Autism spectrum disorders (ASD) are a group of heterogeneous and fairly devastating neurodevelopmental disorders that include autism, Rett and Asperger syndromes and pervasive developmental disorder-not otherwise specified (PDD-NOS). Although several recent retrospective studies of toddlers indicated the presence of abnormalities in social, communication and play behaviour as early as 14 months of age 1 2 3 4 5 6 , autism often remains undiagnosed until the age of 3 years or later. Children with autism have deficits in reciprocal social interaction and communication and display repetitive and stereotyped behaviours 7 . The prevalence of autism, which has seemingly increased dramatically during the last decade 8 is estimated at one or two per 1000 for autistic disorder 9 10 and five or six per 1000 for ASD 11 , although the most recent estimates from the Centers for Disease Control in the USA indicate that as many as 1/150 8-year-olds in the USA may be affected 12 .

The causes of autism are still unclear, although results from twin and family studies provide evidence for a strong genetic contribution, with the probability of multiple genetic loci involved 13 . Less than complete concordance in monozygotic twins reveals the necessary role of non-genetic factors in the aetiology of autism. As the population prevalence of ASD has increased, results from research on fertility in the USA have demonstrated a parallel increase in the population mean age of both maternity and paternity 14 . This trend, coupled with findings that parental age is associated with some genetic disorders, makes parental age a likely candidate for epidemiological investigation of autism.

The first indication that paternal age might be associated with a genetic disorder was revealed in 1912, when Weinberg discovered that dominant achondroplasia occurred mainly in last-born children 15 . However, it was Penrose who first showed that the association was due to advanced paternal age, but not maternal age or the child's birth order 16 . Paternal age at the birth of offspring has been associated with an increasing risk for several congenital disorders 17 , including cleft lip and palate 18 , syndactyly 19 , situs inversus 20 , hydrocephalus 21 , Apert syndrome 22 and craniosynostosis 23 . Some other disorders associated with paternal age are childhood cancer 24 25 , miscarriage 26 , fetal death 27 and some autoimmune disorders 28 . Paternal age also appears to increase risk for both schizophrenia 29 30 31 32 33 34 and decreased intellectual capacity in offspring 35 36 . Advanced maternal age is associated with increased risk for chromosomal abnormalities such as Down's syndrome 37 , and has been associated with brain damage during pregnancy 38 , dyslexia 39 and idiopathic mental retardation 40 .

Results from studies of increasing paternal and maternal age and risk of autism are diverse 41 42 43 44 45 46 47 . Only a few studies have focused primarily on parental age 48 49 50 or adjusted for the other parent's age 46 48 50 . Results include reports that the age of both parents is associated with autism 51 , that paternal, but not maternal age, is associated 48 50 , that maternal but not paternal age matters 45 and that there is no association of autism with parental age 46 .

The present study is a case-cohort study, designed to investigate the association between parental age and risk of ASD in an Iranian population sample. Case and cohort comparison individuals were all preschool children, aged 4-11 (cases) or 5-7 (cohort comparisons), who were identified in the course of the first annual nation-wide screening of all preschool children in Iran for ASD and other developmental disorders. To our knowledge, this is the first Iranian epidemiological study of ASD; retrospective data are available from nearly 900,000 children per year since 1995.

Data for the present study come from the Children's Health and Evaluation Project (CHEP), sponsored by the Special Education Organization (SEO) of Iran. The CHEP conducts multidisciplinary screening and diagnosis of all Iranian preschool children every year in order to identify children with disorders that may adversely affect their educational outcome. This screening is mandatory and children's placement in schools is based on the results of the screening and diagnosis process. In the areas which participated in the CHEP, the coverage rate is close to 100%. However, since many remote villages and tribes were not included, CHEP achieved about 90% coverage nationwide in 2005; this coverage rate has increased in subsequent years.

Since 1995, almost a million preschool children have been screened each year for vision and hearing impairments, cognitive disabilities and musculoskeletal problems. More recently, a subset of these children were also screened for autism spectrum disorders. Children with identified disabilities are referred to specialists for appropriate interventions and to special classes, as indicated. In addition to data on preschool children, the CHEP also collects data on the children's parents, such as age, education, province of residence, whether the residence is in a rural or urban location and consanguinity.

Cases included children with autistic disorder (68%), or Asperger's syndrome, and PDD-NOS combined (32%), based on a child psychiatrist's diagnosis verification.

More than half of the 179 ASD cases were first born, and the male to female ratio was 4:1. As shown in Table 1, children with ASD were significantly more likely than their cohort comparisons to be male, and to have parents who were more highly educated.

In this population-based study of data from the large CHEP in Iran we found that risk of ASD was associated with paternal but not maternal age. Although CHEP was not designed as a prevalence study, the first CHEP conducted in 2005 found a prevalence for ASD of one per 913, which is lower than in the USA and Europe. This is, in part, because the screening relied on the SCQ parent questionnaire. For all other developmental disabilities CHEP screening has always been conducted directly with the children and has not previously relied exclusively on parent reports. We suspect that screening via parent report resulted in an artificially low positive screen for ASD prevalence.

As noted above, the results from different investigations of paternal age and risk of autism are inconsistent. Our result is inconsistent with epidemiological studies that found that both paternal and maternal age, or only maternal age but not paternal age, were associated with increased risk for autism. This inconsistency may be explained by differences in study design, the specific variables investigated and adjusted for, as well as sample characteristics. For example, Glasson et al. 45 found an association of advanced maternal age and increased risk for autism. As this result was obtained as part of their epidemiological study on obstetric factors, no data were available on paternal age. Consequently, maternal age was not adjusted for paternal age in this study.

However, Croen et al. 49 , in a historical birth cohort study in northern California, reported that the risk of autism increased significantly with each 10-year increase in both maternal and paternal age, although the effect was stronger for maternal age. Diagnostic subgroup analyses showed a slightly higher risk with increasing maternal age in the Asperger's syndrome and PDD-NOS subgroups compared to the autistic disorder group and showed an increased risk with paternal age only for autistic disorder but not Asperger's syndrome or PDD-NOS 49 .

The unmatched analysis of our data showed that the risk of having a child with ASD increases 29% for every 10 years of increase in paternal age, after adjusting for all other covariates. However, we found no significant association with maternal age in either the unmatched, adjusted analyses or the matched analyses. Although we were unable to conduct analyses of the effect of paternal and maternal age within the diagnostic subgroups of ASD, due to an inadequate number of cases in each of the subgroups, this inconsistency for the effect of maternal age may be due to a difference in the proportion of diagnostic subgroup cases in the sample studied by Croen et al. and the present study sample. Therefore, a higher proportion of autism cases, compared to Asperger's syndrome and PDD-NOS cases, in our sample may account for our finding of increased risk with paternal, but not maternal, age. On the other hand, a higher proportion of Asperger's syndrome and PDD-NOS cases may account for the results from other studies that have reported either both paternal and maternal age or only maternal age associated with increased risk of autism. Along these lines, it is of interest that a recent study in Japan found that advanced paternal age but not maternal age was associated with increased risk of high-functioning autism without intellectual disability 29 . Thus, the variable distribution of diagnostic subgroups composing a sample of ASD cases, as well as the distribution of intellectual ability among the cases, may be one reason for the inconsistency of results in studies of the effect of paternal and maternal age on ASD. However, it is by no means clear how or why either paternal or maternal age should be associated with any one diagnostic subgroup in particular.

It is clear that, in addition to genetic risk, environmental factors must also be important in the aetiology of ASD. Of course, specific environmental risk factors may be associated with other environmental and/or genetic risk factors which, although they may be unknown or unmeasured, may nonetheless serve as modifiers or confounders. As mentioned earlier, matched analysis has the advantage of being able to control for potential confounders that are difficult or impossible to measure directly and which, therefore, cannot be adjusted for in an unmatched analysis.

An example of a potential confounder that is possibly more important in Middle Eastern populations is consanguinity. The prevalence of consanguineous marriage (between cousins) in Iran is about 20%-30% and is associated with a higher rate of child marriage (marriage before age 18) and a lower socio-economic status. Child marriage is itself associated with: a reduced opportunity to provide education for all family members, including parents; lower parental education level; a higher number of offspring; and a generally lower standard of living. In addition, child marriage (3% prevalence for men and 20% for women in Iran) 60 61 results in lost educational opportunities, interrupted bodily development (due to early pregnancy), less awareness of the importance of self-care and pre-natal care, generally poorer health, a greater compliance with authority figures and a higher exposure to abuse for women. All of these can have an effect on the outcome of pregnancy 62 63 64 . In many cases, child marriage results in a greater age disparity between the two parents, as, for instance, when a 12-year-old girl is married to a 22-year-old man. Each of these variables may have its own confounding or modifying effect on parental age and autism. They may also interact with each other and with other environmental and/or genetic risk factors that are unknown or unmeasured. While most published studies controlled for potential measured confounders, few conducted matched analyses which, because they are better able to control for unknown or unmeasured potential confounders, are more precise. As we were concerned about the effects of known but unmeasured variables, particularly socio-cultural factors, we conducted a matched analysis, matching each case with nine cohort comparison children on parental education, birth order, sex, consanguinity, urbanism and province. Thus, the present study is one of the few to conduct a matched analysis examining the effect of parental age on autism risk.

Larsson and colleagues 46 , in a matched study on prenatal risk factors and autism, found no association of parental age and risk of autism, after matching on all other prenatal factors, socioeconomic status and parental psychiatric history. They matched each case with 25 controls for sex, year of birth and age from a cohort of all children born in Denmark since 1973 who had received a diagnosis of autism not later than the end of December 1999. However, it is notable that when the Larson study matched for only prenatal risk factors but not parental psychiatric history, they found paternal age to be associated with a risk of autism. Thus, parental psychiatric status may be an independent risk factor or confounder, although we were unable to control for this in our study.

Many negative outcomes of pregnancy have been attributed to the age-associated increased risk of spontaneous mutation in male sperm 16 65 . In addition to the many studies that have confirmed the association of advanced paternal age and de novo germ-line mutations, a recent analysis of spermatozoa from men of different ages showed a small increase in mutant sperm with paternal age 61 which, nevertheless, is likely to have a significant disease burden. In addition, another study using fluorescence in situ hybridization analysis of spermatozoa identified several potential paternal risk factors for increasing chromosomal abnormalities, including age, drug use, lifestyle and occupational exposure 66 . However, most epidemiological studies of parental age and autism lack data on such specific potential confounders or modifiers. Finally, another alternative explanation is the potential for the accumulation of toxic chemical exposures in older fathers and/or the potential for inherited epimutation. However, there is very little information about either of these potential causes in Iran.

We also investigated the joint effect of parental age and education. Combining parental age with parental education, we found that, regardless of parental age, a higher risk of having a child with autism was found among couples when the mother had a college or graduate degree. However, the highest risk of having a child with autism was found in couples who were both college graduates, with a dose-response effect of parental age (Figure 2). The increased risk from a combination of advanced parental age and greater level of education may be due to greater educational support seeking behaviours in more educated parents. That is, the more highly educated parents may have been more motivated to participate in this first nationwide screening and diagnosis for autism, whereas those with less education may have been more likely to keep their affected children out of the education system and, thus, did not participate in the screening project. In fact, when we divided our cases into two age categories, those aged 4-7 and those aged 8-11, and compared their parents' education, we found that older cases were significantly more likely than younger cases to have fathers with higher education [χ² ₃ = 8.718 (p = 0.033)], but not more highly educated mothers [χ² ₃ = 1.915 (p = 0.59)].

There were, of course, some limitations to our study design. The main limitation of our study was the small number of cases, which made it impossible for us to conduct diagnostic subgroup analyses. Other limitations include the lack of information on parents' autistic traits, and on environmental risk factors for parents and children.

Our study indicates a clear and significant association of advanced paternal age and the risk of ASD in an Iranian population sample. This finding was observed in both an unmatched data analysis, after adjusting for several potential confounders, as well as a matched analysis, with nine matching factors. De novo germ line mutations may be responsible, at least in part, for this association. Socio-cultural characteristics of the Iranian population, such as a large age difference between the parents of a child, child marriage and consanguineous marriage may, in part, account for the inconsistency between the results of our study and some others. Future studies should attempt to conduct subgroup analyses, particularly for subgroup diagnosis and intellectual ability. These results await replication in a larger case group from the same population. In such a replication sample, it would be ideal to have information on broader autism phenotype traits in the parents, as well as environmental risk exposures for both parents and children.

ADI-R: Autism Diagnostic Interview - revised; ASD: autism spectrum disorder; CG: college graduate; CHEP: Children's Health and Evaluation Project; DSM-IV-TR: Diagnostic and Statistical Manual of Mental Disorder, 4th edition, text revision; HS: high school; PDD-NOS: pervasive developmental disorder not otherwise specified; PS: primary school; SCQ: Social Communication Questionnaire; SEO: Special Education Organization.

The authors declare that they have no competing interests.

RS designed the study, acquired the data, performed most of the data analyses and drafted the manuscript. SH carried out data cleaning and data analysis. AT and MG helped with data acquisition. DY helped with study design and the matched data analysis. SLS contributed to the study design, analysis and interpretation and substantially revised and edited the manuscript.

RS is an Iranian physician, currently working in the USA. She was instrumental in initiating nationwide screening for ASD in Iran, when she worked with the Special Education Organization of Iran. SLS is a genetic epidemiologist with over 16 years of experience in autism research.