unit of measure: Relative abundance of bacterial taxa (%)

The following laboratory tests will be performed on fecal samples obtained from control subjects and children with ASD with and without gastrointestinal symptoms. For the latter group, samples will be collected both prior to the initiation of supplement therapy (T0) and after treatment (T1). Untargeted metabolomic analysis will be performed on fecal samples to identify metabolites of bacterial origin. Metabolites will be detected and quantified using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), followed by bioinformatic analysis.

Unit of measure:

• Relative or absolute metabolite abundance (normalized peak intensity/concentration)

The following laboratory tests will be performed on fecal samples obtained from control subjects and children with ASD with and without gastrointestinal symptoms. For the latter group, samples will be collected both prior to the initiation of supplement therapy (T0) and after treatment (T1). Intestinal permeability will be assessed by quantifying fecal levels of zonulin and albumin using commercially available enzyme-linked immunosorbent assays (ELISA). Increased fecal zonulin levels will be interpreted as indicative of tight junction disassembly and increased epithelial permeability. Elevated fecal albumin concentrations will be interpreted as a marker of intestinal barrier dysfunction associated with inflammatory leakage across the intestinal mucosa . Both biomarkers will be analyzed as indicators of altered intestinal permeability and gut barrier integrity.

Unit of measure:

• Zonulin family peptides: concentration (e.g., ng/mL or ng/g of feces)
• Albumin: concentration (e.g., ng/mL

Correlation analyses will be performed to investigate the relationships between metagenomic and metabolomic profiles and behavioral and gastrointestinal outcomes in children with ASD. This initial analysis will enable the identification of significant associations between microbial diversity, metabolite abundance, and clinical measures potentially modulated by postbiotic treatment.

Metagenomic, metabolomic, and intestinal permeability data will then be integrated with behavioral outcomes using advanced computational multi-omics approaches. Following data normalization and harmonization through standardized pipelines, metagenomic and metabolomic datasets will be combined using network-based integration strategies and multivariate methods, including Canonical Correlation Analysis (CCA), sparse Partial Least Squares (sPLS), and Multi-Omics Factor Analysis (MOFA), to identify shared functional signatures, key microbial-metabolic interactions, and convergent pathways across datasets.

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