Autism spectrum disorder (ASD) comprises a group of neurodevelopmental disorders characterized by impairments in social communication and repetitive behaviors [1, 2]. The prevalence of ASD has risen dramatically in the past 2 decades, with current incidences estimated at approximately 1.70% in children in the United States and 1.55% in Europe [3]. Data from previous systematic reviews and meta-analyses indicated that 54.8% of patients with ASD had at least 1 comorbid psychiatric disorder [4], including anxiety, depression, bipolar and mood disorders, schizophrenia spectrum, and attention-deficit/hyperactivity disorder.

Despite extensive studies, the mechanisms and etiology of ASD have not been clearly elucidated; nevertheless, it is known to be associated with genetic-environmental interactions [5]. Possible risk factors include genetic factors, a family history of psychiatric disorders, parental age, premature birth, fetal exposure to toxins, exposure of pregnant mothers to infections, and the use of psychotropic medications [2, 6, 7]. Data from the Global Burden of Disease Study 2019 indicate that ASD imposes enormous costs on patients’ families and healthcare systems [8]; therefore, identifying more effective treatment options is crucial.

Existing treatment methods for ASD comprise both pharmacological and non-pharmacological therapies. Pharmaceuticals primarily target the comorbid symptoms of ASD [9]. Currently, mainstream drugs include serotoninergic medications, atypical antipsychotics, psychostimulants, and $\alpha$-2-adrenergic agonists [10, 11]. These drugs regulate neurotransmitter levels and related receptor functions in the brain, often showing adverse effects (sedation, weight gain, drowsiness, and vomiting). Some over-the-counter medicationswith few adverse effects andgood tolerability are also being used in ASD [12, 13], including melatonin and N-acetylcysteine. In addition, emerging target medications are being explored for their possible roles in ASD treatment, such as oxytocin, bumetanide, and cannabidiol [14, 15, 16], which may require more credible supporting evidence. Non-pharmacological treatments, including cognitive-behavioral therapy, probiotics and prebiotics, dietary supplements (sulforaphane, omega-3 fatty acids, and vitamin D), overall diet management (gluten- and casein-free diets), music therapy, and herbal medicine [2, 9, 17, 18], show good tolerability and few adverse effects and have, therefore, become increasingly popular in recent years [19].

With the introduction of the concept of the brain-gut axis, the connection between the gut microbiome and the central nervous system (CNS) has become increasingly valued. The gut microbiome influences CNS activity through microbial metabolites, immune mediators, gut hormones, and the vagus nerve [20]. Gut microbiome disturbances are common in patients with ASD [21] and may result in gastrointestinal (GI) symptoms [22]. Recent reviews highlighted the importance of nutritional interventions in reducing GI symptoms by modulating the gut microbiome [20, 23, 24]. Oral probiotic and prebiotic management may reduce ASD-related symptoms through the brain-gut axis. In particular, several clinical studies have revealed that probiotics and prebiotics could be promising therapies for ASD [15, 25, 26, 27].

However, 2 recent systematic reviews have shown that probiotics and prebiotics had an overall limited efficacy in managing GI or behavioral problems in autism patients [28, 29]. To better understand the current therapies and the rationale behind them, the present study summarized the latest clinical research on probiotics and prebiotics for ASD. In the current study, we explained the underlying mechanisms by which probiotics and prebiotics interact with ASD to intuitively demonstrate the potential of probiotics and prebiotics in ASD treatment.

A literature review was conducted until February 2023 using databases, including PubMed, MEDLINE, Embase, Web of Science, and Cochrane Central. The searched terms included ASD, autism, probiotics, prebiotics, therapy, gut microbiome, microbiota, brain-gut axis, and clinical trials. Additionally, sub-references of the selected articles were searched. Studies involving probiotic/prebiotic therapies and ASD, which were human clinical studies published in the last 5 years, were selected for detailed analysis.

Probiotics are living microorganisms that benefit human health by increasing short-chain fatty acids (SCFAs) production, intestinal transit regulation, and enterocyte turnover rate [30]. Some well-studied microbial species, such as Bifidobacterium and Lactobacillus, are widely used as probiotic supplements. Prebiotics, which are organic substances, have the ability to selectively promote the metabolism and proliferation of beneficial microorganisms, thereby improving host health. Some examples of prebiotics include oligosaccharides, fructans, and galactans [31]. Owing to their good tolerability and few adverse effects, probiotics and prebiotics show increasing therapeutic potential for ASD.

Clinical studies suggested that probiotic and prebiotic supplements could improve ASD symptoms and regulate gut microbiome distribution [27, 32]. A longitudinal study demonstrated that after 3 months of probiotic administration, the number of Bifidobacterium spp. and Lactobacillus spp. increased in the stool of children with ASD. In addition, communication skills and social networking improved in these children, whereas childhood autism scale scores, sleep disturbances, and hyperactivity decreased [32]. Shaaban SY et al. [27] also reported that after 3 months of probiotic therapy, the number of Bifidobacteria and Lactobacilli increased in the stool of children with autism, whereas their body weight, autism scale scores, and GI symptoms decreased. A randomized double-blind, crossover study with placebo revealed that after 3 months of supplementation with a probiotic mixture, patients with ASD exhibited decreased GI problems and improved communication skills and maladaptive behaviors, and their parents reported reduced stress levels [33]. A crossover study revealed that colostrum (prebiotic) and Bifidobacterium infantis exhibited good tolerability, improved core symptoms, and decreased GI symptoms in children with ASD [34]. A study by Billeci et al. [35] demonstrated that after 6 months of probiotics therapy, the brain power (electroencephalography) of beta and gamma bands was modified in children with ASD. These waves (beta and gamma) are related to working-memory tasks, analytical thinking, and sensory responses [35].

Combined interventional therapies may play a unique role in reducing autism symptoms. In a previous randomized double-blind, placebo-controlled, 2-stage pilot trial, patients with ASD received probiotics or a placebo for 28 weeks, and both groups received oxytocin at week 16. The findings demonstrated reductions in autism scale scores and GI symptoms only in the group treated with the probiotic and oxytocin combination [15]. Another study demonstrated that probiotics combined with fructooligosaccharide intervention reduced autism and GI symptoms in children with ASD and modulated gut microbiome, fecal SCFAs, and plasma serotonin levels [25]. Grimaldi R et al. [36] focused on combined intervention and observed that exclusion diets (gluten-and casein-free diets) reduced the GI symptoms in children with ASD; however, this therapy may result in high amino acid excretion and nutrient malabsorption. When combining an exclusion diet with prebiotics, patients demonstrated a significant decrease in anti-social behavior and an increase in favorable gut microbiome [36].

In patients with autism, with or without GI symptoms, the response to probiotics may vary. A randomized double-blind, placebo-controlled trial divided children with ASD into 2 groups: one with GI symptoms (GI group) and one without GI symptoms (NGI group). Following 5 months of probiotics therapy, the NGI group exhibited improvement in core autism and GI symptoms, while the GI group exhibited improved adaptive functioning and sensory profiles compared to those of the placebo group [37]. Researchers have also reported that the plasma levels of 25(OH)D may be positively related to the response to probiotic treatment in reducing ASD severity [38].

Owing to the lack of sensitivity of the assessment tools and the limitations of small sample sizes, some studies did not report statistically significant effects. For instance, in a randomized, double-blind, placebo-controlled study [39], probiotics did not decrease Aberrant Behavior Checklist (ABC) scores in children with ASD. However, opposition/defiant behaviors, hyperactivity, and impulsivity significantly decreased. A randomized, crossover trial [40] indicated that after 8 weeks of therapy, the quality of life and emotional stability improved from baseline in children with ASD, but the differences between the probiotic and placebo effects were not statistically significant.

A summary of clinical studies on probiotics/prebiotics for ASD over the last 5 years is presented in Table 1 (Ref. [15, 25, 27, 32, 33, 34, 35, 36, 37, 39, 40]). Although probiotics and prebiotics used in the trials differed in their forms, dosages, and microbiota composition, all these studies demonstrated their benefits in improving ASD-related behavioral problems and GI symptoms. However, owing to the heterogeneity among the trials, identifying the particular strain or therapy that exerts the greatest efficacy in decreasing ASD symptoms is challenging.

The bi-directional crosstalk between the gut microbiome and the brain, also termed the microbiome-gut-brain axis, is widely recognized. Interventions that modulate the gut microbiome may improve CNS disorders via this axis. In recent years, probiotics and prebiotics, which have good tolerability and few side effects, have gradually gained traction in the treatment of various neurodevelopmental disorders.

The clinical evidence presented in this review suggests that prebiotic or probiotic supplementation decreases GI symptoms and/or ASD-related behaviors in patients with ASD. Most of these studies focused on children, while one study examined both children and adults [15]. Bifidobacterium and Lactobacillus were the most common species found in the probiotic products, followed by Streptococcus thermophilus. In these studies, probiotics and prebiotics were typically administered for 4 weeks to 6 months. Among the previously-performed randomized, double-blind, placebo-controlled trials, a trial by Guidetti [33] demonstrated that probiotics decreased GI symptoms, behavioral disorders, and parenting stress levels. These results are consistent with Santocchi’s study [37], which demonstrated a decrease in ASD severity and adaptive function. Wang et al. [25] reported the beneficial effects of probiotics combined with prebiotics (fructooligosaccharide) on gut microbiome composition, hyper-serotonergic state, and dopamine metabolism problems in ASD patients. In addition, Arnold et al. [40] reported no serious adverse events in probiotic therapy for ASD. However, the improvement of the main outcomes (quality of life and anxiety) has not reached statistical significance. The heterogeneity across studies makes it challenging to draw conclusions, and more human studies on probiotics and prebiotics therapies with consistent inclusion and evaluation criteria are required to clarify the future of these treatments. Moreover, most studies have focused on immediate or short-term efficacy and lacked follow-up data for long-term benefits. The variation in species, dosages, and durations of probiotics/prebiotics in these studies suggest the significance of individualized treatment, considering the specific characteristics of ASD and gut microbiome composition [85]. Currently, probiotics have been considered safe for consumption in food and dairy products, but some researchers believe that probiotics may increase the risk of infection in chemotherapy patients [86]. Therefore, systematic reviews on the safety and adverse events associated with probiotic and prebiotic therapies are necessary.

Current evidence suggests that the immune system, microbial metabolites, neurotransmitters, and vagal system mediate the brain-gut communication. Abnormalities in microbiome composition and/or its interactions with the host may lead to GI problems as well as a risk of brain dysfunction through the microbiome-gut-brain axis [87]. Probiotics and prebiotics regulate the function of the microbiome-gut-brain axis by modulating the gut microbiome. This review provided preliminary evidence that probiotic/prebiotic management can downregulate the inflammatory state, as their associated beneficial metabolites stabilize the intestinal barrier, thereby decreasing immune disturbances in ASD. In addition, they can modulate brain activity by directly or indirectly affecting the level of neurotransmitters and the development of microglia and astrocytes. As previously mentioned, the gut microbiome is involved in 5-HT synthesis, transport, and metabolism, and dysfunction in the 5-HT system has been implicated in the development of ASD. This finding is supported by the identification of serotonin transporter and tryptophan hydroxylase 2 gene polymorphisms in ASD patients [88]. However, more direct evidence is needed to clarify how the microbiome influences autism pathology through the serotonin pathway. Furthermore, the available evidence has some limitations. For instance, the effects of oral probiotics/prebiotics on intestinal microbial homeostasis may be short-lived [89], and achieving long-term results through probiotic therapy remains unknown. Furthermore,many studies examining the effects of probiotics and prebiotics on ASD have been conducted using animal models, and the complexity of gut microbiome and variations across species limits the applicability of animal-study findings to humans. Additionally, the bi-directional effect of the brain-gut axis makes it difficult to discern the origin of this problem. The microbiome may also be affected by ASD [29].

Probiotic and prebiotic supplementation may show beneficial effects on ASD, but the heterogeneity across studies makes it challenging to draw conclusions. Although gut microbial dysbiosis is associated with ASD pathogenesis, current knowledge is insufficient to elucidate the interaction between the microbiome and the host and to investigate how their metabolites influence intestinal homeostasis. Regarding microbiome alterations in ASD, which microorganisms are abnormal and whether a single microbe or colony can be identified as a culprit remain unclear.

FF and SZ designed the research study. QW collected the references and FH provided help and advice on the table and figures. SZ and FF wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

The authors gratefully would like to acknowledge the China Academy of Chinese Medical Sciences, Beijing, China.

This research received no external funding.

The authors declare no conflict of interest.