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&(&(&(b;&(&(&(&(��$�	�z"���	z"�������������Sleep characteristics in patients with Autism spectrum disorders.
Fructuoso Ayala-Guerrero*, Graciela Mexicano, Sarah� Huicochea-Arredondo
Laboratorio de Neurociencias, Facultad de Psicolog�a. Universidad Nacional Aut�noma de M�xico.
Avenida Universidad No. 3004, Ciudad Universitaria. C.P. 04510



Corresponding autor*: Tel. 5622-2222 #41243
e-mail: fayala@unam.mx


Abstract
Autism spectrum disorder (ASD) is a heterogeneous, behaviorally defined, neurodevelopmental disorder. Patients with ASD might also have co-morbid disorders such as intellectual impairment, epilepsy and anxiety. Findings from questionnaire studies have revealed the existence of several sleep problems in pediatric patients with ASD 
 While parental sleep concerns have been well documented in children with ASD by means of sleep questionnaires, few studies have related these concerns to polysomnographic (PSG) findings. 
On the other hand, about a third of people with autism also suffer from epilepsy. Therefore long-duration electroencephalograms that include an adequate amount of delta sleep should be carried out in order to detect epileptiform activity.
The aim of this work is to describe the sleep characteristics and to detect EEG disturbances in ASD patients by means of polysomonographic recordings.
Methods. Polysomnographic recordings for two consecutive nights were carried out in 12 autistic children and compared to age and sex-matched controls. Sleep efficiency as well as percentages of each sleep phase were obtained from the two groups of participant children. Distribution of SWS and REM sleep throughout the night was also obtained and compared between both groups. At the same time EEG characteristics were analyzed and compared 
Results. ASD children presented low sleep efficiency, fragmented sleep and reduction in both SWS and REM sleep. Epileptifom brain activity was observed in 50% of ASD children.
In conclusion ASD patients presented quantitative and qualitative sleep disturbances as well as EEG abnormalities.

Keywords:  SWS, REM sleep, Autism spectrum disorders, epileptiform activity.



Introduction
Autism spectrum disorder (ASD) is a heterogeneous, behaviorally defined, neurodevelopmental disorder that occurs in 1 of 150 children (1) Amaral, et al., 2008)1. Individuals with autism have deficits in social interaction and verbal and nonverbal communication, as well as in restricted or stereotyped patterns of behavior (2) DSM-IV 2000)2. These patients might also have co-morbid disorders such as intellectual impairment, epilepsy and anxiety. Epilepsy is associated with pathology of multiple brain regions where the cerebral cortex, amygdale, cerebellum and hippocampal formation are included. These brain regions have also been implicated in autism (1) Amaral et al., 2008)1. Postmortem and magnetic resonance imaging studies have highlighted the frontal lobes, amygdale and cerebellum as pathological in autism. However, there is no unambiguous and consistent pathology that has emerged for this neurodevelopmental disorder. Additionally, recent studies emphasize that disturbance in the time course of brain development is fundamental in ASD (3) Wegiel et al., 20103). It has been suggested that the heterogeneity of both the core and co-morbid features is related to the heterogeneous pattern of neuropathology observed in ASD patients. 
In this context, defined phenotypes in larger samples of children and well-characterized brain damage are indispensable for clarification of the neuroanatomy of ASD.
The neurological and social deficits observed in autistic patients contribute to varying levels of impairment. Recent advancements in autism research have improved the understanding of the disorder; however numerous questions about the pathophysiology of ASD remain without precise answers. 
The expression of ASD is extremely heterogeneous, owing to the complex interactions between genes, the central nervous system, and behavior throughout development. Therefore, some researchers refer to �the autisms� or ASD rather than a single autism phenotype HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0166223608000386%20\\%20bib1"(4) Geschwind and Levitt, 2007)4.
Within the ASD patients, variability in the social domain varies from a near absence of interest in interacting with others, to more slight difficulties managing complex social interactions, where an understanding of other people�s goals and intentions and other cues of social context are implicated. Likewise, stereotyped and repetitive behaviors vary from simple motor stereotypes, to much more sophisticated rituals, accompanied by emotional disruption when these rituals are interrupted. Some patients with ASD lack basic speech abilities, while others can have language deficits that are only limited to pragmatic language. While a majority of individuals with ASD exhibit some level of intellectual impairment, intelligence quotients vary from the severe and profoundly impaired to above average.
On the other hand, sleep disturbances have commonly been described in children with intellectual disabilities (5-7) Quine 19915; Espie and Tweedie, 19916; Stores, 19927). Numerous studies have evidenced that sleep problems in mental retarded children, are frequently related to the specific type of syndrome or to its etiological characteristics (8) Richdale, 19998).
In pediatric patients with ASD, findings from questionnaire studies have revealed the existence of several sleep problems with respect to other groups of pediatric patients who have intellectual disabilities, (9,10) Schreck and Mulick, 20009; Wiggs and Stores, 200410). Parents of ASD children report a high prevalence of sleep problems, such as difficulty falling asleep, fragmented sleep by frequent awakenings and reduced total nocturnal sleep time (11) Gail et al., 200411) These sleep disturbances have been hypothesized to have a predictive role in the presence of autistic behavior during the day (12) Schreck et al., 200412).
While parental sleep concerns have been well documented in children with ASD by means of sleep questionnaires, few studies have related these concerns to polysomnographic (PSG) findings. Describing PSG abnormalities provides objective evidence of sleep disturbances in these patients.
Although sleep disturbances are not part of the diagnostic criteria for ASD, these disturbances are commonly present in autistic children (12-15) Malow et al., 200613; Hering et al., 199914; Richdale, 199915; Schreck, et al., 200412). Additional research is needed to clarify the amount and quality of the sleep problems experienced by these patients compared to healthy children (16) Durand, 200216).
It has been described a variety of several daytime behaviors, related to sleep disturbances for autistic patients. Some authors have suggested that the intensity of developmental disturbances (17) Glascoe et al., 199117) and general ASD symptoms (18) Malow and McGrew, 200618) are increased in relation to sleeping problems. Studies pointing out the relationships among some of the key diagnostic factors in ASD (e.g., communication difficulties, social problems, stereotypic behavior and difficulties breaking routines) have indicated that intensified symptoms in these areas are related to disturbed sleep (12) Schreck et al., 200412). Other general daytime behavior troubles have been reported as related to sleep problems for people with an ASD, such as overactivity, psychopathology, and disruptive behavior (19) O�Brien, 200919).
On the other hand, about a third of people with autism also suffer from epilepsy. Therefore long-duration electroencephalograms that include an adequate amount of delta sleep should be carried out in order to detect epileptiform activity. Tuchman and Rapin (20) described that the incidence of epilepsy in children with autism ranged from 5% to 40% while Hughes and Melyn (21) reported that the electroencephalograms of 75% of the children with autism showed abnormal patterns of electrical activity in the brain and 46% had seizures.�
The relationship between epilepsy and ASD is poorly understood. It is not known whether epileptic activity in these patients is a secondary phenomenon or whether, in some situations, it is responsible for some symptoms that occur in people with ASD. This is partly because epileptic syndromes are often associated with cognitive, language and behavioral dysfunction, all of which are traits of this neurodevelopmental disturbance.

Objective 
The aim of this work is to describe the sleep characteristics and to detect EEG disturbances in ASD patients by means of polysomonographic recordings.
Materials and Methods
We followed the guidelines of the Declaration of Helsinki (22) BMJ 1991)22 in obtaining the consent of parent of children to participate. Besides, the research project was approved by the ethical committee of the institution where the study took place. 
Children with a clinical history of ASD were recruited from a local autism society. Typically developing (TD) children of comparable sex and ages were invited to participate as control subjects. Autistic patients received a clinical diagnosis of ASD from psychologists or clinicians based on parent interviews, children observation, and direct testing. These clinical diagnoses of ASD were verified using the autism diagnostic observation schedule (23) Lord et al., 2000)23. All autistic children, aged between 4 and 10 years, were not taking psychotropic medications, and did not have a history of epileptic seizures or mental retardation.

Laboratory sleep study
A Grass Comet Model 25 Acquisition System was used for obtaining polysomnographic recordings for two consecutive nights from the 12 autistic children and compared to age- and sex-matched control. First night was considered as habituation night; therefore the compared findings were obtained from the second recording night.
All participants had the opportunity to go to bed at their preferred time therefore, sleep latencies were not evaluated. Sleep was recorded and scored using 30-s epochs according to standard methods, including central and occipital EEG (C3-A1, C4-A2, O1-A1 and O2-A2), submental EMG and periorbital EOG (24) HYPERLINK "http://brain.oxfordjournals.org/content/128/5/1049.full%20\\%20ref-54"Rechtschaffen and Kales, 196824). Oronasal airflow and thoracic and abdominal respiratory effort were also monitored throughout the night. 
Statistical analyses
Statistical comparisons were conducted between groups (children with ASD versus their comparison group) using student�s t-test and non-parametric Mann�Whitney u-tests with criteria for significance set at 0.05. 
Analysis of variance (ANOVA) per halves of night were also performed to evaluate the distribution of SWS and REM sleep throughout the night.

Results
During and immediately after electrode placement, some children with ASD showed anxiety and hyperactivity, therefore it was necessary to wait for them to calm down before the polysomnographic recording beginning. 
Control children displayed normal sleep distribution throughout the recording night (Fig. 1).
In contrast to healthy children, atypical characteristics of sleep were displayed by children with ASD. Patients presented fragmented sleep due to frequent interruptions provoked by intermittent awakenings (Fig. 2). They also showed more wakefulness after sleep onset, they woke up and fluctuated between wakefulness and stage 1 sleep more frequently than control participants.
Sleep onset and maintenance problems, as well as early morning awakening resulted in, lower sleep efficiency, increased stage 1 sleep and decreased delta sleep (Stage 3 sleep). Total REM sleep time was also reduced. All these variables were significantly different to those in healthy children (Table 1). Some patients with ASD were observed to lie awake for a long time.
Analyses of sleep stages distribution throughout the night showed that the autistic patients spent significantly more time in stages 1 and 2 sleep during the first half of the night, and less delta sleep than the control group.
Both children with ASD and control participants presented more delta sleep in the first half of the night (p < 0.05).
Difference in REM sleep distribution was significant between the halves of night in patients, and in control participants since REM sleep increased in the second half of the night.
Average duration of REM sleep episodes was shorter in patients with ASD (15.3� 5.8 min) than in controls (18.6 � 3.4 min), and they also showed less REM sleep episodes (Table 1).
On the other hand, although epileptic seizures were absent, 50 % of the studied patients had epileptiform EEG activity during light sleep (Fig. 3). The most frequent EEG abnormalities were observed in the central area (C3-A1 and C4-A2 leads) followed by the occipital area (O1-A1, O2-A2 leads). 

Discussion
A considerable amount of polysomnographic studies carried out in children with ASD have mainly focused on abnormalities related to REM sleep (25,26) Tanguay et al., 197625; Thirumalai et al., 200226). Thus Mahowald and, Schenck (27) diagnosed REM sleep behavior disorder without muscle atonia during REM sleep, in autistic children who had symptoms of disrupted sleep and nocturnal awakenings (26) Thirumalai et al., 200226). Although we noted a decreased in the amount of REM sleep, we did not observe abnormalities in muscle tone during this sleep phase. The absence of such findings in the autistic children of our study may reflect that they were not under medication. The absence of certain drugs, usually administered to patients with ADS, such as serotonin reuptake inhibitors may contribute to the lack of muscle atonia that normally is installed during REM sleep (27) Mahowald and Schenck, 200527).
As in other reports, we observed reduced sleep time; sleep fragmentation provoked by intermittent awakenings, and reduced sleep efficiency. However, our study evidenced a relative absence of other sleep disorders, such as sleep apnea and parasomnias, described by different authors.
Disrupted sleep continuity has been reported, with total sleep time correlating negatively with social and communication abilities (28) Cortesi et al., 201028). Thus it is probable that implementation of techniques for sleep continuity may improve these abilities.
Similarly ASD patients, compared to age and sex-matched controls, have showed prolonged sleep latency, increased duration of N1 and N2 stage sleep and decreased delta sleep.
On the other hand, it has been hypothesized that both epilepsy (29,30) HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0387760408002428%20\\%20bib68"Tuchman et al., 200929; 201030) and autism may result from severe alterations in cortical-subcortical systems connectivity. Alterations in neocortical minicolumns observed in autism support the idea of cerebral under connectivity (31) Tuchman and Cuccaro, 201131). According to Tuchman et al (29), alterations in subcortical systems such as basal ganglia-substantia nigra connectivity, may decrease the seizure threshold, contribute to cognitive impairments and to the motor stereotypes commonly found in autism. However, there are also hormonal influences on cortical and subcortical systems connectivity in the developing brain such as testosterone that have been implicated in both autism and epilepsy HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0387760408002428%20\\%20bib75"(32,33) Knickmeyer et al., 200532; Veliskov�, et al. 200433). These findings have shifted the center of attention from anatomical alterations to functional disturbances within a developmental process. 
Findings from different authors suggest that molecular abnormalities of neurons as well as network abnormalities would be responsible of the epileptogenesis and cognitive and socio-communicative disturbances usually present in autistic patients (3,34) Rubenstein and Merzenich, 200334; Wegiel et al., 20103).
Particularly, alteration in cortical minicolumns with selective sparsity of gabaergic interneurons is likely responsible for the increased seizure susceptibility HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0387760408002428%20\\%20bib72"(35,36) Casanova et al., 200235; HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0387760408002428%20\\%20bib79"Casanova et al., 200236). 
It has been described that epileptic seizures are present in up to one-third of ASD individuals (37) McDermott et al., 200537). On the same way, autistic characteristics are commonly observed in severe forms of epilepsy associated with mental retardation (38) Dravet, 200238). It has been suggested that the association between epileptic and autistic phenotypes is because of these diseases may share common predisposing genes. Some of the known epilepsy-predisposing genes are related to voltage-gated or ligand-gated ion channels (39) Noebels, 200339). Defects in synaptic proteins implicated in neurotransmitter release and synaptic vesicle flow are also involved in this mechanism, (40,41) Rosahl et al., 199540; Janz et al., 199941).
These findings provide the knowledge about the interaction between epilepsy and autism and to have a better understanding of the pathophysiology of these diseases that alter brain development. They will also contribute to the development of new treatment strategies
Interventional studies focused to improving sleep by means of behavioral treatments and pharmacological therapies, should be carried out in order to determine if improving sleep in children with ASD can also improve daytime behavior and autistic symptomatology.
Prognosis of children with autism requires to normalize sleep instability and to obtain a good sleep quality.
The strong association between ASDs and epilepsy suggests that common mechanisms of synaptic dysfunction may underlie both diseases. In the healthy brain, a balance of excitation and inhibition is essential for all functions, from physiological network activity and oscillations to cognitive processes. 
Taking into consideration these findings, monitoring children with ASDs for seizure and subclinical seizure activity is recommended.

Conclusions
The relation of epilepsy to ASD remains unresolved. The present findings suggest that there are common pathophysiological mechanisms involved both in the autism and the epilepsy. It will be necessary to identify specific neurobiological subtypes within the broader autistic spectrum in order to support our understanding of the genetics of some of the components of autism and to elucidate whether epilepsy or specific EEG findings in children with autism correspond to specific subtypes.
Further research should be carried out to determine whether treatment of sleep disturbances in autistic patients significantly improve daytime behaviors and reduce their symptomatology. Acknowledgement


This study was supported the DGAPA/PAPIIT/ UNAM 2009 Grant.





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Legends of figures
Fig. 1.  Hypnogram of a healthy child. Note the normal distribution of sleep.
Fig. 2.  Hypnogram of a child with ASD.  Disorganization of sleep patterns is observed.
Fig.  3.   Epileptiform activity during stage 2 sleep.
 Note that epileptiform discharges are observed on the left of EEG recordings.
Table 1. Means and standard deviations for PSG measures. *P < .05



Figures



Fig. 1




Fig. 2




Fig. 3
        

                           Quantitative data of sleep
Control (10)ASD (10)Total Sleep Time (min) 473.83 (33.26) 369.25 (29.45)* Stage 1 Sleep (%)  11.19 (0.9)19.02 (2.8)*Stage 2 Sleep (%)  48.09 (2.2)52.82 (4.1) Stage 3 Sleep (%)  18.94 (1.8)12.08 (2.1)*REM Sleep (%) 22.36 (1.71)16.14 (2.1)*REM Sleep Duration (min)18.6� 3.4 15.3 � 5.8 REM Sleep episodes5.45 (0.93)3.82 (1.24)*Sleep Efficiency90.94%75.08%*
Table 1.
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