Assessment of Serum Melatonin Levels, Sleep Patterns, and Clinical Symptoms in Children with Autism Spectrum Disorder: A Case-Control Study

Assessment of Serum Melatonin Levels, Sleep Patterns, and Clinical Symptoms in Children with Autism Spectrum Disorder: A Case-Control Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Assessment of Serum Melatonin Levels, Sleep Patterns, and Clinical Symptoms in Children with Autism Spectrum Disorder: A Case-Control Study Çağlar Charles Daniel Jaicks, Perihan Çam Ray, Özlem Görüroğlu Öztürk, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4927190/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: This study aims to evaluate the relationship between serum melatonin levels, sleep habits, and clinical features in children with autism spectrum disorder (ASD) compared to healthy controls. Methods: In accordance with DSM (The Diagnostic and Statistical Manual of Mental Disorders)-V, children aged 2-8 years diagnosed with autism spectrum disorder (n:38) and healthy children (n:33) of similar age and gender in the control group were evaluated. Vitamin D levels, ferritin, serum iron, and iron binding capacity of all participants were examined, as well as endogenous melatonin levels, and a pediatric sleep habits questionnaire was filled out. Autism Behavior Checklist (ABC-Autism Behavior Checklist) and The Modified Checklist for Autism in Toddlers (M-CHAT) were applied to the autism spectrum disorder group. The relationship between the blood values obtained and the completed questionnaire results was evaluated. Results: Melatonin levels were compared between the patients with autism spectrum disorder and the control group. In total, 71 participants were included in it. The gender of 81.7% (n=58) of the participants was male.The mean age of the autism spectrum disorder group was 44.4±20.4 months, and that of the control group was 51.2±20 months (p=0.104). When children’s sleep habits questionnaire data were compared between the two groups, the "daytime sleepiness" subscale score was higher in the control group, while the "bed-wetting" subscale score was higher in the autism spectrum disorder group (p=0.036 and p=0.008, respectively). No significant correlation was found between the questionnaire scores of the patients and their melatonin levels. Melatonin levels were on average 823.2±237.9 U/L in the autism spectrum disorder group and 677.4±254.7 U/L in the control group. This difference between the two groups was found to be statistically significant (p=0.027). When the participants were compared by dividing according to their gender, it was found that there was a difference between the two groups only in male patients (p=0.020). It was observed that there was no significant difference between girls in terms of melatonin levels (p=0.608). Conlucion: In the light of the data we obtained in our study, it was observed that there are sleep problems in autism spectrum disorder and therefore, melatonin levels measured during the daytime are higher than in healthy children. This makes us think that there may be desensitization to melatonin receptors in the ASD group, and the fact that melatonin levels differ according to gender, suggesting that melatonin treatment modalities applied in ASD patients may be regulated according to gender. Biological sciences/Physiology Health sciences/Signs and symptoms Melatonin Autism Spectrum Disorder Autism Spectrum Disorder Sleep Disorders Introduction 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 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 a.m. and is also called the hormone of darkness. Polymorphism in Acetyl-Serotonin O-Methyl Transferase 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 gender, 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 Autism Spectrum Disorder (ASD). This case-control study seeks to determine whether variations in melatonin levels are associated with differences in sleep patterns and the severity of clinical symptoms in ASD patients compared to a control group. Materials and Methods Our study was designed as a controlled and prospective study. Our study was initiated with the approval of Çukurova University Faculty of Medicine Non-Invasive Clinical Research Ethics Committee. 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 Autism Spectrum Disorder according to DSM-V, and a healthy control group matched for gender 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, SSRI, anti-psychotic, SNRI, mood stabilizer, stimulant and benzodiazepine drugs and other drugs known to change sleep patterns. Psychometric Measurements The diagnostic criteria of DSM– V, ABC- Autism Behavior Checklist, The Modified Checklist for Autism in Toddlers / M-CHAT, Children’s Sleep Habits Questionnaire. Laboratory data Vitamin D level, ferritin, serum iron and iron binding capacity, as well as endogenous melatonin levels, which are routinely requested from patients diagnosed with ASD, were examined. Blood samples were collected in ACD-A tubes between 8.30–10.30 in the morning (background light intensity < 2 lx, participants were allowed to sleep between 23:00 and 06:00 in the evening). In addition, the function of the pineal gland can be roughly evaluated in studies where melatonin metabolites are evaluated non-invasively. Therefore, it was decided to measure blood melatonin levels in our study. For the measurement of serum melatonin level, 5 ml blood sample was taken from the brachial vein of the forearm and was centrifuged. The obtained serum samples were stored as 2 ml at -80°C. All patient blood samples and control group blood samples were taken into Becton Dickinson vacuum-gel tubes. Serum was obtained by centrifuging them at 2000 rpm for 20 minutes. Serum MELATONIN (SUNRED brand BT LAB Cat.NO EA0013Ge) level was measured by enzyme-linked immunosorbent assay (ELISA) in accordance with kit protocols. Serum samples were diluted according to the kit procedure. Serial dilutions were made according to the standards of kit procedure. 50 microliters of standard serum or 40 microliters of patient and control serum were added to each determined well. 10 microliters of Biotin Antigen was added to the samples. They were incubated at 37oC at the specified time intervals in accordance with the procedure. 50 mL of stop solution was added. It was immediately read at 450 nm. The resulting concentration values were multiplied by the dilution coefficient. Serum melatonin level concentration was found. Serum melatonin levels were measured as pg/mL. In addition, chemiluminescence method was used with DXI 800 device for ferritin, photometric method was used with AU5800 device for iron and iron binding capacity, HPLC method was used with Thermo Dionex device for vitamin D. Melatonin levels of patients diagnosed with ASD and healthy controls were compared. Statistical Analysis Statistical analysis of the data was performed using the SPSS 25.0 (SPSS Inc., Chicago, Illinois) program. Normality test of numerical variables was determined by Kolmogorov-Smirnov test. Numerical variables were summarized as mean (± standard deviation). Categorical variables were given as numbers and percentages. When comparing numerical variables between the two groups, the Mann Whitney U test was used because the variables did not show normal distribution. Chi-square and Fisher's exact tests were used when comparing categorical variables. The correlation between the measurement variables was compared with the Pearson correlation test and their correlation coefficients were calculated. Statistical significance level was taken as 0.05 for each test. Results 38 patients with autism spectrum disorder and 33 healthy children evaluated in the control group were included in the study. In this study, the mean age of children diagnosed with Autism Spectrum Disorder (ASD) was 44.4 ± 20.4 months, which did not differ significantly from the control group’s mean age of 51.2 ± 20 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 ). Table 1 Demographic, melatonin levels, laboratory data and children's sleep habits questionnaire among groups ASD (n = 38) Control (n = 33) p value Demografic Data Age (months) 44.4 ± 20.4 51.2 ± 20 0.104 Gender, n (%) Female 5(%13.2) 8(%24.2) 0.228** Male 33(%86.8) 25(%75.8) Level of Melatonin (U/L) Female (n = 13) 838.6 ± 221.8 766.2 ± 254.7 0.608 Male (n = 58) 820.8 ± 243.5 649.0 ± 262.8 0.020 Total (n = 71) 823.2 ± 237.9 677.4 ± 254.7 0.027 Laboratory Data RBC (10 12 /L) 4.5 ± 0.8 4.6 ± 0.3 0.773 WBC (10 9 /L) 8.7 ± 2.8 8.5 ± 1.9 0.572 HGB (g/dL) 12.1 ± 2.2 12.1 ± 0.8 0.415 HTC (%) 35.3 ± 6.5 35.6 ± 2.3 0.471 MCV (fL) 75.6 ± 15.6 77.4 ± 3.6 0.199 PLT (10 9 /µL) 336.5 ± 109.2 345.6 ± 109.5 0.904 Iron (g/dL) 68 ± 35.9 77.9 ± 40.1 0.333 Iron binding capacity (g/dL) 282.6 ± 110.8 303.6 ± 77.9 0.421 Ferritin (ng/mL) 25 ± 18.8 24.2 ± 26.5 0.365 Vitamin D (ng/mL) 20 ± 13.7 16.8 ± 9.5 0.278 Sleep habits questionnaire ( Mean ± Sd) Total Sleep Duration 10.2 ± 1.9 10.4 ± 1.7 0.479 Nighttime Awake Time 45.5 ± 54.9 18.3 ± 24 0.052 Difficulty Waking Up 5.7 ± 2.2 5.8 ± 1.9 0.703 Disruption Of Sleep 7.3 ± 2.4 6.9 ± 2 0.538 Sleep Anxiety 5.7 ± 2.1 6 ± 1.8 0.456 Breathing Disturbance During Sleep 3.8 ± 1.5 4 ± 1.4 0.303 Parasomnia 4.3 ± 1.5 4.5 ± 1.4 0.492 Way Of Waking Up in the Morning 2.5 ± 0.8 2.9 ± 1 0.079 Sleep Duration 4.7 ± 1.8 4.5 ± 1.7 0.732 Sleep Latency 3.4 ± 1.3 3.2 ± 1.1 0.531 Needing to Sleep With Others 4.2 ± 1.3 3.6 ± 1.3 0.072 Sleepiness During Daytime 3.2 ± 1.2 3.8 ± 1.3 0.036 Bedwetting 3.3 ± 1.3 2.5 ± 0.8 0.008 * : Mann-Whitney U test **: Chi-square test ASD : Autism spectrum disorder , Mean ± Sd : Mean ± standard deviation , RBC : Erythrocyte/Red Blood Cell Count, WBC : White Blood Cell Count; HGB : Hemoglobine, HCT : Hematocrit, MCV : Mean Corpuscular Volume, PLT : Platelet. Table 2 M-Chat and ABC assessment results of patients with autism spectrum disorder OSB (n = 38) Mean ± Sd Risky questions (M-Chat) 5.3 ± 2.9 Total (M-Chat) 13.2 ± 4.8 Sensorial (ABC) 11.2 ± 5.9 Building a Relationship (ABC) 15.9 ± 8.1 Use of body and object (ABC) 16.6 ± 9.5 Language skills (ABC) 14.1 ± 5.8 Social and self-care (ABC) 12.6 ± 4.7 Total (ABC) 70.4 ± 25.3 ASD : Autism spectrum disorder , ABC : Autism Behavior Checklist M-Chat : Modified Checklist for Autism in Toddlers Mean ± Sd : Mean ± Standard deviation Table 3 Comparison of laboratory data of patients with autism spectrum disorder according to their clinical status Level of Clinical ASD p value Light (n = 17) Mean ± Sd Medium (n = 17) Mean ± Sd Heavy (n = 4) Mean ± Sd RBC (10 12 /L) 4.3 ± 1.1 4.6 ± 0.3 4.8 ± 0.3 0.334 WBC (10 9 /L) 8.6 ± 3.1 8.9 ± 2.8 8.2 ± 0.9 0.907 HGB (g/dL) 11.5 ± 3.1 12.6 ± 1.1 12.7 ± 1 0.361 HTC (%) 33.7 ± 9 36.3 ± 3.1 37.4 ± 2.3 0.393 MCV (fL) 71.2 ± 22.4 79.2 ± 3.8 78.6 ± 7.1 0.308 PLT (10 9 /µL) 313.2 ± 107 350.9 ± 121.2 374.5 ± 35.3 0.471 Iron (g/dL) 68.2 ± 29.9 73.6 ± 37.2 43.5 ± 52.3 0.328 Iron Binding Capacity (g/dL) 296.2 ± 94.7 299.3 ± 92.9 153.5 ± 181.2 0.043 Ferritin (ng/mL) 23.7 ± 13.5 28.7 ± 24.2 14.9 ± 7.8 0.397 Vitamin D (ng/mL) 26.2 ± 15.7 17.9 ± 8.6 3.3 ± 4.3 0.005 Melatonin (U/L) 805.7 ± 244.1 872.1 ± 237.8 690.3 ± 200.0 0.367 * : Mann-Whitney U test , ASD : Autism spectrum disorder , Mean ± Sd : Mean ± standard deviation , RBC : Erythrocyte/Red Blood Cell Count, WBC : White Blood Cell Count; HGB : Hemoglobine, HCT : Hematocrit, MCV : Mean Corpuscular Volume, PLT : Platelet 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 RBC, WBC, HGB, HTC, MCV, and 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 Autism Spectrum Disorder (ASD) revealed no significant correlations. Specifically, melatonin levels showed a weak positive correlation with hemoglobin levels (r = 0.196) and hematocrit (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 Autism Behavior Checklist (ABC) (r = -0.066). These findings suggest that melatonin levels are not strongly associated with these parameters in the ASD population studied (Table 4 ). Table 4 Correlation analysis of melatonin and other parameters with autism spectrum disorders Parameters Melatonin Total (M-Chat) r 0.063 Total (ABC) r -0.066 Vitamin D (ng/mL) r -0.062 Ferritine (ng/mL) r 0.010 Hemoglobine (g/dL) r 0.196 Hematocrit (%) r 0.201 r : Pearson correlation coefficient , ABC : Autism Behavior Checklist , M-Chat : Modified Checklist for Autism in Toddlers. Discussion 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 gender are thought to be due to genetic characteristics 6 . In our study, red blood cell count, white blood cell count, hemoglobin, hematocrit, MCV and platelet 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 autism spectrum disorder 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 7 . 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 8 . In addition, four different studies have shown that melatonin levels decrease as the clinical condition of ASD gets worse 6 . 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 9 , 10 . However, almost all of these studies measured melatonin levels at night. In the study of Ritvo et al., it was observed that melatonin levels measured during the day were higher in patients with ASD 11 . 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 patients with ASD, while elevated melatonin levels were observed in patients with ASD in daytime melatonin levels 12 . 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 13 . 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 gender-related melatonin values in autism spectrum disorder 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. Limitations of the Study One of the biggest limitations of our study is that children’s sleep habits in children were evaluated with a questionnaire method that provides subjective data. In addition, at the time of blood analysis, the number of hours the participant slept, what time he woke up in the morning, or what time he slept at night was disregarded. Since there is less female gender among the applications, in order to conduct future studies with multicenter, cohort and large study groups, with female-weighted participants, and to evaluate the sleep of the children studied more objectively, it is thought that more precise results can be obtained by performing sleep monitoring. Conclusion Our study differs from previous studies in that the participants were selected from patients who did not use any medication that could affect sleep and melatonin, eventhough it has already been stated in the literature that the relationship between autism spectrum disorder and melatonin levels is significant, there may be an inverted oscillation rhythm, and a significant relationship between sleep habits and clinical severity of autism. We keep the idea that evaluation with larger and perhaps 24-hour serum samples to be made in this field, and evaluation with study groups in which the female gender is more predominant, will contribute significantly to the literature. Declarations Data Sharing Statement All the data can be requested from the corresponding author upon reasonable request. Ethics and Approval and Consent to Participate The study was conducted in accordance with the Declaration of Helsinki, and was approved by the ethics committee of Cukurova University Faculty of Medicine Non-Invasive Clinical Research Ethics Committee (decision number: 17, date: 9 July 2021). Individual written consent was required. Consent for Publication Informed consent was obtained from all subjects involved in the study. Author Contributions All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Funding This study was supported by Cukurova University Faculty of Medicine Key Research and Development Program of Anhui Province (202004j07020001). Disclosure The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Maruani A, Dumas G, Beggiato A, Traut N, Peyre H, Cohen-Freoua A, et al. Morning plasma melatonin differences in autism: beyond the impact of pineal gland volume. 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Nir I, Meir D, Zilber N, Knobler H, Hadjez J, Lerner YJJoa, et al. Brief report: circadian melatonin, thyroid-stimulating hormone, prolactin, and cortisol levels in serum of young adults with autism. 1995;25(6):641-54. Bruni O, Alonso-Alconada D, Besag F, Biran V, Braam W, Cortese S, et al. Current role of melatonin in pediatric neurology: clinical recommendations. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society. 2015;19(2):122-33. Shomrat T, Nesher N. Updated view on the relation of the pineal gland to autism spectrum disorders. Frontiers in endocrinology. 2019;10:37. Ritvo ER, Ritvo R, Yuwiler A, Brothers A, Freeman B, Plotkin SJEC, et al. Elevated daytime melatonin concentrations in autism: a pilot study. 1993;2(2):75-8. Yenen AS, Cak HJTPD. Melatonin and Circadian Rhythm in Autism Spectrum Disorders. 2020;31(3). Jonsson L, Ljunggren E, Bremer A, Pedersen C, Landén M, Thuresson K, et al. Mutation screening of melatonin-related genes in patients with autism spectrum disorders. 2010;3(1):1-7. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4927190","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":357086062,"identity":"959c91bf-f265-438c-9e3d-d072776bcf7d","order_by":0,"name":"Çağlar Charles Daniel Jaicks","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYHACxgMMDGxgyPCBgSGBKD1wLYwzSNACAmwMzDzEaOFvP37hwM89fPJ87MeSP9u22eXxszcwfviYg1uLxJmcgoM9z9gM23jSjknntiUXS/YcYJacuQ23FgOGnIQDPAfYGNsk2NuYc9uYEzfcSGBj5sWnhf9NwsE/B9jsgVqaP1u21ROhRSL9wGGgLYltEmwHpBnbDhPWInHjDcNhmQNsyUC/pEn2nDueOLPnYDNev/D3pz98+ObAMdv57ceMP/woq07sZ28++OEjHi0MDDwGQOIYhM3IBiYb8KkHAvYHQKIGyvlDQPEoGAWjYBSMSAAAV1ZVsLWF3ZEAAAAASUVORK5CYII=","orcid":"","institution":"Cukurova University","correspondingAuthor":true,"prefix":"","firstName":"Çağlar","middleName":"Charles Daniel","lastName":"Jaicks","suffix":""},{"id":357086063,"identity":"f95e4a4b-d5ff-450a-9356-d9b4512f6995","order_by":1,"name":"Perihan Çam Ray","email":"","orcid":"","institution":"Cukurova University","correspondingAuthor":false,"prefix":"","firstName":"Perihan","middleName":"Çam","lastName":"Ray","suffix":""},{"id":357086064,"identity":"af03f9c3-7b42-416f-9b68-ea403158a8c5","order_by":2,"name":"Özlem Görüroğlu Öztürk","email":"","orcid":"","institution":"Cukurova University","correspondingAuthor":false,"prefix":"","firstName":"Özlem","middleName":"Görüroğlu","lastName":"Öztürk","suffix":""},{"id":357086065,"identity":"615b06c4-a28e-4647-b319-79f337a36850","order_by":3,"name":"Gonca Gül Çelik","email":"","orcid":"","institution":"Cukurova University","correspondingAuthor":false,"prefix":"","firstName":"Gonca","middleName":"Gül","lastName":"Çelik","suffix":""},{"id":357086066,"identity":"a9558b7a-6d4c-4d60-bc62-bf1d195e868e","order_by":4,"name":"Ayşegül Yolga Tahiroğlu","email":"","orcid":"","institution":"Cukurova University","correspondingAuthor":false,"prefix":"","firstName":"Ayşegül","middleName":"Yolga","lastName":"Tahiroğlu","suffix":""},{"id":357086067,"identity":"e48fc146-ebc7-49c0-ace8-61d9f35d83ab","order_by":5,"name":"Zeliha Haytoğlu","email":"","orcid":"","institution":"Cukurova University","correspondingAuthor":false,"prefix":"","firstName":"Zeliha","middleName":"","lastName":"Haytoğlu","suffix":""},{"id":357086068,"identity":"b98263e2-7ce2-451c-9c52-26f9a4b73b8d","order_by":6,"name":"Yusuf Döğüş","email":"","orcid":"","institution":"Cukurova University","correspondingAuthor":false,"prefix":"","firstName":"Yusuf","middleName":"","lastName":"Döğüş","suffix":""}],"badges":[],"createdAt":"2024-08-16 22:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4927190/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4927190/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69324207,"identity":"fdc88b95-08bc-4e2b-ad4b-5615254718cd","added_by":"auto","created_at":"2024-11-19 07:39:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":787658,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4927190/v1/76c87efb-a500-40f5-a688-08527f5a8f0f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of Serum Melatonin Levels, Sleep Patterns, and Clinical Symptoms in Children with Autism Spectrum Disorder: A Case-Control Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSleep 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 ASD and their families is sleep disorders, ranging from 40 to 86% of the patients\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In ASD cases, it was shown that the serum melatonin level and the level of melatonin metabolites in the urine are low\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The concentration of melatonin is regulated by the circadian rhythm under physiological conditions, which is low during the day and high at night\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The Peak secretion is usually around 2 a.m. and is also called the hormone of darkness. Polymorphism in Acetyl-Serotonin O-Methyl Transferase may be related to low melatonin in ASD\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The Melatonin receptor agonists can treat insomnia and behavioral symptoms by reducing nitrosative/oxidative stress and inflammation in ASD. Melatonin levels also differ by gender, usually 2\u0026ndash;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\u0026ndash;14 age range. In the 4\u0026ndash;14 age range, inverted rhythm, e.g. the opposite of normal rhythm, was found\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe aim of this study is to evaluate the relationship between serum melatonin levels, sleeping habits, and clinical symptoms in children with Autism Spectrum Disorder (ASD). This case-control study seeks to determine whether variations in melatonin levels are associated with differences in sleep patterns and the severity of clinical symptoms in ASD patients compared to a control group.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eOur study was designed as a controlled and prospective study. Our study was initiated with the approval of \u0026Ccedil;ukurova University Faculty of Medicine Non-Invasive Clinical Research Ethics Committee. Our study included; the cases aged 2\u0026ndash;8 years who applied to our outpatient clinic between August 2021 and January 2022 and were diagnosed with Autism Spectrum Disorder according to DSM-V, and a healthy control group matched for gender 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, SSRI, anti-psychotic, SNRI, mood stabilizer, stimulant and benzodiazepine drugs and other drugs known to change sleep patterns.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePsychometric Measurements\u003c/h2\u003e \u003cp\u003eThe diagnostic criteria of DSM\u0026ndash; V, ABC- Autism Behavior Checklist, The Modified Checklist for Autism in Toddlers / M-CHAT, Children\u0026rsquo;s Sleep Habits Questionnaire.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory data\u003c/h2\u003e \u003cp\u003eVitamin D level, ferritin, serum iron and iron binding capacity, as well as endogenous melatonin levels, which are routinely requested from patients diagnosed with ASD, were examined. Blood samples were collected in ACD-A tubes between 8.30\u0026ndash;10.30 in the morning (background light intensity\u0026thinsp;\u0026lt;\u0026thinsp;2 lx, participants were allowed to sleep between 23:00 and 06:00 in the evening). In addition, the function of the pineal gland can be roughly evaluated in studies where melatonin metabolites are evaluated non-invasively. Therefore, it was decided to measure blood melatonin levels in our study. For the measurement of serum melatonin level, 5 ml blood sample was taken from the brachial vein of the forearm and was centrifuged. The obtained serum samples were stored as 2 ml at -80\u0026deg;C. All patient blood samples and control group blood samples were taken into Becton Dickinson vacuum-gel tubes. Serum was obtained by centrifuging them at 2000 rpm for 20 minutes. Serum MELATONIN (SUNRED brand BT LAB Cat.NO EA0013Ge) level was measured by enzyme-linked immunosorbent assay (ELISA) in accordance with kit protocols. Serum samples were diluted according to the kit procedure. Serial dilutions were made according to the standards of kit procedure. 50 microliters of standard serum or 40 microliters of patient and control serum were added to each determined well. 10 microliters of Biotin Antigen was added to the samples. They were incubated at 37oC at the specified time intervals in accordance with the procedure. 50 mL of stop solution was added. It was immediately read at 450 nm. The resulting concentration values were multiplied by the dilution coefficient. Serum melatonin level concentration was found. Serum melatonin levels were measured as pg/mL. In addition, chemiluminescence method was used with DXI 800 device for ferritin, photometric method was used with AU5800 device for iron and iron binding capacity, HPLC method was used with Thermo Dionex device for vitamin D. Melatonin levels of patients diagnosed with ASD and healthy controls were compared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the data was performed using the SPSS 25.0 (SPSS Inc., Chicago, Illinois) program. Normality test of numerical variables was determined by Kolmogorov-Smirnov test. Numerical variables were summarized as mean (\u0026plusmn;\u0026thinsp;standard deviation). Categorical variables were given as numbers and percentages. When comparing numerical variables between the two groups, the Mann Whitney U test was used because the variables did not show normal distribution. Chi-square and Fisher's exact tests were used when comparing categorical variables. The correlation between the measurement variables was compared with the Pearson correlation test and their correlation coefficients were calculated. Statistical significance level was taken as 0.05 for each test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e38 patients with autism spectrum disorder and 33 healthy children evaluated in the control group were included in the study. In this study, the mean age of children diagnosed with Autism Spectrum Disorder (ASD) was 44.4\u0026thinsp;\u0026plusmn;\u0026thinsp;20.4 months, which did not differ significantly from the control group\u0026rsquo;s mean age of 51.2\u0026thinsp;\u0026plusmn;\u0026thinsp;20 months (p\u0026thinsp;=\u0026thinsp;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\u0026thinsp;=\u0026thinsp;0.228). Notably, serum melatonin levels were significantly elevated in the ASD group, averaging 823.2\u0026thinsp;\u0026plusmn;\u0026thinsp;237.9 U/L compared to 677.4\u0026thinsp;\u0026plusmn;\u0026thinsp;254.7 U/L in the control group (p\u0026thinsp;=\u0026thinsp;0.027). This elevation was particularly evident among male participants, with ASD males showing melatonin levels of 820.8\u0026thinsp;\u0026plusmn;\u0026thinsp;243.5 U/L versus 649.0\u0026thinsp;\u0026plusmn;\u0026thinsp;262.8 U/L in control males (p\u0026thinsp;=\u0026thinsp;0.020), while no significant difference was observed among females (838.6\u0026thinsp;\u0026plusmn;\u0026thinsp;221.8 U/L in ASD versus 766.2\u0026thinsp;\u0026plusmn;\u0026thinsp;254.7 U/L in controls, p\u0026thinsp;=\u0026thinsp;0.608). Additionally, children with ASD reported lower daytime sleepiness scores (3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2) compared to the control group (3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3, p\u0026thinsp;=\u0026thinsp;0.036) and exhibited higher bedwetting scores (3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 versus 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8, p\u0026thinsp;=\u0026thinsp;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).\u003c/p\u003e \u003cp\u003ePatients with ASD had a mean score of 5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 on the risky questions section of the M-Chat, with a total M-Chat score averaging 13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8. The ABC assessment revealed that these patients scored 11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 in the sensorial domain, 15.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1 in building relationships, and 16.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5 in the use of body and object. Language skills were assessed with a mean score of 14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8, while social and self-care abilities scored 12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7. The overall ABC score for the ASD patients was 70.4\u0026thinsp;\u0026plusmn;\u0026thinsp;25.3. These results indicate significant challenges in multiple domains of behavior and development in children with ASD (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic, melatonin levels, laboratory data and children's sleep habits questionnaire among groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eASD\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;38)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;33)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eDemografic Data\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge (months)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.4\u0026thinsp;\u0026plusmn;\u0026thinsp;20.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.2\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(%13.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8(%24.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.228**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33(%86.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25(%75.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLevel of Melatonin (U/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale (n\u0026thinsp;=\u0026thinsp;13)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e838.6\u0026thinsp;\u0026plusmn;\u0026thinsp;221.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e766.2\u0026thinsp;\u0026plusmn;\u0026thinsp;254.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.608\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMale (n\u0026thinsp;=\u0026thinsp;58)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e820.8\u0026thinsp;\u0026plusmn;\u0026thinsp;243.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e649.0\u0026thinsp;\u0026plusmn;\u0026thinsp;262.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.020\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal (n\u0026thinsp;=\u0026thinsp;71)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e823.2\u0026thinsp;\u0026plusmn;\u0026thinsp;237.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e677.4\u0026thinsp;\u0026plusmn;\u0026thinsp;254.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.027\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLaboratory Data\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRBC (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e12\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.773\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWBC (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e9\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.572\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHGB (g/dL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.415\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTC (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.471\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMCV (fL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.6\u0026thinsp;\u0026plusmn;\u0026thinsp;15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.199\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePLT (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e9\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e336.5\u0026thinsp;\u0026plusmn;\u0026thinsp;109.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e345.6\u0026thinsp;\u0026plusmn;\u0026thinsp;109.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.904\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIron (g/dL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68\u0026thinsp;\u0026plusmn;\u0026thinsp;35.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77.9\u0026thinsp;\u0026plusmn;\u0026thinsp;40.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.333\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIron binding capacity (g/dL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e282.6\u0026thinsp;\u0026plusmn;\u0026thinsp;110.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e303.6\u0026thinsp;\u0026plusmn;\u0026thinsp;77.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.421\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFerritin (ng/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;18.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.2\u0026thinsp;\u0026plusmn;\u0026thinsp;26.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.365\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVitamin D (ng/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;13.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.278\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSleep habits questionnaire (\u003c/b\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;Sd)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal Sleep Duration\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.479\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNighttime Awake Time\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.5\u0026thinsp;\u0026plusmn;\u0026thinsp;54.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.052\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDifficulty Waking Up\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.703\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDisruption Of Sleep\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.538\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSleep Anxiety\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.456\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBreathing Disturbance During Sleep\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.303\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eParasomnia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.492\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWay Of Waking Up in the Morning\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.079\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSleep Duration\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.732\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSleep Latency\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.531\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNeeding to Sleep With Others\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.072\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSleepiness During Daytime\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBedwetting\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cb\u003e*\u003c/b\u003e: \u003cb\u003eMann-Whitney U test\u003c/b\u003e \u003cem\u003e**: Chi-square test\u003c/em\u003e \u003cb\u003eASD\u003c/b\u003e: \u003cem\u003eAutism spectrum disorder\u003c/em\u003e, \u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;Sd\u003c/b\u003e: \u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation\u003c/em\u003e, \u003cb\u003eRBC\u003c/b\u003e: Erythrocyte/Red Blood Cell Count, \u003cb\u003eWBC\u003c/b\u003e: White Blood Cell Count; \u003cb\u003eHGB\u003c/b\u003e: Hemoglobine, \u003cb\u003eHCT\u003c/b\u003e: Hematocrit, \u003cb\u003eMCV\u003c/b\u003e: Mean Corpuscular Volume, \u003cb\u003ePLT\u003c/b\u003e: Platelet.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eM-Chat and ABC assessment results of patients with autism spectrum disorder\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOSB (n\u0026thinsp;=\u0026thinsp;38)\u003c/p\u003e \u003cp\u003eMean\u0026thinsp;\u003cem\u003e\u0026plusmn;\u0026thinsp;Sd\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRisky questions (M-Chat)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal (M-Chat)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSensorial (ABC)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBuilding a Relationship (ABC)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUse of body and object (ABC)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLanguage skills (ABC)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSocial and self-care (ABC)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal (ABC)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e70.4\u0026thinsp;\u0026plusmn;\u0026thinsp;25.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003cb\u003eASD\u003c/b\u003e: \u003cem\u003eAutism spectrum disorder\u003c/em\u003e, \u003cb\u003eABC\u003c/b\u003e: \u003cem\u003eAutism Behavior Checklist\u003c/em\u003e \u003cb\u003eM-Chat\u003c/b\u003e: \u003cem\u003eModified Checklist for Autism in Toddlers\u003c/em\u003e Mean\u0026thinsp;\u003cb\u003e\u0026plusmn;\u0026thinsp;Sd\u003c/b\u003e: \u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard deviation\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of laboratory data of patients with autism spectrum disorder according to their clinical status\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eLevel of Clinical ASD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLight\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;17)\u003c/p\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;Sd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;17)\u003c/p\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;Sd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeavy\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;Sd\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRBC (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e12\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.334\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWBC (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e9\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.907\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHGB (g/dL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.361\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTC (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e33.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e36.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e37.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.393\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMCV (fL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e71.2\u0026thinsp;\u0026plusmn;\u0026thinsp;22.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e79.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e78.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.308\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePLT (10\u003c/b\u003e\u003csup\u003e\u003cb\u003e9\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e/\u0026micro;L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e313.2\u0026thinsp;\u0026plusmn;\u0026thinsp;107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e350.9\u0026thinsp;\u0026plusmn;\u0026thinsp;121.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e374.5\u0026thinsp;\u0026plusmn;\u0026thinsp;35.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.471\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIron (g/dL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e68.2\u0026thinsp;\u0026plusmn;\u0026thinsp;29.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e73.6\u0026thinsp;\u0026plusmn;\u0026thinsp;37.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e43.5\u0026thinsp;\u0026plusmn;\u0026thinsp;52.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.328\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIron Binding Capacity (g/dL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e296.2\u0026thinsp;\u0026plusmn;\u0026thinsp;94.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e299.3\u0026thinsp;\u0026plusmn;\u0026thinsp;92.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e153.5\u0026thinsp;\u0026plusmn;\u0026thinsp;181.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.043\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFerritin (ng/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e23.7\u0026thinsp;\u0026plusmn;\u0026thinsp;13.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;24.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.397\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVitamin D (ng/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.005\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMelatonin (U/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e805.7\u0026thinsp;\u0026plusmn;\u0026thinsp;244.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e872.1\u0026thinsp;\u0026plusmn;\u0026thinsp;237.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e690.3\u0026thinsp;\u0026plusmn;\u0026thinsp;200.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.367\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003e*\u003c/b\u003e: \u003cb\u003eMann-Whitney U test\u003c/b\u003e, \u003cb\u003eASD\u003c/b\u003e: \u003cem\u003eAutism spectrum disorder\u003c/em\u003e, \u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;Sd\u003c/b\u003e: \u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation\u003c/em\u003e, \u003cb\u003eRBC\u003c/b\u003e: Erythrocyte/Red Blood Cell Count, \u003cb\u003eWBC\u003c/b\u003e: White Blood Cell Count; \u003cb\u003eHGB\u003c/b\u003e: Hemoglobine, \u003cb\u003eHCT\u003c/b\u003e: Hematocrit, \u003cb\u003eMCV\u003c/b\u003e: Mean Corpuscular Volume, \u003cb\u003ePLT\u003c/b\u003e: Platelet\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePatients with severe ASD exhibited a significantly lower iron binding capacity, with a mean value of 153.5\u0026thinsp;\u0026plusmn;\u0026thinsp;181.2 g/dL, compared to 296.2\u0026thinsp;\u0026plusmn;\u0026thinsp;94.7 g/dL in the mild group and 299.3\u0026thinsp;\u0026plusmn;\u0026thinsp;92.9 g/dL in the moderate group (p\u0026thinsp;=\u0026thinsp;0.043). Additionally, vitamin D levels were markedly lower in the severe ASD group, averaging 3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 ng/mL, as opposed to 26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;15.7 ng/mL in the mild group and 17.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6 ng/mL in the moderate group (p\u0026thinsp;=\u0026thinsp;0.005). Although melatonin levels and other hematological parameters (such as RBC, WBC, HGB, HTC, MCV, and 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).\u003c/p\u003e \u003cp\u003eThe correlation analysis between melatonin levels and various clinical and laboratory parameters in children with Autism Spectrum Disorder (ASD) revealed no significant correlations. Specifically, melatonin levels showed a weak positive correlation with hemoglobin levels (r\u0026thinsp;=\u0026thinsp;0.196) and hematocrit (r\u0026thinsp;=\u0026thinsp;0.201), although these were not statistically significant. The analysis also indicated negligible correlations between melatonin and vitamin D (r = -0.062), ferritin (r\u0026thinsp;=\u0026thinsp;0.010), and the scores from the M-Chat (r\u0026thinsp;=\u0026thinsp;0.063) and Autism Behavior Checklist (ABC) (r = -0.066). These findings suggest that melatonin levels are not strongly associated with these parameters in the ASD population studied (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation analysis of melatonin and other parameters with autism spectrum disorders\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMelatonin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal (M-Chat)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.063\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal (ABC)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.066\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVitamin D (ng/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.062\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFerritine (ng/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHemoglobine (g/dL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.196\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHematocrit (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.201\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003cb\u003er\u003c/b\u003e: \u003cem\u003ePearson correlation coefficient\u003c/em\u003e, \u003cb\u003eABC\u003c/b\u003e: \u003cem\u003eAutism Behavior Checklist\u003c/em\u003e, \u003cb\u003eM-Chat\u003c/b\u003e: \u003cem\u003eModified Checklist for Autism in Toddlers.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn 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 gender are thought to be due to genetic characteristics\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In our study, red blood cell count, white blood cell count, hemoglobin, hematocrit, MCV and platelet values were found to be quite similar between children with ASD and healthy children (p\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026thinsp;=\u0026thinsp;0.005 and p\u0026thinsp;=\u0026thinsp;0.043, respectively). In our study, when healthy children and children with autism spectrum disorder were compared in terms of sleep problems, it was observed that the total sleep duration was similar between the two groups (p\u0026thinsp;=\u0026thinsp;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\u0026thinsp;=\u0026thinsp;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\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026thinsp;=\u0026thinsp;0.036 and p\u0026thinsp;=\u0026thinsp;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\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn 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\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In addition, four different studies have shown that melatonin levels decrease as the clinical condition of ASD gets worse\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. 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\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, almost all of these studies measured melatonin levels at night. In the study of Ritvo et al., it was observed that melatonin levels measured during the day were higher in patients with ASD\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In our study, it was observed that serum melatonin levels were significantly higher in ASD patients than in healthy controls (p\u0026thinsp;=\u0026thinsp;0.027). In studies where melatonin levels were measured at night, lower melatonin levels were found in patients with ASD, while elevated melatonin levels were observed in patients with ASD in daytime melatonin levels\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. 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\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMelatonin 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 gender-related melatonin values in autism spectrum disorder 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\u0026thinsp;=\u0026thinsp;0.020). It was observed that melatonin levels were higher in girls with ASD, but this difference was not significant (p\u0026thinsp;=\u0026thinsp;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.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLimitations of the Study\u003c/h2\u003e \u003cp\u003eOne of the biggest limitations of our study is that children\u0026rsquo;s sleep habits in children were evaluated with a questionnaire method that provides subjective data. In addition, at the time of blood analysis, the number of hours the participant slept, what time he woke up in the morning, or what time he slept at night was disregarded. Since there is less female gender among the applications, in order to conduct future studies with multicenter, cohort and large study groups, with female-weighted participants, and to evaluate the sleep of the children studied more objectively, it is thought that more precise results can be obtained by performing sleep monitoring.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study differs from previous studies in that the participants were selected from patients who did not use any medication that could affect sleep and melatonin, eventhough it has already been stated in the literature that the relationship between autism spectrum disorder and melatonin levels is significant, there may be an inverted oscillation rhythm, and a significant relationship between sleep habits and clinical severity of autism. We keep the idea that evaluation with larger and perhaps 24-hour serum samples to be made in this field, and evaluation with study groups in which the female gender is more predominant, will contribute significantly to the literature.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Sharing Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data can be requested from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki, and was approved by the ethics committee of Cukurova University Faculty of Medicine Non-Invasive Clinical Research Ethics Committee (decision number: 17, date: 9 July 2021). Individual written consent was required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Cukurova University Faculty of Medicine Key Research and Development Program of Anhui Province (202004j07020001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaruani A, Dumas G, Beggiato A, Traut N, Peyre H, Cohen-Freoua A, et al. Morning plasma melatonin differences in autism: beyond the impact of pineal gland volume. Frontiers in psychiatry. 2019;10:11.\u003c/li\u003e\n\u003cli\u003eRossignol DA, Frye RE. Melatonin in autism spectrum disorders. J Current clinical pharmacology. 2014;9(4):326-34.\u003c/li\u003e\n\u003cli\u003eLiebrich LS, Schredl M, Findeisen P, Groden C, Bumb JM, N\u0026ouml;lte ISJJoMRI. Morphology and function: MR pineal volume and melatonin level in human saliva are correlated. 2014;40(4):966-71.\u003c/li\u003e\n\u003cli\u003eDoyen C, Mighiu D, Kaye K, Colineaux C, Beaumanoir C, Mouraeff Y, et al. Melatonin in children with autistic spectrum disorders: recent and practical data. 2011;20(5):231-9.\u003c/li\u003e\n\u003cli\u003eKulman G, Lissoni P, Rovelli F, Roselli MG, Brivio F, Sequeri PJNL. Evidence of pineal endocrine hypofunction in autistic children. 2000;21(1):31-4.\u003c/li\u003e\n\u003cli\u003eMelke J, Goubran Botros H, Chaste P, Betancur C, Nygren G, Anckars\u0026auml;ter H, et al. Abnormal melatonin synthesis in autism spectrum disorders. 2008;13(1):90-8.\u003c/li\u003e\n\u003cli\u003eTordjman S, Anderson GM, Pichard N, Charbuy H, Touitou YJBp. Nocturnal excretion of 6-sulphatoxymelatonin in children and adolescents with autistic disorder. 2005;57(2):134-8.\u003c/li\u003e\n\u003cli\u003eNir I, Meir D, Zilber N, Knobler H, Hadjez J, Lerner YJJoa, et al. Brief report: circadian melatonin, thyroid-stimulating hormone, prolactin, and cortisol levels in serum of young adults with autism. 1995;25(6):641-54.\u003c/li\u003e\n\u003cli\u003eBruni O, Alonso-Alconada D, Besag F, Biran V, Braam W, Cortese S, et al. Current role of melatonin in pediatric neurology: clinical recommendations. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society. 2015;19(2):122-33.\u003c/li\u003e\n\u003cli\u003eShomrat T, Nesher N. Updated view on the relation of the pineal gland to autism spectrum disorders. Frontiers in endocrinology. 2019;10:37.\u003c/li\u003e\n\u003cli\u003eRitvo ER, Ritvo R, Yuwiler A, Brothers A, Freeman B, Plotkin SJEC, et al. Elevated daytime melatonin concentrations in autism: a pilot study. 1993;2(2):75-8.\u003c/li\u003e\n\u003cli\u003eYenen AS, Cak HJTPD. Melatonin and Circadian Rhythm in Autism Spectrum Disorders. 2020;31(3).\u003c/li\u003e\n\u003cli\u003eJonsson L, Ljunggren E, Bremer A, Pedersen C, Land\u0026eacute;n M, Thuresson K, et al. Mutation screening of melatonin-related genes in patients with autism spectrum disorders. 2010;3(1):1-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Melatonin, Autism Spectrum Disorder, Autism Spectrum Disorder, Sleep Disorders","lastPublishedDoi":"10.21203/rs.3.rs-4927190/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4927190/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eThis study aims to evaluate the relationship between serum melatonin levels, sleep habits, and clinical features in children with autism spectrum disorder (ASD) compared to healthy controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eIn accordance with DSM (The Diagnostic and Statistical Manual of Mental Disorders)-V, children aged 2-8 years diagnosed with autism spectrum disorder (n:38) and healthy children (n:33) of similar age and gender in the control group were evaluated. Vitamin D levels, ferritin, serum iron, and iron binding capacity of all participants were examined, as well as endogenous melatonin levels, and a pediatric sleep habits questionnaire was filled out. Autism Behavior Checklist (ABC-Autism Behavior Checklist) and The Modified Checklist for Autism in Toddlers (M-CHAT) were applied to the autism spectrum disorder group. The relationship between the blood values obtained and the completed questionnaire results was evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Melatonin levels were compared between the patients with autism spectrum disorder and the control group. In total, 71 participants were included in it. The gender of 81.7% (n=58) of the participants was male.The mean age of the autism spectrum disorder group was 44.4±20.4 months, and that of the control group was 51.2±20 months (p=0.104). When children’s sleep habits questionnaire data were compared between the two groups, the \"daytime sleepiness\" subscale score was higher in the control group, while the \"bed-wetting\" subscale score was higher in the autism spectrum disorder group (p=0.036 and p=0.008, respectively). No significant correlation was found between the questionnaire scores of the patients and their melatonin levels. Melatonin levels were on average 823.2±237.9 U/L in the autism spectrum disorder group and 677.4±254.7 U/L in the control group. This difference between the two groups was found to be statistically significant (p=0.027). When the participants were compared by dividing according to their gender, it was found that there was a difference between the two groups only in male patients (p=0.020). It was observed that there was no significant difference between girls in terms of melatonin levels (p=0.608).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConlucion: \u003c/strong\u003eIn the light of the data we obtained in our study, it was observed that there are sleep problems in autism spectrum disorder and therefore, melatonin levels measured during the daytime are higher than in healthy children. This makes us think that there may be desensitization to melatonin receptors in the ASD group, and the fact that melatonin levels differ according to gender, suggesting that melatonin treatment modalities applied in ASD patients may be regulated according to gender.\u003c/p\u003e","manuscriptTitle":"Assessment of Serum Melatonin Levels, Sleep Patterns, and Clinical Symptoms in Children with Autism Spectrum Disorder: A Case-Control Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-27 08:16:15","doi":"10.21203/rs.3.rs-4927190/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3acb65ca-fe58-4489-bc59-b9b2e9435506","owner":[],"postedDate":"September 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":37988676,"name":"Biological sciences/Physiology"},{"id":37988677,"name":"Health sciences/Signs and symptoms"}],"tags":[],"updatedAt":"2024-11-19T07:38:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-27 08:16:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4927190","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4927190","identity":"rs-4927190","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}