132 Male Gender Bias and Levels of Male Hormones During Fetal Development
in the EMB hypothesis of Baron‐Cohen [3–10] which postulates that elevated
fetal testosterone is a risk factor for ASD (summarized in the preceding sec-
tion); (2) genetic mechanisms which involve X or Y chromosome inactivation
[1]; (3) and recently, Hu et al. [69] have shown that retinoic acid‐related orphan
receptor alpha (RORA) is reduced in the brain and lymphoblastoid cell lines of
multiple cohorts of individuals with ASD. This gene targets CYP19A1 (the
gene that codes for aromatase enzyme), in a gender‐dependent manner that
can also lead to elevated testosterone levels, a proposed risk factor for autism
as mentioned in the above three hypotheses. These hypotheses have been
comprehensively described in our recent review [2]. To date, none of these
hypotheses have been either proven or disproven. Given the high clinical het-
erogeneity of ASD, it is possible that each of these mechanisms for gender bias
may apply to specific cohorts of individuals with ASD, considering that ASD is
a spectrum [1,2].Male and Female Brains in a Test Tube
Since all of the above theories relate to early fetal brain development (weeks
4–24 of gestation), we hypothesize that the scientific community needs a
robust in vitro model that can reveal the fundamental mechanism for gender
bias in ASD. For the last decade we have been evaluating an in vitro model,
utilizing human neuroblastoma cell lines (NBCs). A detail rationale and infor-
mation is provided in Chapter 7. Briefly, these cell lines represent early fetal
brain progenitor neurons, as has been shown previously [2,5]. In our prelimi-
nary studies we have evaluated two highly plausible hypotheses: (1) if testoster-
one levels that are found in the amniotic fluids of normotypic, classical autistic
and Asperger syndrome children (as determined by Baron‐Cohen et al. [3])
affect the neurodevelopment of NBCs; and (2) examined expression levels of
RORA and CYP19A1 (aromatase) genes in male and female NBCs exposed to
three different levels of testosterone, equivalent to what Baron‐Cohen’s team
found in the amniotic fluids of Danish children [3,4].
We have carried out preliminary studies to determine the feasibility of
whether: (1) NBCs exposed to three different levels of testosterone (represent-
ing normal, autistic and Asperger syndrome children) differ at morphologic
levels and exhibit signs of “connectome” dysregulation; (2) there is a differen-
tial expression of RORA and CYP19Aa?; and (3) there is a downregulation of
oxytocin‐ and AVP‐receptor neurons in the exposed cell lines compared with
the unexposed (control cell lines). For this purpose, we analyzed one pair of
NBCs that represents and behaves similarly to developing fetal brain progeni-
tor cells in vitro (i.e., CCL‐2266 (female) and CRL‐2267 (male)] to determine
whether exposure to different levels of testosterone induces differential mor-
phologic and immunologic changes (2). We also determined the differential