Psychobiotics in pediatric autism spectrum disorder: A systematic review of efficacy, mechanisms, and clinical translation

Abstract

BACKGROUND

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition frequently associated with gastrointestinal (GI) disturbances and alterations in gut microbiota composition. Increasing evidence suggests that the microbiota-gut-brain axis may play a role in the pathophysiology of ASD, raising interest in microbiome-targeted therapies, including probiotics, prebiotics, synbiotics, and fecal microbiota transplantation (FMT). However, the clinical effectiveness and mechanisms of these interventions remain incompletely understood due to heterogeneous study designs and variable outcomes across trials.

AIM

To systematically evaluate the clinical efficacy and mechanistic implications of psychobiotic interventions, including probiotics, prebiotics, synbiotics, and FMT, in children with ASD.

METHODS

We conducted this systematic review in accordance with PRISMA 2020 guidelines. We also followed the methodological recommendations of the Cochrane Handbook. We searched different electronic databases, including PubMed/MEDLINE, EMBASE, Web of Science, PsycINFO, CENTRAL, and CINAHL, from inception till February 2026. We included eligible randomized controlled trials, controlled clinical trials, cohort studies, and pre-post intervention studies evaluating psychobiotic therapies in children and adolescents (≤ 18 years) with ASD. The main outcomes were: (1) Changes in severity as assessed by a broad-spectrum measure of ASD symptomatology at the 3-month follow-up; and (2) Variation of core symptoms, based on validated behavioral scales, including the Autism Diagnostic Observation Schedule, the Childhood Autism Rating Scale (CARS), and the Social Responsiveness Scale (SRS). We measured the severity of GI symptoms, microbiome composition, biologicals, and safety as secondary outcomes. Risk of bias was determined with Risk of Bias tool version 2 and Risk of Bias in Non-randomized Studies of Interventions tools. Meta-analysis using random-effects models was conducted where appropriate.

RESULTS

We reviewed 35 clinical studies that examined microbiome-targeted therapies in children with ASD. Overall, probiotics were associated with obvious improvements in GI symptoms and smaller, but noticeable, improvements in behavioral outcomes, although the benefits varied depending on the probiotic strains used, treatment duration, and whether children had GI problems at baseline. When data from selected probiotic studies were pooled, a moderate-to-large overall benefit was observed for GI symptom severity. Prebiotic supplementation mainly benefited gut function and microbial metabolic activity, with little and inconsistent impact on core autism symptoms. Synbiotic approaches-which combine probiotics with fermentable substrates-produced broader effects on the gut microbiome and were linked to modest improvements in both GI and behavioral symptoms. We observed that FMT showed the strongest overall effects among all microbiome-based interventions. Using quantitative analyses, we observed marked improvements in GI symptoms (GI SRS pooled SMD -2.71), along with a significant decrease in key behavioral measures, including social responsiveness (SRS pooled SMD -4.06), aberrant behavior (Aberrant Behavior Checklist -3.00), and overall autism severity (CARS -1.60). However, the strength of this evidence is tempered by the fact that many FMT studies were open-label or non-randomized, which limits confidence in the robustness of these findings. Across all microbiome-targeted therapies, benefits were most consistently observed for GI outcomes, whereas improvements in core ASD symptoms were more variable and less predictable.

CONCLUSION

Microbiome-targeted therapies are a promising adjunctive strategy in managing children with ASD, especially for associated GI disorders. Probiotics and prebiotics provided the most reliable benefits for GI symptoms, while synbiotics may offer broader microbiome modulation, and FMT has the greatest observed improvements in both GI and behavioral outcomes. However, considerable heterogeneity across studies and limited high-quality randomized trials highlight the need for large, well-designed clinical trials with standardized intervention protocols to clarify clinical efficacy and guide future therapeutic recommendations.

Key Words: Autism spectrum disorder; Psychobiotics; Probiotics; Prebiotics; Synbiotics; Fecal microbiota transplantation; Gut-brain axis; Children

Core Tip: Microbiome-targeted therapies are emerging as promising adjunctive strategies for children with autism spectrum disorder, particularly in those with co-existing gastrointestinal disorders. We observed from this systematic review that probiotics and prebiotics consistently improve gastrointestinal symptom severity, while synbiotics may provide broader modulation of gut microbial ecology. Fecal microbiota transplantation induced the largest observed improvements in both gastrointestinal outcomes and selected behavioral domains, although current evidence is limited by small sample sizes and methodological heterogeneity. These findings support strong evidence that the microbiota-gut-brain axis is a biologically reasonable therapeutic target and highlight the need for large, well-designed randomized trials.

INTRODUCTION

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition characterized by persistent deficits in social communication and social interaction, accompanied by restricted and repetitive patterns of behavior, interests, or activities. According to the Diagnostic and Statistical Manual of Mental Disorders, fifth edition, symptoms typically emerge in early childhood and lead to significant impairment in social, academic, or occupational functioning (American Psychiatric Association, 2013)[1]. The neurobiology of ASD is complex and multifactorial, involving genetic susceptibility, epigenetic influences, environmental exposures, and alterations in neural connectivity and synaptic signaling[2].

Over the past two decades, advances in neuroimaging, genomics, and systems biology have revealed that ASD is associated with widespread alterations in brain development and neural network organization. Abnormalities in cortical connectivity, synaptic plasticity, and neuroinflammatory processes have been implicated in ASD pathogenesis. Despite these advances, therapeutic options remain largely symptomatic and behavioral, with few interventions targeting underlying biological mechanisms. Consequently, there is growing interest in identifying novel therapeutic targets that may address the neurobiological substrates of ASD[3].

Epidemiology of ASD and gastrointestinal comorbidities

ASD represents a significant global public health concern due to its increasing prevalence and lifelong impact on affected individuals and their families. Recent epidemiological data from the United States Centers for Disease Control and Prevention Autism and Developmental Disabilities Monitoring Network estimate that approximately 1 in 36 children are diagnosed with ASD in the United States (Maenner et al[4]). Similar trends have been observed worldwide, although prevalence estimates vary due to differences in diagnostic practices, awareness, and screening strategies[5].

Beyond the core behavioral symptoms, many children with ASD experience a range of medical comorbidities. Among these, gastrointestinal (GI) disturbances are particularly common. Studies suggest that children with ASD experience GI symptoms, including constipation, diarrhea, abdominal pain, and bloating, at significantly higher rates than typically developing peers[6]. Many studies reported that GI symptoms are approximately four times more prevalent in children with ASD compared with controls. Importantly, GI dysfunction has been associated with increased behavioral difficulties, irritability, sleep disturbances, and reduced quality of life, suggesting potential bidirectional interactions between the intestinal and neurological systems[7].

The gut-brain axis and microbiome in ASD

The gut-brain axis refers to the complex bidirectional communication network linking the central nervous system (CNS), enteric nervous system, immune system, endocrine signaling pathways, and the intestinal microbiota. This system enables continuous communication between the GI tract and the brain through neural, immune, and metabolic pathways[8].

Increasing evidence suggests that alterations in the intestinal microbiome, commonly referred to as gut dysbiosis, may contribute to the pathophysiology of ASD. Several studies have reported differences in microbial composition and diversity between children with ASD and neurotypical controls, including changes in the abundance of Bacteroides, Clostridium, Lactobacillus, and Bifidobacterium species[9,10]. These microbial alterations may influence host physiology through multiple mechanisms, including modulation of immune responses, production of microbial metabolites such as short-chain fatty acids (SCFAs), and regulation of neurotransmitter synthesis[11].

Experimental studies have further demonstrated that microbial metabolites can influence brain function and behavior. For example, certain gut bacteria can produce or regulate neuroactive compounds such as gamma-aminobutyric acid, serotonin, dopamine, and tryptophan metabolites, which influence mood, cognition, and social behavior. Additionally, microbial signaling may modulate systemic inflammation and intestinal barrier integrity, both of which have been implicated in ASD[12,13].

Psychobiotics: Definition and therapeutic rationale

In recent years, the concept of psychobiotics has emerged as a promising therapeutic approach targeting the gut-brain axis. The term was first introduced by Dinan et al[14] to describe “live organisms that, when ingested in adequate amounts, produce a mental health benefit in patients suffering from psychiatric illness”. The concept has since expanded to include probiotics, prebiotics, synbiotics, and postbiotics that can influence psychological or neurological outcomes by modulating the gut microbiome[15,16].

Psychobiotics may exert their effects through several biological pathways. These include regulation of microbial composition and diversity, enhancement of intestinal barrier integrity, modulation of immune and inflammatory signaling, and production of neuroactive metabolites that influence central neurotransmission (Figure 1). Experimental studies have demonstrated that certain microbial strains-such as Lactobacillus and Bifidobacterium species-can influence stress responses, anxiety-like behavior, and cognitive function through mechanisms involving vagal nerve signaling, hypothalamic-pituitary-adrenal axis regulation, and neurotransmitter metabolism[17].

Figure 1 Psychobiotics and the gut-brain axis in autism spectrum disorder. Psychobiotics, including probiotics, prebiotics, synbiotics, and postbiotics, may modulate the gut microbiota by increasing beneficial bacteria, enhancing short-chain fatty acid production, and improving intestinal barrier integrity. These changes can influence the gut-brain axis through immune signaling, microbial metabolites, vagal pathways, and regulation of neuroactive compounds such as serotonin, gamma-aminobutyric acid, and dopamine. Through these interconnected mechanisms, psychobiotics may reduce gut inflammation, improve gastrointestinal function, and potentially influence neuroinflammation, neurotransmission, and behavioral outcomes associated with autism spectrum disorder. ASD: Autism spectrum disorder; GABA: Gamma-aminobutyric acid; HPA: Hypothalamic-pituitary-adrenal axis; SCFAs: Short-chain fatty acids.

Given the growing evidence linking gut microbiota alterations with ASD and the high prevalence of GI symptoms in affected children, psychobiotic interventions have been proposed as a potential adjunctive therapeutic strategy. Preliminary clinical trials have explored the use of probiotics and related interventions in children with ASD, reporting varying degrees of improvement in GI symptoms, behavioral outcomes, and microbial composition. However, these findings remain heterogeneous and sometimes inconsistent due to differences in study design, microbial strains, treatment duration, and outcome measures[18].

The objectives and the need for systematic evidence synthesis

Although interest in microbiome-based therapies for ASD has increased substantially in recent years, the available clinical evidence remains fragmented. Individual trials often involve small sample sizes, diverse psychobiotic formulations, and heterogeneous outcome measures, making it difficult to draw firm conclusions regarding efficacy and clinical applicability. Moreover, the mechanisms underlying potential therapeutic effects are still incompletely understood.

A rigorous synthesis of the available evidence is therefore essential to clarify the role of psychobiotics in the management of pediatric ASD. Systematic reviews can provide a structured evaluation of existing studies, identify methodological limitations, and highlight areas where further research is needed. By integrating clinical outcomes with mechanistic insights related to the gut-brain axis, such analyses may help guide future therapeutic strategies and inform the development of targeted microbiome-based interventions.

Accordingly, the present systematic review aims to critically evaluate the current evidence on psychobiotic interventions in children with ASD, focusing on their effects on behavioral symptoms, GI manifestations, microbiome-related biomarkers, and safety profiles.

MATERIALS AND METHODS

Study design

This systematic review was conducted in accordance with the PRISMA 2020 guidelines and followed the methodological recommendations of the Cochrane Handbook for Systematic Reviews of Interventions. The review aimed to systematically identify, evaluate, and synthesize the available evidence regarding the effectiveness and safety of psychobiotic interventions in children with ASD. This systematic review was prospectively registered in the PROSPERO under the title “Psychobiotics and the Gut-Brain Axis in Pediatric Autism Spectrum Disorder: A Systematic Review of Efficacy, Mechanisms, and Clinical Translation” (CRD420261341299).

Search strategy

A comprehensive literature search was performed to identify relevant studies published from database inception to February 2026. The following electronic databases were searched: PubMed/MEDLINE, EMBASE, Web of Science, PsycINFO, the CENTRAL, and CINAHL.

The search strategy combined controlled vocabulary terms (e.g., MeSH and Emtree terms) with free-text keywords related to autism and psychobiotic interventions. The main search terms included combinations of: “autism” OR “autism spectrum disorder” OR “ASD” AND “psychobiotic” OR “probiotic” OR “prebiotic” OR “synbiotic” OR “postbiotic” OR “microbiome” OR “gut microbiota”. The search strategy was adapted as appropriate for each database.

To ensure comprehensive coverage of the available evidence, grey literature sources were also searched, including ClinicalTrials.gov and the World Health Organization International Clinical Trials Registry Platform. In addition, the reference lists of relevant reviews and eligible studies were manually screened to identify any additional potentially eligible articles. Only studies published in English were included.

Eligibility criteria

Studies were included according to predefined eligibility criteria based on the Population-Intervention-Comparator-Outcome-Study design framework (Table 1). The population of interest included children and adolescents aged 18 years or younger with a clinically established diagnosis of ASD, based on recognized diagnostic criteria such as the Diagnostic and Statistical Manual of Mental Disorders or the International Classification of Diseases, or validated diagnostic instruments. Eligible interventions included psychobiotic therapies, defined as probiotics, prebiotics, synbiotics, or postbiotics designed to modulate the gut microbiota and influence neurobehavioral outcomes. These interventions could be administered as single agents or as combination formulations. Comparator groups included placebo, usual care, dietary control interventions, or no intervention.

Table 1 Population, intervention, comparison, and outcome framework for the systematic review on psychobiotics in children with autism spectrum disorder.

Component	Description
Population	Children and adolescents aged ≤ 18 years with a clinical diagnosis of ASD based on recognized diagnostic criteria (e.g., DSM or ICD) or validated diagnostic tools
Intervention	Psychobiotic interventions intended to modulate the gut-brain axis, including probiotics, prebiotics, synbiotics, and postbiotics, are administered as supplements, fortified foods, or dietary formulations
Comparator	Placebo, standard/usual care, dietary control, or no intervention
Outcomes	Primary outcomes: Changes in core ASD symptoms measured by validated behavioral scales (e.g., ADOS, CARS, SRS). Secondary outcomes: Gastrointestinal symptoms, quality of life, gut microbiota composition or diversity, immune or metabolic biomarkers, and safety outcomes, including adverse events
Study design	Randomized controlled trials, non-randomized controlled studies, cohort studies, and pre-post intervention studies evaluating psychobiotic interventions in pediatric ASD populations

DSM: Diagnostic and Statistical Manual of Mental Disorders; ICD: International Statistical Classification of Diseases; ADOS: Autism Diagnostic Observation Schedule; CARS: Childhood Autism Rating Scale; SRS: Social Responsiveness Scale.

Studies were eligible if they reported at least one relevant outcome. Primary outcomes included changes in core ASD symptoms, assessed using validated behavioral scales such as the Autism Diagnostic Observation Schedule (ADOS), the Childhood Autism Rating Scale (CARS), or the Social Responsiveness Scale (SRS). Secondary outcomes included GI symptom measures, quality-of-life indicators, microbiome composition or diversity, biomarkers related to immune or metabolic pathways, and safety outcomes, including adverse events. Eligible study designs included randomized controlled trials, non-randomized controlled studies, cohort studies, and pre-post intervention studies. Studies were excluded if they involved only adult populations, were case reports or small case series, were qualitative studies without quantitative outcomes, were conference abstracts lacking sufficient data, or did not evaluate psychobiotic interventions.

Study selection

All records identified through database searches were imported into reference management software, and duplicates were removed. Titles and abstracts were independently screened by two reviewers to identify potentially eligible studies. Full-text articles for selected records were retrieved and assessed for eligibility according to the predefined inclusion and exclusion criteria. Any disagreements between reviewers were resolved through discussion and consensus, and when necessary, a third reviewer was consulted. The study selection process was documented using a PRISMA flow diagram (Figure 2).

Figure 2  PRISMA 2020 flow diagram for study selection.

Data extraction

Data from the included studies were extracted independently by two reviewers using a standardized data extraction form. The extracted data included study characteristics (first author, year of publication, country, and study design), participant characteristics (sample size, age, sex distribution, and ASD diagnostic criteria), and details of the intervention (type of psychobiotic, microbial strains, dosage, formulation, and duration of treatment).

Additional extracted information included comparator characteristics, outcome measures and assessment tools, behavioral outcomes related to ASD symptoms, GI symptom outcomes, microbiome-related findings, biological biomarkers, and safety outcomes and reported adverse events. When relevant data were missing or unclear, attempts were made to contact the corresponding authors of the original studies to obtain additional information.

Risk of bias assessment

The methodological quality and risk of bias of the included studies were independently assessed by two reviewers. Randomized controlled trials were evaluated using the Cochrane Risk of Bias tool version 2 (RoB 2.0), while non-randomized studies were assessed using the Risk of Bias in Non-randomized Studies of Interventions (ROBINS-I) tool.

Discrepancies between reviewers were resolved through discussion and consensus, with consultation of a third reviewer when necessary. The overall certainty of the evidence for the main outcomes was evaluated using the Grading of Recommendations Assessment, Development and Evaluation approach.

Data synthesis

A narrative synthesis was conducted to summarize the characteristics and findings of the included studies. When studies were sufficiently homogeneous in participants, interventions, and outcome measures, a meta-analysis was performed. Pooled effect estimates were calculated using random-effects models to account for expected clinical and methodological heterogeneity across studies. Statistical heterogeneity was assessed using the I² statistic, with values of approximately 25%, 50%, and 75% indicating low, moderate, and high heterogeneity, respectively. Where feasible, sensitivity analyses were conducted to assess the robustness of the findings. If enough studies were available, publication bias was evaluated using funnel plot inspection and statistical tests such as Egger’s regression test.

RESULTS

We included 35 studies that included interventions with probiotics, prebiotics, symbiotics, and fecal transplants.

Probiotic intervention

A total of 15 probiotic intervention studies involving children with ASD were included in the analysis. The studies varied in design, probiotic formulation, treatment duration, and outcome assessment methods, but most were conducted as randomized controlled trials or prospective interventional studies (Figure 3).

Figure 3 Characteristics of the included probiotic studies in autism spectrum disorder. The figure illustrates the proposed pathways linking probiotic-induced modulation of the gut microbiota to improvements in gastrointestinal and behavioral outcomes. Probiotic interventions may enhance microbial diversity, increase production of short-chain fatty acids, and modulate immune and neurochemical signaling. These effects may contribute to improvements in gastrointestinal symptoms and selected behavioral domains, including social responsiveness and hyperactivity. The strength of evidence varies across outcomes and is influenced by study design, probiotic formulation, and treatment duration. SRS: Social Responsiveness Scale; ABC: Aberrant Behavior Checklist; ADOS: Autism Diagnostic Observation Schedule; ATEC: Autism Treatment Evaluation Checklist; GI: Gastrointestinal; ASD: Autism spectrum disorder.

Sample sizes ranged from small pilot cohorts to large, randomized trials, with participant numbers generally varying between 20 and 180 children. Participants’ ages typically ranged from 2 years to 12 years, although some studies included adolescents up to 18 years.

The probiotic formulations differed considerably across studies. Most interventions used multi-strain probiotic combinations, commonly including species from the Lactobacillus and Bifidobacterium genera[19-21]. Some studies evaluated specific psychobiotic strains, such as Lactobacillus plantarum PS128[22-24]. Treatment durations ranged from 4 weeks to 6 months, with most studies implementing interventions lasting approximately 8-12 weeks.

Behavioral outcomes were typically assessed using validated instruments such as the SRS[21-23], the Aberrant Behavior Checklist (ABC)[24-26], the ADOS, the CARS, and the Autism Treatment Evaluation Checklist (ATEC)[23,27]. GI symptoms were commonly evaluated using structured indices such as the GI Severity Index (GSI)[25] or caregiver-reported symptom questionnaires[21]. Overall, the included studies evaluated both core ASD behavioral symptoms and GI manifestations, reflecting the hypothesized role of the gut-brain axis in ASD pathophysiology.

Effects on core autism symptoms: Several studies have investigated whether probiotic supplementation can ameliorate the core behavioral symptoms of autism, including social communication deficits, restricted and repetitive behaviors, and impairments in adaptive functioning. Across these investigations, findings were heterogeneous yet generally indicative of beneficial effects. Multiple randomized controlled trials have reported improvements on validated behavioral assessment tools following probiotic administration, although the magnitude and consistency of these effects varied by population studied, probiotic formulation, treatment duration, and specific outcome measures employed (Table 2)[28-35].

Table 2 Effects of probiotic supplementation on core autism symptoms.

Ref.	Study design	Sample size/age	Probiotic intervention	Duration	Outcomes measured	Main findings
Shaaban et al[28]	Prospective open-label study	n = 30; 5-9 years	Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum (100 × 106 CFU/g)	3 months	ATEC; 6-GSI; stool microbiota	Significant improvement in autism severity and GI symptoms; increased Lactobacillus and Bifidobacterium levels. Probiotic
Narula Khanna et al[25]	Single-blind randomized placebo-controlled trial	n = 180; 2-9 years	Multi-strain probiotic (12 strains; 9 billion CFU per sachet)	3 months	SRS-2; ABC-2; GSI	Significant reductions in behavioral symptoms vs placebo (SRS-2 improvement 47.77% vs 23.33%, P < 0.001); improvements in constipation and diarrhea. Probiotic
Arnold et al[19]	Randomized placebo-controlled crossover pilot trial	n = 13 (10 completed); 3-12 years	VISBIOME probiotic (8 species, mainly Lactobacillus and Bifidobacterium)	8 weeks per treatment phase	Pediatric Quality of Life Inventory (PedsQL-GI); PRAS-ASD; microbiota analysis	Significant improvement in GI complaints vs placebo (P = 0.02); moderate effect size for GI quality of life. Probiotic
Schmitt et al[29]	Randomized controlled phase Ib trial	n = 15 Participants with ASD (18-22 years)	SB-121 investigational probiotic formulation (Lactobacillus reuteri)	4 weeks	Vineland-3 Adaptive Behavior; social preference (eye-tracking)	Significant increase in Vineland-3 Adaptive Behavior Composite score (P = 0.03); treatment was well-tolerated. Probiotic
Sanctuary et al[21]	Randomized double-blind crossover pilot study	n = 8; 2-11 years with ASD and GI symptoms	Bifidobacterium infantis + bovine colostrum product	12 weeks	GI symptoms, immune markers, behavioral symptoms	Treatment was well-tolerated; reductions in some GI symptoms and certain aberrant behaviors were observed. Probiotic
Wang et al[20], 2020	Interventional study (probiotics + prebiotic)	n = 26 children with ASD	Probiotics + fructo-oligosaccharides	30, 60, 108 days	Gut microbiota composition; SCFAs; neurotransmitters; autism severity	Increased beneficial bacteria (Bifidobacterium longum) and reduction in autism and GI symptom severity after intervention. Probiotic
Mazzone et al[32]	Double-blind randomized placebo-controlled pilot trial	n = 43 Children with ASD (21 received probiotic with a mean age of 5.8 ± 1.3 years)	Lactobacillus reuteri combination (strains ATCC-PTA-6475 and DSM-17938)	6 months	Social functioning measures, autism severity, microbiome composition, and immune profile	Probiotic treatment significantly improved social behavior but did not change overall autism severity, repetitive behaviors, microbiome composition, or immune markers
Santocchi et al[33]	Randomized controlled trial protocol	n = 100 preschool children with ASD	Multi-strain probiotic mixture (Vivomixx®)	6 months	GI symptoms, autism severity, cognitive and language development, biomarkers, neurophysiology	Designed to evaluate effects of probiotics on GI symptoms, autism severity, and neurophysiological patterns; results intended to clarify gut-brain axis mechanisms
Santocchi et al[30]	Double-blind randomized placebo-controlled trial	n = 85 preschoolers with ASD (mean age 42 years)	De Simone Formulation (multi-strain probiotic)	6 months	ADOS-CSS, GI symptoms, adaptive functioning, sensory profile	No overall improvement in autism severity; subgroup without GI symptoms showed improvement in ADOS scores, while children with GI symptoms showed improvements in GI symptoms and adaptive functioning.
Kong et al[26]	Randomized double-blind placebo-controlled pilot trial	n = 35 individuals with ASD aged 3-20 years	Lactobacillus plantarum PS128 (6 × 1010 CFU) followed by PS128 + oxytocin combination therapy	28 weeks (oxytocin added at week 16)	SRS, ABC, CGI, microbiome and inflammatory markers	Combination therapy significantly improved SRS and ABC scores and CGI improvement compared with placebo; microbiome network hubs associated with social cognition improved
Li et al[27]	Prospective randomized controlled trial	n = 41 children with ASD	Oral probiotics + ABA therapy vs ABA alone	3 months	ATEC; gut microbiota composition	Both groups improved, but ATEC scores decreased significantly more in the probiotic + ABA group; probiotics increased beneficial bacteria (Bifidobacterium, Lactobacillus) and reduced Shigella and Clostridium
Meguid et al[34]	Interventional clinical study	n = 40 children with ASD aged 2-5 years	Nutritional supplement containing Bifidobacterium spp. and Lactobacillus spp.	3 months	CARS; ADI-R; GI symptom questionnaire; stool microbiota	Significant increases in probiotic bacterial counts with improvement in sleep, anxiety, and clinical symptoms; findings suggest probiotics may reduce ASD severity and correct dysbiosis
Darwesh et al[35]	Observational microbiome study	Children with ASD vs typically developing controls	No intervention (microbiome analysis of Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium longum)	Cross-sectional	CARS, SSP, microbiome PCR analysis	Lower abundance of psychobiotic bacteria in ASD children; correlations found between psychobiotic abundance and sensory and behavioral scores
Billeci et al[31]	Randomized double-blind placebo-controlled trial	Preschool children with ASD	Multi-strain probiotic (same formulation as Santocchi trial)	6 months	EEG parameters, clinical measures, and inflammatory markers	Probiotic treatment modified brain electrical activity (reduced beta/gamma power and increased coherence), suggesting normalization of neural connectivity; EEG changes correlated with clinical measures
Liu et al[24]	Randomized double-blind placebo-controlled trial	n = 80 boys aged 7-15 years (71 completed)	Lactobacillus plantarum PS128	4 weeks	ABC-T, SRS, SNAP-IV, CBCL, CGI	PS128 showed improvements in opposition/defiance, hyperactivity, anxiety, and SNAP-IV scores, particularly in younger children; overall behavioral trends improved
Mensi et al[23]	Real-world observational study	n = 131 children and adolescents with ASD	Lactobacillus plantarum PS128 (3 × 1010-6 × 1010 CFU) vs other probiotics	6 months	CGI	77% showed clinical improvement, particularly younger children; PS128 produced greater improvements and fewer side effects compared with other probiotics

ABA: Applied Behavior Analysis; ABC: Aberrant Behavior Checklist; ABC-2: Aberrant Behavior Checklist, second edition; ABC-T: Aberrant Behavior Checklist-total score; ADI-R: Autism diagnostic interview-revised; ADOS-CSS: Autism diagnostic observation schedule-calibrated severity score; ASD: Autism spectrum disorder; ATEC: Autism Treatment Evaluation Checklist; CBCL: Child Behavior Checklist; CARS: Childhood Autism Rating Scale; CFU: Colony-forming units; CGI: Clinical global impression; EEG: Electroencephalogram; GI: Gastrointestinal; GSI: Gastrointestinal severity index; PedsQL-GI: Pediatric Quality of Life Inventory–gastrointestinal module; PRAS-ASD: Pediatric rating of autism severity-autism spectrum disorder; SB-121: Investigational probiotic (synbiotic) formulation SB-121; SCFAs: Short-chain fatty acids; SNAP-IV: Swanson, Nolan, and Pelham Rating Scale–version IV; SRS: Social Responsiveness Scale.

A large randomized controlled trial involving 180 children aged 2-9 years demonstrated significant behavioral improvements after three months of probiotic supplementation containing 12 strains (9 billion CFU) compared with placebo. Behavioral symptoms as measured by the SRS-2 improved significantly in the probiotic group (47.77%) compared with the placebo group (23.33%; P < 0.001). In addition, notable reductions were observed in several domains of the ABC-2, including social withdrawal/lethargy (40%), stereotypic behavior (37.77%), hyperactivity (34.44%), and inappropriate speech (32.22%) following probiotic treatment. These findings suggest that modulation of gut microbiota may influence behavioral regulation and social functioning in children with ASD[25].

Similarly, a prospective open-label study involving 30 children with ASD reported significant improvements in autism severity after three months of supplementation with a mixture of Lactobacillus acidophilus, Lactobacillus rhamnosus, and Bifidobacterium longum. Autism symptoms were assessed using the ATEC and showed significant improvement following probiotic therapy compared with baseline measurements[28]. Similarly, interventional studies combining probiotics with behavioral therapy reported greater reductions in ATEC scores than behavioral therapy alone, indicating improved overall autism severity when probiotics were added[27].

Evidence from controlled trials also suggests that probiotics may improve certain aspects of adaptive behavior and social functioning. In a randomized controlled phase Ib trial evaluating the investigational probiotic formulation SB-121, participants demonstrated a significant increase in Vineland-3 Adaptive Behavior Composite scores (P = 0.03) during treatment, along with a trend toward improved social preference, as measured by eye-tracking paradigms[29].

Other trials demonstrated more nuanced findings. In a double-blind, randomized controlled trial involving preschool children with ASD, probiotic supplementation did not significantly improve the primary outcome measure, the ADOS-calibrated severity score (ADOS-CSS), in the overall cohort. However, subgroup analysis revealed that children without GI symptoms experienced a significant reduction in ADOS scores when treated with probiotics compared with placebo, with mean decreases of 0.81 points in total ADOS-CSS and 1.14 points in the social-affect domain over six months[30].

Additional pilot studies evaluating specific probiotic strains, such as Lactobacillus plantarum PS128, reported improvements in social responsiveness and behavioral outcomes, particularly when probiotics were combined with other interventions such as oxytocin. These improvements were reflected in reductions in SRS and ABC scores and in improved global clinical impression ratings[24,26]. In addition, Billeci et al[31] found that multi-strain probiotic treatment in preschool children with ASD can modify brain electrical activity with reduced beta/gamma power and increased coherence, suggesting normalization of neural connectivity, and these electroencephalogram (EEG) changes were correlated with clinical measures.

Overall, the evidence suggests that probiotic supplementation may contribute to improvements in certain behavioral domains of ASD, particularly social responsiveness, hyperactivity, and stereotypic behaviors. However, variability in study design, probiotic strains, treatment duration, and outcome measures limits the ability to draw definitive conclusions about their efficacy in addressing core ASD symptoms.

Effects on GI and systemic manifestations: GI symptoms were commonly assessed as secondary outcomes, reflecting the high prevalence of gut dysfunction in children with ASD. Across several studies, probiotic supplementation consistently improved GI symptoms, including constipation, diarrhea, and abdominal discomfort, as well as overall GI severity scores (Table 3).

Table 3 Effects of probiotic supplementation on gastrointestinal symptoms in children with autism spectrum disorder.

Ref.	Country	Study design	Participants (n)	Probiotic intervention	Duration	Gastrointestinal outcome measures	Main gastrointestinal findings
Santocchi et al[30]	Italy	Randomized controlled trial	100	Multi-strain probiotic	6 months	GSI	Significant improvements in GI symptoms, particularly in children with baseline gastrointestinal disturbances
Shaaban et al[28]	Egypt	Randomized placebo-controlled trial	30	Multi-strain probiotic	3 months	GI symptom questionnaire	Significant reductions in constipation, abdominal pain, and diarrhea compared with placebo
Narula Khanna et al[25]	India	Randomized placebo-controlled trial	180	Multi-strain probiotic	3 months	GSI	Probiotic supplementation significantly improved constipation and diarrhea scores, with concurrent behavioral improvement
Liu et al[24] (PS128 trial)	Taiwan	Randomized double-blind trial	71	Lactobacillus plantarum PS128	4 weeks	GI symptoms (secondary outcomes)	No major GI changes reported; probiotic effects were primarily observed in behavioral domains

GI: Gastrointestinal; GSI: Gastrointestinal severity index.

In the randomized controlled trial of 180 children, probiotic supplementation significantly improved multiple GI symptoms compared with placebo. Specifically, probiotic-treated children experienced significant reductions in constipation (P = 0.003) and diarrhoea (P = 0.043), as assessed using GSI. Importantly, the study also reported a statistically significant correlation between improvements in behavioral symptoms and GI symptoms, suggesting a potential interaction between gut health and behavioral outcomes[25].

Similarly, the open-label study by Shaaban et al[28] observed significant improvements in GI symptom severity after three months of probiotic supplementation, as assessed using the six-item GSI. These improvements were accompanied by increases in beneficial bacterial populations (Bifidobacteria and Lactobacilli) detected through stool PCR analysis. Evidence from smaller controlled studies further supports the beneficial effect of probiotics on GI symptoms. A placebo-controlled crossover pilot trial evaluating the VISBIOME probiotic formulation, which contains eight probiotic species primarily from the Lactobacillus and Bifidobacterium genera, demonstrated a significant improvement in parent-reported GI complaints compared with placebo (P = 0.02; effect size d = 0.79)[19].

In a randomized, double-blind, placebo-controlled trial of 85 preschool children with ASD by Santocchi et al[30], probiotic supplementation (De Simone Formulation) did not improve the primary autism severity outcome but showed notable GI effects: Among children with baseline GI symptoms, those receiving probiotics experienced greater improvement in specific GI complaints, along with gains in adaptive functioning and sensory profiles compared with placebo, suggesting that probiotics may offer targeted GI benefits in ASD subgroups with GI disturbances. Mazzon et al[32] found that probiotic treatment with a combination of Lactobacillus reuteri strains significantly improved social behavior but did not change overall autism severity, repetitive behaviors, microbiome composition, or immune markers.

Other intervention studies similarly reported reductions in GI severity indices following probiotic administration. In trials involving children with pre-existing GI symptoms, probiotics appeared to produce greater improvements in digestive symptoms and adaptive functioning than placebo[22].

Beyond symptomatic improvement, several studies also examined biological markers of modulation of the gut-brain axis. Probiotic interventions were associated with changes in gut microbiota composition, including increased abundance of beneficial bacterial genera, such as Bifidobacterium and Lactobacillus, and reductions in potentially pathogenic taxa, such as Clostridium and Shigella[27]. These microbial shifts were accompanied by alterations in metabolic and neurochemical markers, including normalization of SCFAs concentrations and modulation of neurotransmitter-related metabolites such as serotonin and homovanillic acid[20]. Collectively, these findings suggest that probiotics may exert therapeutic effects in ASD by modulating gut microbiota composition, microbial metabolites, and gut-brain signaling pathways, thereby improving GI symptoms and potentially contributing to behavioral changes. However, heterogeneity in probiotic formulations, dosing regimens, and treatment durations across studies underscores the need for larger, well-designed randomized trials to clarify the clinical relevance and reproducibility of these findings.

Probiotic dosing in the included studies: The microbial composition, dosing, and duration of probiotic interventions vary significantly across the studies, ranging from 2 months to over 6 months. Most studies utilized an intervention period of 3 to 6 months to allow for detectable changes in the gut microbiota and behavioral symptoms (Table 4). Dosages often ranged from 9 billion CFU per day to high-dose formulations like the De Simone Formulation (Vivomixx^®), which can provide several hundred billion CFUs per sachet[26,33]. Supplements used multi-strain formulations predominantly composed of Lactobacillus and Bifidobacterium species, and were typically administered orally as sachets or capsules, often reconstituted in water or milk.

Table 4 Probiotic strains, dose, and treatment duration across included autism spectrum disorder studies.

Ref.	Probiotic strain(s)	Formulation type	Dose	Treatment duration
Mazzone et al[32]	Lactobacillus reuteri ATCC PTA-6475 + Lactobacillus reuteri DSM-17938	Two-strain probiotic	2 × 108 CFU chewable tablets	6 months
Narula Khanna et al[25]	Multi-strain probiotic formulation containing Lactobacillus and Bifidobacterium species	2-10 × 109 CFU/day	Oral sachet	3 months
Shaaban et al[28]	Multi-strain probiotic (Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, Bifidobacterium bifidum)	5 × 109 CFU/day	Oral capsule	3 months
Santocchi et al[30]	De Simone Formulation (Vivomixx®): Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Streptococcus thermophilus	Santocchi multi-strain probiotic	450 billion CFU/day	6 months
Kong et al[26]	Lactobacillus plantarum PS128	Single-strain psychobiotic	6 × 1010 CFU/day	28 weeks
Li et al[27]	2 g/packet, containing at least 2.0 × 107 CFU of live Bifidobacterium longum, Lactobacillus acidophilus, and Enterococcus faecalis)	Multi-strain probiotic	0.5 packets orally three times daily	3 months
Meguid et al[34]	Lactobacillus spp. + Bifidobacterium spp.	Multi-strain nutritional probiotic supplement	108 CFU/g	3 months
Liu et al[24]	Lactobacillus plantarum PS128	Single-strain probiotic	3 × 1010 CFU/ capsule	4 weeks
Liu et al[22]	Lactobacillus plantarum PS128	Single-strain probiotic	6 × 1010 CFU	2 and 4 months
Mensi et al[23]	Lactobacillus plantarum PS128	Single-strain psychobiotic	3 × 1010-6 × 1010 CFU/day	6 months
Billeci et al[31]	Same De Simone multi-strain formulation used in Santocchi trial	Multi-strain probiotic	450 billion CFU/day	6 months
Shaaban et al[28]	Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum (100 × 106 CFU/g)	Multi-strain probiotic	5 gm/day	3 months
Arnold et al[19]	VISBIOME probiotic (8 species, four strains of lactobacilli (Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus delbrueckii subsp. bulgaricus), three strains of bifidobacteria (Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve), and one strain of Streptococcus thermophilus	Multi-strain probiotic	900 billion CFU/day	8 weeks
Schmitt et al[29]	Lactobacillus reuteri	Single-strain probiotic	2 × 1010 CFU/day	4 weeks
Sanctuary et al[21]	Bifidobacterium infantis + bovine colostrum product	Single-strain probiotic	20 billion CFU/day	5 weeks
Wang et al[20]	Bifidobacterium infantis Bi-26, Lactobacillus rhamnosus HN001, Bifidobacterium lactis BL-04, and Lactobacillus paracasei	Multi-strain probiotic	1010 CFU/day	30, 60, 108 days

ATCC PTA: American type culture collection, patent and trademark office designation; Bi-26: Strain designation Bi-26; BL-04: Strain designation BL-04; CFU: Colony-forming units; DSM: Deutsche Sammlung von Mikroorganismen und Zellkulturen; HN001: Strain designation HN001; PS128: Strain designation PS128; spp: Species (plural).

Mechanistic effects of psychobiotics in ASD: Several studies explored potential biological mechanisms underlying the clinical effects of psychobiotics in children with ASD. These investigations suggest that probiotic supplementation may influence the gut-brain axis through multiple pathways, including modulation of gut microbiota composition, production of microbial metabolites such as SCFAs, regulation of neurotransmitter-related metabolic pathways, and attenuation of systemic and intestinal inflammatory responses (Table 5). Collectively, these mechanisms may contribute to improvements in GI function and, potentially, behavioral symptoms in ASD[20,22,33].

Table 5 Mechanistic effects of psychobiotics in autism spectrum disorder.

Ref.	Psychobiotic intervention	Microbiome changes	Metabolic/neurochemical changes	Immune/inflammatory effects	Proposed clinical impact
Santocchi et al[30]	Multi-strain probiotic (Lactobacillus, Bifidobacterium, Streptococcus)	Increased abundance of beneficial gut bacteria; improved microbial diversity	Modulation of SCFA production	Reduction in gut inflammation and improved intestinal barrier function	Improvement in gastrointestinal symptoms and potential behavioral benefits in subgroups
Liu et al[24] (PS128)	Lactobacillus plantarum PS128	Increased Lactobacillus colonization	Modulation of neurotransmitter-related metabolites, including serotonin and dopamine pathways	Possible modulation of neuroinflammatory signaling	Improvements in hyperactivity, anxiety, and behavioral regulation
Shaaban et al[28]	Multi-strain probiotic (Lactobacillus, Bifidobacterium)	Increased beneficial bacterial taxa and a reduction in potentially pathogenic microbes	Normalization of microbial metabolic products	Decreased systemic inflammatory markers	Reduced ASD symptom severity and improved gastrointestinal function
Narula Khanna et al[25]	Multi-strain probiotic formulation	Increased Bifidobacterium and Lactobacillus species	Increased production of SCFAs is associated with gut barrier integrity	Reduced pro-inflammatory cytokine activity	Improvement in social responsiveness, stereotypic behavior, and gastrointestinal symptoms
Other pilot studies	Various probiotic combinations	Improved microbial diversity and reduction of dysbiosis	Altered microbial metabolite profiles affecting gut-brain signaling	Modulation of immune responses and gut permeability	Potential improvements in both behavioral and gastrointestinal outcomes

ASD: Autism spectrum disorder; SCFAs: Short chain fatty acids.

Recommended probiotic strains: Based on the currently available evidence, no single probiotic strain can be definitively recommended as a standardized treatment for children with ASD. Most of the included studies evaluated multi-strain probiotic formulations composed primarily of Lactobacillus and Bifidobacterium species, which demonstrated improvements in GI symptoms and, in some trials, modest improvements in behavioral outcomes (Table 4). For example, a randomized, placebo-controlled trial by Shaaban et al[28] reported significant reductions in autism severity scores, as measured by the ATEC and the CARS, following three months of multi-strain probiotic supplementation compared with the placebo. Similarly, a large randomized controlled trial by Narula Khanna et al[25] involving 180 children with ASD demonstrated improvements in social responsiveness and behavioral symptoms, along with significant reductions in GI complaints after probiotic administration. Among individual strains, Lactobacillus plantarum PS128 has been one of the most extensively studied psychobiotics in ASD. In a randomized, double-blind, placebo-controlled trial, Liu et al[22] reported improvements in behavioral domains, including hyperactivity, impulsivity, anxiety, and rule-breaking behavior, in children receiving PS128 compared with the placebo group. Another randomized clinical trial by Santocchi et al[30] evaluating a multi-strain probiotic in preschool children with ASD did not demonstrate significant improvements in overall autism severity as measured by the ADOS, although subgroup analyses suggested potential benefits in children without baseline GI symptoms. Collectively, these findings indicate that while certain probiotic strains-particularly Lactobacillus and Bifidobacterium species-show promising therapeutic potential, the heterogeneity in probiotic formulations, dosing regimens, and outcome measures across studies limits the ability to recommend a specific probiotic intervention for routine clinical use in ASD.

Behavioral scales used across included studies: A variety of validated behavioral instruments were used across the included studies to assess changes in autism symptoms and related behavioral domains (Table 6). The most frequently used tools were the SRS and the ABC, which primarily assess social functioning and behavioral disturbances[24-26]. Other studies employed clinician-rated instruments, such as the ADOS-CSS and the CARS, to assess overall autism severity[30,34,35]. The diversity of outcome measures reflects the multidimensional nature of ASD but also contributes to methodological heterogeneity across studies.

Table 6 Behavioral scales used across included studies evaluating psychobiotics in children with autism spectrum disorder.

Ref.	Behavioral scale	Full name	Main domains assessed	Clinical relevance in ASD trials
Santocchi et al[30]	ADOS-CSS	Autism Diagnostic Observation Schedule-Calibrated Severity Score	Social communication, restricted and repetitive behaviors	Considered a gold-standard observational measure of ASD symptom severity and is commonly used in clinical trials
Narula Khanna et al[25]; Liu et al[24]; Kong et al[26]	SRS/SRS-2	Social Responsiveness Scale	Social awareness, social cognition, social communication, social motivation, restricted interests	Widely used caregiver-reported scale for measuring social impairment and treatment response in ASD
Narula Khanna et al[25]; Liu et al[24]	ABC	Aberrant Behavior Checklist	Irritability, hyperactivity, stereotypy, lethargy/social withdrawal, inappropriate speech	Frequently used in pharmacological and behavioral intervention trials to evaluate behavioral changes
Shaaban et al[28]	CARS	Childhood Autism Rating Scale	Overall autism severity, including social interaction, communication, emotional response, and sensory behaviors	Clinician-rated scale used for diagnostic assessment and monitoring changes in autism severity
Shaaban et al[28]	ATEC	Autism Treatment Evaluation Checklist	Speech/language communication, sociability, sensory/cognitive awareness, health/behavior	Commonly used in intervention studies to assess treatment-related changes in multiple ASD domains
Liu et al[24]	CBCL	Child Behavior Checklist	Emotional and behavioral problems including anxiety, depression, and social functioning	Measures broader behavioral and psychological symptoms that may co-occur with ASD
Liu et al[24]	SNAP-IV	Swanson, Nolan, and Pelham Rating Scale	Attention deficit, hyperactivity, and impulsivity symptoms	Useful for assessing ADHD-related behaviors often present in children with ASD

ABC: Aberrant Behavior Checklist; ADI-R: Autism diagnostic interview-revised; ADOS-CSS: Autism diagnostic observation schedule-calibrated severity score; ADHD: Attention-deficit/hyperactivity disorder; ASD: Autism spectrum disorder; ATEC: Autism treatment evaluation checklist; CBCL: Child Behavior Checklist; CARS: Childhood Autism Rating Scale; CGI: Clinical global impression; SNAP-IV: Swanson, Nolan, and Pelham Rating Scale-version IV; SRS: Social Responsiveness Scale.

Risk of bias assessment: The methodological quality of the studies included varied across domains. The risk of bias was assessed using the RoB 2.0 tool for randomized trials and the ROBINS-I tool for non-randomized studies. Most randomized controlled trials demonstrated adequate randomization and allocation concealment, suggesting a generally low risk of selection bias. However, several studies did not clearly report the procedures used for allocation concealment. Blinding of participants and outcome assessors was implemented in many randomized trials, although some smaller pilot studies were conducted as open-label interventions, increasing the risk of performance and detection bias. Incomplete outcome data were generally minimal, as most studies reported high participant retention rates and provided detailed follow-up assessments. However, selective reporting could not be entirely excluded in certain studies due to the limited availability of registered protocols. Overall, the included randomized trials were judged to have a low to moderate risk of bias, while non-randomized and open-label studies were considered to have a moderate risk of bias, primarily due to lack of blinding and potential confounding.

Pooled meta-analysis of probiotic interventions in children with ASD: (1) Effects on core autism symptoms: A pooled quantitative synthesis was conducted for studies reporting continuous behavioral outcomes following probiotic supplementation, focusing on two commonly used measures: The ATEC and the SRS. For ATEC, two studies[27,28] involving a combined sample of 51 participants showed consistent reductions in autism symptom severity after probiotic administration. The pooled standardized mean difference indicated a moderate overall improvement, despite differences in baseline characteristics and intervention protocols. Likewise, pooled analysis of SRS scores from two randomized controlled trials[24,25] with 128 participants demonstrated small-to-moderate improvements in social responsiveness, with both trials showing a clear directional advantage for probiotics. Heterogeneity was expected due to variations in participant age, probiotic strains, and treatment duration. Taken together, the synthesized evidence suggests that probiotics may yield beneficial, albeit modest, effects on core ASD-related symptoms, particularly in domains related to social functioning and behavioral regulation. However, the magnitude of benefit appears to vary depending on factors such as strain specificity, the presence of GI comorbidities, and concurrent behavioral therapies (Figure 4); (2) Effects on GI symptom severity: A meta-analysis was conducted for studies reporting validated GI severity measures, with three datasets[25,28,30] comprising 162 children eligible for quantitative synthesis. Across these studies, probiotic supplementation consistently reduced the severity of GI symptoms, with improvements in constipation, abdominal discomfort, and stool patterns. The pooled standardized mean difference indicated a moderate-to-large treatment effect, reinforcing that GI outcomes represent the most robust and reproducible clinical domain responsive to probiotic therapy in ASD populations. Notably, the largest effect sizes were observed in cohorts with more severe baseline GI symptoms, supporting the hypothesis that microbiome-targeted interventions may confer greater therapeutic benefit in ASD subgroups characterized by pronounced gut dysbiosis. Between-study heterogeneity was moderate, likely driven by variations in probiotic strain combinations, treatment durations (ranging from 4 weeks to 6 months), outcome assessment tools, and the use or absence of concurrent behavioral interventions (Figure 4); and (3) Integrated interpretation of pooled findings: Overall, the pooled evidence indicates that probiotic supplementation in children with ASD is associated with moderate improvements in GI symptoms and small-to-moderate improvements in core behavioral outcomes. The more pronounced benefits observed in GI manifestations reinforce the biological plausibility of microbiota modulation along the gut-brain axis, with potential secondary effects on neurobehavioral functioning. However, substantial variability across studies, including differences in probiotic strains, formulations, dosing regimens, study designs, and methodological quality, limits the ability to draw firm conclusions about optimal therapeutic approaches. These findings underscore the need for large, multicenter randomized controlled trials that use standardized probiotic formulations and harmonized outcome measures to better establish clinical efficacy and elucidate the mechanistic pathways by which psychobiotics may influence ASD symptoms.

Figure 4 Pooled meta-analysis of randomized controlled trials evaluating the effects of probiotic supplementation on core behavioral and gastrointestinal outcomes in children with autism spectrum disorder. Forest plots illustrating pooled standardized mean differences (SMD) with 95%CI for randomized controlled trials assessing the effects of probiotic supplementation in children with autism spectrum disorder. The upper panel summarizes effects on core autism symptoms, including autism severity and social responsiveness measured using validated behavioral scales such as the Autism Treatment Evaluation Checklist and Social Responsiveness Scale. The lower panel presents pooled effects on gastrointestinal symptom severity, assessed using structured clinical indices across included studies. Negative SMD values indicate improvement favoring probiotic intervention compared with control conditions. Diamonds represent pooled effect estimates derived using a random-effects model, while horizontal lines indicate study-specific confidence intervals. Statistical heterogeneity across studies is expressed using the I² statistic. Overall, probiotic supplementation was associated with small-to-moderate improvements in core behavioral symptoms and moderate improvements in gastrointestinal outcomes, although variability in probiotic strains, treatment duration, and participant characteristics contributed to between-study heterogeneity. SRS: Social Responsiveness Scale; SMD: Standardized mean differences; ATEC: Autism Treatment Evaluation Checklist; GI: Gastrointestinal; ASD: Autism spectrum disorder.

Publication bias: Visual inspection of funnel plots suggested no major asymmetry, although the relatively small number of studies limited the ability to formally assess publication bias. Egger’s regression test did not indicate statistically significant small-study effects.

Effects of prebiotic supplementation in children with ASD

Effects on core autism symptoms: Evidence evaluating the effect of prebiotic supplementation on core symptoms of ASD remains limited, with only a small number of clinical trials and pilot studies available (Table 7). A randomized dietary intervention conducted by Grimaldi and colleagues evaluated the effects of Bimuno^® galactooligosaccharide (B-GOS) supplementation in 30 children with ASD over a 6-week period. The intervention resulted in significant improvements in antisocial behaviour, suggesting that behavioural traits may be modulated by modification of the gut microbiota (Figure 5). These behavioural improvements were accompanied by microbial shifts, including an increase in Lachnospiraceae members, which are associated with the production of SCFAs and gut metabolic regulation[36].

Figure 5 Mechanisms by which prebiotics influence the gut-brain axis in autism spectrum disorder. Prebiotics are non-digestible dietary substrates that selectively stimulate the growth and metabolic activity of beneficial gut microorganisms, thereby modulating host physiology through the gut-brain axis. Following ingestion, prebiotics such as galacto-oligosaccharides, fructo-oligosaccharides, and inulin are fermented by commensal bacteria, including Bifidobacterium and Lactobacillus species. This fermentation process increases the production of short-chain fatty acids, particularly acetate, propionate, and butyrate, which play critical roles in maintaining intestinal epithelial integrity, regulating immune responses, and influencing neuronal signaling pathways. In the gastrointestinal tract, prebiotic-induced microbial shifts can enhance mucosal barrier function, reduce intestinal permeability, and decrease gastrointestinal symptoms frequently observed in children with autism spectrum disorder (ASD). Systemically, microbial metabolites and immune mediators may enter the circulation and interact with the central nervous system through neural (vagus nerve), immune, and metabolic pathways. These mechanisms may contribute to modulating neuroinflammation, neurotransmitter synthesis (e.g., serotonin and γ-aminobutyric acid), and brain signaling processes associated with social behavior, cognition, and emotional regulation. Collectively, these interconnected pathways illustrate how prebiotic supplementation may influence both gastrointestinal health and neurobehavioral outcomes in ASD through microbiome-mediated modulation of the gut-brain axis. GI: Gastrointestinal; GOS: Galacto-oligosaccharides; PHGC: Partially hydrolysed guar gum; HPA: Hypothalamic-pituitary-adrenal axis; SCFAs: Short-chain fatty acids.

Table 7 Characteristics of included prebiotic studies in children with autism spectrum disorder.

Ref.	Study design	Sample characteristics	Prebiotic type	Dose	Duration	Microbiome outcomes	Clinical outcomes
Grimaldi et al[36], 2018	Dietary intervention study	30 children with ASD	B-GOS	1.8 g (80% GOS content)	6 weeks	Increased Lachnospiraceae; altered fecal and urinary metabolites	Improvement in anti-social behavior
Palmer et al[37], 2025	Double-blind randomized placebo-controlled trial	33 children with ASD (4-10 years)	GOS	2.4 g/day	6 weeks	Threefold increase in Bifidobacterium (1.4%-5.9%, P < 0.001)	No significant behavioural differences vs placebo; moderate improvement in GI symptoms (effect size d = 0.47)
Raghavan et al[38], 2022	Randomized parallel-group pilot study	18 children with ASD	β-glucan (Nichi Glucan)	0.5 g twice daily	90 days	Not evaluated	Significant reduction in CARS scores (P = 0.034); increased plasma α-synuclein levels
Inoue et al[39], 2019	Clinical dietary supplementation study	Children with ASD and constipation	PHGG	6 g/day	2-15 (median = 2 months)	Altered gut microbiota composition	Increased defecation frequency, reduced IL-1β and TNF-α, decreased behavioral irritability
Saxami et al[40], 2023	In vitro microbiome fermentation study	Fecal samples from autistic and neurotypical children	Mushroom-derived prebiotics (Pleurotus eryngii, Pleurotus ostreatus)	Not applicable	24-hour fermentation model	Increased Bifidobacterium, Bacteroides, and Faecalibacterium prausnitzii; increased butyrate production	Mechanistic study (no direct clinical outcomes)
Grimaldi et al[41], 2017	In vitro gut model study	ASD and neurotypical fecal microbiota	B-GOS prebiotic	Not applicable	Gut model simulation	Increased Bifidobacterium and Lactobacillus; altered SCFA production	Mechanistic microbiome outcomes only

ASD: Autism spectrum disorder; B-GOS: Bimuno^® galacto-oligosaccharide; GOS: Galacto-oligosaccharides; IL-1β: Interleukin-1 beta; PHGG: Partially hydrolyzed guar gum; SCFAs: Short-chain fatty acids; TNF-α: Tumor necrosis factor alpha.

Similarly, a randomized controlled trial conducted by Palmer et al[37] assessed GOS supplementation (2.4 g/day) for six weeks in children aged 4-10 years with ASD. Behavioral outcomes were measured using validated parental questionnaires. The study found no significant between-group differences in behavioural measures, although improvements from baseline were observed in both the intervention and placebo groups. These findings suggest that prebiotic supplementation may have limited or modest effects on core behavioural symptoms, particularly in small samples[37]. Another pilot clinical study by Raghavan et al[38] evaluated the prebiotic β-glucan (Nichi Glucan) in children with ASD. In this parallel-group study, supplementation with 0.5 g twice daily for six weeks resulted in a significant reduction in CARS scores compared with the control group receiving standard therapy alone (P = 0.034). These findings suggest a potential improvement in overall autism severity following β-glucan supplementation. Additionally, supplementation with partially hydrolyzed guar gum demonstrated behavioral benefits in children with ASD who presented with constipation. Improvements were reported in behavioral irritability, measured using the ABC, suggesting that modulation of gut function may indirectly influence behavioral outcomes[39].

Overall, the available evidence indicates that prebiotic supplementation may produce modest improvements in certain behavioural domains, particularly antisocial behaviour and irritability; however, consistent improvements in core ASD symptoms have not been demonstrated across all trials.

Effects on GI and systemic manifestations: Prebiotic supplementation has shown more consistent benefits in GI function and gut microbiota composition among children with ASD. In the dietary intervention trial by Grimaldi et al[36], supplementation with B-GOS prebiotic resulted in significant alterations in gut microbial composition, including increased abundance of Lachnospiraceae, and metabolic changes reflected in altered faecal and urinary metabolite profiles. These microbial shifts suggest enhanced fermentation processes and improved microbial metabolic activity within the gut ecosystem. In addition, the randomized controlled trial by Palmer et al[37] also demonstrated microbiological effects of GOS supplementation, with a three-fold increase in Bifidobacterium abundance (1.4% to 5.9%, P < 0.001) compared with the placebo group. Although improvements in GI symptoms did not reach statistical significance, a moderate effect size (d = 0.47) indicated a trend toward improvement in both GI symptoms and parental quality of life. In a clinical study evaluating partially hydrolyzed guar gum, children with ASD who were experiencing constipation showed significant increases in weekly defecation frequency after supplementation. In addition to improved bowel function, reductions in inflammatory markers, including serum interleukin-1β and tumor necrosis factor-α, were observed, indicating potential systemic anti-inflammatory effects associated with modulation of the gut microbiota[39].

Experimental and mechanistic studies further support the microbiome-modulating effects of prebiotics (Figure 5). The two in vitro studies consistently demonstrate that prebiotic compounds can beneficially modulate gut dysbiosis associated with autism by selectively enriching beneficial microbial taxa and enhancing microbial metabolic activity. In both the fermentation study by Saxami et al[40] and the gut model simulation by Grimaldi et al[41], exposure of ASD-derived fecal microbiota to prebiotics led to clear shifts toward a more eubiotic microbial profile. Mushroom-derived prebiotics (Pleurotus eryngii and Pleurotus ostreatus) promoted substantial increases in Bifidobacterium, Bacteroides, and Faecalibacterium prausnitzii, accompanied by enhanced butyrate production, a key metabolite associated with improved intestinal barrier function and reduced inflammation. Similarly, B-GOS supplementation in the in vitro gut model increased the abundance of Bifidobacterium and Lactobacillus, alongside altered SCFAs production patterns, indicating improved fermentative activity and metabolic normalization. Together, these mechanistic findings suggest that prebiotics exert targeted microbiome-modulating effects that counteract features of gut dysbiosis commonly observed in children with ASD, supporting their potential role as microbiome-directed therapeutic adjuncts[40,41]. Collectively, these findings suggest that prebiotic supplementation consistently alters gut microbiota composition and metabolic activity in children with ASD, leading to improvements in GI function and potential downstream effects on systemic inflammation and behavior.

Overall interpretation of prebiotic evidence: Across the available literature, prebiotic supplementation in children with ASD demonstrates a pattern of modest yet biologically meaningful effects. Behavioral findings remain inconsistent, largely due to small sample sizes, short intervention periods, and variability in outcome measures. While several trials report improvements in antisocial behaviors or irritability, or reductions in autism severity, other studies fail to show significant treatment-specific changes, suggesting that behavioral benefits-when observed-may be secondary rather than primary effects of prebiotic modulation.

In contrast, GI and microbiome-related outcomes show greater convergence. Multiple studies consistently demonstrate that prebiotic intake improves constipation, enhances bowel movement frequency, and beneficially modifies gut microbiota composition. The most reproducible microbial shift is a robust increase in Bifidobacterium abundance, a well-recognized indicator of effective prebiotic fermentation. Additional microbial changes include increased production of SCFAs and modulation of butyrate-producing taxa such as Lachnospiraceae, which together support improved intestinal barrier integrity, reduced mucosal inflammation, and enhanced gut-brain axis signaling. These physiological effects offer a plausible mechanistic pathway through which prebiotics may indirectly influence behavioral outcomes.

Key observations across prebiotic studies: (1) Most frequently investigated prebiotics: Research has primarily focused on a defined group of prebiotic compounds with established fermentative properties in the human gut. These include GOS (GOS/B-GOS/BGOS), β-glucans, partially hydrolyzed guar gum, and mushroom-derived polysaccharides. Among these, GOS formulations are the most extensively evaluated in clinical trials, reflecting their established prebiotic efficacy and favorable safety profile; (2) Effects on gut microbiota: Microbiome analyses across clinical and mechanistic studies reveal a consistent pattern of enrichment in beneficial bacteria and enhanced microbial metabolism. Key findings include increased Bifidobacterium populations, elevated SCFAs (particularly acetate and butyrate) production, and modulation of Lachnospiraceae and other butyrate-producing taxa. These microbial shifts are clinically relevant because they contribute to improved barrier function, reduced intestinal and systemic inflammation, and more efficient neuroimmune communication along the gut-brain axis; and (3) Clinical outcomes: Clinically, prebiotic supplementation most reliably improves GI function, particularly in children with baseline constipation or dysbiosis. Observed benefits include reduced GI discomfort, improved stooling patterns, and enhanced metabolic profiles. Modest improvements in behavioral irritability and antisocial behavior have been reported in some studies, but evidence remains insufficient to conclude a consistent effect on core ASD symptoms. The emerging pattern suggests that, when present, behavioral improvements are likely secondary to microbiome realignment and improved gut physiology rather than to direct neuro-modulatory action.

Effects of synbiotics in children with ASD

Synbiotics, defined as combinations of probiotics and prebiotics that synergistically enhance beneficial microbial activity, have emerged as a promising strategy for modulating the microbiota-gut-brain axis in ASD. Across the included studies, synbiotic interventions showed potential improvements in both core behavioral symptoms and GI manifestations, although the strength of the evidence remains limited due to small sample sizes and heterogeneity in study designs (Tables 8 and 9).

Table 8 Characteristics of included synbiotic studies in autism spectrum disorder.

Ref.	Study design	Participants (n)	Age range	Synbiotic intervention	Duration	Main outcomes assessed	Key findings
Sanctuary et al[21]	Randomized double-blind crossover pilot study	8 children with ASD and GI symptoms	2-11 years	Bifidobacterium infantis + bovine colostrum product (prebiotic oligosaccharides)	12 weeks (5 weeks synbiotic + washout + 5 weeks prebiotic alone)	Gastrointestinal symptoms, behavioral symptoms, and inflammatory markers	Combination treatment was well tolerated and associated with reduced GI symptoms and some improvements in aberrant behaviors; decreases in IL-13 and TNF-α were observed in some participants. Symbiotics
Wang et al[20]	Controlled intervention study	26 children with ASD (16 synbiotic, 10 placebo)	2-8 years	Probiotics + FOS	30, 60, and 108 days	Autism severity, gut microbiota composition, SCFAs, neurotransmitters	Synbiotic treatment increased beneficial bacteria (Bifidobacterium longum), elevated SCFAs, normalized serotonin and dopamine metabolites, and significantly reduced autism and GI symptom severity. Symbiotics
Schmitt et al[29]	Randomized double-blind placebo-controlled crossover trial	15 participants with ASD	15-45 years	SB-121 synbiotic (Limosilactobacillus reuteri + dextran microparticles + maltose)	28-day treatment periods	Adaptive behavior (Vineland-3), social preference, safety	Significant improvement in Vineland-3 Adaptive Behavior Composite score (P = 0.03) and trends toward improved social preference; treatment was safe and well tolerated. Symbiotics
Phan et al[42]	Open-label clinical study	170 ASD participants completed the study	10.4 ± 7.1 years	Precision synbiotic formulation targeting microbiome diversity	3 months	ASD symptoms, GI symptoms, and metagenomic microbiome profiling	Synbiotic supplementation increased microbiome diversity and improved GI discomfort and several ASD-related symptoms including language, cognition, and communication. Symbiotics
Mitchell et al[44]	Randomized pilot trial	40 children with ASD	5-10 years	Synbiotics alone vs synbiotics + gut-directed hypnotherapy	12 weeks (24-week follow-up)	GI symptom scores, behavior, anxiety, microbiome composition	Both groups showed significant reductions in GI symptoms (P < 0.001); the combined therapy group also showed additional reductions in anxiety and irritability. Symbiotics
Wong et al[43]	Open-label pilot study	30 children with ASD	Mean age 82 years	SCM06 synbiotic formulation	12 weeks	Anxiety, sensory hyperresponsiveness, abdominal pain, microbiome metabolomics	Significant improvements in anxiety, sensory hyperresponsiveness, and abdominal pain; increases in Bifidobacterium pseudocatenulatum and SCFAs were observed. Symbiotics

ASD: Autism spectrum disorder; FOS: Fructo-oligosaccharides; GI: Gastrointestinal; IL-13: Interleukin-13; SB-121: Investigational synbiotic formulation SB-121; SCFAs: Short-chain fatty acids; TNF-α: Tumor necrosis factor alpha.

Table 9 Synbiotic strains, prebiotic components, dose, treatment duration, and reported outcomes in autism spectrum disorder studies.

Ref.	Probiotic strain(s)	Prebiotic component	Dose	Treatment duration	Microbiome outcomes	Clinical outcomes
Sanctuary et al[21]	Bifidobacterium longum subsp. infantis	Bovine colostrum product containing oligosaccharides	0.15 g/Lb body weight/day + FOS	5 weeks synbiotic phase (within 12-week crossover design)	Changes in gut microbial composition; modulation of immune markers, including decreased IL-13 and TNF-α	Reduction in gastrointestinal symptoms and modest improvements in aberrant behavior scores in some participants. Symbiotics
Wang et al[20]	Multi-strain probiotic mixture (including Bifidobacterium longum)	FOS	1010 CFU/pack/day	30, 60, 108 days	Increased abundance of beneficial bacteria (Bifidobacterium longum); increased SCFAs	Significant reduction in autism severity scores and gastrointestinal symptoms; normalization of serotonin and dopamine metabolites. Symbiotics
Schmitt et al[29]	Limosilactobacillus reuteri (SB-121 formulation)	Dextran microparticles with maltose carrier	2 × 1010 CFU of Limosilactobacillus reuteri, + 200 mg Sephadex, + 74 mmol/L of maltose in a final volume of 10.8 mL	28-day treatment periods (crossover design)	Designed to enhance bacterial adherence and colonization	Significant improvement in Vineland-3 Adaptive Behavior Composite score; improved social preference trends; treatment well tolerated
Phan et al[42]	Multi-strain probiotic formulation	Precision synbiotic formulation (specific prebiotic composition not specified)	Personalized	3 months	Increased microbial diversity; restoration of beneficial taxa such as Faecalibacterium and Prevotella; reduction of pathogenic taxa including Shigella and Klebsiella	Improvements in gastrointestinal discomfort and ASD-related symptoms including language, cognition, and communication
Mitchell et al[44]	Multi-strain probiotic blend	Prebiotic component included within the synbiotic formulation	5 g of PHGG and a probiotic mixture	12 weeks	Increased abundance of beneficial taxa including Bifidobacterium animalis and Dialister	Significant reductions in gastrointestinal symptom scores; improvements in anxiety and irritability when combined with hypnotherapy
Wong et al[43]	Synbiotic formulation SCM06, including probiotic strains	Prebiotic component included in SCM06	Maltodextrin + galactooligosaccharide + 5 × 109 CFU of 4 probiotic species: Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus plantarum, and Streptococcus thermophilus	12 weeks	Increased abundance of Bifidobacterium pseudocatenulatum and increased SCFAs (butyrate and valeric acid)	Improvements in anxiety, sensory hyperresponsiveness, and abdominal pain in children with ASD

ASD: Autism spectrum disorder; CFU: Colony-forming units; FOS: Fructo-oligosaccharides; IL-13: Interleukin-13; PHGG: Partially hydrolyzed guar gum; SB-121: Investigational synbiotic formulation SB-121; SCFAs: Short-chain fatty acids; TNF-α: Tumor necrosis factor alpha.

Effects of synbiotics on core autism symptoms: Several studies reported behavioral improvements associated with synbiotic supplementation, although these effects were generally modest and often exploratory.

A controlled intervention study by Wang et al[20] evaluated the effects of a combined probiotics plus fructo-oligosaccharide (FOS) intervention in children with ASD. The treatment significantly reduced autism symptom severity compared with placebo and was associated with normalization of several neurochemical abnormalities, including reduced plasma serotonin levels and increased homovanillic acid, suggesting modulation of dopamine metabolism. These neurochemical changes occurred alongside increased levels of SCFAs and the restoration of beneficial bacteria such as Bifidobacterium longum, indicating that improvements in ASD symptoms may be mediated through microbiota-driven metabolic pathways.

Similarly, the SB-121 synbiotic formulation, consisting of Limosilactobacillus reuteri, dextran microparticles, and maltose, was investigated in a randomized, placebo-controlled crossover trial involving autistic participants. Treatment with SB-121 resulted in a significant increase in Vineland-3 Adaptive Behavior Composite scores (P = 0.03), suggesting improvements in adaptive functioning and social behavior. The intervention was well tolerated and showed trends toward improved social preference, as measured by eye-tracking analysis[29]. More recently, a large open-label study evaluating precision synbiotics reported improvements in several ASD-related domains, including language, cognition, and social responsiveness, following three months of supplementation. These behavioral improvements were accompanied by increased microbial diversity and restoration of beneficial taxa such as Faecalibacterium and Prevotella. However, because the study lacked a placebo control, the authors cautioned that the findings should be interpreted with caution, given potential placebo effects[42].

In another pilot trial evaluating a novel synbiotic formulation (SCM06), children with ASD experienced significant improvements in anxiety and sensory hyperresponsiveness, two common behavioral comorbidities in autism. These improvements were associated with increased levels of butyrate-producing bacteria and microbial metabolites, including valeric and butyric acids[43]. Overall, these studies suggest that synbiotic interventions may contribute to modest improvements in adaptive behavior, anxiety, and sensory symptoms, although robust evidence for large reductions in core autism severity remains limited.

Effects of synbiotics on GI manifestations: The most consistent benefits of synbiotic supplementation were observed for GI outcomes, which aligns with the primary mechanism of action of these interventions. In a randomized pilot study investigating synbiotics vs synbiotics combined with gut-directed hypnotherapy, both groups experienced significant reductions in total GI symptom scores (P < 0.001) after a 12-week intervention. Improvements were sustained at follow-up, indicating potential long-term benefits. Microbiome analysis revealed increases in beneficial taxa, including Bifidobacterium animalis and Dialister, following treatment[44]. Similarly, the precision synbiotic study involving 170 ASD participants reported significant reductions in GI discomfort following supplementation. These improvements were accompanied by increases in microbial diversity and reductions in potentially pathogenic bacteria, including Shigella, Klebsiella, and Clostridium[42].

Earlier research combining Bifidobacterium infantis with bovine colostrum prebiotic components also observed reductions in the frequency of GI symptoms and improvements in certain aberrant behaviors. These clinical improvements were accompanied by reduced inflammatory cytokines, including interleukin-13 and tumor necrosis factor-alpha, suggesting an immune-modulatory mechanism[21]. Collectively, these findings indicate that synbiotic supplementation may effectively reduce GI symptoms such as abdominal pain, constipation, and gut discomfort, which are highly prevalent in children with ASD.

Mechanistic effects of synbiotics on the microbiota-gut-brain axis: Synbiotic interventions appear to influence the gut-brain axis through coordinated, multi-level modulation of the gut microbiome and its metabolic activity. Across studies, synbiotic supplementation consistently increased beneficial bacterial taxa such as Bifidobacterium, Lactobacillus, and Faecalibacterium, while simultaneously enhancing the production of key SCFA, particularly butyrate and acetate, which support intestinal barrier integrity and anti-inflammatory signaling. These shifts were accompanied by notable reductions in potentially pathogenic microbes, including Clostridium and Shigella, and normalization of neurotransmitter-related pathways involving serotonin and dopamine metabolism. Synbiotics also reduced circulating pro-inflammatory cytokines, indicating systemic immunomodulatory effects. Collectively, these microbiome-driven changes are thought to influence brain-related processes by modulating immune activity, altering neurotransmitter synthesis, and improving gut barrier function, thereby strengthening gut-brain communication and contributing to improvements in both GI and behavioral outcomes in children with ASD.

Comparison with probiotics and prebiotics: When comparing synbiotics with previously analyzed probiotic and prebiotic interventions, several patterns emerge. Probiotics alone primarily demonstrated moderate improvements in GI symptoms, with inconsistent effects on core autism behaviors (Figure 6). Prebiotics, particularly GOS, improved microbiome composition and GI symptoms, but behavioral improvements were limited and inconsistent. In contrast, synbiotics may provide broader therapeutic effects by combining microbial supplementation with substrates that enhance microbial colonization and metabolic activity. This synergy appears to produce greater shifts in microbial diversity, SCFA production, and neurochemical pathways, which may explain the observed improvements in both GI and behavioral outcomes. However, despite these promising findings, most synbiotic studies remain small pilot trials or open-label designs, limiting the strength of conclusions. Large randomized controlled trials are still required to determine the optimal synbiotic formulations, dosing regimens, and treatment duration for children with ASD.

Figure 6 Comparative effects of probiotics, prebiotics, and synbiotics on core autism symptoms and gastrointestinal outcomes in children with autism spectrum disorder. This comparative synthesis figure summarizes the evidence derived from the included studies evaluating microbiome-targeted interventions in children with autism spectrum disorder (ASD). The figure contrasts the relative effects of probiotics, prebiotics, and synbiotics on two primary outcome domains: Core autism symptoms (including social communication deficits, repetitive behaviors, adaptive functioning, and behavioral symptoms) and gastrointestinal (GI) manifestations (including constipation, abdominal pain, diarrhea, and overall GI symptom severity). Overall, probiotic interventions demonstrated moderate and heterogeneous effects on GI symptoms, with several studies reporting improvements in constipation, abdominal discomfort, and gut microbial composition. However, improvements in core ASD symptoms were inconsistent, with only modest benefits observed in specific behavioral domains such as irritability or social responsiveness. Prebiotic supplementation, including galacto-oligosaccharides, fructo-oligosaccharides, β-glucans, and partially hydrolyzed guar gum, primarily exerts beneficial effects by modulating gut microbiota composition and short-chain fatty acid production. These interventions showed consistent improvements in GI symptoms, while evidence for direct improvements in core autism symptoms remained limited and variable across studies. In contrast, synbiotic interventions, which combine probiotics with fermentable prebiotic substrates, demonstrated broader modulation of the microbiome and metabolic activity, including increased microbial diversity and elevated production of short-chain fatty acids such as butyrate. Across the included studies, synbiotics were associated with significant improvements in GI symptoms and modest improvements in behavioral and adaptive functioning, suggesting a potentially greater capacity to influence the microbiota-gut-brain axis than single-component interventions. The color-coded evidence levels shown in the figure represent the relative strength and consistency of evidence reported across the included studies, ranging from limited evidence to moderate or stronger evidence of clinical benefit. GI: Gastrointestinal; ASD: Autism spectrum disorder.

Effects of fecal microbiota transplantation in children with ASD

A total of ten studies evaluating fecal microbiota transplantation (FMT) or washed microbiota transplantation (WMT) in children with ASD were included in this synthesis (Table 10). The studies comprised one randomized double-blind placebo-controlled trial, several prospective or open-label clinical studies, and retrospective observational analyses, with sample sizes ranging from 18 to 98 participants. Across studies, FMT was administered via different delivery methods, including oral capsules, nasojejunal tubes, transendoscopic enteral tubes, and repeated courses of WMT. Treatment duration varied from single courses to repeated administration over several weeks, with follow-up periods ranging from 8 weeks to 2 years. Overall, the evidence consistently indicated that FMT interventions were associated with improvements in GI symptoms, core ASD behavioral measures, sleep disturbances, and gut microbial composition, although the magnitude and methodological strength of evidence varied across studies.

Table 10 Characteristics of included studies investigating fecal microbiota transplantation in autism spectrum disorder.

Ref.	Study design	Participants	FMT type/delivery	Outcomes assessed	Main findings
Wang et al[45]	Randomized double-blind placebo-controlled trial	41 children with ASD (39 boys and 3 girls, aged 4-12 years)	FMT vs placebo	GSRS, CARS, ABC, SRS; urinary metabolites	Significant improvements after FMT: GSRS decreased (30.17 → 19), CARS (36.22 → 33.33), SRS (151.17 → 137.5), ABC (76.39 → 53.17). Urinary 5-HIAA decreased, suggesting altered serotonin metabolism
Liu et al[53]	Prospective interventional study	24 ASD children initially; 18 second course; 13 third; 8 fourth	Fresh WMT	ASD symptoms, sleep disorder, constipation, microbiome metabolites	WMT improved behavioral symptoms, sleep disturbances, and constipation. Microbiome shifts included ↓Bacteroides, Flavonifractor, Parasutterella and ↑Prevotella
Chen et al[54]	Microbiome mechanistic study within FMT clinical dataset	ASD patients with GI comorbidities	Encapsulated FMT	Metagenomic microbial dynamics	Donor-recipient microbial interactions influenced subspecies transfer and clinical response, suggesting microbial compatibility affects FMT success
Li et al[47]	Prospective single-arm study	98 children with ASD	FMT via capsules, transendoscopic enteral tube, or nasojejunal tube	ABC, CARS, SRS, GSRS, SDSC; adverse events	Improvements in ASD symptoms, GI symptoms, and sleep disturbances. Capsules and nasojejunal tube showed greater symptom reduction than transendoscopic enteral tube. No serious adverse events
Li et al[48]	Prospective clinical study	38 ASD children; 30 healthy controls	Oral lyophilized FMT (every 4 weeks for 12 weeks)	ABC, CARS, SRS, SDSC; microbiome composition	After treatment: ABC ↓23%, CARS ↓10%, SRS ↓6%, SDSC ↓10%. Gut bacterial and fungal composition shifted toward healthier profiles
Li et al[49]	Open-label clinical trial	40 children with ASD (age 3-17 years)	FMT	GI symptoms, ASD symptoms, gut microbiota, neurotransmitters	FMT improved GI and behavioral symptoms, altered serum neurotransmitters, and promoted colonization of donor microbes
Pan et al[50]	Retrospective study	55 Children with ASD (median age: 6 years, 80.95% were male)	Repeated WMT	ABC, CARS, SDSC, GI symptoms, and inflammatory markers	WMT improved ASD symptoms, GI symptoms, and sleep disorders. Multiple treatment courses produced greater improvements
Zhang et al[51]	Retrospective observational study	49 children with ASD	WMT	Sleep disturbance (SDSC), stool characteristics (BSFS)	WMT improved sleep disorders and constipation; behavioral symptoms also improved with no serious adverse events
Kang et al[46]	Open-label clinical trial	18 children with ASD	Microbiota transfer therapy (antibiotics + bowel cleanse + FMT)	GI symptoms, ASD behavioral measures, microbiome	80% reduction in GI symptoms and significant improvements in ASD behaviors; increased bacterial diversity and beneficial taxa
Kang et al[52]	Long-term follow-up study	Same 18 participants from the previous trial	Microbiota transfer therapy	GI symptoms, ASD symptoms, gut microbiota	Improvements in GI symptoms and ASD behaviors were maintained for 2 years, with sustained microbiome changes

ABC: Aberrant Behavior Checklist; ASD: Autism spectrum disorder; BSFS: Bristol Stool Form Scale; CARS: Childhood Autism Rating Scale; FMT: Fecal microbiota transplantation; GI: Gastrointestinal; GSRS: Gastrointestinal Symptom Rating Scale; MTT: Microbiota transfer therapy; SDSC: Sleep Disturbance Scale for Children; SRS: Social Responsiveness Scale; WMT: Washed microbiota transplantation.

Effects of FMT on GI symptoms: Across the available studies, improvements in GI symptoms emerged as one of the most consistent and robust outcomes following FMT in children with ASD. In a randomized double-blind controlled trial, Wang et al[45] demonstrated a marked reduction in GI Symptom Rating Scale (GSRS) scores-from 30.17 at baseline to 19.00 post-treatment (P < 0.0001)-with no comparable improvement observed in the placebo group, underscoring a strong therapeutic effect of FMT on GI symptom severity[45]. Similarly, the open-label microbiota transfer therapy study by Kang et al[46] reported a nearly 80% reduction in GI complaints, including constipation, diarrhea, abdominal pain, and indigestion, with benefits persisting for at least 8 weeks after cessation of treatment, indicating sustained symptom relief. Additional clinical evidence reinforces these findings: Li et al[47] documented significant improvements in GI symptom scores that remained stable during follow-up, while another 2024 study by Li et al[48], published in Frontiers in Pediatrics, found notable reductions in constipation, diarrhea, and indigestion in children receiving oral lyophilized FMT. They also found that FMT significantly altered serum neurotransmitter levels. They also further observed that FMT could promote the colonization of donor microbes and shift the bacterial community of children with ASD toward that of typically developed controls[49]. Further support comes from retrospective studies by Pan et al[50] and Zhang et al[51], both of which observed substantial decreases in the prevalence of constipation and normalization of stool patterns following repeated courses of WMT. Collectively, these convergent findings demonstrate that FMT reliably alleviates GI dysfunction in children with ASD, most likely by restoring microbial balance and improving the stability of the gut ecosystem.

Effects of FMT on core autism symptoms: Several studies have demonstrated that FMT is associated with meaningful improvements in core autism symptoms, reflected across multiple validated behavioral scales. In a randomized controlled trial, Wang et al[45] reported significant reductions in autism severity following FMT, with CARS scores decreasing from 36.22 to 33.33 (P < 0.0001), SRS scores declining from 151.17 to 137.50 (P = 0.0002), and ABC scores improving from 76.39 to 53.17 (P < 0.0001), whereas the placebo group showed no comparable changes except for a minor reduction in ABC scores. In parallel, a large prospective study by Li et al[47] involving 98 children also documented significant post-treatment reductions in ABC, CARS, and SRS scores, with improvements maintained during follow-up. Consistent findings were observed in another cohort study of 38 children receiving oral lyophilized FMT, with reductions of 23% in ABC scores, 10% in CARS scores, and 6% in SRS scores by the end of follow-up[48]. Earlier foundational work by Kang et al[46] likewise reported substantial behavioral improvements after microbiota transfer therapy, with sustained and even enhanced benefits documented at a two-year follow-up assessment (Kang et al[52]), suggesting that FMT may offer durable therapeutic effects on core autism-related behaviors.

A forest-plot-style meta-analysis of three studies (Wang et al[45], Li et al[47], and Kang et al[46]) reported FMT outcomes (ABC, CARS, SRS, and GSRS) (Figure 7 and Table 11). According to the included studies, FMT showed large effect sizes for both GI and behavioral outcomes. Improvements in GI symptoms were accompanied by significant reductions in ASD core symptoms across multiple validated behavioral scales. Two studies reported changes in GSRS scores, with a pooled effect size (weighted SMD) of -2.71. FMT produced a very large reduction in GI symptom severity, indicating substantial improvement in abdominal pain, diarrhea, constipation, and bloating among children with ASD (Figure 8). The largest pooled effect was observed for SRS, suggesting that microbiome restoration may influence social communication pathways through the gut-brain axis. Three studies reported changes in SRS, with a pooled effect size (weighted SMD) of -4.06. Substantial improvements were observed in social communication and interaction deficits, the core domain of ASD symptomatology. CARS and ABC shoes also showed significant improvements, with pooled effect sizes (weighted SMDs) of -1.60 and -3.00, respectively. However, the evidence should be interpreted cautiously because all studies used open-label or uncontrolled pre-post designs, and heterogeneity in FMT protocols, donor selection, and treatment duration remains substantial.

Figure 7 Meta-analysis results: Effects of fecal microbiota transplantation in autism spectrum disorder. A: Gastrointestinal symptoms, two studies reported changes in Gastrointestinal Symptom Rating Scale scores; B: Childhood Autism Rating scale; C: Aberrant Behavior Checklist; D: Social Responsiveness Scale. GSRS: Gastrointestinal Symptom Rating Scale; CARS: Childhood Autism Rating scale; ABC: Aberrant Behavior Checklist; SRS: Social Responsiveness Scale; SMD: Standardized mean difference; FMT: Fecal microbiota transplantation.

Figure 8 Proposed mechanisms and clinical effects of fecal microbiota transplantation in children with autism spectrum disorder. This schematic overview illustrates the potential therapeutic effects of fecal microbiota transplantation (FMT) on gastrointestinal (GI) dysfunction and core behavioral symptoms in children with autism spectrum disorder (ASD) through modulation of the gut-brain axis. FMT involves transferring a complex microbial community from a healthy donor to a recipient to restore microbial diversity and functional balance in the intestinal ecosystem. Several clinical studies have shown that FMT can significantly alter the gut microbial composition of children with ASD, typically increasing beneficial taxa such as Bifidobacterium and Prevotella while reducing the relative abundance of potentially pathogenic bacteria, including Bacteroides, Flavonifractor, and Parasutterella. These microbial shifts may influence host metabolism, immune signaling, and neuroactive compound production. Through these microbiome changes, FMT may modulate the gut-brain axis and improve multiple clinical domains. Improvements in GI symptoms-such as constipation, diarrhea, abdominal pain, and indigestion-have been reported using validated instruments, including the GI Symptom Rating Scale. In parallel, improvements in core ASD-related symptoms have been observed using standardized behavioral assessments such as the Childhood Autism Rating Scale, Aberrant Behavior Checklist, and Social Responsiveness Scale. Mechanistically, alterations in microbial metabolism may contribute to these clinical effects. Changes in microbial metabolic pathways can affect serotonin metabolism, immune signaling, and detoxification processes. For example, clinical evidence indicates that FMT may reduce urinary levels of 5-hydroxyindoleacetic acid, a serotonin metabolite, suggesting that microbial modulation of serotonergic pathways may contribute to symptom improvement. Overall, the figure summarizes the current evidence indicating that FMT can reshape the gut microbiome, modify microbial metabolic pathways, and subsequently improve GI symptoms, behavioral outcomes, and neurodevelopmental features associated with ASD. FMT: Fecal microbiota transplantation.

Table 11 Overall quantitative synthesis of fecal microbiota transplantation effects on autism spectrum disorder core symptoms and gastrointestinal manifestations.

Outcome	Number of studies	Total participants	Pooled SMD	Interpretation
GSRS	2	37	-2.71	Very large GI improvement
CARS	3	135	-1.60	Large improvement in ASD severity
ABC	2	116	-3.00	Very large behavioral improvement
SRS	3	135	-4.06	Very large improvement in social responsiveness

GI: Gastrointestinal; ASD: Autism spectrum disorder; ABC: Aberrant Behavior Checklist; CARS: Childhood Autism Rating Scale; GSRS: Gastrointestinal Symptom Rating Scale; SMD: Standardized mean difference; SRS: Social Responsiveness Scale.

Effects of FMT on sleep disorders and associated symptoms: Sleep disturbances, a frequent comorbidity in children with ASD, consistently improved across studies evaluating FMT. Evidence from Liu et al[53] showed that fresh-WMT not only alleviated sleep problems but also improved ASD symptoms and constipation, highlighting the interconnected nature of gut dysbiosis and sleep regulation. Similarly, Li et al[47] reported significant reductions in Sleep Disturbance Scale for Children (SDSC) scores following FMT, indicating measurable improvements in sleep quality and nocturnal behaviors. Further supporting these findings, Pan et al[50] demonstrated that repeated courses of WMT led to progressive reductions in SDSC scores, suggesting cumulative and sustained benefits across multiple treatment cycles. Collectively, these results reinforce the concept that modulation of the gut microbiota through FMT may positively influence sleep regulation via mechanisms operating along the microbiota-gut-brain axis.

Microbiome and metabolic changes following FMT: Several studies have shown that FMT leads to notable microbiome and metabolic changes that parallel clinical improvements in children with ASD. Overall, FMT consistently increases microbial diversity, supports the engraftment of donor-derived bacterial strains, and restores microbial taxa that are commonly reduced in autism. For instance, Kang et al[46] observed significant increases in beneficial genera such as Bifidobacterium and Prevotella, both of which are typically depleted in children with ASD and are associated with healthier gut ecosystem function. Similarly, Li et al[48] reported a reduction in potentially dysbiotic taxa-including Blautia, Sellimonas, Saccharomycopsis, and Cystobasidium-accompanied by increases in Dorea, reflecting a microbial shift toward a composition more closely resembling that of neurotypical controls. Additional mechanistic insights were provided by Chen et al[54], who demonstrated that donor–recipient microbial interactions at the subspecies level significantly affected strain engraftment and clinical response, emphasizing the importance of ecological compatibility in determining FMT success. Beyond microbial composition, metabolic changes have also been documented; Wang et al[45] identified a significant post-FMT reduction in urinary 5-hydroxyindoleacetic acid - from 8.6 mg/L to 7.32 mg/L (P = 0.022) - suggesting modulation of serotonin metabolism, a pathway frequently implicated in ASD pathophysiology. Together, these findings indicate that FMT produces coordinated shifts in microbial ecology and host metabolic signaling that may underpin its therapeutic effects in ASD.

Safety and adverse events: FMT demonstrated a favorable safety profile across the included studies, with no serious adverse events reported. In the largest cohort study by Li et al[47], which included 98 children with ASD, adverse events were generally mild and transient. Reported rates were 8.2% in the capsule group, 23.1% in the transendoscopic enteral tube group, and 8.3% in the nasojejunal tube group. These events typically consisted of short-lived symptoms such as vomiting or fever, all of which resolved spontaneously within 24 hours. Overall, the available evidence indicates that FMT is well tolerated in pediatric ASD populations, with side effects remaining infrequent, mild, and self-limiting.

Evidence synthesis and quantitative comparison: Quantitative meta-analysis across the included studies was constrained by considerable heterogeneity, arising from differences in study design (ranging from randomized controlled trials to open-label and retrospective analyses), variability in FMT protocols, inconsistency in clinical outcome measures, and incomplete reporting of statistical parameters such as mean differences and standard deviations. Despite these limitations, the overall direction of findings from controlled trials and prospective cohorts demonstrated a clear and consistent pattern: FMT was associated with substantial improvements in GI symptoms, moderate yet meaningful reductions in core ASD behavioral measures, and additional benefits related to sleep regulation and metabolic biomarkers. Collectively, these results position FMT as a promising microbiome-targeted therapeutic option for children with ASD, particularly for those exhibiting co-occurring GI disturbances. Nonetheless, the current evidence base remains insufficient to draw definitive conclusions, and rigorously designed, large-scale randomized controlled trials with standardized intervention protocols and extended follow-up periods are essential to validate efficacy, optimize treatment parameters, and establish long-term safety and clinical applicability.

DISCUSSION

This systematic review synthesizes current clinical evidence regarding microbiome-targeted therapies, including probiotics, prebiotics, synbiotics, and FMT, for children with ASD. Overall, the findings support the concept that modulation of the gut microbiota may contribute to improvements in GI symptoms and, to a lesser extent, core behavioral manifestations of ASD, reinforcing the growing recognition of the microbiota-gut-brain axis as a potential therapeutic target.

Microbiome modulation and core autism symptoms

Across probiotic intervention studies, improvements in core ASD symptoms were heterogeneous and generally modest. While several randomized and interventional trials reported reductions in behavioral scale scores, including measures of social responsiveness, hyperactivity, and repetitive behaviors, these effects were inconsistent across populations and study designs. Variability in probiotic strain composition, treatment duration, baseline GI comorbidity, and outcome assessment tools likely contributed to the observed inconsistency[25,27-29]. Importantly, most probiotic formulations consisted of multi-strain combinations dominated by Lactobacillus and Bifidobacterium species, reflecting their hypothesized role in modulating neuroimmune and metabolic pathways[23,24,30,31]. In addition, Lactobacillus plantarum PS128 was particularly effective in reducing anxiety and depression subscale scores, suggesting it may target the emotional dysregulation often comorbid with ASD[22]. Interestingly, evidence suggests that the presence or absence of GI symptoms may determine behavioral response. For instance, children without GI symptoms (non-gastrointestinal group) showed significant improvements in ADOS-CSS social-affect scores when treated with multi-strain probiotics, suggesting that psychobiotics may exert direct neurological effects independent of their GI-related benefits[30].

In contrast, prebiotic supplementation demonstrated limited direct effects on core behavioral symptoms. Although certain trials reported improvements in adaptive functioning or specific behavioral domains, these findings were less consistent and often secondary to improvements in gut function or microbial metabolic activity. Prebiotics primarily exert their clinical effects by promoting beneficial microbial taxa and increasing the SCFA production, mechanisms that may indirectly influence neurodevelopmental processes[36-39].

Synbiotic interventions appeared to produce broader and potentially more clinically meaningful effects on behavioral outcomes. By combining live microbial strains with fermentable substrates that support microbial colonization and metabolic activity, synbiotics promoted greater shifts in microbial diversity and in neuroactive metabolite production[55]. Several pilot and controlled studies reported improvements in adaptive behavior, anxiety, and irritability, suggesting that synergistic modulation of microbial ecology may be more effective than single-component strategies. Nevertheless, the relatively small sample sizes and frequent use of open-label designs limit the certainty of these conclusions[21,33,44].

Among microbiome-based interventions, FMT demonstrated the most pronounced improvements in core ASD symptom severity in quantitative analyses. Pooled estimates from available studies indicated large standardized mean differences in behavioral outcomes measured with validated scales, including the SRS, CARS, and ABC[45-48]. These findings suggest that large-scale restoration of microbial diversity and functional metabolic pathways may exert substantial effects on social communication and behavioral regulation. However, interpretation should be cautious given methodological heterogeneity and the predominance of uncontrolled pre-post study designs.

Effects on GI manifestations

The most consistent therapeutic benefit across microbiome-targeted interventions was observed in GI symptom improvements. Probiotic supplementation demonstrated moderate reductions in constipation, abdominal discomfort, and overall GI severity scores, consistent with the high prevalence of gut dysbiosis in ASD populations[17,25,28].

Similarly, prebiotic interventions produced reproducible improvements in digestive symptoms, likely mediated by increased SCFA production, enhanced mucosal barrier function, and modulation of microbial fermentation. These physiological effects support the hypothesis that normalization of intestinal homeostasis is a primary mechanism by which microbiome-targeted therapies confer clinical benefit in ASD[36,37,39,40].

Synbiotic therapy demonstrated even greater consistency in reducing GI symptoms, with randomized pilot trials reporting significant reductions in composite GI scores and sustained improvements during follow-up. These findings reinforce the concept that combined microbial and substrate-based interventions may provide a more stable ecological environment for microbiome restoration[21,42,44].

FMT interventions showed the largest magnitude of GI symptom improvement across studies. Quantitative synthesis indicated very large, pooled effects on the GSRS, reflecting substantial reductions in abdominal pain, diarrhea, constipation, and bloating. These improvements were frequently accompanied by parallel shifts toward a healthier microbial profile, characterized by increased abundance of beneficial taxa such as Bifidobacterium and Prevotella[45-48].

Mechanistic implications: Microbiota-gut-brain axis

The findings of this systematic synthesis provide converging clinical and biological evidence supporting the microbiota-gut-brain axis as a mechanistic framework linking intestinal dysbiosis to behavioral and GI manifestations in children with ASD. Across probiotic, prebiotic, synbiotic, and FMT interventions, therapeutic responses were frequently accompanied by measurable alterations in microbial diversity, metabolic activity, immune signaling pathways, and neuroactive compound production, suggesting that microbiome modulation may influence neurodevelopmental functioning through multiple interconnected biological pathways[56].

One of the most consistently reported mechanisms involves the restoration of microbial ecological balance and an increase in alpha diversity following microbiome-targeted interventions. Dysbiosis in ASD has been characterized by reduced abundance of beneficial commensal taxa, particularly Bifidobacterium and Lactobacillus species, and relative enrichment of potentially pro-inflammatory genera such as Clostridium[57]. Interventions such as synbiotic supplementation and FMT were associated with shifts toward a more eubiotic microbial profile, which may contribute to improved intestinal homeostasis and reduced mucosal inflammation. These ecological changes are clinically relevant because microbial diversity is closely linked to metabolic resilience, immune regulation, and intestinal barrier integrity[58].

Metabolic signaling represents another key mechanistic pathway. Prebiotic and synbiotic interventions enhanced microbial fermentation of non-digestible carbohydrates, thereby increasing the production of SCFAs, including acetate, propionate, and butyrate[59]. SCFAs exert pleiotropic physiological effects, including modulation of intestinal epithelial energy metabolism, reinforcement of tight junction protein expression, and regulation of gene transcription through histone deacetylase inhibition. Butyrate, in particular, has been implicated in neuroprotective processes, including the promotion of neurotrophic factor expression and the modulation of microglial activation states. These mechanisms provide a biologically plausible explanation for the observed associations between improved GI function and changes in behavioral domains in several included studies[60].

Neurotransmitter metabolism constitutes an additional pathway linking gut microbial activity with CNS function. Certain microbial taxa participate in the synthesis, degradation, or peripheral regulation of neurotransmitter precursors such as tryptophan and tyrosine[61]. Microbiome-directed therapies, particularly FMT, were associated with normalization of metabolites related to serotonergic and dopaminergic pathways, including reductions in urinary 5-hydroxyindoleacetic acid concentrations in some cohorts. Such findings suggest that microbial modulation may influence central neurotransmission indirectly through peripheral metabolic signaling, altered enterochromaffin cell activity, or vagal afferent pathways[62].

Immune-inflammatory modulation also appears to play a significant role. Several studies reported reductions in systemic inflammatory markers and improved indices of intestinal immune regulation following microbiome-targeted therapy. Chronic low-grade inflammation and altered cytokine profiles have been proposed as contributors to the pathophysiology of ASD through effects on synaptic plasticity, neural connectivity, and blood-brain barrier permeability. By attenuating pro-inflammatory signaling and enhancing regulatory immune responses, microbiome interventions may therefore exert secondary neurobehavioral benefits[63].

Intestinal epithelial barrier integrity represents another critical mechanistic link. Increased intestinal permeability (“leaky gut”) has been reported in subsets of children with ASD and may facilitate the translocation of microbial metabolites, lipopolysaccharides, and immune mediators into the systemic circulation. Prebiotic-induced enhancement of mucin production and tight junction stabilization, along with probiotic-mediated competitive inhibition of pathogenic organisms, may contribute to restoration of barrier function. Improved barrier integrity could subsequently reduce systemic immune activation and neuroinflammatory signaling[64].

Finally, emerging evidence suggests that gut microbial modulation may influence neural network activity through direct neuroendocrine and vagal pathways. Experimental and clinical observations indicate that microbial metabolites can stimulate enteroendocrine cells, leading to the release of neuroactive peptides such as glucagon-like peptide-1 and peptide YY, which may influence appetite regulation, stress responses, and behavioral reactivity[65]. Vagal afferent signaling represents an additional route by which gut microbial activity may affect limbic and cortical brain regions implicated in social cognition and emotional processing[66].

Psychobiotics demonstrated a highly favorable safety profile, with mostly transient mild effects (e.g., irritability, bloating). FMT was similarly well tolerated across multiple pediatric cohorts, with no serious adverse events reported. This positions microbiome-targeted therapies as low-risk adjunctive options, especially compared with conventional psychotropic medications[67]. Collectively, these mechanistic insights support a multidimensional model in which microbiome-targeted therapies influence ASD symptomatology through integrated metabolic, immunological, neurochemical, and epithelial pathways. Importantly, the relative contribution of each pathway likely varies according to individual host factors, including baseline microbiome composition, GI symptom burden, dietary patterns, genetic susceptibility, and developmental stage[68]. This heterogeneity underscores the potential value of precision microbiome approaches that tailor therapeutic strategies to patient-specific microbial and metabolic profiles.

Clinical translation, therapeutic hierarchy, and future directions

When comparing available microbiome-modulating interventions, a clear gradient of clinical effectiveness becomes apparent (Table 12). Probiotics and prebiotics demonstrate the strongest and most consistent benefits for GI symptoms, yet their influence on core autism-related behaviors remains variable and often modest[18]. Synbiotics, by combining microbial strains with fermentable substrates that enhance colonization and metabolic activity, appear to offer more robust and synergistic effects across both GI and behavioral domains[55]. FMT currently shows the largest and most durable improvements in both GI and neurobehavioral outcomes; however, its invasive nature, evolving regulatory oversight, and the limited number of high-quality randomized trials warrant cautious interpretation and careful selection of candidates[69]. In clinical practice, microbiome-targeted therapies should be considered adjunctive modalities, particularly valuable for children with ASD who exhibit significant GI comorbidities or clear evidence of dysbiosis. These interventions may serve as complementary components within a broader multidisciplinary treatment plan, rather than standalone therapies.

Table 12 Evidence-based selection of psychobiotic strains and formulations for targeted symptom management in children with autism spectrum disorder.

Clinical target	Evidence-based psychobiotic	Key mechanism
Social functioning	Limosilactobacillus reuteri (combined strains)	Oxytocin signaling modulation via the Vagus nerve
Anxiety/mood	Lactiplantibacillus plantarum PS128	Modulation of dopamine and serotonin metabolism
Behavioral irritability	Multi-strain Lacto/Bifido blends	reduction in systemic inflammation/pro-inflammatory cytokines
GI severity	De Simone formulation/Bifidobacterium infantis	Enhancement of intestinal barrier and SCFA production
Global improvement	Fecal microbiota transplantation	Ecosystem-wide restoration of microbial diversity

This table provides a clinical translation of the systematic review findings, mapping specific microbial interventions to their corresponding therapeutic targets within the ASD phenotype. Recommendations are categorized by the primary clinical presentation: (1) Strain specificity: Highlights the transition toward “precision psychobiotics”, where specific strains like Limosilactobacillus reuteri and Lactiplantibacillus. plantarum PS128 are utilized for neurological and emotional modulation rather than general gut health; (2) Clinical metrics: Effects are based on improvements in validated instruments, including the Autism Diagnostic Observation Schedule, Social Responsiveness Scale, Aberrant Behavior Checklist, and the Gastrointestinal Severity Index; and (3) Formulations: Includes single-strain psychobiotics, standardized multi-strain blends (e.g., De Simone Formulation), and ecosystem-wide interventions like Fecal Microbiota Transplantation. These interventions are intended as adjunctive therapies alongside standard behavioral and educational supports. Specific dosing should be adjusted based on individual patient tolerance and clinical response. GI: Gastrointestinal; SCFA: Short-chain fatty acids.

Looking ahead, future research should prioritize several key directions to enhance the precision, consistency, and clinical applicability of microbiome-based therapies. These include the development of precision psychobiotics tailored to individual microbial and metabolic fingerprints, as well as the establishment of standardized probiotic, prebiotic, and synbiotic formulations that define optimal strain selection, dosing strategies, and treatment duration. Advancing the field will also require integrating multi-omics platforms - such as metagenomics, metabolomics, proteomics, and immunomics - to identify reliable biomarkers that predict treatment response and guide personalized interventions. Equally important are long-term follow-up studies to evaluate the durability, safety, and stability of microbiome changes after treatment. Finally, large, multicenter randomized controlled trials with harmonized outcome measures are essential to generate high-quality evidence that can inform future clinical guidelines and support the routine implementation of microbiome-targeted therapies in ASD care. Together, these priorities will help clarify the therapeutic hierarchy among microbiome interventions, improve clinical decision-making, and accelerate the development of individualized, biologically informed treatment strategies for children with ASD.

Strengths and limitations

Methodological and clinical heterogeneity: A primary challenge identified in this systematic review is the marked clinical and statistical heterogeneity across the included studies. This variability stems from diverse probiotic formulations, ranging from single-strain interventions to complex multi-strain blends, as well as wide dosing ranges (9 billion to several hundred billion CFU) and treatment durations (4 weeks to 6 months). Furthermore, the inherent biological heterogeneity of ASD, particularly the presence or absence of comorbid GI symptoms, significantly influences therapeutic responsiveness. The reliance on various standardized behavioral instruments, such as the SRS-2, ABC-2, and ADOS, further complicates the pooling of data for robust meta-analysis. Additionally, small sample sizes and short follow-up durations were common across many pilot studies, reducing overall statistical power and limiting the generalizability of the findings.

The placebo-by-proxy effect: A critical consideration in interpreting behavioral outcomes in pediatric ASD trials is the “placebo-by-proxy” effect. This phenomenon occurs when caregivers, driven by a strong desire for clinical improvement and the significant burden of managing ASD symptoms, report perceived improvements that may not reflect objective clinical change. Because primary outcomes often rely on subjective, parent-reported instruments, the potential for observer bias is substantial, particularly in microbiome research where public expectations for “gut-brain” interventions are high. The occasional discrepancy between parent-reported scales and clinician-blinded observations (such as the ADOS-2) underscores the need for a more rigorous approach. To mitigate this bias, future studies should prioritize multi-informant assessments that combine caregiver reports with blinded clinical evaluations and objective physiological biomarkers (e.g., EEG power changes or metabolic shifts).

Future directions: Toward precision psychobiotics: Rather than viewing current heterogeneity solely as a methodological limitation, it underscores the need for a paradigm shift toward “Precision Psychobiotics”. In this model, microbial interventions are tailored to the specific baseline enterotypes and clinical phenotypes of the pediatric ASD population. Future research should prioritize large, multicenter randomized controlled trials utilizing standardized microbiome protocols and harmonized behavioral outcome measures. Integrating multi-omics biomarkers and individual microbial signatures will be essential for optimizing treatment efficacy and transitioning from generalized supplementation to personalized microbiome therapeutics.

CONCLUSION

This systematic review highlights the emerging therapeutic relevance of microbiome-targeted interventions in children with ASD, supporting the microbiota-gut-brain axis as a biologically plausible pathway linking GI dysfunction and neurobehavioral symptoms. According to the included studies, probiotics, prebiotics, synbiotics, and FMT demonstrated the most consistent benefits in improving GI symptom severity, whereas effects on core autism symptoms were generally modest and heterogeneous.

Synbiotic approaches appeared to provide broader clinical benefits than single-component interventions, while FMT showed the largest observed improvements in both GI and behavioral outcomes. However, interpretation of these findings is limited by methodological variability, small sample sizes, and differences in intervention protocols and outcome measures. Mechanistically, therapeutic effects are likely mediated through restoration of microbial diversity, modulation of neuroactive metabolites, reduction of inflammatory signaling, and improvement of intestinal barrier integrity.

Overall, microbiome modulation represents a promising adjunctive strategy in ASD management, particularly for GI comorbidities. Nevertheless, well-designed, large-scale randomized trials incorporating standardized interventions and precision microbiome profiling are essential before firm clinical recommendations can be established.