This study was an open-label clinical trial involving 40 children with ASD (age 3-17 years) who were diagnosed by the Autism Diagnostic Interview-Revised (ADI-R) and accompanied with symptoms of the gastrointestinal tract (such as constipation, diarrhea, etc.), whose custodian can fully understand and accept the informed consent, all participants were able to undergo follow-up examination. Moreover, children with fever, have reliance on tube feeding, and accompanying emergent gastrointestinal disease that needs prompt medication, diagnosed with severe malnutrition or excessive weight loss or severe immunodeficiency disease, have a history of severe allergies, monogenic diseases, mental disorder or depression were exclude from the study. Besides, children who were undergoing probiotics or had antibiotics 7 days before screening were also excluded.

Apart from ASD children, 16 sex- and age-matched typically developing control (TD) children without gastrointestinal (GI) disorders were included in this trial as well. TD control children had no gastrointestinal symptoms in the last 1 month and had no antibiotics therapy for the last 3 months or any medication that would affect gut microbiota (such as proton pump inhibitors, gastrointestinal stimulants, steroids, and aspirin). Meanwhile, the Bristol stool score was 4 for each TD children.

All children participated in the study for 12 weeks, which included a 4-week FMT treatment phase and an 8-week follow-up observation phase after the treatment. The TD children were monitored for 12 weeks without any intervention. There were two routes of administration for FMT, 27 children received oral route of FMT through freeze-dried capsules while 13 children received colonoscopic FMT. In this trial, participants who were not capable of swallowing capsules were chosen for colonoscopy, while the remaining participants could choose oral capsules, moreover, all participants were allowed to switch to another intervention if they had a strong preference regarding the route of administration. The researchers were not blinded to the group allocation or outcome assessment. Figure 1 illustrates the study design. Neither vancomycin nor proton pump inhibitors (PPIs) was given before FMT. All participants received 2 liters of GOLYTELY (polyethylene glycol) before FMT and remained fasting until the scheduled treatment. To improve the tolerance of patients to GOLYTELY, we applied it by fractionated dose: 1 L was given one day before enema and the rest was given on the day of enema. For patients with poor tolerance (such as abdominal distention and vomiting), saline was added for bowel preparation. All the participants had clinic visits at weeks 0, 4, 8, and 12.

This clinical study was reviewed and approved by the Ethics Committee at the Army Medical Center of PLA. Written informed consent was obtained from each participant before enrollment. In addition, the study was registered at the Chinese Clinical Trial Registry (www.chictr.org.cn); the trial registration number was ChiCTR1800014745, through which the trial protocol can be accessed.

One rigorously screened donor volunteered to provided stool for all participants. The screening involved review of medical history, serological examinations to screen infectious disease, stool examination, gut microbiota sequencing, and confirmation of the absence of gastrointestinal disorders and other neurodevelopmental problems, meanwhile, Helicobacter pylori was also detected through C13 breath tests. The serological examination was performed to exclude hepatitis A, B, and C infections, human immunodeficiency virus-1 infection, human immunodeficiency virus-2 infection, TB infection of T cells, TORCH virus infection, and syphilis infection. Fasting glucose levels, lipid levels, liver function, renal function, and C-reactive protein levels were also assessed. The stool used for the preparation was tested for the presence of bacterial pathogens (Escherichia coli O157, Shigella, Salmonella, Campylobacter, Staphylococcus aureus, Yersinia, Vibrio parahaemolyticus, and Vibrio cholera), infection with viruses (rotavirus A, adenovirus, and norovirus), infection with fungi (Candida albicans) and the presence of parasites (Giardia, Cryptosporidium, Cyclospora, and Isospora).

The donated stool samples were collected under anaerobic and sterile conditions. The samples were mixed with sterile normal saline and then homogenized immediately. The homogenates were then filtered through 20 μm nylon filters to remove large particles and fibrous matter. The filtered suspensions were then centrifuged at 6000 g for 5 min at 4°C with a centrifuge (Sorvall SS-34). The supernatant was removed, and the precipitate was dissolved in normal saline, finally, a fecal bacterial solution of 60 mg/mL was obtained and used for future manufacture of FMT capsules or colonoscopic transplantation (10 g/50 kg per child). FMT capsules were prepared as previously reported (Xu et al., 2021), lyophilized protectant was added to the fecal bacterial solution and frozen at -80°C, which was then freeze-dried into powder using a cryogenic lyophilizer and placed in a capsule for further use.

The participants received 2 liters of GOLYTELY (polyethylene glycol) the night before transplantation. Both the oral capsule administration group and the rectal administration group received the same dose (approximately 2 × 10¹⁴ CFU per patient) once a week for 4 weeks.

During the study, the physical examination of participants was carried out at week 0. Blood samples were collected at week 0, 4, 8, and 12. Stool samples from the participants were collected at week 0, 1, 2, 3, 4, 6, 8, 10, and 12 for 16s rRNA sequencing to determine the composition and abundance of gut microbiota. For each participant, an interview by phone was conducted after FMT treatment. If the patients had any questions or adverse symptoms after FMT, more interviews or consultations would be carried out.

To evaluated the gastrointestinal symptoms of each participant, their parents were asked to complete the Gastrointestinal Symptoms Rating Scale (GSRS) and the Bristol Stool Form Scale at week 0, 4, 8, and 12. The GSRS is a self-management questionnaire that is a 7-point Likert scale with the following descriptive indicators: 1 (asymptomatic), 2 (slight), 3 (mild), 4 (moderate), 5 (moderate to severe), 6 (severe), and 7 (very severe). It was originally a hierarchical scale used for face-to-face questioning, mainly to evaluate common gastrointestinal symptoms, and was modified to generate a self-management questionnaire. The 15 items can be divided into 5 dimensions: abdominal pain (3 items), reflux (2 items), dyspepsia (4 items), diarrhea (3 items), and constipation (3 items). The questions were all regarding the severity of symptoms in the last 2 weeks. The score for each dimension was expressed as the average score of all items in the dimension, with the lowest score being 1 and the highest score being 7. The Bristol Stool Form Scale was used to assess the condition of the participant’s recent stool.

Autism and ASD related symptoms were assessed by the Childhood Autism Rating Scale (CARS), Social Responsiveness Scale (SRS) and Autism Behavior Checklist (ABC). The CARS is a 15-item scale that can be used to both diagnose ASD and assess the overall severity of the symptoms. The ABC assesses problem behaviors in five areas that are common in children with ASD, including irritability, lethargy, stereotypy, hyperactivity, and inappropriate speech. Additionally, we assessed parents’ levels of anxiety with the Self-Rating Anxiety Scale (SAS). The SAS is a 20-item scale that measures the severity of anxiety in parents of children with ASD. The CARS, ABC, SRS, and SAS were administered at baseline (week 0), at the end of treatment (week 4), and at the end of the observation period (week 12).

We collected fecal samples, stored them in a refrigerator at -80°C, and then delivered them to the G-Bio Sequencing Company (www.igeneseq.com) in dry ice, for further 16S rRNA sequencing. Microbial DNA was extracted from the fecal samples from ASD children, TD controls, and donors by using the PowerSoil DNA Isolation Kit (Mobio Carlsbad, CA). The purified PCR products were used to prepare a sequencing library using the TruSeq DNA Kit from Illumina following the manufacturer’s manual and were subjected to gene sequencing using Reagent Kit v3 on the MiSeq sequencer (Illumina, San Diego, CA, USA). The Quantitative Insights into Microbial Ecology 2 (QIIME2, version 2019.7) platform within a condo environment was used to process the sequencing data on our Linux server. Bioinformatics analysis was performed according to the official “Moving Pictures” tutorial provided by the QIIME2 website (https://docs.qiime2.org/2019.7/tutorials). Linear discriminant analysis effect size (LEfSe) was used to select significantly enriched candidates at the genus level. We compared the relative abundance of taxa between the two groups and within each group at different periods using the nonparametric Mann-Whitney U test followed by linear discriminant analysis (LDA) to estimate the effect size of each microbial feature with differential abundance. A taxon was considered significantly enriched if it had an LDA score greater than 2.0 and p value < 0.05.

Blood samples were taken from ASD children on week 0, 4, 8, and 12 in vacutainer tubes without anticoagulant, and were centrifuged at 3000 r/min for 10 min at room temperature. After centrifugation, the serum (supernatant) was transferred to clean tubes and was immediately stored at -80°C for future experiments. Each serum sample was kept at 4°C for 12 hours for thawing before use. In this study, ELISA kits (Thermo Fisher Scientific, USA) were used to quantify the serum concentration of 5-hydroxytryptamine (5-HT), dopamine (DA), and γ-aminobutyric acid (GABA) of each ASD child, based on the manufacturer’s instruction.

For statistical analysis, SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA) was used in this study. Measurement data are validated by a normality test and presented as mean ± SD. Comparisons of the measurement data were performed by t-test, Mann-Whitney U test, and one-way ANOVA (analysis of variance), and comparisons of categorical data were performed by chi-square test. Unless otherwise indicated, P < 0.05 was considered statistically significant.

Forty children who were diagnosed with ASD based on critical inclusion criteria, and 16 typically developing (TD) children from different families were enrolled in this study. All children with ASD received FMT treatment for consecutive 4 weeks and then underwent 8 weeks of follow-up study (the TD children were not treated during the whole trial) (Figure 1). The TD children had no relatives with any mental disorders. All participants enrolled in this trial were comparable in most demographic characteristics at baseline (Table 1), including ages, gender distributions, body mass indexes (BMIs). We also evaluated the consumption of the nutrients among participants, the consumption of carbohydrates and fat was comparable between children with ASD and TD cohort, while protein consumption was higher in ASD children (51.95 ± 9.84 g) than TD controls (48.44 ± 11.36 g) (p < 0.05). Moreover, children in the ASD cohort took more rounds of antibiotics during the first 3 years of life (5.75 ± 1.86 vs 10.35 ± 3.36, p<0.001), and suffer from a higher rate of food allergy incidence (6.25% vs 60.00%, p < 0.001).

During the follow-up phase, we found that children after FMT showed obvious improvement on symptoms like abdominal pain, constipation, or diarrhea, some children relived a lot from reflux as well. The overall GI symptoms of participants after treatment were measured using the GSRS. The average GSRS scores of ASD children decreased 35% after 4 weeks of FMT treatment and last for the next 8 weeks (Figure 2A), indicating that symptoms including abdominal pain, reflux, indigestion, diarrhea, and constipation were significantly improved after FMT. The Bristol Stool Form Scale and Daily Stool Record (DSR) were used to evaluate changes in stool properties. The occurrence of no stool, hard stool (type 1 or 2), and soft/liquid stool (type 6 or 7) was significantly decreased at the end of treatment compared to baseline, and this improvement persisted 8 weeks after FMT (Figure 2B and Table 2).

In addition to GI symptoms, ASD symptoms were also improved after FMT treatment. Scores on the ABC, which includes 57 items that assess mood, behavior, emotion, and language, were significantly alleviated by the treatment, and no obvious reversion was observed during 8 weeks after FMT (Figure 2C). Scores on the CARS, which evaluates core ASD symptoms, were decreased by 10% at the end of the treatment and remained decreased by 6% after 8 weeks (Figure 2D). The SAS was used to assess the level of anxiety in the parents of children with ASD. We found that parents’ anxiety levels decreased with the improvement of gastrointestinal symptoms and autism-like symptoms in the children with autism but returned to baseline levels by 8 weeks after the end of treatment (Figure 2E). Furthermore, children with ASD exhibited improvements in scores on the SRS, which assesses social skill deficits, at the end of treatment, but these improvements were reversed after 8 to 12 weeks without further treatment (Figure 2F).

Moreover, FMT was generally safe and only induced minimal adverse effects, including hyperactivity and aggression. Nausea/vomiting, major changes in blood chemistry, or long-term adverse effects were observed during follow-up (Table 3), suggesting that FMT was well tolerated. Thus, our data implied that FMT could improve GI symptoms and ASD symptoms without eliciting any severe complications and that the beneficial effects were gradually lost within a few weeks of the end of therapy, suggesting that extended treatment with FMT is needed.

In this trial, FMT treatment was performed through oral or rectal (colonoscopic) route based on the condition of participants. To understand whether the administration route would lead to a different outcome on FMT recipients, we also analyzed the clinical indicators of oral and rectal FMT subgroups (Supplementary Figure 1).

Participants in the rectal FMT subgroup had a higher Bristol stool score at week 0, showing that children in the rectal subgroup tended to be suffered from diarrhea while the oral subgroup suffered more from constipation. At week 4, 8 and 12, the Bristol score of the two subgroups was also statistically different, interestingly, the discrepancy between the oral and the rectal subgroup decreased after 4 weeks of FMT treatment, and the score of the two subgroups get closer to 3 to 4 after FMT simultaneously, indicating that both oral and rectal FMT improved stool characteristics in ASD children. As for GSRS, the score of the rectal subgroup was significantly higher than the oral subgroup at week 0, this is consistent with the fact that children in the rectal subgroup had more severe symptoms and were not capable of swallowing capsules, however, after 4 weeks of FMT treatment, GSRS score of oral and rectal subgroup both decreased remarkably and showed no significance in comparison, this effect continued during the whole follow-up phase after FMT. This result implies that both oral and rectal routes of FMT could induce significant improvement in GI symptoms, and no significant difference was observed between oral or rectal FMT administration.

Consistent with GI symptoms, autism in participants in both oral and rectal subgroups showed similar change after FMT. At week 0, children in the rectal subgroup had a higher score of ABC and CARS, showing the severity of autism in rectal children, while children in the oral subgroup had a higher SRS score. After 4 consecutive weeks of FMT treatment, ABC, CARS, and SRS score of children in both subgroups decreased significantly and had no statistical difference at week 4, which sustained to week 8 and 12.

Neurotransmitters are important in neurological regulation and can affect mood, cognition, and behavior in human beings, abnormality of neurotransmitters would lead to autistic behavior and other neurodevelopmental disorders, alteration of several neurotransmitters are also found in serum or plasma samples of ASD children (Marotta et al., 2020). To further evaluated the effect of FMT on ASD children, we next measured the serum levels of neurotransmitters after FMT. We found that 5-HT and GABA concentrations in the serum decreased after treatment, while the level of DA increased after 4 weeks of FMT (Figure 3). Similar to the GI symptom scores, the change of these neurotransmitters in serum was maintained weeks after FMT. During the whole follow-up, the level of 5-HT, DA and GABA changed most significantly at week 4 when FMT treatment was just completed and did not go further since then. Moreover, correlation analysis between the serum level of neurotransmitters and scales for clinical outcomes reveal that 5-HT had a negative correlation with Bristol stool score (r = -0.434, p = 0.001) while GABA had a positive correlation with Bristol score (r = 0.527, p < 0.001) (Supplementary Table 1), these results suggested that alteration of serum neurotransmitters could have a therapeutic outcome on clinical symptoms on ASD children. Furthermore, at week 8 and 12, these neurotransmitters were still at an altered level significantly but tend to get closer to the original level (Figure 3).

We first evaluated alpha diversity and found that there were no significant differences in alpha diversity between TD and ASD groups at baseline (Supplementary Figure 2). To examine the differences in the microbiota between the two groups, we calculated beta diversity and found significant differences in beta diversity between the two groups (Supplementary Figure 3). At both the phylum and genus levels, the microbiota composition of patients with ASD differed from that of the TD controls (Supplementary Figure 4). To further identify the bacterial taxa with differential abundance between the ASD group and TD controls at baseline, we performed LEfSe analysis and identified eight genera with significant difference, we found that the abundance of gut microbiota in ASD children differs from TD controls, children in ASD group had a relative higher abundance of Christensenellaceae, Akkermansiaceae in family level, and Christensenellaceae R 7 group, Akkermansia, Coprococcus 2, Eisenbergiella, and Tyzzerella 3 in genus level, while TD cohorts had a higher abundance of Peptostreptococcaceae in family level, and Romboutsia, Fusicatenibacter, Eubacterium eligens group in genus level (Supplementary Figure 5).

After FMT treatment for 4 weeks, although the gut microbiota diversity in ASD children remained unaltered, suggesting that FMT did not affect the overall structure of gut microbial communities (Figure 4A), the unweighted UniFrac distances of the recipient patient samples were significantly decreased compared with those of the TD controls and donor after 4 weeks of FMT (Figures 4B, C). Eight weeks after the FMT treatment (week 12), the distances increased to the level observed before FMT (Figures 4B, C), suggesting that FMT could promote the colonization of donor microbes and shift the bacterial community of patients with ASD toward that of TD controls or donor.

Because the pre-existing intestinal landscape can dramatically affect the response to FMT (Ericsson et al., 2017; Zuo et al., 2018), we next assessed the composition of the gut microbiota pre-FMT and analyzed its correlations with clinical outcomes post-FMT therapy. Children with ASD who achieved a less than 50% reduction in average GSRS score were defined as nonresponders. We found that FMT responders and nonresponders clustered separately based on orthogonal partial least squares discriminant analysis (OPLS-DA) (Figure 5A). The variable importance in projection (VIP) score for the gut microbiota showed that several genera contributed significantly to group separation (Figure 5B). Further comparisons of the relative abundance of all the significant bacteria between responders and nonresponders were performed. A significantly lower relative abundance of Eubacterium coprostanoligenes was reported in the responder group than in the nonresponder group (Supplementary Figure 6), whereas no significant differences in the abundance of the other bacterial genera were found (Supplementary Figure 6).

Furthermore, we observed a significant negative correlation between the pre-FMT relative abundance of Eubacterium coprostanoligenes and GSRS scores post-FMT in the FMT recipients (Figure 5C). We further assessed the changes in Eubacterium coprostanoligenes abundance after FMT treatment and found that the reduction in Eubacterium coprostanoligenes abundance was positively correlated with a reduction in GI symptoms, as indicated by the change in GSRS score (Figure 5D). Besides, the correlation analysis of neurotransmitters between gut microbiota showed that Eubacterium coprostanoligenes had a negative correlation at week 12 (r = 0.341, p = 0.048) with serum concentration of GABA. This result indicated that FMT might act partially to treat ASD by reducing the abundance of Eubacterium coprostanoligenes and that the genus Eubacterium coprostanoligenes is a potential regulator of FMT treatment response in children with ASD.