Sleep disorder affects the daily lives of the cases negatively due to cognitive functions such as working memory and attention. One of the most frequent complains of individuals with autism spectrum disorder (ASD) and their families is sleep disorders, ranging from 40% to 86% of the patients [1]. In ASD cases, it was shown that the serum melatonin level and the level of melatonin metabolites in the urine are low [2]. The concentration of melatonin is regulated by the circadian rhythm under physiological conditions, which is low during the day and high at night [3]. The peak secretion is usually around 2:00 AM and is also called the hormone of darkness. Polymorphism in acetylserotonin O-methyltransferase may be related to low melatonin in ASD [4]. The melatonin receptor agonists can treat insomnia and behavioral symptoms by reducing nitrosative/oxidative stress and inflammation in ASD. Melatonin levels also differ by sex, usually 2-3 times higher in girls than boys. Therefore, ASD may be less common in girls (as it prevents oxidative damage to DNA). In a study conducted with 14 ASD cases, the 24-hour circadian rhythm concentration of serum melatonin was examined, and it was found to be lower at night in ASD compared to healthy controls. No circadian variation was detected in the 10-14 age range. In the 4-14 age range, inverted rhythm, e.g., the opposite of normal rhythm, was found [5].

The aim of this study is to evaluate the relationship between serum melatonin levels, sleeping habits, and clinical symptoms in children with ASD through a case-control design. We selected this design to allow direct comparisons between children with ASD and age-matched typically developing peers, controlling for key confounding factors. Additionally, by focusing on both sleep patterns and melatonin levels alongside clinical severity, we aim to address gaps in the current literature. While previous studies have highlighted the association between melatonin and disrupted sleep in ASD [6,7], relatively few have employed a robust case-control framework to clarify the role of melatonin in modulating core ASD symptoms. Our approach not only extends prior findings but also provides a novel perspective by incorporating detailed clinical assessments and age-specific evaluations, thus offering new insights into the interplay between melatonin, sleep, and ASD symptomatology.

This study was approved by the Non-Invasive Clinical Research Ethics Committee of Çukurova University Faculty of Medicine at its meeting dated July 9, 2021, with decision number 17. Informed consent was obtained from all subjects involved in the study. The study was evaluated and deemed to comply with ethical principles and relevant regulations. Our study included; the cases aged 2-8 years who applied to our outpatient clinic between August 2021 and January 2022 and were diagnosed with ASD according to Diagnostic and Statistical Manual of Mental Disorders-Fifth Edition (DSM-5), and a healthy control group matched for sex and age. Exclusion criteria are those who have medical diseases known to affect cognitive and brain function, cancer, bipolar disorder, psychotic disorders, epilepsy, having any diagnosis of neurological diseases, having received electroconvulsive therapy, intraocular surgery or laser therapy in the last 3 months, ocular diseases such as retinitis pigmentosa, traveling between continents in the last 1 month, sleep apnea and sleep disorders due to a medical condition, autoimmune diseases, severely impaired hearing or severe mental retardation, who use psychotropic, neurotropic, selective serotonin reuptake inhibitor, anti-psychotic, serotonin-norepinephrine reuptake inhibitor, mood stabilizer, stimulant and benzodiazepine drugs and other drugs known to change sleep patterns.

Inclusion criteria for the ASD group were children aged 2-8 years with a clinical diagnosis of ASD based on DSM-5 criteria, without any additional uncontrolled or severe comorbid neurological or psychiatric disorders (e.g., active epilepsy, severe anxiety disorder) that could interfere with sleep or cognitive assessments. For the control group, age- and sexmatched children with no history of ASD or any significant neurological or psychiatric disorders were included. Additional exclusion criteria for both groups consisted of chronic medical conditions affecting the central nervous system, ongoing treatment with medications known to alter sleep patterns or cognitive function, and any acute or uncontrolled chronic illnesses. Specifically, children diagnosed with epilepsy or significant anxiety disorders under ongoing pharmacological treatment were excluded to minimize confounding factors.

Thirty eight patients with ASD and 33 healthy children evaluated in the control group were included in the study. In this study, the mean age of children diagnosed with ASD was 44.4±20.4 months, which did not differ significantly from the control group’s mean age of 51.2±20.0 months (p=0.104). The ASD cohort exhibited a higher proportion of males (86.8%) compared to the control group (75.8%), although this difference was not statistically significant (p=0.228). Notably, serum melatonin levels were significantly elevated in the ASD group, averaging 823.2±237.9 U/L compared to 677.4± 254.7 U/L in the control group (p=0.027). This elevation was particularly evident among male participants, with ASD males showing melatonin levels of 820.8±243.5 U/L versus 649.0± 262.8 U/L in control males (p=0.020), while no significant difference was observed among females (838.6±221.8 U/L in ASD versus 766.2±254.7 U/L in controls, p=0.608). Additionally, children with ASD reported lower daytime sleepiness scores (3.2±1.2) compared to the control group (3.8±1.3, p=0.036) and exhibited higher bedwetting scores (3.3±1.3 versus 2.5±0.8, p=0.008). These findings highlight significant differences in melatonin levels and specific sleep-related behaviors between children with ASD and their healthy counterparts (Table 1).

Patients with ASD had a mean score of 5.3±2.9 on the risky questions section of the M-Chat, with a total M-Chat score averaging 13.2±4.8. The ABC assessment revealed that these patients scored 11.2±5.9 in the sensorial domain, 15.9±8.1 in building relationships, and 16.6±9.5 in the use of body and object. Language skills were assessed with a mean score of 14.1±5.8, while social and self-care abilities scored 12.6±4.7. The overall ABC score for the ASD patients was 70.4±25.3. These results indicate significant challenges in multiple domains of behavior and development in children with ASD (Table 2).

Patients with severe ASD exhibited a significantly lower iron binding capacity, with a mean value of 153.5±181.2 g/dL, compared to 296.2±94.7 g/dL in the mild group and 299.3±92.9 g/dL in the moderate group (p=0.043). Additionally, vitamin D levels were markedly lower in the severe ASD group, averaging 3.3±4.3 ng/mL, as opposed to 26.2±15.7 ng/mL in the mild group and 17.9±8.6 ng/mL in the moderate group (p=0.005). Although melatonin levels and other hematological parameters (such as erythrocyte/red blood cell [RBC], white blood cell [WBC], hemoglobine [HGB], hematocrit [HCT], mean corpuscular volume [MCV], and platelet [PLT]) did not show statistically significant differences across the clinical severity groups, the trend indicates varying degrees of impact on these laboratory markers depending on the severity of ASD (Table 3).

The correlation analysis between melatonin levels and various clinical and laboratory parameters in children with ASD revealed no significant correlations. Specifically, melatonin levels showed a weak positive correlation with hemoglobin levels (r=0.196) and HCT (r=0.201), although these were not statistically significant. The analysis also indicated negligible correlations between melatonin and vitamin D (r=-0.062), ferritin (r=0.010), and the scores from the M-Chat (r=0.063) and ABC (r=-0.066). These findings suggest that melatonin levels are not strongly associated with these parameters in the ASD population studied (Table 4).

When examining melatonin levels, some values in the control and ASD groups were observed to be relatively higher or lower compared to each other. These outliers may stem from individual differences in participants’ circadian rhythms, the timing of melatonin measurement, or the heterogeneity of the sample population. To minimize the impact of outliers on statistical analysis, normality checks (Kolmogorov-Smirnov test) were conducted, and appropriate non-parametric tests, such as the Mann-Whitney U test, were applied for non-normally distributed data. Additionally, inclusion and exclusion criteria for participants helped to partially limit the effect of extreme outliers.

In the ASD patients included in our study, the male sex ratio was observed as 86.8%, and the male sex ratio was 6.6 times higher. The differences in ASD according to sex are thought to be due to genetic characteristics [11]. In our study, RBC count, WBC count, HGB, HCT, MCV, and PLT values were found to be quite similar between children with ASD and healthy children (p>0.05). Vitamin D level and iron binding capacity; were found to be significantly lower in the severe ASD group compared to the other groups (p=0.005 and p=0.043, respectively). In our study, when healthy children and children with ASD were compared in terms of sleep problems, it was observed that the total sleep duration was similar between the two groups (p=0.479). In terms of staying awake at night, it was determined that patients with ASD had significantly longer awake times, but this difference was not statistically significant (p=0.052). There were no significant differences between the ASD and control groups in terms of difficulty in waking up in the morning, sleep fragmentation, sleep anxiety, sleep disturbance, parasomnia, morning awakening style, sleep duration, sleep transition, and the need to sleep with others (p>0.05). It was determined that the sleepiness problem experienced during the day was observed less frequently in ASD patients compared to the control group, but bedwetting problems were observed more frequently (p=0.036 and p=0.008, respectively). It was determined that melatonin levels are high in ASD patients and melatonin levels decrease as the clinical condition worsens. In addition, in this study, although melatonin levels were lower in measurements made at night than in the control group, it was found that melatonin levels were higher in measurements made during the day than in healthy children [12].

In the seven different studies compiled in a meta-analysis study examining the relationship between ASD and melatonin, melatonin levels were found to be lower in ASD patients than in healthy individuals [13]. In addition, four different studies have shown that melatonin levels decrease as the clinical condition of ASD gets worse [11]. There are studies claims that the potential to lead to an effective and relatively simple treatment and maybe prevention procedure for ASD, using exogenous melatonin even for infants [14,15]. However, almost all of these studies measured melatonin levels at night. In the study of Ritvo et al. [16], it was observed that melatonin levels measured during the day were higher in patients with ASD. In our study, it was observed that serum melatonin levels were significantly higher in ASD patients than in healthy controls (p=0.027). In studies where melatonin levels were measured at night, lower melatonin levels were found in pa-tients with ASD, while elevated melatonin levels were observed in patients with ASD in daytime melatonin levels [17]. This is an indication that the circadian rhythm of melatonin is impaired in patients with ASD. Although there are still missing data today, some genes that cause disruption of melatonin synthesis are thought to play a role in the etiology of ASD. In addition, the presence of some gene mutations that play a role in melatonin receptor desensitization is also accused in the etiology of ASD [18].

Previous studies report that melatonin levels in individuals with ASD are generally irregular, negatively impacting the sleep-wake cycle [19,20]. Furthermore, reductions in endogenous melatonin levels or disruptions in its rhythm have been shown to play a significant role in initiating and maintaining sleep in individuals with ASD [21,22]. Meta-analyses have revealed that melatonin supplementation has particularly beneficial effects on sleep duration, sleep latency, and sleep efficiency [23]. Similarly, the literature reports that 67%-89% of children with ASD experience various sleep disorders, such as short or fragmented sleep, prolonged sleep onset latency, and frequent nighttime awakenings, which in turn exacerbate ASD symptoms like daytime irritability and stereotypic behaviors [24,25]. Additionally, it has been suggested that low maternal melatonin levels during the intrauterine period may increase the fetus’s susceptibility to ASD [13] and that sex-based differences in melatonin levels (e.g., relatively higher levels in females) may contribute to the higher prevalence of ASD in males [26]. However, environmental factors, such as seasonal transitions (particularly the transition from winter to spring), are reported to have the potential to exacerbate sleep disturbances, including late bedtimes and fragmented sleep patterns [27]. In our study, while total sleep duration in the ASD group was found to be similar to that of the control group, significant differences were observed in specific subdomains, particularly daytime sleepiness and nocturnal enuresis. These findings highlight the impact of endogenous melatonin levels and circadian rhythm on the specific sleep habits of children with ASD. Furthermore, the observation that melatonin levels in male ASD participants were higher compared to the control group appears to align with previous findings regarding sex-based melatonin differences. In conclusion, our findings support the view emphasized in the literature that “melatonin deficiency or disruptions in its rhythm may exacerbate sleep problems accompanying ASD” and demonstrate that sleep disturbances are a significant modifying factor in the clinical course of ASD.

Melatonin receptor desensitization, which is thought to be observed in ASD patients, may also play a role in the ASD clinic. The fact that the daytime melatonin levels were increased in our study is in parallel with the literature. There is no existing study examining the sex-related melatonin values in ASD patients in the literature. Studies in healthy people have shown that women’s melatonin levels are higher than men, and the difference can sometimes be up to 3 times higher. In our study, melatonin levels were found to be significantly higher in male patients between ASD patients and the control group (p=0.020). It was observed that melatonin levels were higher in girls with ASD, but this difference was not significant (p=0.608). High melatonin levels observed in women are thought to be a protective factor against ASD. In particular, it has been found as a data that has been revealed in previous studies that the circadian rhythm of melatonin works differently in ASD patients and that it is an inverted rhythm.