Introduction

      Autism is a neurodevelopmental disorder characterized by impaired social interaction, verbal and non-verbal communication, and by restricted and repetitive behavior. The diagnostic criteria (1,2) require that symptoms become apparent before a child is three years old (3). Autism affects information processing in the brain by altering how nerve cells and their synapses connect and organize; how this occurs is not well understood (4). It is one of three recognized disorders in the autism spectrum (ASDs), the other two being Asperger syndrome, which lacks delays in cognitive development and language, and pervasive developmental disorder, not otherwise specified (commonly abbreviated as PDD-NOS), which is diagnosed when the full set of criteria for autism or Asperger syndrome are not met (5-7).

Autism has a strong genetic basis, although the genetics of autism are complex and it is unclear whether ASD is explained more by rare mutations, or by rare combinations of common genetic variants (8). In rare cases, autism is strongly associated with agents that cause birth defects (9). Controversies surround other proposed environmental causes, such as heavy metals, pesticides or childhood vaccines (10); the vaccine hypotheses are biologically implausible and have been disproven in scientific studies (11). The prevalence of autism is about 1–2 per 1,000 people worldwide, and it occurs four to five times more often in boys than girls(12,13). The Centers for Disease Control and Prevention (CDC) report 1.5% of children in the United States (one in 68) are diagnosed with ASD as of 2014 a 30% increase from one in 88 in 2012 (11-15).  The number of people diagnosed with autism has been increasing dramatically since the 1980s, partly due to changes in diagnostic practice and government-subsidized financial incentives for named diagnoses (16); the question of whether actual prevalence has increased is unresolved (12).

Parents usually notice signs in the first two years of their child's life (17).The signs usually develop gradually, but some autistic children first develop more normally and then regress (18). Early behavioral, cognitive, or speech interventions can help autistic children gain self-care, social, and communication skills (17). Although there is no known cure (17), there have been reported cases of children who recovered (19).

Results shown a comprehensive understanding of the etiology of ASD remains elusive (20); however reports of excess ASD diagnoses among males, approximately 4:1 compared with females (21), suggest an important role for X-linked risk factors (22-24), such as prenatal androgen exposure. This suggests that ASDs may comprise ‘hyper-male’ phenotypes (25) and motivate the so-called ‘hyperandrogenic’ etiologic hypothesis (Fig.1). Digit ratio, defined as the length of the 2nd digit divided by the length of the 4th digit (2D:4D), is purported to be a marker for androgen exposure in utero (26). During gestation, development of the bones of the hands, the metacarpals and phalanges, are governed by the Hox/Homeobox genes, which also orchestrate the development of the male reproductive tract, and respond to circulating androgens (27). Excess androgen exposure secondary to fetal testis synthesis at approximately seven weeks of gestation (28) elicits elongation of the 4th digit relative to the 2nd digit, and thus lower 2D:4D ratios are generally indicative of higher in utero androgen exposure (29). Investigators have previously employed the 2D:4D ratio as a marker of exposure to test hypotheses concerning androgen exposure in utero as an etiologic factor for ASD. One report found children with autism or Asperger's syndrome, along with their 1st-degree relatives (unaffected siblings, mothers, and fathers), to have significantly lower 2D:4D ratios than sex-matched controls (30). More recently, lower 2D:4D ratios from the right and left hands of boys with autism, as well as other disorders of the autism spectrum, were reported in comparison to a reference group (31). A contemporary review of the literature underscores the consistency of these reports describing inverse associations between 2D:4D ratio, indicating a more masculine pattern among subjects receiving ASD diagnoses (32).

The most important element of research on autistic children,  is avoided  of harming to the child  while  the research; because the autistic children can be severe distress  with using the simplest  tests  and damage to the body.

The method that are considered today, is the morphometric characteristics of autistic children compared with normal children (33). These  methods according to keep calm the child  at the time of study and research ethics, is  valuable.

The properties of morphometric characteristics, we can cite to check the size of the head (34, 35), to check the status of curling hair (36) and evaluation of finger length ratio 2D (Index finger) finger length 4D (Ring finger) in autistic children compared to normal children, noticed (37). The aim of this study was to measurement 2D and 4D finger length in ASD children with with healthy children as a control group.

Fig.1: Hand with index finger being shorter than the ring finger, resulting in a small 2D:4D ratio, pointing to a high exposure to testosterone in the uterus

Methods and Materials

 This study was a causal-comparative with 48 children in ages 7 to 8 years old (10 girls and 38 boys) with ASD,  who were reffered to Rehabilitation Center of Noor Hedayat, Mashhad-Iran  and 41 normal children in ages 7 to 8 years old (10 girls and 31 boys) of 3 kindergartens of Mashhad,  and children were elected  with random sampling. Also,kindergartens were selected of areas (1, 4, 6) of Mashhad. The test used in this study was Childhood autism rating scale (CARS).

The diagnosis was made by a child psychiatrist based on DSM-IV-TR criteria and using Autism diagnostic interview-revised (ADIR) and Autism diagnostic Observation Schedule (ADOS). In this research, Autism rating scale, Childhood autism rating scale was (CARS) used to assess the severity of symptoms in children. The CARS is a behavior rating scale that is utilized to assess symptoms of autistic disorder. In addition, it is able to differentiate children with autistic disorder from other developmental delays. It is conducted as an observation which can be completed during a testing session or in classrooms. However, some items can be answered from parent report or from reports of history. The CARS contains 15 items that are utilized to compare the behavior of the child being assessed to the behavior of typically developing children. The 15 items include: (1) Relating to people, (2) Imitation, (3) Emotional response, (4) Body use, (5) Object use, (6) Adaptation to change, (7) Visual response, (8) Listening response, (9) Taste, smell, and touch response and use, (10) Fear or nervousness, (11) Verbal communication, (12) Nonverbal communication, (13) Activity level, (14) Level and consistency of intellectual response, and (15) General impressions. Each item is rated on a Likert scale as follows: 1 (within normal limits for a child that age), 2 (mildly abnormal), 3 (moderately abnormal), and 4 (severely abnormal). Midpoints between these values can also be utilized (e.g., 2.5). A total score is computed from the sum of the 15 items. Those with a total score of 30 or above fall within the autistic range (yields an agreement rate of 87%). Psychometric studies on the CARS yield promising results with an internal consistency of α = .94, interrater reliability average of .71 (good agreement), and coefficient kappa of .64 for test retest reliability. In regards to validity, there was high criterion related validity, r = .84 ( Schopler et al., 1988). This scale (CARS) can be completed by a clinician or teacher or parent, based on subjective observations of the child's behavior. Each of the fifteen criteria listed above is rated with a 7-point score. Lower scores indicate less severity of Autism. Total CARS score indicates total score of severity about autism spectrum disorders(ASD) (6,7).

2D Finger length   and 4D finger length in both hands in ASD children was measured by a ruler. The reason for not using a digital caliper to measure, was that autistic children  while saw the caliper, had a extreme fear and were not willing to cooperate. 2D Finger length   and 4D finger length in both hands in normal children, also was measured by a ruler. Data were analyzed using SPSS 16 software and p value less than 0.05 was statistically considered significant.

Results

   In this study was 48 children in ages 7 to 8 years old (10 girls and 38 boys) with ASD,  and 41 normal children in ages 7 to 8 years old (10 girls and 31 boys) were participated. The mean age of children in the normal group was (7.46+0.3457) and the ASD group was (7.38+0.2816).

 Mean  right  and  left hand 2D:4D across the  groups is given  in (Table.1) and (Fig.2).

Fig.1: Radiograph of right hand.

Table 1: 2D:4D finger length ratio between left and right hand in ASD group and control group

Paired t-tests showed the average ratio of 2D:4D in ASD children were lower than the ratio in control group (P<0.05(. Results also showed that 2D to 4D finger-length ratio of the right of children with autism was lower than the normal children (P<0.05(;  bute this difference was not significant between the two groups in the left hand (P>0.05) (Table.1).

Discusion

    Children from ASD group had lower 2D:4D digit ratio than the control group. This finding reflects higher prenatal testosterone levels in ASD children (22-32). The results show 2D to 4D finger of the right hand of children with ASD was significantly lower than that the normal children. These results are consistent with the findings of Manning, Hönekopp and Milne (38-41). The results of the comparison of 2D to 4D left hand of children with ASD with normal children, Showed that despite the lower the proportion of children with autism spectrum disorder, there was no significant difference between the two groups. The lack of significant differences, may be because  that the ratio of 2D to 4D finger of the left hand  compared with the right hand, the secretion of testosterone in utero  is less sensitive (42). Hönekopp and Watson on their research showed that the ratio of finger 2D:4D finger of the right hand  is particularly associated with sex differences.

Conclusion

    The results showed that the mean 2D to 4D finger length ratio in autistic children is lower than that of normal children. It was concluded that 2D:4D ratio may be a possible marker for autism wich could implicate prenatal testosterone in its aetiology.

We are aware of the  fact that our sample is small to generalization results but it is in focus of our future research to collect more data. Another limitation of the study sampling process,  some autistic children  because  of distress, were not willing to cooperate.

Conflict of Interest

  The authors declare that they have no competing interests.

Acknowledgment
    The authors  would like to thank the  staff of Animal Developmental of Applied Biology Research Center, Mashhad Branch, Islamic Azad University, and the staff of Noor Hedayat, Center of Autism Spectrum Disorders, Mashhad, Iran.