Studies suggest that the bidirectional relationship existent between the gut microbiome (GM) and the central nervous system (CNS), or so-called the microbiome–gut–brain axis (MGBA), is involved in diverse neuropsychiatric diseases in children and adults. In pediatric age, most studies have focused on patients with autism. However, evidence of the role played by the MGBA in attention deficit/hyperactivity disorder (ADHD), the most common neurodevelopmental disorder in childhood, is still scanty and heterogeneous. This review aims to provide the current evidence on the functioning of the MGBA in pediatric patients with ADHD and the specific role of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in this interaction, as well as the potential of the GM as a therapeutic target for ADHD. We will explore: (1) the diverse communication pathways between the GM and the CNS; (2) changes in the GM composition in children and adolescents with ADHD and association with ADHD pathophysiology; (3) influence of the GM on the ω-3 PUFA imbalance characteristically found in ADHD; (4) interaction between the GM and circadian rhythm regulation, as sleep disorders are frequently comorbid with ADHD; (5) finally, we will evaluate the most recent studies on the use of probiotics in pediatric patients with ADHD. They and their genes (“microbiome”) are involved in different biological functions, some of them are essential for our survival ,2,3. in an genes . The gutan genes . This rian genes . Regardian genes ,9,10,11 an genes  and neuran genes ,14. As aan genes ,15. The an genes .The relationship between the gut microbiome and their host is bidirectional , so thatModern life and the consequent impact on the gut microbiome have also made fundamental changes in the pattern of human illnesses, which has shifted from traditional infectious diseases towards increasingly frequent autoimmune diseases, such as asthma and allergies; cardiovascular diseases, such as hypertension; metabolic diseases, like diabetes; mental diseases, such as depression and anxiety; and a variety of neurological and neurodevelopmental disorders, such as multiple sclerosis, Alzheimer’s, Parkinson, autism spectrum disorders (ASD) and attention-deficit and/or hyperactivity disorder (ADHD). All of these conditions have been associated with an imbalance in the gut microbiome composition ,24.In this review, we will focus on the participation of the gut microbiome into the pathophysiological mechanisms of ADHD and the therapeutic potential of these microorganisms in pediatric patients with this disorder.The term “gut–brain axis” has been coined to describe the bidirectional communication between the gut microbiome and the central nervous system (CNS) ,26. ThisCampylobacter jejuni and Citrobacter amalonaticus induced anxiety-like behavior [Lactobacillus rhamnosus and Bifidobacterium longum alleviated these anxiety/depression-like conducts [Lactobacillus reuteri in genetic mouse models of autism were halted in vagotomized mice [The gut is innervated by the hepatic and celiac branches of the vagus nerve. Depending on their location and type, vagal afferents detect a variety of mechanic  and chemical stimuli  . The vagbehavior , whereasconducts ,34. Intezed mice . 4) receptors [Another important network of neurons lying between the microbiota and the host is configured by the enteric nervous system (ENS), which is composed of the myenteric and submucosal plexus. The ENS communicates with the CNS via afferent neurons with sensory information that follow spinal and vagal routes, and it is responsible for the coordination of gut functions, such as motility and fluid movement . Maturateceptors .Clostridium perfringens modulates the synthesis of 5-HT via the expression of tryptophan hydroxylase-1, which is the rate-limiting enzyme in its synthesis [Campylobacter jejuni and Bacteroides vulgatus was found to influence cognitive function in patients with Alzheimer’s disease via Glu metabolism [The gut microbiome may impact on the generation of neurotransmitters, either by synthesizing them “de novo” or by influencing the neurotransmitter-related metabolism pathways. Bacterial species of ynthesis . In factynthesis ,42, wereynthesis . As 5-HTynthesis . The gutynthesis . It has ynthesis ,47. Therynthesis ,49. Otheynthesis ,51. In atabolism .L. reuteri and increased serum levels of kynurenines in mice exposed to chronic stress, which was secondary to bacterial regulation of kynurenine enzymes via the production of H2O2 [The intestinal microbiome is also involved in the kynurenine pathway, which is in turn implicated in neuroinflammation processes associated with schizophrenia and depression . Mice th of H2O2 .Lactobacillus plantarum for 12 weeks, which were associated with an increase in BDNF levels [As reported in animal studies, the gut microbiome regulates the expression of the brain-derived neurotrophic factor (BDNF), which is involved in neurogenesis ,60. In aF levels . Lower lF levels . In a moF levels . The apoF levels . AlteratF levels , whereasF levels ,66.Bifidobacterium infantis at an early stage reversed this response [The gut microbiome is essential in the development and function of the hypothalamic–pituitary–adrenal (HPA) axis, which represents the crux of the neuroendocrine transmission and the stress response system . In GF mresponse ,14. In hresponse , togetheresponse . The gutresponse .L. reuteri [The gut microbiome modulates the secretion of gastrointestinal peptides that mediate metabolic functions related to energy homeostasis, such as insulin, leptin, ghrelin, neuropeptide Y family (NPY) and glucagon-like-peptide 1 (GLP-1) . Microbi reuteri . Functio reuteri . Opioids reuteri ,79.The intestinal microbiome produces short-chain fatty acids (SCFAs), particularly butyrate, propionic acid and acetate, mostly derived from the degradation of fibres and undigested saccharides . They seThe gut microbiome also creates other substances derived from the metabolism of proteins and amine acids, such as ammonia (NH3) , which iThe development and integrity of the gut barrier and the blood-brain barrier (BBB) are dependent on the gut microbiome. Alterations of the gut microbiome downregulate the expression of the tight junctions (TJs) , exposinThe intestinal microbiome regulates the differentiation and maturations of innate immunocytes, such as macrophages, innate lymphoid cells and dendritic cells . Highly L. reuteri, together with a tryptophan-rich diet, to reprogram intraepithelial CD4-positive T cells into immunoregulatory T cells [Clostridium [Bifidobacteria and Enterobacteriaceae, whereas IgG levels were associated with Haemophilus [Acquired immunity develops and matures during exposure to the gut microbiota. Intraepithelial lymphocytes  were reported to require the presence of  T cells . In a ststridium . Absencestridium . A recenmophilus .ADHD is the most common neurodevelopmental disorder in children and adolescents, affecting 5% of individuals younger than 18 years ,105. It Numerous etiological factors have been attributed to ADHD: genetic factors, which represent around 70–80% ; and divNowadays, there is increasing evidence that the aforementioned etiological factors and catecholaminergic dysfunction may lead to a neuronal state predominantly characterized by oxidative stress and inflammation, which could perpetuate the neurochemical alterations responsible for ADHD . IncreasBifidobacterium was significantly increased in the ADHD cohort. The authors also predicted bacterial gene function in relation to the metabolic pathways involved in the synthesis of phenylalanine, tyrosine and tryptophan. Interestingly, the relative abundance in the genus Bifidobacterium was correlated with a significant increase in the enzyme cyclohexadienyl dehydratase (CDT), which is involved in the synthesis of a dopaminergic precursor . In a subset of 28 participants, independent of diagnosis, Aarts et al. also performed functional magnetic resonance imaging (fMRI) analysis to correlate the differences in microbial composition with neural reward responses. They observed a negative association between the relative abundance of CDT and reward anticipation responses in bilateral ventral striatum. Reward anticipation, which is dependent on DA neurotransmission [Aarts et al.  were thesmission , is crucsmission . Aarts esmission .Faecalibacterium (from the family Ruminococcaceae) was reported in the ADHD group. These authors also found a negative association between the abundance of Faecalibacterium and parental reports of ADHD symptoms. Interestingly, low levels of Faecalibacterium have been reported in atopic diseases such as asthma, eczema and allergic rhinitis [In order to avoid the potential interference of ADHD medication, Jiang et al.  analyzedrhinitis , which srhinitis . Jiang erhinitis  could beRuminococcaceae_UGC_004, which was found to be associated with inattention symptoms. This association was not affected by the intake of ADHD medication. Szopinska-Tokov et al. compared the ADHD population with other two cohorts of patients: subthreshold ADHD group, composed of individuals who did not reach the criteria for ADHD but scored too high to be considered healthy controls; and the control group, which included unaffected siblings of ADHD patients, although the family relatedness was a factor considered in the statistical analysis. The alpha diversity was not significantly different between groups, although the beta diversity was reported to be significantly reduced among patients with ADHD, and this correlated with inattention scores. The Basic Local Alignment Search Tool (BLAST) available at the National Center for Biotechnology Information (NCBI) was used to search for similarities between protein and nucleotide regions. Consequently, the authors found that the genus Ruminococcaceae_UGC_004 shared sequences with microbial species with the ability to consume the GABA neurotransmitter. The novelty of this study was to provide new evidence on the role of the gut microbiome in neurotransmitter systems related to ADHD pathophysiology, via similarity between biological sequences. However, the main limitations of Szopinska-Tokov et al.’s study were related to the selection and comparability between samples: no information in relation to the recruitment process of the control and subthreshold ADHD samples was reported; information on lifestyle and dietary patterns, which may have interfered with the results, was not collected by the authors; intake of ADHD medication was registered through self-reports or parental reports on the day of measurement, which could have introduced a recall bias.A recent study, conducted by Szopinska-Tokov et al.  on a DutPrevotella and Parabacteroides were detected as markers for the control group, whereas Neisseria was identified as marker for the ADHD group. At the family level, a significantly higher abundance of Bacteroidaceae was found in ADHD samples in comparison with controls. Interestingly, the reduction in alpha diversity was also observed in the mothers of ADHD patients, but not between the fathers of ADHD patients and controls. This finding suggests that alterations in the microbiome composition might be passed on maternally to the children. However, the study of Prehn-Kristensen et al. had several limitations: first, the small sample size; second, as no female patients were included in this study, it was not possible to explore if sex could influence the differences found; third, most ADHD patients were on medication, which was interrupted 48 h before being tested. This, however, might not have allowed enough time to avoid the potential impact of medication on microbial patterns [Prehn-Kristensen et al.  analyzedpatterns . The oriFaecalibacterium (family Ruminococcaceae) was significantly decreased in ADHD patients, whereas the concentrations of Odoribacter  and Enterococcus were significantly higher. At the species level, Bacteroides caccae, Odoribacter splanchnicus, Paraprevotella xylaniphila and Veillonella parvula were significantly increased in the ADHD group. The authors also analyzed the metabolic pathways associated with the microbial genes that were significantly different between the two samples of patients. Alterations in genes encoding enzymes involved in the dopaminergic synaptic pathways were found in the ADHD group. Faecalibacterium may exert anti-inflammatory effects, and their abnormal levels may lead to a higher expression of inflammatory factors that could contribute to ADHD pathogenesis [Enterococcus has been reported to be associated with neurotransmitter release. It could lead to excessive intestinal conversion of levodopa into DA. However, peripheral DA is not able to cross the BBB to enter the CNS, therefore reducing the effectiveness of levodopa [Enterococcus have been found in mice lacking the 5-HT transporter, which can lead to decreased 5-HT concentrations [Odoribacter found by Wan et al. in ADHD patients were in line with a previous study that found increased levels of this genus in pediatric acute-onset neuropsychiatric syndrome (PAN) and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) [In a Chinese pediatric population, Wan et al.  comparedogenesis ,145. Entlevodopa . Additiotrations , a neuro(PANDAS) . The mai(PANDAS) .n = 226) were selected from the Influence of Life-style factors on the development of the Immune System and Allergies in East and West Germany (LISA) birth cohort. This was a population study in which healthy full-term neonates were recruited from different hospitals in Germany and their bedrooms’ floor dust samples were collected at the age of 3 months. Indoor bacterial and fungal diversity were respectively analyzed by 16S rDNA gene sequencing and high-throughput sequencing. Fungal and bacterial alpha diversity metrics were calculated . The Strength and Difficulty Questionnaire (SDQ) was used to evaluate hyperactivity/inattention behavior at the ages of 10 and 15 years. A total of 23 children at the age of 10 years reached criteria for ADHD, while this number increased to 50 individuals at the age of 15 years. At the age of 10, bacterial richness (number of different taxa) was found to be inversely correlated with ADHD prevalence, whereas the number of fungal species was positively associated with a high prevalence. However, at the age of 15, only the Shannon index was significantly associated with hyperactivity/inattention symptoms, directly for bacteria and inversely for fungi. Therefore, this study suggests that early life microbial environment may be associated with the development of behavioral problems during childhood. However, the direction of the associations observed was heterogeneous and the observational design of the study did not allow establishing causality. Furthermore, the authors did not consider other confounding factors which could have influenced the results, such as certain lifestyles or the exposure to phthalates. With the collection of dust samples at one single time-point (3 months of age), changes in the indoor microbial environment across time were not considered either. An additional limitation of the study conducted by Casas et al. was in relation to the representativeness of the sample: the assessment of inattention/hyperactivity symptoms was only based on scores obtained in the parent-completed and self-completed Strengths and Difficulties Questionnaire (SDQ), which could have introduced a reporting bias.Casas et al.  conducten = 7) or a blend of vitamins, minerals and antioxidants (n = 10) for a period of 10 weeks. The aim was to investigate the effect of micronutrient supplementation on the human fecal microbiome composition. Sequencing of 16S rDNA was performed on fecal samples collected at baseline and after supplementation. Micronutrient supplementation was associated with 50% responder rates vs. 29% responder rates in the placebo group. The response resulted in enhanced overall function, improved attention, emotional regulation and aggression. However, no significant differences in the scores obtained in either the Children’s Global Assessment Scale (CGAS) or the ADHD Rating Scale-IV (ADHD-RS-IV) were found between the treatment and the placebo group. There were no significant changes in the alpha or beta diversity between the placebo and the micronutrient group, although patients receiving micronutrients showed increased community richness after 10 weeks supplementation in comparison with placebo. Micronutrient supplementation caused a significant decrease of the phylum Actinobacterium, particularly a 25% reduction in the order Bifidobacteriales, which was attributed to the genus Bifidobacterium. This was accompanied by higher levels of the genus Collinsella. A pairwise correlation was detected between lower ADHD-RS-IV scores and decreased Actinobacteria abundance, as well as between lower concentrations of Actinobacteria and higher scores in the CGAS (in which higher results indicate better functioning). The results highlight a potential effect of micronutrient supplementation to modulate the abundance of putative probiotic bacterial species. Nevertheless, the role of Bifidobacterium in ADHD is contradictory. Several studies reported a protective effect of Bifidobacterium longus on several neuropsychiatric disorders, including ADHD [In a double-blind placebo-controlled trial carried out by Stevens et al. , 17 maleing ADHD ,152. On ing ADHD  found anBacteroides coprocola was significantly lower in the ADHD group, whereas Bacteroides uniformis, Bacteroides ovatus and Sutterella stercoricannis were significantly increased. The genus Fusobacterium was relatively enriched in ADHD patients, while the relative abundance of Lactobacillus was higher in control subjects. Additionally, dietary patterns differed between the two cohorts of participants. Participants with ADHD showed a higher intake of refined grains and a lower proportion of vitamin B2 and dairy. The amount of S. stercoricannis was correlated with the intake of dairy, nuts/seeds/legumes, ferritin and magnesium. B. uniformis was associated with fat and carbohydrate intake, whereas no correlations were observed between B. ovatus and B. coprocola with any of the items included in the food frequency questionnaire. A positive correlation was found between ADHD symptoms and both S. stercoricannis and B. ovatus. The sample selection process was carefully described by the authors, who also considered some confounding factors, such as the use of probiotics, antibiotics, special dietary patterns and the presence of neuropsychiatric comorbidities. However, the main limitations of this study were found in relation to the representativeness and comparability of samples: ADHD patients with no neuropsychiatric comorbidities who scored lower than typical ADHD patients in the Swanson, Nolan, and Pelham Version IV Scale (SNAP-IV); and cases and controls who significantly differed in their dietary habits. These differences in the dietary pattern could have been the only reason for distinct microbial profiles.In a Taiwanese study, Wang et al.  comparedDesulfovibrio and the order Clostridiales were significantly associated with ADHD. However, Desulfovibrio was also associated with ASD, which suggested that the abundance of this genus was not specific of ADHD. The novelty of this study consists in screening candidate gut microbiome for subsequent functional studies investigating the role of the gut microbiota in the pathophysiology of diverse neuropsychiatric disorders. The authors also used DNA level GWAS data, so that the results were less likely to be affected by environmental and dietetic factors. However, an important limitation of this study was that the gut microbiome-related gene sets were collected from previously published GWAS of gut microbiome, which are still quite scarce. Therefore, only a limited number of datasets was available for analysis.Cheng et al.  applied The main findings and particular aspects of the aforementioned studies are summarized in The previous evidence argues for a different microbial composition in patients with ADHD. However, the results are too heterogeneous to draw confident conclusions regarding whether a specific microbial profile is associated with ADHD. Several factors may have contributed to the differences found between studies: diverse geographic, cultural, demographic and dietary characteristics of study populations; differences in relation to the selection of control groups (siblings or non-related controls) or the intake of ADHD medication; methodological differences associated with the sampling and storing, microbiome sequencing and the choice of bioinformatics pipeline and reference databases ,156.Bifidobacterium, which belongs to the phylum Actinobacterium. The improvement of ADHD symptoms after supplementation could have been influenced by these microbial changes, as a correlation was found between a reduction in ADHD-RS-IV scores and a decreased abundance of Actinobacteria. Previously, Aarts et al. had showed a significant increase of the genus Bifidobacterium in young patients with ADHD, establishing a correlation between this genus and a particular DA-related enzyme (CDT) associated with altered neural reward responses. In both studies, no differences in alpha/beta diversities were found between patients and controls.The results obtained by Stevens et al.  agreed wBacteroidaceae in adolescents with ADHD. This was corroborated later by Wang et al. [Bacteroides in the ADHD group. However, the results obtained in relation to alpha and beta diversity were discordant: alpha diversity was reduced and significant differences in beta diversity were found in the ADHD group in the study carried out by Prehn-Kristensen et al. By contrast, no significant differences in beta diversity between groups were found by Wang et al., who also highlighted higher values of Shannon and Chao1 indices in the ADHD group. This could account for methodological differences between studies: sample size, gender, intake of ADHD medication, ethnicity, and dietary habits.Prehn-Kristensen et al.  revealedg et al. , who fouClostridiales and ADHD. Interestingly, other three studies are in line with this finding: a significant increase of the genus Ruminococcaceae_UGC_004  in the ADHD group was shown by the study conducted by Szopinska-Tokov et al. [Faecalibacterium in patients with ADHD, which belongs to the same family. In any case, these results are not mutually exclusive, as the increase in a genus can be accompanied by a lower abundance of a different genus from the same family. For instance, in the study performed by Stevens et al. [Bifidobacterium occurred along with an increase of the genus Collinsella, from the same family study. Differences in alpha and beta diversity between studies could again be attributed to demographic and clinical differences between participants , and divergences in sampling and storing.Cheng et al.  showed sv et al. . Jiang ev et al.  and Wan v et al.  found a s et al. , the decNotwithstanding heterogeneity, we attempted to group studies according to the differential microbial features in patients with ADHD:Bacteroides spp. and Clostridiae spp. [According to the previous studies, it seems clear that the gut microbiome could play a role in the pathophysiological mechanisms leading to ADHD symptoms. As explained before, the gut microbiome may interfere in the catecholaminergic neurotransmission system by either influencing their metabolic pathways or the expression of the genes encoding these neurotransmitter transporters . Furtheriae spp. . SCFAs ciae spp. . Changesiae spp. , which wiae spp. . An addiA schematic representation of bacterial taxonomy to facilitate comprehension is shown in Omega-3 PUFAs, and particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), play an important role in membrane fluidity, neurotransmission and receptor function ,164. TheTotal omega-3 PUFAs are significantly decreased in pediatric patients with ADHD, who showed a significant higher omega-6:omega-3 fatty acids ratio in comparison with controls . Omega-3Bifidobacterium breve (NCIMB 702258 and DPC 6330) yielded different fatty acid profiles in the host tissues. Mice fed with B. breve NCIMB 702258 showed significantly higher concentrations of arachidonic acid (AA) and DHA in the brain in comparison with those fed with B. breve DPC 6330 and the non-supplemented control group. Nevertheless, both groups of B. breve-supplemented mice presented significantly lower brain concentrations of the omega-6 dihomo-γ-linolenic acid (20:3 n-6). The results were not due to differences in dietary patterns others than the supplementation received. They were also accompanied by changes in the gut microbial composition: at the family level, there was a higher abundance of Clostridiaceae and lower concentrations of Eubacterium in the supplemented groups, particularly in those receiving B. breve DPC 6330 [There is growing evidence to support a bidirectional relationship between omega-3 PUFAs and the gut microbiome. In mice, an 8-week dietary supplementation with different strains of DPC 6330 .Lactobacillus and Bifidobacterium during adulthood in comparison with the other two groups [On the other hand, omega-3 PUFAs also seem to influence the gut microbial composition. A group of pregnant female mice and their male offspring were fed with a control diet, omega-3 (DHA + EPA) enriched diet or omega-3 deficient diet. All three diets only differed in the fatty acid content. Cognitive performance and social behavior were assessed in male offspring at adolescence and adulthood, in parallel with the microbial profile, analyzed through 16S rDNA sequencing. Animals receiving the omega-3 deficient diet showed impaired communication, social and depression-related behaviors, whereas the mice fed with omega-3 enriched diet displayed improved cognition. On a microbial level, a significant increase in the Firmicutes:Bacteroidetes ratio was observed in the mice following a diet deficient in omega-3 fatty acids. At the genus level, mice following a diet abundant in omega-3 showed a significant increase in o groups . In a reo groups . These rBifidobacterium, Roseburia and Lactobacillus in fecal samples. No significant changes in alpha or beta diversity were found after supplementation. However, the authors found no correlation between the microbial changes and the concentrations of EPA/DHA measured in red blood cells after supplementation. The recruitment process was carefully described by the authors, who also stratified for confounding factors such as the current use of omega-3 PUFA supplements, concomitant use of non-steroidal anti-inflammatory medication, previous surgery for bowel resection, current treatment for any chronic inflammatory condition or malignancy, pregnancy or smoking. As limitations, this study included a small sample size and no information on dietary habits was recorded.Similarly, in a randomized, open-label, cross-over trial of 8-week supplementation with omega-3 PUFAs (DHA/EPA) to 22 healthy volunteers, Watson et al.  reportedA representation of the interaction between the gut microbiome and omega-3 PUFAs, and how this relationship could interfere with ADHD pathophysiological mechanisms, is shown in Bmal:Clock, Per:Cry, Rorα, Rev-erbα, Chrono) [Sleep disorders represent one of the most frequent comorbidities in children with ADHD. They can be present in up to 70% of patients , affecti Chrono) . Indeed, Chrono) ,184. Bacteroidetes and Clostridia, swayed during the light-dark period [Bacteroidetes, whereas bacterial load was reduced during the rest phase, with a predominance of the phylum Firmicutes [Per1/2, attenuated these oscillations [Firmicutes:Bacteroidetes ratio in mice [Akkermansia, Bacteroides and Faecalibacterium, together with an increase of Aeromonas, was found in mice with sleep deprivation. Goblet cells and the expression of TJ proteins were also significantly reduced by sleep deprivation. By contrast, melatonin administration restored the richness and diversity indices, as well as the Firmicutes:Bacteroidetes ratio, to values similar to the control group. After treatment with melatonin, a significant decrease of Aeromonas was observed, along with a rise in the content of Akkermansia, Bacteroides and Faecalibacterium. Furthermore, melatonin significantly increased the number of goblet cells and the expression of TJ proteins. These changes were in parallel with a neutralization of the disrupting effects provoked by sleep deprivation in the balance between pro-inflammatory/anti-inflammatory cytokines and the redox status.The diversity and composition of the gut microbiome oscillate during the 24 h light-dark cycle, suggesting that the gut, as the rest of the tissues, has its own peripheral circadian clocks, which ultimately depend on the central clock. In animal studies, the diversity and abundance of certain species, such as k period . The numrmicutes ,187. Knollations ,188. Add in mice . Recentl in mice  evaluaten = 60) and those without sleep problems (n = 60). Sleep disorders were assessed through the Children Sleep Habits Questionnaire (CSHQ). Microbial DNA was extracted from stool samples and analyzed through next-generation sequencing of 16S rDNA. No information on medication or dietary patterns was included. Abundance and richness of the gut microbiota were significantly higher in ASD children with sleep problems. No significant differences were observed in the Firmicutes:Bacteroides ratio, but at the genus level, a significant reduction in the butyrate-producing bacteria Faecalibacterium and Agathobacter was found in the sleep disorder group. The abundance of each of the two genera was negatively correlated with CSHQ scores. Three differential metabolites were observed between sleep disorder and no sleep disorder groups: concentrations of 3-hydroxybutyric acid (a butyrate-derived acid) and melatonin were significantly lower among patients with ASD and sleep problems, whereas levels of 5-HT were significantly higher. The concentrations of 3-hydroxybutyric acid were correlated with melatonin levels and Faecalibacterium abundance. Additionally, a positive correlation was also found between melatonin concentrations and the abundances of Faecalibacterium and Agathobacter. Due to the design of the study, it was not possible to elucidate if the differences observed in the gut microbiome were due to a circadian dysregulation in patients with ASD, or if a previous alteration in the microbial composition contributed to the changes observed in melatonin concentrations. Another limitation was the absence of a control group to be compared with the populations with ASD, apart from the absence of information on dietary patterns and medication intake. Notwithstanding, this study provided compelling evidence for an association between microbial metabolites (butyrate) and melatonin. Studies on the bidirectional relationship between the gut microbiome and the sleep/wake circadian rhythm in ADHD are lacking. However, given the evidence provided and the genetic overlap between ASD and ADHD [In a Chinese population of 120 children with ASD, Hua et al.  exploredand ADHD , it seemLactobacillus and Bifidobacterium represent the most investigated strains [Given the documented involvement of the MGBA in the pathophysiological mechanisms of ADHD, it may be reasonable to consider the gut microbiome as a potential therapeutic target for this disorder. In this regard, probiotics are living non-pathological microorganisms that provide a health benefit by improving physiological conditions in the host when they are administered in adequate amounts, well as a food ingredient, supplement or as a drug . To date strains .Lactobacillus rhamnosus GG (1 × 1010 colony-forming units (CFU)) or placebo, starting 4 weeks before expected delivery. The intervention was continued for 6 months after delivery, given either to the children or to the mother, if they were breastfed. Behavioral patterns and fecal microbiome composition of the children were periodically analyzed until 13 years of age. The diagnosis of ADHD or ASD was established by an experienced child psychiatrist or neurologist in accordance with the International Classification of Diseases 10th Edition (ICD-10). Techniques of fluorescein in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) were used to analyze the gut microbiome. Seventy-five subjects completed the study, 40 in the intervention group, and 35 participants in the placebo group. Significant differences were found in the percentage of patients who were diagnosed with ADHD or ASD at the age of 13: 17.1% children in the placebo group vs. none in the probiotic group. At the genus level, the content of Bifidobacterium at 6 months of life was significantly lower among children who later developed neuropsychiatric disorders, although this difference was not present at 13 years of age. All children diagnosed with neurodevelopmental disorders were male, but the differences found were not influenced by gender when controlling for this factor in a logistic regression analysis. Perinatal factors, such as delivery mode, birth/length weight and infant feeding mode were comparable between placebo and intervention groups. The recruitment process was detailed by the authors, who also used accurate techniques of FISH and qPCR. However, some limitations should be taken into account when interpreting these results: a considerable percentage of drop-outs (around 50%) may have biased the results; no information on maternal dietary habits during pregnancy and after delivery (in the case of breastfed infants) was recorded; additional information on risk factors for ADHD, such as maternal smoking, nutritional deficiencies or psychosocial factors was not reported either; finally, no association were established between the abundance of Bifidobacterium at 6 months of life and ADHD psychometric scores.A recent systematic review included randomized controlled trials, published between 1990 and 2018, that had evaluated the effects of probiotic supplementation on the cognitive function in children and adolescents. Seven studies were found to meet the inclusion criteria, but only one of them reported a significant reduction in the risk to develop ADHD or ASD . This waLactobacillus rhamnosus GG ATCC53103 (once-daily dose of 1 × 1010 CFU). Thirty-two naïve-medication patients with ADHD (4–17 years old) were randomized to take the probiotic (n = 18) or placebo capsules (n = 14). The serum profile of pro/anti-inflammatory cytokines was assessed at baseline and after 3 months, as well as the scores obtained in the following ratings: the ADHD Parent Report Rating Scale-IV, which is based on the diagnostic criteria for ADHD as established by the Diagnostic and Statistical Manual of Mental Disorders 4th Edition (DSM-IV); the Child Self-Report and Parent Proxy-Report of the Pediatric Quality of Life InventoryTM (PedsQLTM) 4.0 Generic Core Scale; the Parent Form (CBCL/6-18) and the Teacher Report Form (TRF) of the Child Behavior Checklist (CBCL) of the Achenbach System of Empirically Based Assessment (ASEBA). The most interesting finding was a significant improvement in the scores obtained in the PedsQLTM Child-Self Report after 3 months only in the probiotic group. This may reflect that these patients felt an improvement in physical, emotional and social terms, as well as in their school functioning. Mixed results were obtained in relation to the other tests: the PedsQLTM Parent-Proxy Report showed significantly better scores only in the placebo group, whereas a significant improvement was reflected by the scores obtained in the ADHD Rating Scale and the CBCL Parent Form in both groups of participants. The differences observed in the serum levels of cytokines were also ambiguous: the proinflammatory IL-12 p70 and TNF-α significantly decreased in the probiotic group only, whereas significantly lower concentrations of the proinflammatory IL-6 was found in both groups; a significant reduction of the anti-inflammatory IL-10 was also observed, and only in the probiotic group. The authors attributed these controversial results to both the small sample size and the short observation period [By contrast, less consistent results were shown by a more recent double-blind pilot randomized placebo-controlled trial on children and adolescents with ADHD who received a 3-month supplementation with n period . This pi11 CFU per dose of three lactic acid bacteria  and the fermentable fibers betaglucan, inulin, pectin and resistant starch. The pediatric sample was composed of 68 children and adolescents with ADHD (10–14 years) who were allocated to take Synbiotic 2000 (n = 34) or placebo capsules (n = 34) for a period of 9 weeks. The following questionnaires were completed before and after intervention: the Swanson, Nolan and Pelham-IV scale (SNAP-IV) parent rating scale to measure ADHD symptoms; the Weiss Functional Impairment Rating Scale-parent-reported for child (WFIRS-PC) to assess functional impairment; and the Social Communication Questionnaire (SCQ) to evaluate autistic symptoms. Given that Synbiotic 2000 has documented anti-inflammatory properties [In a double-blind randomized placebo-controlled trial, Skott et al.  aimed atoperties , the autBifidobacterium infantis (1–1.5 × 109 CFU) and Lactobacillus acidophilus (1–3 × 109 CFU), administered once-daily from days 1–3 of life until days 14–35 of life. Neurocognitive outcome was assessed by considering the scores in the parent-reported Strength and Difficulties Questionnaire (SDQ), and the intelligence quotient obtained in the Wechsler Preschool and Primary Scale of Intelligence 3rd Edition (WPPSI-III) test. This represents the first large-scale study on neurocognitive outcome after probiotic administration during the neonatal period. The sample was carefully selected, stratifying for potential confounding factors, such as gestational age, motor impairment, maternal age, maternal educational level, multiple birth and bronchopulmonary dysplasia. However, the study exhibited several limitations that should be taken into account when interpreting the results: first, the design was observational, so it was not possible to draw causal conclusions based on data; second, breastfeeding was considered as either exclusive breastfeeding or mixed breastfeeding combining breast milk and infant formula; third, information on the duration of breastfeeding was collected by a parent-reported questionnaire at the age of 5 years, which may have introduced a recall bias; finally, a combination of biological and psychological factors that could have influenced neurodevelopment in the time lapse between perinatal period and 5–6 years of age were not included in the study [An observational study on 2467 very low birth-weight (VLBW) infants who were followed until the age of 5–6 years, showed no association between probiotic treatment and neurocognitive outcome of the study population. However, breastfeeding for a minimum of 3 months was associated with lower scores of conduct disorder and inattention/hyperactivity. The probiotic formulation consisted of a standardized combination of he study .Bifidobacterium at 6 months of age was significantly lower among those who developed neuropsychiatric disorders. This would contrast with the results obtained by Aarts et al. [Bifidobacterium referred by Pärtty et al. at 6 months was not present at 13 years of age, when the diagnosis was made. The study of Pärtty et al. was only slightly corroborated by the randomized placebo-controlled trial conducted by Kumperscak et al. [TM Child-Self Report scores after supplementation. This study was conducted on a clinical population of children and adolescents with ADHD using exactly the same probiotic as Pärtty et al., but for a shorter period. However, the results obtained in the serum cytokine profile and in ADHD-RS-IV scores, a psychometric scale specifically designed to detect ADHD core symptoms, were mixed. Similar mixed results were also offered by the clinical trial carried out by Skott et al. [In general, the aforementioned studies provide some evidence to suggest a therapeutic role of the gut microbiome in pediatric patients with ADHD. However, notable inconsistencies were found among the three supplementation clinical trials on children with ADHD. The most robust results were provided by Pärtty et al. , who reps et al.  and Stevs et al. , as prevk et al. . They sht et al.  on a pedAs suggested by Cerdó et al. , there aIn the present review, we have aimed to provide the current evidence on: the role of the gut microbiome in ADHD pathophysiology and possible association with symptoms; implication of the gut microbiome in the omega-3/omega-6 PUFA imbalance found in patients with ADHD; interaction between the gut microbiome and circadian rhythms; and the therapeutic role of probiotics in pediatric patients with ADHD. Certainly, we have carefully selected and described the most recent studies on the aforementioned research lines. However, this is a narrative review, as such, our work is essentially informative and has limited capacity to assess the robustness of the results provided by the current literature in these topics. We have tried to improve this by: highlighting the limitations of the different studies, making a judgement of their novelty, quality and reliability; grouping studies according to their results, in order to show controversies and inconsistencies among them; and including final remarks with suggestions on the aspects that require to be improved in future studies. On the other hand, we cannot discard a possible selection bias, given by the absence of a detailed search strategy designed a priori. Therefore, a systematic review, with a previous comprehensive plan to identify all relevant studies on the topic and the use of statistical analysis to measure effect sizes, would have provided more solid conclusions to our initial hypothesis. A systematic assessment of the quality of the studies could also have been included, through the use of the Newcastle–Ottawa Quality Assessment scale for Cohort and Case-Control Studies (NOS)  and the The gut microbiome maintains a bidirectional relationship with its host through neurological, hormonal and immune mechanisms, configuring the microbiome–gut–brain axis (MGBA). Alterations of the MGBA are responsible for the appearance of diverse neurological and neurodevelopmental disorders in pediatric populations. In children and adolescents with ADHD, the MGBA is involved in the pathophysiological mechanisms of neuroinflammation and oxidative stress that give rise not only to the ADHD core symptoms, but also to associated comorbidities, such as sleep disorders. Furthermore, changes in the gut microbiome may also constitute the basis for the efficacy of new alternative therapies currently under investigation, such as omega-3 PUFAs. Thus, the gut microbiome could represent a potential therapeutic target in children and adolescents with ADHD.Nevertheless, current studies still offer heterogeneous results due to substantial methodological differences in relation to sample size, participant selection criteria, neuropsychological assessment of participants, identification of confounding factors, microbiome analysis techniques, dosage and combination of probiotics, and duration of the intervention period. This could be improved by conducting studies that: include a formal calculation of the required sample size and clearly defined recruitment procedures, including information on the intake of ADHD medication and dietary patterns; use standardized psychometric scales to evaluate ADHD symptoms in accordance with the current diagnostic criteria (DSM-5); have designs that allow inferring causality; use high-resolution techniques beyond 16S rDNA sequencing for taxonomy and functional analysis, such as whole-metagenome shotgun sequencing, metatranscriptomics and metaproteomics. Future research lines should be focused on the design of protocols to standardize the supplementation with probiotics in pediatric populations: composition, dose and time of supplementation.


This comprehensive review examined sex differences in prescription rates and efficacy or effectiveness of pharmacotherapy treatment in girls and women with attention deficit hyperactivity disorder (ADHD), while identifying gaps in the scientific knowledge on this topic.A rigorous electronic database search was carried out in order to identify all published studies on female-specific effects of stimulants and non-stimulants in the treatment of ADHD. In total, 2672 studies were screened of which 21 studies  met the inclusion criteria and were included for analysis.In all seven studies on ADHD prescription rates, girls received significantly less prescriptions than boys, a difference however no longer seen in adults with the exception of one study. Each of the 14 studies on effectiveness / efficacy found at least one sex-difference in the effects of ADHD pharmacotherapy.Several sex-differences are demonstrated in the prescription, usage and efficacy /effectiveness of both stimulant and non-stimulant ADHD pharmacotherapy. A single daily use of MPH may possibly not be optimal for girls with ADHD and ATX may be a promising medication for girls and women with ADHD. The robustness of this result requires further investigation. Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder that often persists into adulthood and involves a persistent pattern of inattention and/or hyperactivity-impulsivity that interferes with functioning or development . The impFemales with ADHD were underdiagnosed for many years, partly due to their unique presentation leading them to meet fewer ADHD diagnostic criteria –19. GirlThis increased recognition of ADHD in females is partly due to increasing awareness of sex differences in ADHD presentations e.g. . Males wPharmacotherapy is currently the first-line treatment in the management of ADHD symptoms –29. ObseWith regard to ADHD pharmacotherapy, two classes of medication exist; stimulants and non-stimulants . StimulaEven though stimulants are generally effective in most patients with ADHD, non-stimulant medication is also prescribed, because in approximately 25–30% of patients, ADHD symptoms are not adequately controlled by stimulant therapy, and not all individuals respond well to stimulant treatment , 52. AtoOnly in recent years the medical sciences have become increasingly aware of the impact of sex on the effects of pharmacotherapy in general. Female sex is increasingly seen as a risk factor for clinically relevant adverse drug reactions (ADRs) –59, withAs early as two decades ago, an important conclusion of the National Institute of Mental Health's Conference on Sex Differences in ADHD was that inferences drawn from studies in boys with ADHD cannot be applied routinely to girls with ADHD . In receth, 2019. The primary keywords ‘ADHD’ or ‘attention deficit hyperactivity disorder’ were combined with the secondary keywords ‘pharmacotherapy’, ‘effectiveness’, and ‘efficacy’. These keywords were then combined with the sex-related keywords ‘females’, ‘girls’, ‘women’, ‘gender’ or ‘sex’, as well as with keywords relating to ADHD pharmacotherapy, such as ‘methylphenidate’, ‘MPH’, ‘amphetamine’, ‘AMP’, dextroamphetamine’, ‘dAMP’, ‘levoamphetamine’, ‘lAMP’,‘lisdexamphetamine’, ‘lisdexAMP’, ‘atomoxetine’, ‘ATX’, clonidine’ or ‘guanfacine’. In order to be included in this systematic literature review, studies had to meet the following inclusion criteria: (a) the study was written in English; (b) the ADHD sample was formally diagnosed with this disorder; (c) the study provided data explicitly sorted by sex; and (d) the study provided data on either prescription rates of ADHD pharmacotherapy or on the efficacy or effectiveness of stimulant or non-stimulant ADHD pharmacotherapy. Due to the scarce body of literature, no strict excluding criteria for the measurement of efficacy / effectiveness were set. The combination of the primary keywords with the keywords related to either sex or pharmacotherapy yielded an overall total of 2671 records. These initial results were then filtered to exclude duplicates and articles not fulfilling the inclusion criteria. Most articles were excluded for not explicitly reporting the data by sex. Following this procedure, eleven relevant articles remained. Subsequently, reference lists of these relevant articles were scanned for further potential articles. This search generated another three articles that fulfilled all set criteria and were therefore included. Hence, 14 articles were included in the review on the efficacy or effectiveness of stimulant or non-stimulant ADHD pharmacotherapy. The results were grouped by ADHD pharmacotherapy type; i.e. stimulants and non-stimulants, and age; i.e. children and adolescents (<18 years), and adults (≥ 18 years). Next, the same search method was conducted for studies on prescription rates, with the exception of the secondary keywords, which were ‘prescription’ or ‘prescription rate’. After completing the search process, seven studies were found and the results were grouped by age; i.e. children and adolescents (<18 years), and adults (≥ 18 years). See This systematic literature review was performed conforming to the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) , medium (d = 0.5) and large (d = 0.8) effects Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: N/A**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This comprehensive review examined sex differences in prescription rates and efficacy or effectiveness of pharmacotherapy in girls and women with ADHD. The authors found sex-differences are demonstrated in the prescription, usage and efficacy/effectiveness of both stimulant and non-stimulant ADHD pharmacotherapy. The authors suggest that a single daily use of MPH may possibly not be optimal for girls with ADHD and ATX may be a promising medication for girls and women with ADHD. This paper is generally well-written and has several merits. There are some minor issues to be addressed:1. The authors suggest that a single daily use of MPH may possibly not be optimal for girls with ADHD and ATX may be a promising medication for girls and women with ADHD. I think this statement is somewhat over-concluded. There are only 5 studies about ATX recruited in this study. The robustness of the results should be further investigated.2. The recruitment criteria were not limited to randomized, controlled, double-blinded trials. Because the prescription for ADHD patients were generally based on clinicians’ discretion and patients’/caregivers’ willingness in the clinical settings, the effect were likely influenced by selection bias or rating bias.3. If the authors considered that observational studies, not only randomized controlled trials, are also important for assessing efficacy or effectiveness of pharmacotherapy. I suggest some epidemiological studies using claims data should also be considered in this study, and the indicators of effectiveness may be set as discontinuation rates or co-occurrence of comorbidity.Reviewer #2: This paper intends to examine an interesting topic summarizing differences between males and females with ADHD, regarding their clinical presentation, and response to pharmacotherapy, based on a literature review. The authors appropriately suggest two main aims, which include reporting on differences between females when compared to males regarding rates of prescriptions of ADHD meds, and responses to such medications regarding effectiveness or efficacy. The authors also review the literature in order to compare the sexes based on their tolerability to medications. I liked the topic, but found the paper hard to read due to its typographical and other errors. Please see the attached review.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentPlosOneSexDifferencesADHDMeds 06-14-20.docxSubmitted filename: Click here for additional data file. 6 Jul 2020Please see attached Rebuttal letters.AttachmentRebuttal letter Reviewer #2.docxSubmitted filename: Click here for additional data file. 3 Sep 2020The female side of pharmacotherapy for ADHD – a systematic literature review.PONE-D-19-29154R1Dear Dr. Kok,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Gerard Hutchinson, MDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have adequately address my questions. The authors make a great contribution for this research field. I have no further comment.**********what does this mean?). If published, this will include your full peer review and any attached files.7. 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Conclusions: The majority of male primary school teachers in Abha City, Saudi Arabia, have inadequate knowledge about ADHD. Applying a knowledge improvement program can substantially improve their knowledge of ADHD. Consideration should be given to the integration of ADHD knowledge improvement program into teachers’ educational programs.Background: Adequately knowledgeable schoolteachers can play an essential role in early detection and proper treatment of children with attention-deficit/hyperactivity disorder (ADHD) at school. Objectives: To assess the schoolteachers’ knowledge of ADHD and to evaluate the effectiveness of the ADHD knowledge improvement program. Methods: A randomized controlled trial study design was followed on 100 primary school teachers from Abha City, Saudi Arabia. Teachers were randomly selected and allocated into trial and control groups. A self-administered questionnaire was used to evaluate teachers’ knowledge about ADHD. After the baseline assessment, participants in the trial group received a two-day ADHD knowledge improvement program. Teachers’ knowledge about ADHD was reassessed using the same questionnaire immediately after finishing the intervention program and after three months in both groups. Results: Only 16% and 22% of trial and control groups, respectively, showed adequate baseline knowledge about ADHD. Teachers’ main sources of information about ADHD were the internet (67%), friends (47%), TV (34%), and reading books (23%). Knowledge of teachers in the trial group significantly improved immediately after the intervention. After three months, these knowledge benefits slightly declined but remained more adequate than those of teachers in the control group. The logistic regression model revealed that being a participant in the trial group and of higher qualification were associated with significant improvement in teachers’ retained knowledge on ADHD ( Attention-deficit/hyperactivity disorder (ADHD) is one of the most common chronic mental disorders that can affect school-aged children. It is characterized by a persistent pattern of impulsivity, hyperactivity, and inattention problems ,2. ADHD Worldwide, the prevalence rates of ADHD ranged from 2.2% to 17.8% . A more Primary school teachers play an essential role in the assessment of children’s behaviors and can be the first ones to identify children with ADHD . AdditioDifferent questionnaires and self-reports are available to assess teachers’ impressions of a potential student with ADHD. The most common tool used was Conners’ rating scales . In SaudRecently, some tools invented to help schoolteachers in the diagnosis and behavior management of students having ADHD. Teachers should be aware of their existence, as well as their usefulness. These tools include Aula Nesplora in primary schools  and AquaSeveral studies have recommended educational program intervention to improve teachers’ knowledge about ADHD and consequently their practice with children having ADHD ,12,17,18The objectives of the present study were to assess the schoolteachers’ knowledge regarding ADHD and to evaluate the effectiveness of the ADHD knowledge improvement program among male primary school teachers’ in Abha City, Saudi Arabia.The present study is quantitative community-based research. It followed a randomized controlled trial research design and carried out during the period from September 2019 to January 2020 on governmental and private male primary schools in Abha city. Abha city is the capital of the Aseer region, which located in the southwest of Saudi Arabia. It incorporated 51 public schools and 12 private schools with a total of 1341 male teachers: 1091 in public schools, and 250 in private schools.The target population was male teachers at primary schools in Abha city. The sample size was calculated based on Zhong , with thA stratified two-stage cluster sampling technique was used. Firstly, schools were stratified by type of schools into public and private. Then, eight public schools and two private schools were selected by a simple random technique. Next, from each school, the teachers were enlisted, and 10 male teachers were selected by a systematic random sampling method from each school. None of the schools involved in the study has school counseling services.To avoid contamination, the selected schools were randomly allocated to either the trial group or control group using computer-based randomization. Each group included 50 teachers from four public primary schools and one private school. A self-administered questionnaire, which was developed by Awadalla et al. , was useThe Data collection tool was used to assess socio-demographic characteristics and the baseline knowledge about ADHD for both experimental and control groups. After the baseline assessment, participants in the trial group received a two-day ADHD knowledge improvement program. Teachers’ knowledge about ADHD was reassessed in both groups using the same questionnaire immediately after finishing the program sessions and after three months interval to detect retained knowledge.The program was developed by the first author after reviewing the current literature, the World Health Organization’s Mental Health Gap Action Program Intervention Guide (MhGAP-IG)  and the It was conducted during a two-day workshop. On the first day, participants attended a one-hour lecture about ADHD covering the following: an overview of ADHD; the prevalence in Saudi Arabia; causes, symptoms and risk factors; associated impairment and prognosis; different treatment approaches, including behavioral interventions and medication. This lecture was done using Microsoft PowerPoint with audiovisual aids. At the end of the session, open discussion with the participants was done to answer there inquires. A hard copy of the lecture was given to the participants and the school director. On the second day, they attended a workshop about the teachers’ toolkit for ADHD, which contained how to identify ADHD and strategies for dealing with ADHD students in the classroom. Brochures about this disorder were distributed at the end of the program. Teachers attended the program in their schools using the library or the learning resource room with a maximum of ten teachers for each session. Educational materials were offered to the control group after the third assessment.p-values < 0.05 were considered as statistically significant.Data entry and analysis were performed using the Statistical Package for Social Sciences . Quantitative variables were presented as means and standard deviations, while categorical variables were presented as frequencies and percentages. Chi-square test and Friedman test were applied to assess differences between the trial and control groups. The Mann–Whitney U test was used to compare the overall knowledge score of trial and control groups at different points of assessment. Hierarchical multivariate logistic regression analysis was used to assess the individual effect of intervention after controlling all co-variance. The dependent variable was teachers’ knowledge at the third month post intervention, and the independent variables were: study group , age (less than 40 years old vs. above 40 years), nationality (non-Saudi vs. Saudi), qualification (postgraduate vs. university), experience (above 10 years vs. below), school (private vs. public). The research proposal was submitted and approved by the Research Ethical Committee in General Directorate of Health Affairs, Aseer Region (REC-NO:2-8-2019). An official approval was obtained from the Directorate of Education in Abha city and the schools’ authorities. An informed consent was obtained from all participant before data collection. The informed consent included their voluntary participation, the right to withdraw from the study at any time without penalty or loss of any benefit from the institute/organization and assured confidentiality and privacy of participants.The present study included 100 male primary school teachers selected from eight public schools and two private schools (10 teachers from each school). They were randomly allocated into trial and control groups (four public and one private schools in each group). To minimize the possibility of contamination, different schools were assigned for trial and control groups. Most of the teachers (51%) were less than 40 years old, Saudi (81%), having a university degree (86%), and 10–19 years of experience (48%). All of them have a Bachelor of Education and 14% have a postgraduate degree. They represented different teaching classes at primary schools. Most of them teaching Arabic (24%), religion (21%), and mathematics (17%) courses. There were no statistically significant differences between the trial and the control groups regarding participant’ characteristics. All participants did not previously receive any training about ADHD.All teachers in both groups were aware about ADHD. The teachers’ main sources of information about ADHD were the internet (67%), their friends (47%) and the television (34%). Books were the sources of information for only 23%.p > 0.05).According to p > 0.05). However, significant improvements were detected only in two items, i.e., the progress of the disease and treatment lines .Based on p = 0.001) and after three months (p = 0.01).Regarding the overall knowledge score, immediately after the intervention, the overall knowledge of teachers in the trial group became more adequate than that in the control group . After three months, knowledge of teachers in the trial group remained more acceptable than that in the control group . Furthermore, p < 0.001 and p = 0.040, respectively).Result of multiple hierarchical logistic regression model shown in Teachers spend a long time at school with their students. They are usually aware of students’ behaviors in different classroom situations. Therefore, they expected to be the first to observe and detect ADHD among their schoolchildren . UnfortuThe present study revealed that the most important sources of ADHD information, in order, were as follows: internet, friends, television, and books. Similar results were reported in Riyadh, Saudi Arabia , and in The baseline status of teachers’ knowledge regarding ADHD in the present study confirms the need for effective schoolteachers’ intervention . The curThe results of our study showed that the overall knowledge of teachers in the trial group about ADHD was significantly improved immediately after the intervention. Then, these knowledge benefits were slightly declined but could last for three months and remained more adequate than those in the control group. The substantial improvement in teachers’ knowledge about ADHD following the intervention in the present study is in agreement with the findings reported by several studies using educational or training methods, which have all shown rapid improvement in teachers’ knowledge about ADHD, with benefits remaining for up to 6 months ,33,34,35In comparison with previous interventional studies where training has to proceed throughout several days  and by tThe results of the multivariate logistic regression model revealed that attending the educational program on ADHD was the most significant predictor for teachers’ post-intervention retained knowledge about ADHD. The two-day face-to-face workshop implemented in the present study is effective in improving ADHD knowledge among attended teachers. However, the observed decline in the retained knowledge score at three months among the trial group highlights the need for periodic reminder training . UpdatinThe multivariate logistic regression model revealed that having postgraduate qualification was associated with significant improvement of teachers’ retained knowledge. These results are in accordance with findings of previous research from Saudi Arabia, which have consistently shown more qualified teachers and those who received educational programs on ADHD had more adequate knowledge about ADHD ,38. ThesAlthough acquiring enough knowledge about ADHD is necessary to have appropriate an attitude and proper practice , the preAlthough the present study confirms the effectiveness of the intervention program in improving teachers’ knowledge. Few limitations should be considered in interpreting our results. Firstly, due to strict time constraints, the duration of the intervention and follow up was only three months. Therefore, the long-term impact of the applied knowledge improvement program could not be assessed. However, the decline of retained knowledge in the third month may indicate the need to renew training and long-term impact assessment. Secondly, we used a knowledge assessment tool to explore the program’s effectiveness. An on-site practice assessment may be also necessary to confirm teachers’ performance improvement. Another limitation in questionnaire coding, we combined both incorrect and do not know responses together and gave them the same score. This issue hardens the distinction between teachers’ misconceptions and lack of knowledge. Finally, the study involved only male primary school teachers in Abha city. This issue may minimize the generalizability of our results.The study highlighted the inadequacy of primary school teachers’ knowledge about ADHD in Abha city, Saudi Arabia. A two-day ADHD education program is effective in improving teachers’ knowledge. Consideration should be given for integrating the ADHD knowledge improvement program into teachers’ educational and training programs. This program should be part of their continuous professional development. Periodic reminder training is suggested to update and refresh their knowledge. Field assessment may be necessary to confirm teachers’ practice improvement. Further studies are needed in various regions of Saudi Arabia, with a longer follow-up duration to evaluate the long-term impact of the program. Building up trustworthy ADHD online resources is recommended to improve teachers’ capacity and to protect them from unreliable information.


We investigated infant’s manual motor behaviour; specifically behaviours crossing the body midline. Infants at elevated likelihood of Autism Spectrum Disorder (ASD) and/or Attention Deficit Hyperactivity Disorder (ADHD) produced fewer manual behaviours that cross the midline compared to infants with a typical likelihood of developing these disorders; however this effect was limited to 10-month-olds and not apparent at age 5 and 14 months. Although, midline crossing did not predict ASD traits, it was related to ADHD traits at 2 years of age. We rule out motor ability and hand dominance as possible explanations for this pattern of behaviour, positing that these results may be a consequence of multisensory integration abilities, and the neurobehavioural shift period, in the first year of life.The online version of this article (10.1007/s10803-020-04489-1) contains supplementary material, which is available to authorized users. Transdiagnostic approaches to neurodevelopmental disorders have gained increasing traction over the last decade  and Attention Deficit Hyperactivity Disorder (ADHD). Both are early-onset neurodevelopmental disorders that can be diagnosed by early to mid-childhood. Both significantly co-occur, with 20–50% of children diagnosed with ASD also meeting the diagnostic criteria for ADHD . For example, it may be possible that midline crossings in the first year of life could potentially predict later ASD/ADHD traits at 2 years of life.The current study examined the emergence and development of spontaneous midline crossing behaviours in infants with a first degree relative (older sibling or parent) with ASD and/or ADHD. Infants with an older sibling with ASD have an approximately 20% chance of developing ASD themselves  or on the caregiver’s lap, with the caregiver holding their trunk (5 month protocol). A box of foam (5 month protocol) or wooden (10 and 14 month protocol) blocks were placed on the table top in front of the infants. The blocks were always placed within reach of the infants, if they were ever positioned out of reach, either the researcher or the parent would place them back within reach. Three custom built cameras recorded infant’s behaviours . At the time of enrolment, none of the infants had a known medical or developmental condition. Informed written consent was provided by the parent(s) prior to the commencement of the study. The testing only took place if the infants were in a content and alert state. Ethical approval was granted by the National Research Ethics Service and the Research Ethics Committee of the Department of Psychological Sciences, Birkbeck, University of London. Participant families were reimbursed expenses for travel, subsistence and overnight stay if required. Infants were given a certificate and t-shirt after each visit.confirmed presence of the relevant condition. Ratings were primarily based on the presence of a clinical diagnosis of ASD or ADHD. This approach allowed us to test the effect of familial likelihood of ASD, familial likelihood of ADHD, and their interaction .Each infant in the study was assigned a rating for familial likelihood of ASD and ADHD. A rating of 1 for ASD indicates the presence of ASD in a parent or older sibling; a rating of 1 for ADHD indicates the presence of ADHD in a parent or older sibling; and a rating of 0 for either category indicates no Our sample for this paper includes: 81 infants with an elevated likelihood of ASD (ASD-L), 31 infants with an elevated likelihood of ADHD (ADHD-L), 20 infants with an elevated likelihood of both ASD and ADHD (ASD + ADHD-L) and 29 infants with a Typical Likelihood for either ASD or ADHD (TL). A number of participants were excluded from the final analyses for fussy behaviour (n = 5 at the 5-month timepoint) or technical difficulties with the video recording . See Table Infant’s manual motor behaviours were coded for as long as the blocks were in front of them. Broadly, behaviours fell into three categories: ‘reaches’, ‘hand movements’ and ‘object manipulation’. A motor behaviour was considered a reach if infants extended their hand to grasp an object. If, however, the infant extended their arm and placed their hand on top of the object, without the grasping motion, this was considered a ‘hand movement’. Other ‘hand movements’ included sub categories such as: waving hand/arm, placing hand in mouth, and sliding/swiping hand where the infant is moving their hand in the space around them without grasping or contacting a block. Any instance of the infant acting upon the block was considered ‘object manipulation’, e.g., banging blocks, throwing blocks, putting blocks in the mouth. Every behaviour was coded; for example, if the infant banged a block three times in a row, this would be considered three distinct ‘object manipulation’ behaviours. Every behaviour was also coded for which hand (left or right) carried it out. If both hands worked in unison, with actions starting and ending at the same time , this was coded as ‘both hands’ for the one action. If, however, the hands were not working in unison (but were engaging in similar actions asynchronously), this was considered to be two distinct actions and the handedness was assigned to each action separately. Crucially, every behaviour was also coded for whether infant’s crossed their body midline to engage in the behaviour. For example, did the right hand pass into the left side of space when the infant was executing a reach? If the hand crossed the body midline, this was considered a ‘contralateral’ action. If the hand did not pass the body midline and stayed within the same side of space, this was considered an ‘ipsilateral’ action. Every action was also marked with the time stamp in the video for when the behaviour occurred.All videos were coded by one researcher (JBA). Twelve percent of the videos were randomly selected and double coded by a second researcher who was blind to the group status of each infant. Intraclass correlations  were conducted on the coded variables. A high level of reliability was found for the following coded behaviours: ‘Total midline crossings’  ‘Contralateral hand movements’ (ICC = 0.73), ‘Contralateral object manipulations’ (ICC = 0.79), ‘Ipsilateral reaches’ (ICC = . 84), ‘Ipsilateral object manipulations’ (ICC = 0.89) and ‘Total behaviours’ (ICC = .92). A moderate level of reliability was found for the following coded behaviours: ‘Contralateral reaches’ (ICC = 0.7) and ‘Ipsilateral hand movements’ (ICC = 0.67); all ps ≤ 0.001).As we were also interested in determining how related midline crossing behaviours were to phenotypical characteristics of ASD and/or ADHD, we also included data from a number of parent report measures at 2 years. At this age, we do not make clinical determinations about the presence or absence of ASD/ADHD. We are currently re-assessing this sample at an older age, and when that process is complete we will be able to show how the motor behaviours reported here relate to categorical diagnostic outcome.Parents completed the Quantatitive Checklist for Autism in Toddlers , we calculated the “rate per minute” for each behaviour (frequency of the behaviour/duration of task).types of behaviour; reaches, hand movements and object manipulations), to examine if there was a type of motor behaviour that especially contributed to ipsilateral or contralateral behaviours.Separate linear mixed models (LMMs) with the following dependent variables were run: ‘Total contralateral behaviours’, ‘Contralateral reaches’, ‘Contralateral hand movements’, ‘Contralateral object manipulations’, ‘Total ipsilateral behaviours’, ‘Ipsilateral reaches’, ‘Ipsilateral hand movements’, ‘Ipsilateral object manipulations’ and ‘Total behaviours’. We decided to decompose the ‘Total behaviours’ variable , Sex  and Likelihood . The Likelihood factor was computed as follows: each infant was given a score of ‘0’ or ‘1’ for ASD and a score of ‘0’ or ‘1’ for ADHD, depending on familial history of these disorders. As such, an infant with a first degree relative with ASD, but not ADHD would be assigned a ‘1’ for ASD-L and a ‘0’ for ADHD-L. An infant with a first degree relative with ADHD, but not ASD would be assigned a ‘0’ for ASD-L and a ‘1’ for ADHD-L. An infant with familial history of both disorders would be assigned ‘1’ for ASD-L and ‘1’ for ADHD-L and an infant with no familial history of either disorder would be assigned a ‘0’ for ASD-L and a ‘0’ for ADHD-L. We computed likelihood in this way so we could examine the interaction of the two disorders and if there were any additive/protective effects of having an elevated likelihood of both disorders.To account for slight variations in age across infants, infants’ exact age (in days) was added as a covariate to each model. The repeated covariance type was set as ‘compound symmetry’ and the maximum likelihood estimate was used for each model.For follow up analyses (where LMMs indicated interactions between elevated likelihood groups) and figures, the more traditional elevated likelihood approach was taken and infants were split into four groups: EL-ASD, EL-ADHD, EL-ASD + ADHD and TL.ηp2 = 0.06], with means indicating that the frequency of crossing the body midline to conduct a manual action in the contralateral side of space increased with age  = 5.17, p = 0.006, ηp2 = 0.04]. No other significant effects were found.The LMM revealed a main effect of Time  or 14 months . However, there was a significant interaction at 10 months . Follow-up analyses showed that, at 10 months of age, the TL group engaged in more contralateral behaviours that involved crossing the midline than both the EL-ASD  and the EL-ADHD groups  and the EL-ASD + ADHD group, though the latter did not survive Bonferroni correction . Further, no differences were found when comparing the EL-ASD group with EL-ADHD , EL-ASD with EL-ASD + ADHD  and the EL-ADHD group compared with the EL-ASD + ADHD group  at 10 months of age.To examine the contributions of ASD and ADHD likelihood on contralateral behaviours, we conducted separate LMMs at each time point (using the same model parameters as above but removing Time point as a repeated and fixed factor). These showed there were no interaction effects of ASD-L*ADHD-L at 5 , with means indicating that this group produced overall fewer reaches that involved crossing the midline  = 4.64, p = 0.03, ηp2 = 0.03], with means indicating that those with a Typical Likelihood of ASD performed a greater number of contralateral hand movements compared to those with an elevated likelihood of ASD  performed a greater number of contralateral hand movements compared to those with an elevated likelihood of ASD (EL-ASD) at the 10 month time point  but not at the 5  or 14  time points.The LMM analysis revealed a significant main effect of ASD-L  and Sex , with means indicating that there is an increase in these behaviours as infants age and that male infants carry out a greater number of object manipulations respectively.The LMM revealed a significant main effect of Time . Examining the data at each time point, LMMs indicated that the above interaction was only significant at the 10 month time point  and not the 5  or 14 month time points .Further to this, the analysis showed a significant interaction between Time*ASD-L*ADHD-L   = 0.3, p = 0.59, ηp2 = 0.004], ADHD-L  and no interaction of ASD-L*ADHD-L . We also examined ipsilateral behaviours by category.Control analyses of ipsilateral behaviours across Time Fig.  indicateηp2 = 0.03]  = 2.07, p = 0.048, d = 0.8].Ipsilateral reaches increased with Time , but was not influenced by ASD-L , ADHD-L  or ASD-L*ADHD-L ,  = 18.12, p < 0.001, see Fig. c.ηp2 = 0.06], but did not interact with ASD-L , ADHD-L  or ASD-L*ADHD-L ,  = 8.8, p < 0.001, ηp2 = 0.25] with means indicating that male infants perform more manual behaviours compared to female infants. Figure The LMM showed a main effect of Sex  or Fine motor scales  respectively  = 3.79, p = 0.054, ηp2 = 0.03]. However, total midline crossings did not significantly predict Gross motor skills at the 10 month time point  or Fine motor skills at 24 months of age .Further, we also conducted a number of linear regression analyses to determine whether total midline crossings at 10 months predicted Gross and Fine motor skills at the 10 and 24 month time points (see Table ηp2 = 0.1] with means indicating that participants performed more behaviours with their right hand compared to their left hand (indicating a right hand dominance across participants). However, there were no main or interaction effects of Group . Further, the total number of Right/Left hand behaviours did not correlate with the proportion of total behaviours that involved crossing the midline .A further potential explanation could relate to infants’ handedness. To explore this, we first calculated the laterality quotient for each infant at the 10 month time point (proportion of ‘Right’ hand behaviours – proportion of ‘Left’ hand behaviours) and then used this quotient to classify hand dominance . Considering that we were only investigating this at the 10 month time point (where significant differences between groups had been shown previously), there was no need to conduct LMMs as above. Instead, we conducted a mixed ANOVA to examine group differences in handedness. Here, we found a found a main effect of Handedness .To examine whether TL infants perform more contralateral behaviours with their dominant hand, an analysis contrasting the proportion of contralateral behaviours performed with the dominant hand across group was conducted. A univariate ANOVA found no significant differences in the proportion of contralateral behaviours performed with the dominant hand across Group , with more contralateral behaviours related to greater ADHD traits. However, total contralateral behaviours did not significantly predict ASD traits on the QChat . Including the entire EL sample and conducting nonparametric bivariate correlations, we found a significant relationship between total midline crossings and the CBCL ADHD subscale  and a trend between total midline crossings and the QChat .We examined the relationship between the frequency of contralateral behaviours and whether they were related to ASD and/or ADHD traits. Examining scatter plots of total midline crosses by the various measures indicated 2 outliers  = 0.44, p = 0.51, ηp2 = 0.005]. In order to examine the relationship between total midline crossings and the Total CBCL score, we conducted a multiple hierarchal regression analysis  and found that Total contralateral behaviours and ADHD CBCL scores explained a significant amount of variance in the CBCL Total score . However, much of this explained variance was a result of the ADHD subscale score , with no relationship between total contralateral behaviours and CBCL Total scores . Further to this, if we conduct the same multiple hierarchal regression analysis with the PDP subscale , we find similar results; Total contralateral behaviours and ADHD CBCL scores explained a significant amount of variance in the CBCL PDP score ; however it is only the ADHD subscale that significantly explains the variance in the model .To investigate whether midline crossing behaviours show specificity to one neurodevelopmental disorder , we also examined the Pervasive Developmental Problems (PDP) subscale of the CBCL as well as the Total CBCL score. The PDP maps well onto DSM-5 criteria for ASD, whilst the Total CBCL score encompasses a range of internalising/externalising problem behaviours in children. A linear regression found no relationship between total contralateral behaviours and raw scores on PDP subscale  or the Total Calibrated Severity .The above measures were parent report questionnaires to examine ASD and ADHD traits. We also conducted a number of clinical/research based assessments to investigate the relationship between contralateral motor behaviours and ASD symptoms. For example, examining the ADOS-T, we found that total midline crosses performed at 10 months did not significantly predict ASD traits on the Calibrated Severity Score for Restrictive and Repetitive Behaviours [RThe current study was the first to examine midline crossing behaviours in a naturalistic, longitudinal design. We demonstrated, for the first time, that (at 10 months of age), infants with an Elevated Likelihood of ASD or ADHD produced fewer manual actions that involved their hand passing the body midline and crossing into the contralateral side of space. However, it must be noted this pattern of findings was only exhibited at the 10 month time point and not at either the 5 or 14 month time point. In comparison, our Typical Likelihood sample demonstrated ‘n shaped’ development, with midline crossing behaviours increasing significantly at 10 months (compared to the Elevated Likelihood groups) before decreasing at 14 months of age and becoming more in line with the EL groups. A number of control analyses ruled out differences in hand dominance and motor ability as possible explanations for this finding. Indeed, midline crossing did not seem to be related to large scale motor development. We discuss a number of potential accounts for our findings below.usually lies in space, perhaps they adopt more ‘usual’ postures and less ‘unusual’ postures, such as crossing over of the arm into the opposite side of space.Firstly, a possible suggestion is that our EL groups are more rigid in the way they segment peri-personal (near the body) space. It may be that infants with an Elevated Likelihood of ASD or ADHD segment space according to left/right parameters and are much less likely to cross the midline boundary with an ‘incongruent’ limb . Indeed, research with children with ASD has suggested that this population relies much more heavily on an anatomical reference frame when locating the hands in space  and demonstrate a very similar profile to that of ASD likelihood. Indeed, attention shifting and multisensory integration atypicalities have also been found in ADHD populations . It is important to thus consider that EL groups may be engaging in more ‘efficient’ manual behaviours through completing primarily ipsilateral behaviours. When differences are found between EL groups and typical likelihood infants, researchers can often view these differences as challenges or difficulties for the EL groups. However, it is important to recognise that these differences may illustrate early strengths or compensatory mechanisms within the EL groups. This stresses the importance of examining neurodevelopmental disorders with the perspective of both strengths and limitations.In terms of the TL group, we must ask what could explain the ‘n shaped’ developmental trajectory demonstrated in this group, with midline crossing behaviours peaking at 10 months (and significantly greater than the EL groups) and then decreasing dramatically to be more in line with the EL groups at 14 months? Here, we must reiterate that this was a fairly surprising finding given that previous research indicated incremental increases in midline crossing behaviours as typically developing infants aged  examined visual foraging behaviours in infants with an older sibling with ASD and either low or high levels of hyperactivity and inattention. The researchers showed that, at 8 months of age, the EL-ASD low hyperactivity and inattention group were more likely to return to areas they had previously visited in the visual scene, as compared to the EL-ASD high hyperactivity and inattention group, which was more in line with results from the TL group. This study both demonstrates that ADHD traits mitigates EL-ASD behaviours and that ADHD traits may be linked to less rigid patterns of spatial exploration. However, these reduced levels of spatial rigidity may not be enough to completely overcome differences in midline crossing behaviours, which is why we see a significant difference between our EL groups with ADHD and the TL group.Taken together, the present study found that infants with an Elevated Likelihood of ASD or ADHD  demonstrated significantly fewer manual behaviours that involved the hand crossing the body midline and passing into the contralateral side of space. We have ruled out motor abilities and hand dominance as possible accounts of our findings. Importantly, we have found the same pattern of behaviour for two EL groups: EL-ASD and EL-ADHD, which points to subtle changes in motor behaviours that are not specific to either disorder. As such, midline crossing behaviours may potentially be a transdiagnostic early risk factor. This reiterates the necessity to examine behaviours across multiple disorders; as otherwise, we would have interpreted midline crossing behaviour differences to be syndrome specific to ASD or ADHD. Examining the relationship between this index and ASD/ADHD traits demonstrated a predictive relationship with ADHD traits; this would suggest that whilst midline crossing behaviour manifests in infancy in the same way in these EL cohorts (in that it is reduced), it may be more specifically related to ADHD traits later on in toddlerhood.Furthermore, the current study employed a nuanced coding scheme that decomposed a broad behaviour (crossing the midline) into categories that tapped into subtly different types of behaviours. As a result of this nuanced coding scheme, one sub-category of crossing the midline behaviours (object manipulations) may lend itself to be a specific early risk marker, potentially differentiating between ASD and ADHD; however this requires further investigation.Supplementary file1 (DOCX 711 kb)Below is the link to the electronic supplementary material.