Oral Microecology in Children With Autism
Conditions: Autism, Caries,Dental
Sponsor: The Dental Hospital of Zhejiang University School of Medicine

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Research Proposal 
I. Research Background 
Research Significance of the Project 
Autism spectrum disorder (ASD) is a pervasive developmental disorder that has become the 
leading cause of mental disability among children in China [1], characterized by early social 
dysfunction, repetitive behaviors, and impaired interests, often coexisting with epilepsy, 
depression, anxiety, attention deficit hyperactivity disorder, as well as sleep and self-injurious 
behaviors [2]. According to the Centers for Disease Control and Prevention in the United 
States, approximately 1 in 44 children is diagnosed with ASD, with boys being about 4 times 
more likely to be affected than girls. The onset of autism is closely linked to genetic factors 
and environmental factors during early development. Recent studies have found that changes 
in the gut microbiome are associated with the pathogenesis of ASD [3]. 
cavities are a chronic, progressive disease that causes destruction of hard tooth tissue under 
the influence of multiple factors, primarily bacteria. Dental plaque in the mouth is closely 
related to cavity disease, and the dental community has continuously focused on studying 
the bacterial composition and characteristics in plaque. cavities have three main traditional 
etiological hypotheses: the specific plaque hypothesis, the non-specific plaque hypothesis, 
and the ecological plaque hypothesis. The specific plaque hypothesis suggests that only 
certain specific bacterial species are associated with the disease, such as Streptococcus 
mutans and Streptococcus sobrinus. The non-specific plaque hypothesis indicates that 
cavities are the result of the collective action of plaque microorganisms. The ecological plaque 
hypothesis suggests that cavities are the result of an imbalance caused by changes in the 
resident microbial flora. 
Saliva Immunoglobulin A（IgA）is the main protective antibody in oral mucosal immunity, 
working with the innate immune system to inhibit the adhesion of microorganisms to the 
mucosa and tooth surface, and promoting the elimination of cariogenic microorganisms such 
as Streptococcus mutans[4].IgA deficiency makes patients more susceptible to oral mucosal 
infections and cavities[5]. Compared to individuals without ASD, previous studies have 
observed poorer oral health conditions in the ASD cohort[6,7]. Given the changes in oral 
health conditions in ASD patients, as well as the correlation between oral microbiota dysbiosis 
and mucosal immunity, saliva IgA levels may change in ASD subjects. 
Microorganisms that cause oral diseases may enter brain tissue through various pathways, 
directly damaging the central nervous system, affecting neuroimmune activity and 
inflammation, thereby influencing the occurrence of ASD. On one hand, the brain 
sends 5 cranial nerves that participate in the sensory and motor innervation of the oropharynx. 
These can serve as direct channels for oral bacteria to reach the central nervous system, or as 
transmitters of neural signals from the oral cavity to the brain. On the other hand, oral 
microorganisms can enter the bloodstream through damaged oral mucosa or tooth roots 
due to conditions like gingivitis or periodontitis, and then breach the blood-brain barrier to 
enter the brain. It has been reported that mice infected with Pseudomonas aeruginosa exhibit 
reduced blood-brain barrier integrity [8]. 
The indirect effects of oral microorganisms on the brain may be mediated by systemic 
inflammation and metabolism. ASD patients exhibit central nervous system inflammation in 

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their brains, and elevated levels of cytokines and chemokines in cerebrospinal fluid [9], which 
may be due to the pro-inflammatory activity of lipopolysaccharides abundant in Gram-
negative bacteria and periodontal-related pathogenic bacteria, leading to synaptic 
dysfunction [10]. Hicks et al. [11] found that children with ASD exhibit upregulation of 
microbial RNA related to lysine degradation in their oral cavity. Lysine degradation produces 
glutamate, which is a key neurotransmitter involved in learning and memory. Therefore, the 
oral microorganisms of ASD children may be connected to the brain, suggesting the existence 
of a "microbiome-brain-mouth axis" -. 
Analysis of the current situation domestically and internationally: 
Currently ASD research on the imbalance of pediatric microecology primarily focuses on gut 
microbiota, with the "microbiota-gut-brain axis" being considered by many scholars to be 
related to ASD [12–14].Chaidez et al.[15]reported that46%–84%ofASD patients exhibit 
gastrointestinal symptoms, which are associated with the severity of their core symptoms and 
can exacerbate social withdrawal, stereotyped behaviors, and other behaviors in ASD children. 
Iglesias et al.[16]found that the abundance of Bacteroides, Parabacterium, Clostridium, 
Faecalibacterium, and Coprococcus genera in the gut microbiota of ASD patients is 
significantly higher, while the abundance of Faecalibacterium and Bifidobacterium genera is 
lower. Desbonnet et al.[17]experimentally demonstrated that when the fecal bacteria of 
neurobehaviorally normal mice are used to colonize autistic male mice raised in a sterile 
environment, many social avoidance and repetitive behavior defects, as well as a lack of 
interest in social novelty and social motivation, were reversed. 
②The mouth is the only entrance to the gut. In the Human Microbiome Project, oral bacteria 
and fecal bacteria overlap in nearly half of the participants. Olsenet al.[18]found that 
Porphyromonas gingivalis, which enters the gut through swallowing, causes gut microbiota 
dysbiosis, further increases intestinal epithelial permeability, and induces immune activation, 
leading to systemic inflammation. Narengaowa et al.[19]believe that Porphyromonas 
gingivalis surviving in the gut and the gut microbiota or its metabolites may enter the 
bloodstream and ultimately reach the brain via the enteric nervous system. Hicks et 
al.[11]studied2-6year-oldASDchildren, non-autistic developmental delayed children, and 
normally developing children's saliva microbiota. Through metagenomic shotgun sequencing, 
they found5types of oral microbiota that could distinguish ASD children from normal children, 
and3types of microbiota that could distinguish ASD children from globally developmental 
delayed children, with an accuracy close to80%. Qiao et al.[6]compared the bacterial diversity 
between normally developing children and ASD children using high-throughput sequencing. 
They found that the abundance of pathogens such as Haemophilus and Streptococcus in the 
saliva and plaque of ASD children was higher, while Prevotella, Sebacibacterium, Actinomyces, 
Porphyromonas, and Fusobacterium were less abundant. 
③Gonget al.[20] demonstrated that Streptococcus mutans can regulate the downregulation 
of polymeric immunoglobulin receptor (PIGR) in human salivary gland (HSG) cells. The impact 
of other bacteria with altered abundance on PIGR. Meanwhile, they also found that the saliva 
IgA levels in mice models of autism spectrum disorder (ASD) established by sodium valproate 
(VPA) were reduced and correlated with autistic-like behaviors in mice. Guo et al.[21] studied 
the blood IgA levels of 75 children with ASD and 75 normally developing children. They found 
that the blood IgA levels of children with ASD were significantly lower than those of the 

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control group and showed a significant negative correlation with the severity of ASD in 
children. 
This project aims to address the scientific questions and the main research approach 
Since oral health is influenced by multiple factors, previous studies have observed 
ASD patients exhibiting poorer oral health conditions. We propose the main scientific 
question of this project: What are the changes in the oral microbiome of autistic children? 
This study will collect saliva and plaque samples from both ASD children and healthy children, 
while also distributing oral hygiene questionnaires. Subsequently, we will utilize RNA-
seq technology to analyze the distribution of oral microbiota and compare the dominant 
microbial species in ASD children to those in typically developing children. Using the 
Elisa method, we will measure saliva IgA levels to validate the host's immune response under 
changes in oral microbiota. Additionally, we will analyze the correlation between the severity 
of autism and the oral microbiome. This research aims to provide experimental evidence for 
the screening of cariogenic microorganisms, the assessment of immunoglobulin A levels, the 
prediction of the relationship between oral microbiota and brain development, and the 
establishment of active disease indicators for caries, saliva immunity, and autism. 
 
II. Research Objectives 
① To clarify the changes in ASD children's IgA levels and oral microbiota abundance across 
different severity groups; 
② To determine the differences in ASD children's and healthy children's IgA levels and oral 
microbiota abundance between groups; 
③ To determine the host immune response under oral microbial changes. 
 
III. Research Design and Methods 
[Research Type] 
This study is a multicenter retrospective cohort study. The main center is the Affiliated Hospital 
of Zhejiang University School of Medicine(Department responsible: Department of 
Conservative Dentistry; Investigator: Tu Yan). Other participating centers include the Seventh 
People's Hospital of Hangzhou (Department responsible: Department of Child Psychology; 
Investigator: Zhou Guoling). 
[Research Period] 
Research Duration:3 years 
Start Date: From the date of approval 
End Date:2025 year9 month 
【Research Methods】 
An oral health status assessment questionnaire was released to patients with ASD aged 6-18 
and their families to collect information on the oral hygiene habits, dietary habits, bad habits, 
and oral hygiene maintenance of children with ASD. Additionally, it investigated the degree 
of concern parents have regarding the oral hygiene of children with ASD and their level of 
knowledge about oral hygiene. Simultaneously, children with ASD aged 6-18 and their 
corresponding control normally developing children were recruited, and questionnaires were 
issued to their parents. 
（1）Inclusion Criteria: This project is supported by Hangzhou Seventh People's Hospital 

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and the Stomatological Hospital Affiliated to Zhejiang University School of Medicine; all 
patients' ages must be clearly recorded6-18 years old, gender unrestricted; Participants 
or/ their families must understand the trial objectives, be willing to cooperate, and voluntarily 
or/ with their families' consent to participate in the trial and sign an informed consent form. 
（2）Exclusion Criteria: Obvious brain structural abnormalities detected by MRI; Severe 
sensory 
organ 
damage 
(blindness, 
hearing 
loss); 
organic 
gastrointestinal 
problems;1month of antibiotic or immunosuppressive medication use; confirmed 
autism associated with genetics; obvious oral mucosal abnormalities; wearing various 
orthodontic appliances and accessories. 
（3）Exit Criteria：The patient, after being informed, does not consent to the use of their 
information for research; Unexpected circumstances arise during the study, causing the 
patient to no longer meet the inclusion/exclusion criteria. 
Children had not brushed or rinsed their mouths the night before and on the morning of 
sample collection; they needed to rinse gently with sterile distilled water half an hour before 
sampling. Saliva collection: Keep saliva in the mouth1min, gently spit into the collection funnel, 
and collect5mLwith a sterile tube3mLnon-irritating saliva sample, mark it, and immediately 
store in a dry ice bucket, then transfer to a-80℃ freezer for storage. Plaque collection: Isolate 
the sampling area from saliva with sterile gauze, use sterilized swabs to scrape supragingival 
plaque from the subject’s11、46labialpalatal/buccal lingual supragingival plaque, place the 
sample in1mL Tris-EDTA(TE)buffer solution, mark it, and store in a dry ice bucket, then transfer 
to a-80℃ freezer for storage. 
Under natural light, conduct an oral health survey using visual and tactile examination, 
employing a specialized examination record form to record simplified oral hygiene indices 
such as the OHI-S and DMFS/dmefs for oral hygiene status. OHI-S: Record the indices of soft 
plaque ( DI-S ) and calculus ( CI-S ) on the buccal side of 16, 26, 36, the lingual side of 46, the 
labial side of 11, and 31, as well as the soft plaque ( DI-S ) and calculus ( CI-S ). DI-S: 0 = no 
soft plaque on the tooth surface; 1 = soft plaque covering an area of less than 1/3 of the 
tooth surface; 2 = soft plaque covering an area of 1/3 to 2/3 of the tooth surface; 3 = soft 
plaque covering an area of more than 2/3 of the tooth surface. CI-S: 0 = No dental calculus 
on or below the gum; 1 = Dental calculus on the gum covers less than 1/3 of the tooth surface; 
2 = Dental calculus on the gum covers between 1/3 and 2/3 of the tooth surface, or scattered 
subgingival dental calculus on the tooth neck; 3 = Dental calculus on the gum covers more 
than 2/3 of the tooth surface, or continuous and thick subgingival dental calculus on the tooth 
neck. 
DMFS/dmefs：D refers to teeth in the oral cavity that exist due to caries that have not been 
repaired. M refers to teeth lost due to caries. Some statistics classify M into two categories, 
using Mi—teeth that need to be extracted due to severe caries; Me—teeth already lost due 
to caries. F represents teeth that have been filled due to caries (excluding fillings done for 
non-caries conditions).At age 9 or younger, if a child loses deciduous teeth that should not 
fall out, such as molars or cuspids, it is considered lost due to caries. For those over 9 years 
old, there is no longer a distinction between teeth lost due to caries or physiological shedding; 
they can be treated as unerupted teeth (corresponding permanent teeth).d: Carious teeth 
requiring treatment; f:Already treated carious teeth; e:Deciduous teeth that should be 
extracted due to caries; m:Teeth lost due to caries. 

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The Childhood Autism Rating Scale (CARS) is used by professionals to assess children with 
autism. This scale consists of 15 items, each scored on a seven-level scale from 1 to 4, with 
increments of 0.5 points. A score of 1 indicates normality, while 4 indicates the most severe 
level, and the total score is calculated by summing the individual item scores. For CARS total 
scores, ages 6-12 with ≤29.5 points are considered normal, 30-36.5 points indicate mild-to-
moderate autism, and ≥37 points indicate severe autism; for ages 13 and older, ≤27.5 points 
are normal, 28-34.5 points indicate mild-to-moderate autism, and ≥35 points indicate 
severe autism. 
DNA extraction and PacBio SMRT high-throughput sequencing: Refer to the instructions of 
the genomic DNA extraction kit to perform genomic DNA extraction on the samples. 
Bacterial16S universal 
primers27F 
(5-AGAGTTTGATCCTGGCTCAG-3′),1492R 
(5-GGT-
TACCTTGTTACGACTT-3′) are used. To distinguish between different samples, each pair of 
primers is separately added with a16 base pair length barcode sequence, and 
PCR amplification is performed on the products, followed by purification, quantification, and 
normalization to form a sequencing library. 
Sequencing and bioinformatics analysis: Quality control of the constructed libraries was 
performed, and qualified libraries were sequenced using the PacBio SMRT high-throughput 
sequencing technology. After quality control of the offline data, raw data were subjected to 
quality 
control 
and 
subsequent 
bioinformatics 
analysis 
using 
the 
Mothur andQIIME2 platforms. Qualified sequences were split into different samples based on 
barcode sequences, and barcode and primer sequences were removed for subsequent 
analysis. Bioinformatics analysis was performed on high-quality sequences, including: (1) 
clustering to obtain operational taxonomic units (Operational taxonomic units, OTU)) using 
the Usearch software at97% similarity; (2) calculating α diversity indices for each sample using 
the Mothur(v.1.30） software, including Shannon index, Simpson index,Chao1 index, and 
abundance-based coverage estimate (ACE） index, and plotting sample dilution curves and 
rank abundance curves; (3) β diversity analysis to assess differences in microbial community 
structure between the two groups, including principal coordinate analysis based on the binary 
jaccard matrix; (4) classifying feature sequences using a naive Bayes classifier combined with 
alignment based on the SILVA reference database to obtain taxonomic classification 
information for each feature, generating community composition statistics for each sample at 
the phylum, class, order, family, genus, and species levels using the QIIME software, and using 
the R language tool to plot community structure diagrams at each taxonomic level for each 
sample, comparing species differences between the two groups. 
According to the manufacturer's instructions, use an enzyme-linked immunosorbent assay 
(ELISA) kit (Bayerin, Wuhan, China) to measure the IgA levels in human saliva samples. 
Data processing: First, determine normality through the Shapiro-Wilk test and check for 
homogeneity of variances using the Brown-Forsythe test. If the two continuous variables are 
normally distributed, use the Student t test; otherwise, use the Mann-Whitney U test. If the 
three continuous variables are normally distributed and meet the homogeneity of variance 
conditions, use one-way analysis of variance (ANOVA); otherwise, use the Kruskal-Wallis test. 
Use the chi-square test to analyze categorical data. Use the Student t test (for age) and the 
chi-square test (for gender) to compare the demographic information of ASD children and 
normal control children. Use the Student t test to analyze human saliva IgA content and 

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evaluate the diagnostic performance of human saliva IgA through receiver operating 
characteristic (ROC) analysis. 
 
IV Sample size 
This study is a multicenter retrospective cohort study, with sample size determined primarily 
based on saliva IgA content as the main observation index. According to the average 
concentration of the control group (161.61 ± 193.37 μg/mL) and the average IgA 
concentration in saliva of children with ASD (116.55 ± 90.97 μg/mL) [22], with a significance 
level of bilateral 0.05, power of 90%, the formula was used for calculation. Considering the 
dropout rate, approximately 50 participants (total of 150 people) are needed in each group 
to detect statistically significant differences between the groups. 
 
V: Data Management and Confidentiality 
The original data involved in the study were obtained from the database of the Stomatological 
Hospital Affiliated to Zhejiang University School of Medicine and the database of the Seventh 
People's Hospital of Hangzhou. The data were entered and stored only by the research 
personnel. Missing data, unused data, and illogical data will be excluded. All eligible 
participants will be included in the participant dataset for statistical analysis. All records related 
to participant identities will be kept confidential, and the data will not be disclosed outside 
the scope permitted by relevant laws and regulations. 
 
VI: Informed Consent 
This study will be conducted by Tu Yan team to obtain informed consent from participants 
through signature before the evaluation. The informed consent process adheres to the 
principles of full disclosure, full understanding, and autonomous choice; the wording of the 
informed consent is easy to understand and aligns with the comprehension level of the 
participant group. This study involves vulnerable populations including children, children with 
autism spectrum disorder. Before the study begins, child psychiatry doctors assess whether 
children with ASD children have self-awareness based on self-perception ability evaluation 
and provide conclusions and signatures; other relevant clinical department physicians (such 
as neurology, neurosurgery, critical care neurology, etc.) evaluate the consciousness status of 
participants with other disorders of consciousness ;if the ASD child has self-awareness, the 
participant signs and dates it; if they have partial self-awareness, both the participant and the 
legal guardian sign and date it; if they do not have self-awareness, the legal guardian of the 
participant signs and dates it 
Before the routine oral examination and collection of saliva and plaque must inform the 
patient and their family of relevant precautions, potential risks, etc., and sign an informed 
consent form, informing that clinical examination data may be used for non-commercial 
medical research, and the content of the informed consent form includes permission for the 
non-commercial use of clinical data in scientific research, sign the informed consent form, 
complete the informed consent. When obtaining informed consent, follow the principles of 
full disclosure, full understanding, and autonomous choice, ensuring that the personal 
information and medical information of the data source are not disclosed, and do everything 
possible within the law to protect the personal medical records and privacy of the data source. 

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VII, Adverse event reporting 
All patients participating in this study need to undergo routine oral examinations and 
collection of saliva and plaque، which is one of the contents for understanding the specific 
condition before treatment，not an invasive treatment，and almost no risk。Due to poor 
cooperation from children and patients with autism spectrum disorders，in clinical procedures 
and follow-upshouldstrictly adhere to the diagnosis and treatment specifications ，
standardized operations。 
If an adverse event occurs，we will report it according to the following procedures： 
Various adverse events：Take timely measures to handle them，record them in the case report 
form. 
Severe adverse events（SAE）：Take timely measures to handle them，record them in the case 
report form，decide to discontinue or reduce medication by the investigator，immediately 
report to the ethics committee、drug clinical trial institutions and sponsor，24 hours to report 
to the national and provincial food and drug administration。 
 
 
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