**12. Etiology and mechanisms of sexual differences**

It is well known that ASD affects more males at a ratio of 4 boys:1 girl. Some studies suggest that gender differences in phenotypic presentation, including less severe manifestations of restricted and repetitive behaviors in girls, seem to affect this ratio. Genetic studies show that girls seem to be more "protected" from the effects of inherited and *de novo* variants that cause ASD than boys. This suggests that genes that participate in sexual development and/or sex hormones, particularly testosterone, may modulate the effects of genetic variations in the autistic phenotype [157].

Despite the fact that ASD is more prevalent in boys *de novo* mutations are also more frequent in girls with ASD. The female protection theory fits in with the model of loss of robustness. According to this model, besides the variations between individuals, there is a difference in the average degree of robustness of the brain between males and females. According to this hypothesis the development of the female brain is more robust, and this may explain the higher rate of severity of behaviors when *de novo* mutations are present in girls with ASD. The brain can also be more vulnerable compared to other organs because it is a complex organ that develops later in life and is mainly composed of terminally differentiated cells. In the first year of life, the brain may be particularly sensitive to reduced robustness because social develop‐ ment depends on signals from the environment [138].

changes in the global balance of the entire microbial community, while others may be exercised by certain bacteria. More studies are needed to clarify whether the gut microbio‐ ta in fact plays a role in ASD. These include prospective studies to address the issue of cause and consequence, and intervention studies aimed at modulating the microbiota with probiotics or dietary interventions. The right profile, the categorization of patients and control groups, with the application of molecular techniques to verify the profile of fecal microbiota and urinary metabolome will corroborate the underlying relationship between

Some authors believe that the action of microbiota may have much wider effects on the physiology of the host than originally thought, and emerging evidence shows that it may include modulation of brain activity and behavior. Differences in the composition of the microbiota between individuals with ASD and healthy controls were identified in several studies, both based on bacterial cultures and on molecular methods [18]. However, changes in bacterial diversity that were reported in one study [51] were not confirmed by another [158]. The direct comparison between studies is complicated because different methodologies are employed and study groups may not be directly comparable due to the heterogeneous nature of ASD [89]. However, if the differences in gut microbiota between autistic children and controls are one of the causes of the disorder or the result, could have implications on the

Nevertheless, despite the substantial amount of data, it is not possible to clarify whether the results represent the presence of a causative agent, or reflect a consequence of treatment, or whether they are nothing more than confounders. This has to be considered and caution is needed before recommending "miracle diets" to the child and family that may only increase

It is well known that ASD affects more males at a ratio of 4 boys:1 girl. Some studies suggest that gender differences in phenotypic presentation, including less severe manifestations of restricted and repetitive behaviors in girls, seem to affect this ratio. Genetic studies show that girls seem to be more "protected" from the effects of inherited and *de novo* variants that cause ASD than boys. This suggests that genes that participate in sexual development and/or sex hormones, particularly testosterone, may modulate the effects of genetic variations in the

Despite the fact that ASD is more prevalent in boys *de novo* mutations are also more frequent in girls with ASD. The female protection theory fits in with the model of loss of robustness. According to this model, besides the variations between individuals, there is a difference in the average degree of robustness of the brain between males and females. According to this hypothesis the development of the female brain is more robust, and this may explain the higher rate of severity of behaviors when *de novo* mutations are present in girls with ASD. The brain can also be more vulnerable compared to other organs because it is a complex organ that

gut microbes and the host [34].

332 Autism Spectrum Disorder - Recent Advances

diagnosis, treatment and prevention [37].

autistic phenotype [157].

the anxiety in respect to symptom improvement.

**12. Etiology and mechanisms of sexual differences**

Although neurodevelopmental systems evolved to be robust, they can be vulnerable to disturbances in a specific subset of genes called phenotypic capacitors [85]. Phenotypic capacitors are genes that act against disturbances and thus contribute to the robustness of the phenotype. Thus, phenotypic capacitors, when operating normally, can prevent the develop‐ ment of diseases such as ASD, even in patients exposed to genetic and environmental risks. This means that girls are less likely to develop ASD, but when they have, they tend to have a more severe phenotype [138].

There seems to be unknown factors that "protect" females from ASD [157]. Recent genome studies showed that, on average, women with ASD have more mutations than men, including single nucleotide variants (SNVs) and CNVs [74, 84, 105]. Furthermore, *de novo* CNVs in females were larger and included significantly more genes than males [84].

Moreover, not all genes in the inactive X chromosome are inactivated, and the genes that escape X inactivation in females are revealing some interesting insights into gender differences related to chromatin. *KDM5C* and *JARID1C* are genes that escape X chromosome inactivation (160). In addition, the gene encoding O-linked N-acetylglucosamine (O-GlcNAc) transferase (*OGT*) which regulates chromatin remodeling factors is less expressed in males than in females and the expression is reduced further by prenatal stress [70]. These sexual and epigenetic differ‐ ences must be investigated further in respect to the protective effect of the female gender in autism [81].

Recently, gender-specific gene expression obtained from the transcriptome of normal human brain development using a bioinformatics approach suggested that male-biased genes are enriched for the processes of extracellular matrix formation/glycoproteins, immune response, chromatin, and cell cytoskeleton. These pathways have been repeatedly implicated in autism and demonstrate that autism candidate genes are also enriched for these pathways. Further‐ more, the development of the male brain may be naturally more susceptible to environmental factors as its normal development is more strictly dependent on the immune system [167].

A gender-dependent difference in the incidence of neural tube defects has been described. It was speculated that these defects are due to a synergistic effect between the gene expression of *SOX9* and *Barx1*. *SOX9* is an essential transcription factor for skeletal development, but it is also involved in the development of the male phenotype [14], thus contributing to the increased risk of autism in males. Interestingly, a study showed up-regulation of *SOX9* in autism [55].

On the other hand, altered behaviors in ASD are often related to a "more masculine" pattern of behavior linked to testosterone. However, although current levels of steroid hormones appear to be altered in patients with autism, the data indicate that prenatal testosterone by itself does not seem to be sufficient for the disorder to develop [125].

Differences between individuals with an atypical karyotype (monosomy X or Y or X-polyso‐ my) and those with a typical karyotype are often interpreted as being significant for the difference in susceptibility between genders. However, it is likely that many of the differences arise because of hyperexpression or hypoexpression of genes in pseudo autosomal regions that escape X inactivation, thereby being similarly expressed in men and women with a typical karyotype. The addition or loss of an X or Y chromosome, which leads to altered protein levels that are atypical or typical for both men and women, cannot explain the gender difference in ASD. However, some genes on the X chromosome are not in these regions and have no corresponding functional alleles on the Y chromosome, and escape X inactivation in some circumstances [125].

Moreover, the *SRY* gene is mapped in a region on the Y chromosome and can also directly regulate the gene monoamine oxidase A (MAOA) that is located in the Xp11.3 region and encodes a key enzyme in the breakdown of catecholamines and other monoamines. SRY can directly affect transcription in the brain [159]. As individuals diagnosed with ASD are found with changes in catecholamine and metabolite levels in the dependent activity MAO-A and autism severity is associated with child and maternal MAO-A genotypes, the disruption of the synthesis of catecholamines may be modulated by the gender-specific *SRY* gene associated with ASD. Several genes on the Y chromosome are expressed in the brain. Since this leads to a specific expression of gender in the brain, these genes, some of which play a role in catecho‐ laminergic functions, are candidate genes for the increased susceptibility to ASD in males. An investigation of the role of these genes will possibly clarify the gender-specific mechanisms underlying ASD and thus help in the understanding of the etiology of ASD [31, 125].

Another factor that may contribute to the skewed gender ratio in ASD is parental age. Increased parental age is known to increase the risk of a child with ASD [45] and there are some indications that parental age affects the gender ratio of children diagnosed with ASD [125]. The male-female ratio dropped from 6.2:1 in under 30-year-old parents to 1.2:1 in parents older than 44 years old [8]. This finding may be related to the higher frequency of *de novo* mutations which are more common in older men and seem to play a minor role in the gender bias in the incidence of familial ASD. However, it is still not clear whether this effect is true for simplex families, which are most representative of ASD in the general population [125].
