Table 3.

Biological studies on melatonin in ASD

References	Participants	Materials	Main findings	Strengths and limitations
Rossignol et al. [36]	Initial studies review: 35 reviewed independently by two reviewers Five of them were investigated through meta-analysis	Database used: PubMed, Google Scholar, CINAHL, EMBASE, Scopus, ERIC Quality of studies was assessed through Downs and Black checklist	Nine studies measured melatonin or its metabolites in ASD: all reported at least one alteration (four studies: abnormal melatonin circadian rhythm; seven studies: below average physiological levels; four studies: positive correlation between melatonin/derivatives levels and autistic behaviors). Five studies reported gene abnormalities that could decrease melatonin production or impair melatonin receptor function in a small percentage of ASD children	Limitations of the review: small sample size; variation in protocols for measuring changes in sleep parameters; five studies contained a mixture of individuals with ASD and other developmental disabilities
Wang et al. [40]	ASD children (N = 398, M = 367, F = 31, range 2–17 years) and healthy controls (N = 437, M = 406, F = 31)	Genotyping sequences in ASMT, DNA analysis and prediction the effects of coding non-synonymous variants on protein function	Four rare ASMT mutations were found only in ASD group (p.R115W, p.V166I, p.V179G, and p.W257X)	Limitations: low sample size for a rare genetic mutations investigation; important disproportion between F and M composition (367/31 in ASD group vs. 406/31 in control one); lack of information on the clinical and biochemical impacts of the ASMT deleterious variants and/or SNPs; other genes in melatonin pathway were not sequenced Strengths: controlled study
Veatch et al. [41]	ASD individuals (N = 29, M = 24 and F = 5, 15 of them underwent analysis for ASMT sequences, while 14 of the total for CY1A2 genotypes)	Examination of variation in two melatonin pathway genes, ASMT and CYP1A2	Higher frequencies than currently reported for variants evidenced to decrease ASMT expression and related to decreased CYP1A2 enzyme activity; a relationship between genotypes in ASMT and CYP1A2 was revealed; expression of sleep onset delay relates to melatonin pathway genes	Limitations: lack of a well-defined control group; all 11 individuals who participated in the melatonin trial were responsive to treatment; minimalization of the environmental effect of poor sleep habits through parent sleep education
Pagan et al. [32]	Unrelated patients with ASD (N = 278), first-degree relatives (129 unaffected siblings, 377 patients) and controls (N = 416)	Serotonin, melatonin and the intermediate NAS measured through whole-blood serotonin, platelet NAS and plasma melatonin	In patients the melatonin deficit was only significantly associated with insomnia. Impairments of melatonin synthesis in ASD may be linked with decreased 14-3-3 proteins. Disruption of the serotonin-NAS-melatonin pathway is a very frequent trait in ASD patients and may be a useful biomarker for a large subgroup of these individuals	Limitations: not equally subdivision between three groups Strengths: two groups of possible controls (unaffected siblings and other controls)
Pagan et al. [44]	ASD patients (N = 239), ASD parents (N = 303), unaffected siblings (N = 78), controls (N = 278)	Examination of melatonin synthesis in post-mortem pineal gland, serotonin synthesis in gut samples, blood platelets	Melatonin deficits in ASD depends on reduction activity of both enzymes implicated in melatonin synthesis (AANAT and ASMT)	Limitations: small number of patient samples Strengths: three different groups
Braam et al. [33]	Mothers of an ASD child (N = 60, mean age: 42.9 ± 5.7 years), control group of mothers (N = 15, mean age = 44.3 ± 9.7 years)	6-SM concentration	6-SM levels were significantly lower in mothers with an ASD child than in controls, and so, low parental melatonin levels could be one of the contributors to ASD and possibly ID etiology	Limitations: differences between number composition of both groups and small sample size; CYP1A2 activity not measured; different children ASD etiologies Strength: presence of a control group
Maruani et al. [45]	ASD individuals (N = 81), unaffected relatives (N = 90), control participants (N = 48)	PGV estimation based on magnetic resonance imaging; blood sampling and plasma melatonin measurement	Patients had both morning melatonin levels and PGV lower than controls; plasma melatonin was correlated to the group of the participant, but also to the PGV melatonin; variations in ASD could be mainly driven by melatonin pathway dysregulation	Limitations: melatonin was detected only in mornings; difficulties in exactly detecting PGV; numeric differences between the first two groups and the third Strengths: three experimental groups

ASD autism spectrum disorder, 6-SM 6-sulphatoxy-melatonin, ASMT acetyl-serotonin-O-methyl-transferase, AANAT aryl-alkylamine-N-acetyltransferase, MTNR1A melatonin receptor 1A, MTNR1B melatonin receptor 1B, GPR50G protein-coupled receptor 50, CYP1A2 cytochrome P450 1A2, NAS N-acetyl-serotonin, PGV pineal gland volume