**3. Evidence for the effect of elevated androgens in ASD**

Investigators have undertaken extensive evaluation of various measurements of elevated androgens in individuals diagnosed with an ASD. They described, among individuals diagnosed with an ASD, significantly increased frequency of genetic changes in multiple genes involved in sex steroid synthesis, transport, and/or metabolism, testosterone-related medical conditions (e.g., polycystic ovarian syndrome, breast and ovarian cancers, acne, etc.), and testosterone-related characteristics (e.g., tomboyism, etc.)[6, 7]. It was also reported that individuals diagnosed with an ASD had significant alterations in the timing of puberty (boys diagnosed with an ASD were observed to enter "male" puberty earlier, while girls diagnosed with an ASD were observed to enter "female" puberty later). Elevated androgen levels and significantly lower second to fourth digit ratios in comparison to neurotypical controls (a known marker of elevated fetal testosterone) [6, 7].

Investigators recently measured fetal steroidogenic activity in amniotic fluid samples for individuals diagnosed with an ASD in comparison to neuroptypical controls. Amniotic fluid samples taken from individuals subsequently diagnosed with an ASD in comparison to neurotypical controls revealed significant elevations in androgen levels [11].

Investigators previously described evaluating blood androgen levels among a large cohort of individuals diagnosed with an ASD using routine laboratory testing from the Laboratory Corporation of American (LabCorp) [12]. It was observed that individuals diagnosed with an ASD were observed to have significant increases in their blood levels of testosterone, free testosterone, dehydroepiandrosterone (DHEA), and androsternedione relative to laboratory provided reference ranges. Overall, it was observed among the various blood androgen attributes examined, that over 80% of the individuals diagnosed with an ASD examined were found to have at least one of the blood androgen attributes examined that exceeded the ageand sex-specific reference ranges provided by the laboratory.

Subsequently, other investigators evaluated the potential role of androgens among individuals diagnosed with an ASD in comparison to neurotypical controls by examining salivary levels of hormones among children from 3-4 years-old and 7-9 years-old [13]. These investigators observed significantly higher salivary concentrations of androgens among individuals diagnosed with an ASD relative to controls, and the anomalies were prominent in older male children diagnosed with an ASD. Among the specific types of androgens observed to be increased among individuals diagnosed with an ASD in comparison to neurotypical controls were androstenediol, DHEA, and androsterone, which, the investigators concluded were indicative of precocious andrenarche and predictive of early puberty. These investigators also commented that some of the androgens observed were significantly increased among the individuals diagnosed with an ASD relative to the neurotypical controls are known to neuroactive and modulate GABA, glutamate, and opioid neurotransmission with the potential consequence of affecting brain development and function. They may also contribute to ASDassociated pathobiology and symptoms such as elevated anxiety, sleep disturbances, sensory deficit, and stereotypic behaviors.

Similarly, other investigators examined hyperandrogenemia in male children and adolescents diagnosed with an ASD in comparison to neuroptyical controls and in relation to ASD severity by assessing serum androgen levels [14]. These investigators observed that androgen levels were significantly higher among individuals diagnosed with an ASD in comparison to neurotypical controls, and the elevations were observed to significantly correlate with ASD severity. Overall, it was observed among individuals diagnosed with an ASD that 36.66% had high serum free testosterone, 30% had high DHEA, 40% had high androstenedione, and 26.66% showed elevation of all androgen levels in comparison to neurotypical controls. These investigators concluded that hyperandrogenemia is prevalent among individuals diagnosed with an ASD, correlate with ASD severity, and studies should explore the use of anti-androgen therapy to treat such patients.
