According to the Diagnostic and Statistical Manual of Mental Disorders (5th edition) criteria, autism spectrum disorder (ASD) is defined as a group of neurodevelopmental disorders characterized by deficits in social communication, social interaction, restricted or repetitive behavior, and interests (American Psychiatric Association, 2013). The global prevalence of ASD has progressively climbed within the past years, with the World Health Organization projecting that ASD will affect one in every 36 people worldwide by 2022 (Maenner et al., 2023). Caring for a child with ASD can be extremely challenging for caregivers, resulting in significant psychological, emotional, and financial hardships. Additionally, caregivers often lack adequate social support. According to a survey conducted in Saudi Arabia in 2022, only 31% of children with ASD had access to ASD facilities in their vicinity, while 72% had no local private schools catering to ASD (Khan et al., 2020). It is now understood that caregivers of children with ASD face a greater burden on their family resources and overall functioning than those caring for children with other developmental disabilities (Rogge and Janssen, 2019). Therefore, ASD has become a major public health concern in many countries (Yousef et al., 2021; Alenezi et al., 2022; De Jonge et al., 2023). The exact causes and factors that increase the risk of ASD remain poorly understood. Still, various potential contributors have been recognized, encompassing prenatal and perinatal periods, as well as exposure to specific environmental factors (Weir et al., 2021).

It is well-established that people with ASD suffer from various developmental abnormalities and experience additional symptoms such as gastrointestinal issues, seizures, anxiety, sleep disturbances, and immune system dysfunction (Kohane et al., 2012; Ballini et al., 2019; Hossain et al., 2020). Besides, children with ASD may be prone to experiencing gastrointestinal problems like diarrhea, constipation, and abdominal pain (Ferguson et al., 2019). There is an increasing consensus suggesting an association between gastrointestinal microbiota and ASD. In this respect, significant heterogeneity has been documented in the composition of the intestinal flora of ASD patients (Ferguson et al., 2019). Numerous prior investigations have indicated the potential role of the gut microbiome in ASD. The underlying mechanisms involve intricate and bidirectional interactions between the gut microbiota and the brain, which occur along the microbiota-gut-brain axis (Yu et al., 2021). Due to the predominantly observational nature of existing studies and the potential influence of confounding variables, the current evidence only establishes an association between gut microbiota and ASD without establishing a cause-and-effect relationship. Besides, the effects of this association on the incidence of ASD and its specific physiological mechanisms are still unclear, and whether this association is violated by reverse causality remains undetermined.

Mendelian randomization (MR) analysis is a novel genetic epidemiological study design that harnesses genetic variation to address causal questions related to the influence of modifiable exposures on different outcomes (Davey Smith et al., 2020; Sanderson et al., 2022). The MR approach draws on Mendel’s first and second laws of genetic inheritance: the law of segregation and the law of independent assortment avoids bias from unobserved confounding of exposure and outcome and can infer causal effects in the presence of unobserved confounding (Davey Smith and Ebrahim et al., 2003). Genome-wide association studies (GWAS) provide vast datasets with numerous single nucleotide polymorphisms (SNPs) and large sample sizes. MR, which employs one or more genetic polymorphisms as the exposure variable of interest, capitalizes on the Mendelian laws of inheritance, making GWAS-based MR an appealing approach to assess causality (Thanassoulis and O’Donnell, 2009; O’Donnell and Sabatine, 2018; Davey Smith et al., 2020; Sanderson et al., 2022). Kantan originally proposed the MR causation hypothesis, he stating that the APOE2 gene variant occurs randomly at birth, that the variation is not affected by confounding factors, and is associated with lower cholesterol levels. If there is a clear causal relationship between low cholesterol levels (exposure factors) and cancer (outcome), then people with APOE2 variants (instrumental variables) will be more susceptible to cancer. The link between APOE2 and cancer may provide indirect evidence of whether there is a causal relationship between serum cholesterol and cancer (Katan, 1986). A large-scale GWAS investigating the impact of host genetic loci on the gut microbiota has facilitated MR studies of gut flora (Kurilshikov et al., 2021). Moreover, the MR analysis is commonly described as a “naturally occurring randomized controlled trial” due to the random assortment of genetic variation during meiosis (following Mendel’s first and second laws), thus it has recently become popular in investigating the potential causal effects of the gut microbiota on different disease (Li et al., 2023; Luo et al., 2023; Sha et al., 2023; Yuan et al., 2023). This study utilized the latest GWAS data on ASD and gut microbiota to ascertain the causal correlation between gut microbiota and ASD. The reverse MR analysis was also conducted to bolster the findings’ dependability. Finally, we performed gene ontology (GO) analysis based on lead SNPs to explore the biological role of gut microbiota on ASD. We have identified specific gut microbiota in increasing or reducing the risk of ASD. Some of them have not been incorporated into the observational studies of ASD. The GO study provided more insights into the underlying physiology mechanism. Our study provides new directions and original insights into future studies on ASD.

In our research, we have discovered through conducting a bidirectional two-sample MR analysis that specific gut microbiota had correlation with ASD. Using IVW, MR Egger, simple mode, weighted median, and weighted model analysis methods, we successfully identified that the family Prevotellaceae, genus Turicibacter had a potential positive link with ASD, while genus RuminococcaceaeUCG005, Ruminiclostridium5, Ruminococcus1, and Sutterella had potential protective effects against ASD. After FDR multiple-testing correction we further observed that the family Prevotellaceae exhibited a significant positive correlation with ASD, while the genus RuminococcaceaeUCG005 displayed a strong negative correlation. Overall, our findings pinpoint specific gut microbiota that had correlation with ASD, which elucidates the potential biological processes of gut microbiota in the development of ASD from a genetic perspective by using GO analysis. This finding may help lead to microbiological interventions in future ASD clinical trials.

According to the recent study, autistic children have dissimilar gut microbiome than normal developing children (Liu et al., 2019a). The gut microbiota, the “second human genome,” might play a key role in the development of ASD by significantly influencing the gut microbiota-brain axis (Grice and Segre, 2012, Yu and Zhao, 2021). The present studies have determined the involvement of chemical, metabolic, and endocrine pathways in the pathogenesis of ASD (Sharon et al., 2019; Needham et al., 2021). Notably, individuals with ASD exhibit signs of low-grade inflammation, which suggests that gut microbiota may have some effect on the immune system and consequently induces neuroimmune responses in the central nervous system (Kim et al., 2017). The significant influence of microbial metabolites including bile acid derivatives and short-chain fatty acids (SCFAs), on patient’s symptoms with ASD cannot be overlooked (Morais et al., 2021). Innovative therapeutic approaches, encompassing diet therapy, probiotic therapy, fecal microbiota transplantation, and targeted antimicrobial therapy, might serve as promising strategies to alleviate the linguistic, cognitive, and behavioral deficiencies associated with ASD (Kang et al., 2017; Karhu et al., 2020). Thus, investigating the pathogenesis of ASD from the perspective of gut microbiota remains crucial.

Our research demonstrates a substantial correlation between the presence of the family Prevotellaceae and the incidence of ASD. Research indicates that the family Prevotellaceae can promote inflammation, with its presence in the gut being associated with autoimmune diseases and chronic intestinal inflammation (Zhou et al., 2020; Kim et al., 2021). Upon colonization in the gastrointestinal tract, these bacteria infiltrate the mucosal layer of the large intestine, and eventually settle in the colonic crypts (Elinav et al., 2011). This penetration into ordinarily sterile areas can escalate the activation of the innate immune system, trigger specific T-cell and antibody responses, and consequently foster chronic intestinal inflammation or exacerbate existing colitis (Elinav et al., 2011; Scher et al., 2013; Palm et al., 2014). Additionally, the activation of a pro-inflammatory environment can lead to neuroinflammation, potentially impacting the development and differentiation of microglial cells within the nervous system (Morais et al., 2021). Given the prevalent mild inflammatory responses observed in children with ASD (Morais et al., 2021), it is hypothesized that the impact of the family Prevotellaceae on the immune system may influence neuroimmune interactions in ASD patients, subsequently leading to neurological symptoms.

Genus Turicibacter is a critical component of the gut microbiota, and recent research suggests its potential role in influencing intestinal health (Zhong et al., 2015). Enhanced serotonin signaling has been observed to reverse detrimental symptoms in various ASD mouse models (Walsh et al., 2021). However, genus Turicibacter fermentation enhances the inhibitory effects of serotonin signaling proteins, Wnt signaling proteins, and the expression of downstream genes (Lin et al., 2023). These findings corroborate our hypothesis that genus Turicibacter might adversely affect ASD through chemical, metabolic, and endocrine pathways.

Beyond the aforementioned gut microbiota associated with ASD, our investigation has pinpointed five gut microbiota that exhibit protective properties: genus RuminococcaceaeUCG005, genus Ruminiclostridium5, genus Dorea, genus Sutterella, and genus Ruminococcus1. Our results indicate a significant negative correlation between RuminococcaceaeUCG005 and ASD. This correlation is noteworthy since this bacterium can produce the SCFAs, butyrate, and is widely considered beneficial for gut health (Min et al., 2023). Literature indicates a reduced concentration of butyrate in children diagnosed with ASD (Liu et al., 2019b). Butyrate possesses neuroprotective qualities, modulating the production of catecholamines and neurotransmitters within the nervous system (De Angelis et al., 2015). Additionally, it promotes memory consolidation through epigenetic mechanisms and maintains neural plasticity (Mohajeri et al., 2018). Research has demonstrated that a sustained low-butyrate environment can diminish PPARα expression in colonic macrophages, consequently inhibiting NF-κB signaling and promoting the release of pro-inflammatory cytokines IL-1β and TNF-α (Chen et al., 2022). Thus, we hypothesize that the protective effect of genus RuminococcaceaeUCG005 against ASD is mediated through a synergistic interaction of microbial metabolites and neuroimmune responses.

Moreover, existing literature suggests that genus Dorea may contribute to mitigating tropomyosin (Tm)-induced allergic reactions (Fu et al., 2017). Genus Dorea possesses the potential to modulate mucosal metabolism, thereby affecting intestinal permeability. A negative correlation has been observed between genus Dorea and food allergies in infants (Savage et al., 2018; Zhou et al., 2021). Given the increased incidence of food allergies in children with ASD, we hypothesize that the protective effect of genus Dorea against ASD may be related to its capacity to alleviate Tm-induced allergic responses (Schieve et al., 2012; Xu et al., 2018). Research indicates that the abundance of genus Sutterella is higher in individuals without any diseases compared to those with Crohn’s disease (Gevers et al., 2014). Cohort studies have shown that aggregation of Suterella is inversely proportional to the host inflammatory response (Morgan et al., 2015). Consequently, it is plausible that the protective role of genus Sutterella against ASD may be achieved through reducing intestinal inflammation and modulating neuroimmune responses.

In our study, we identified previously unreported gut bacteria, genus Ruminiclostridium5 and genus Ruminococcus1, associated with ASD. In addition to the above two newly discovered gut microbiota, the mechanism of action of the microbiota (positive and negative) in ASD identified by the MR method has been briefly illustrated in Figure 5 . The pathophysiological roles of these bacteria in ASD patients warrant further in-depth investigation. While current observational studies have not yet established a connection between these bacteria and ASD, our findings provide essential guidance for subsequent research. Furthermore, the potential effects of the aforementioned gut microbiota and their metabolites on the nervous system remain to be elucidated, necessitating additional observational studies for clarification.

Additionally, enrichment analysis demonstrated that several GO-related biological processes were significantly involved in the association between gut microbiota and ASD. The absence of certain microorganisms has been linked to enduring effects on neurogenesis, synaptogenesis, axonal and dendritic growth, and the establishment of neural connections, all of which can influence the development of ASD (Rogers et al., 2016). Research indicates that infants at higher risk of ASD suffer different gut microbiota composition during a crucial developmental period for the gut microbiota and brain (Zuffa et al., 2023). This indicates that alterations in gut microbiota during early development may have persistent impacts on the formation of neuronal pathways. Recent research highlights that a more diverse gut microbiome in infants at birth can foster stem cell proliferation through microbial metabolites, influencing infant development (Socha-Banasiak et al., 2021). This is consistent with the findings of our gene GO analysis. Besides, abnormalities of key genes in the cell junction assembly pathway can cause the development of ASD (Zhang et al., 2022). Through the action of bile acids and SCFAs, gut microbiota can also alter the energy metabolism of intestinal epithelial mitochondria, activate immune system cells, and modulate intestinal epithelial barrier function (Dunislawska et al., 2023). Changes in early-life gut microbiota can impact host neuroimmunoregulation (Morais et al., 2021). It is now understood that ASD patients typically exhibit a dysregulated intestinal epithelial barrier (Gonzales et al., 2021). Similarly, an observational study found that SCFAs produced by gut microbiota metabolism could modulate the production of interleukin-2 (IL-2), and autistic patients exhibited an abnormally high level of IL-2, consistent with our GO analysis results (Vinolo et al., 2011; Cao et al., 2021). Therefore, we hypothesize that intestinal microorganisms may cause ASD via their influence on early neurodevelopment, metabolic and endocrine pathways and induction of intestinal inflammation mediated by SCFAs.

Currently, employing mathematical algorithms to investigate the origins of diseases is a prevalent approach (Morton et al., 2023). Several studies have made notable advancements in etiological exploration using MR methods (Li et al., 2023; Luo et al., 2023; Sha et al., 2023; Yuan et al., 2023). MR offers an optimal research design, utilizing genetic variants as instrumental variables to elucidate causal relationships between potential risk factors (exposures) and diseases of interest (outcomes), effectively circumventing biases caused by confounding factors (Burgess and Labrecque, 2018). Our research introduces several innovations: 1. By leveraging bidirectional MR, we bypass common biases inherent in observational experiments and, from a genetic perspective, identify specific gut microbes that correlation with ASD. 2. Our study identifies new gut bacteria related to ASD that have not been recognized in observational research, providing a reference for further investigation into the interplay between gut microbiota and ASD. 3. Through GO analysis, we indirectly infer the biological processes through which gut microbiota influences ASD, shedding light on the potential physiological mechanisms underlying ASD from another perspective.

However, our study does have inherent limitations. First, the GWAS data related to gut microbiota were derived from a diverse cohort of 18,340 participants from multiple ethnic backgrounds, while the GWAS summary statistics for ASD were solely from individuals of European descent. Nevertheless, given that nearly 80% of the gut microbiota GWAS data originate from European populations, and that the dataset is extensive, diverse, and broadly representative, we proceeded to use this GWAS data for our research. Moreover, this dataset has been widely employed in existing MR studies. Lastly, due to insufficient data, we were unable to conduct detailed stratified analyses, such as those based on age and gender. As a result, we could not investigate potential disparities across different populations. Future research will need to address these limitations and shortcomings.

In this work, by leveraging bidirectional MR, we bypass common biases inherent in observational experiments and, from a genetic perspective, identify specific gut microbes that correlation with ASD. we successfully identified that the family Prevotellaceae was shown to be substantially positively connected with ASD, whereas the genus RuminococcaceaeUCG005 was found to be strongly negatively correlated with ASD. The genera Dorea, Ruminiclostridium5, Ruminococcus1, Sutterella and Turicibacter have potential effect on ASD. Our study opens up new avenues for therapeutic interventions in this patient population. However, we need to do more study to explore the specific physiological mechanisms and significance of individual bacterial taxa in ASD pathophysiology.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

All participating studies involved in the GWAS obtained informed consent from the study populations. As we utilized publicly available datasets to conduct MR, no additional ethics approval was required.

ZL: Data curation, Formal Analysis, Project administration, Software, Writing – original draft. SL: Data curation, Formal Analysis, Supervision, Validation, Writing – review & editing. FL: Supervision, Validation, Writing – review & editing. ND: Data curation, Project administration, Writing – original draft. RL: Data curation, Writing – original draft. SL: Data curation, Writing – original draft. LB: Data curation, Writing – original draft.