eligibility criteria
All trials will be selected that have published on our research topic,and eligibility criteria were set according to PICOS : the participants (P), the interventions (I), the comparison (C), the outcome (O),and the study design (S).Only trials with eligible data for a meta-analysis will be included.
Step 1: inclusion criteria for trials
The Participants
Neonates, children or adolescents of ASD and pregnant women.
The Interventions
We will focus on trials that used vitamin D3 in any dose and any regimen as the intervention. However, we should pay attention to the time of the 25(OH)D supplemention,because it takes 3 to 6 months for 25(OH)D levels to reach homeostasis after initiating supplementation. Besides, we will also include studies using vitamin D3 bioequivalent substances .
The Comparison
We will include only studies, which diagnosed ASD without vitamin D supplemention as the comparator.
The Outcomes
To be eligible for inclusion in a meta-analysis trials need
to have assessed the outcome of the level of 25(OH)D, the score of CARS,the score of ABC,the score of SRS and the score of M-CHAT. We also intend to record studies with the outcomes of ASD incidence and contact the authors if they have data for the outcomes needed for the planned meta-analyses in an intermediate step of our systematic review, The contents of outcomes are shown in table 2.
Table 2 The contents of outcomes
Outcome contents
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25(OH)D levels testing serum 25(OH)D level
Childhood Autism Rating Scaleas(CARS) CARS is a standardized diagnostic scale for
ASD, which was compiled by E.Schopler, R.J.Reichler and B.R.Renner in 1980.It evaluates children with autism in 15 aspects and suitable for high risk children over 2 years old.26
Autism Behavior Checklist (ABC) ABC, prepared by Krug in 1978, primarily to assess the severity and change of autism symptoms,There are 57 items in the scale, including five aspects: feeling, communication, body movement, language and self-care.27
Social Responsiveness Scale(SRS) SRS was compiled in 2005, which is suitable for screening autism in children aged 4-18. The SRS questionnaire consists of 65 items and is divided into five dimensions: social perception, social cognition, social communication, social motivation and autism behavior; The total score is ≥60 points, indicating that the primary screening is positive. This scale is the first ASD screening tool to quantitatively score social functions. 28
M-CHAT M-CHAT is one of the main screening scales for ASD in the early stage, which is suitable for children between 16 months and 30 months. The scale includes 23 items and can screen children with ASD in the early stage.29
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Study design
We will include randomised controlled trials (RCTs) , case–control, cohort,
and nested case–control studies. No restrictions are applied for the form and time of vitamin D.Studies need to report the odds ratio (OR)/relative risk (RR) for ASD incidence,and mean and standard deviation of vitamin D concentration .We will exclude studies if involved participants were comorbid with any other disease that affect the level of vitamin D .
Step 2: inclusion criteria for pooling in meta-analysis
Studies will be included for pooling in the meta-analysis if the risk ratio and 95% CI for at least one outcome of interest 25(OH)D levels,the score of CARS,the score of ABC,the score of SRS and the score of M-CHAT) were reported. We will include the largest number of information in the meta-analysis when several publication reported the same trial.
Information sources and search strategy
We will search in MEDLINE, PubMed, EMBASE, Web of Science,and the Cochrane Library from database to identify studies on vitamin D and ASD. The search terms we used were medical subject headings(MeSH) phrases combined with text words relating to vitamin D (“vitamin D” OR “25 hydroxyvitamin d3” OR “d3,1,25 dihydroxyvitamin” OR “1,25 dihydroxyvitamin d3” OR “1,25 dihydroxy 20 epi vitamin d3”OR“cholecalciferol”) and autism (“autistic” OR “autism” OR “ASD”OR“Autism Spectrum Disorder”) . We will search for previous systematic reviews in the KSR Evidence and Cochrane Database of Systematic Reviews (CDSR, OVID interface).No restrictions were applied for the languages, time, and geographical position of the studies. The search string for MEDLINE is shown in table 3.
Table 3 Search string for MEDLINE
Step Search string
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1 ‘vitamin D’(tw) OR ‘vitamin D’(MeSH) OR
cholecalciferol*(tw) OR ‘vitamin d 3’(tw) OR ‘25 hydroxyvitamin d3’(MeSH) OR‘25 hydroxyvitamin d3’*(tw) OR
1,25 dihydroxyvitamin d3*(tw) OR 1,25 dihydroxyvitamin d3(MeSH) OR calcitriol(tw) OR ‘1,25 dihydroxy 20 epi vitamin d3’(tw) OR 1,25 dihydroxy 20 epi vitamin d3l(MeSH)
2 autistic(tw) OR autistic(MeSH) OR autism(MeSH)
OR autism(tw) OR ASD(tw) OR ASDl(MeSH)ORAutism Spectrum Disorde(tw) OR Autism Spectrum Disorde(MeSH)
3 (((((((((‘randomized controlled trial’(pt)) OR ‘controlled clinical trial’(pt)) OR randomized(tiab)) OR placebo(tiab)) OR randomly(tiab)) OR trial(tiab)) OR groups(tiab))) NOT ((animals(mh) NOT humans(mh)))
4 placebos(MeSH) OR placebo(tw)
5 2
6 1 AND 3 AND 4 AND 5
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Data collection and management
Data were selected from eligible studies by two reviewers. They cannot know each
other’s decision . References will be stored and managed by The EndNote software.The two reviewers will extract data include first author, year, country, study design, sample size,number of samples, sex, age, vitamin D measurement method,25(OH)D levels, the score of CARS,the score of ABC,the score of SRS and the score of M-CHAT.
Quality assessment
Two independent reviewers will assess all studies according to the Newcastle–Ottawa Scale (NOS). 30 This NOS includes three aspects: study-participant; the comparability of study participants; the exposure or outcome of studies.he results fall into low-quality and high-quality . Two reviewers (WZQ and DR) independently
evaluated the eligible studies. Any discrepancies were resolved by discussion.If there is a disagreement, they will be discuss the key points until a consensus is reached.
Descriptive analysis and meta-analysis
Measures of treatment effect
The 25(OH)D levels and the score of scales be addressed by estimating the overall effect size and 95% confidence interval (95% CI).We will describe the difference of mean 25(OH)D levels between ASD and control groups by Mean difference (MD).
Data synthesis
Due to differences between the studies, all fit the following four major meta-analysis data will be composed deriving random effects results with the Laird
and DerSimonian method and fixed-effects summary estimates using the Mantel-Haenzel method :
- Association of vitamin D3 levels and ASD occurrence;
- study of the effect of vitamin D supplementation of ASD children;
- study of vitamin D supplementation during pregnancy ;
- Outcomes including incidence of ASD, vitamin D levels,and the score of scales.
For the categorical variable, we will calculate the pooled OR/RR and 95% CI of eligible studies. we will perform a meta-analysis on case–control studies with OR if there are studies did not report vitamin D concentration but provided OR and 95% CI for the possibility of ASD which is vitamin D insufficiency or deficiency. To further explore the effect factors of the treatment by methodological or patient characteristics differences of the studies,we will performed subgroup analyses with eligible data.
Subgroup analyses
1. Age (0-1years vs1-3 years vs ≥3 years))
2. Sex (male vs female)
3. Vitamin D3 supplementation duration (<6 vs ≥6 months)
4. Vitamin D3 dosing (<1000 IU vs 1000–2000 IU vs >2000 IU/day).
Assessment of heterogeneity
We will measure heterogeneity by Cochran’s Q test and I2statistic.. I2
shows the ratio of heterogeneity, when I2 ≥ 50% indicated high heterogeneity.Meanwhile,We will perform subgroup and meta-regression analyses to explore sources of heterogeneity.
Assessment of publication bias
We will access Publication bias by visually in funnel plots and tested for with Egger’s test.
The quality of the body of evidence
We will evaluated the quality of all outcomes by the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach.
Patient and public involvement
Since this is a systematic review scheme, no patients and participants need to be recruited.
Ethics and dissemination
This systematic review is a summary of already published data. All studies had ethics approvals ,so,we don’t need an ethics approval .