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309-60

REVIEW ARTICLE
Understanding Environmental Contributions to Autism: Causal
Concepts and the State of Science
Irva Hertz-Picciotto , Rebecca J. Schmidt, and Paula Krakowiak

The complexity of neurodevelopment, the rapidity of early neurogenesis, and over 100 years of research identifying
environmental influences on neurodevelopment serve as backdrop to understanding factors that influence risk and
severity of autism spectrum disorder (ASD). This Keynote Lecture, delivered at the May 2016 annual meeting of the
International Society for Autism Research, describes concepts of causation, outlines the trajectory of research on non-
genetic factors beginning in the 1960s, and briefly reviews the current state of this science. Causal concepts are intro-
duced, including root causes; pitfalls in interpreting time trends as clues to etiologic factors; susceptible time
windows for exposure; and implications of a multi-factorial model of ASD. An historical background presents early
research into the origins of ASD. The epidemiologic literature from the last fifteen years is briefly but critically
reviewed for potential roles of, for example, air pollution, pesticides, plastics, prenatal vitamins, lifestyle and family
factors, and maternal obstetric and metabolic conditions during her pregnancy. Three examples from the case-control
CHildhood Autism Risks from Genes and the Environment Study are probed to illustrate methodological approaches
to central challenges in observational studies: capturing environmental exposure; causal inference when a random-
ized controlled clinical trial is either unethical or infeasible; and the integration of genetic, epigenetic, and environ-
mental influences on development. We conclude with reflections on future directions, including exposomics, new
technologies, the microbiome, gene-by-environment interaction in the era of –omics, and epigenetics as the interface
of those two. As the environment is malleable, this research advances the goal of a productive and fulfilling life for
all children, teen-agers and adults. Autism Res 2018, 0: 000–000. V C 2018 International Society for Autism Research,

Wiley Periodicals, Inc.

Lay Summary: This Keynote Lecture, delivered at the 2016 meeting of the International Society for Autism Research,
discusses evidence from human epidemiologic studies of prenatal factors contributing to autism, such as pesticides,
maternal nutrition and her health. There is no single cause for autism. Examples highlight the features of a high-
quality epidemiology study, and what comprises a compelling case for causation. Emergent research directions hold
promise for identifying potential interventions to reduce disabilities, enhance giftedness, and improve lives of those
with ASD.

Keywords: autism spectrum disorder; environmental risk factors; causal inference; pre- and peri-natal risk factors; pes-
ticides; nutrition; diabetes; gene-environment interaction; epigenetics

Introduction on the mother, despite prominent autism researchers

Over the last half century, varying conceptual models
for etiologic factors in autism spectrum disorder (ASD)
have been proposed, most emphasizing single cause
hypotheses. A predominant theory persisting for several
decades argued that emotionally unresponsive parent-
ing led children to withdraw into their own ‘worlds’
and seek comfort in repetitive behaviors [Bettelheim,
1967]. The term “refrigerator mom” laid responsibility
who published opposing views that drew attention to
biologic factors and predicted genetic underpinnings
[Folstein & Rutter, 1977a; Rimland, 1964]. Twin and
family recurrence studies bolstered the evidence [Fol-
stein & Rutter, 1988; Pickles et al., 1995]. By the 1990’s,
neuroanatomic observations, neuroimaging data
[Courchesne, Yeung-Courchesne, Press, Hesselink, &
Jernigan, 1988; Piven et al., 1990], cytogenetics and
links with genetic syndromes [Cohen et al., 1991;

From the Department of Public Health Sciences, MIND Institute (Medical Investigations of Neurodevelopmental Disorders), University of California,
Davis, Davis, California
Grant sponsor: National Institutes of Health; Grant numbers: UG3-OD023365; UL1-TR000002; Grant sponsor: National Institute of Environmental
Health Sciences; Grant numbers: P01-ES011269; P30-ES023513; R01-ES015359; R01-ES020392; R01-ES028089; R01-ES025574; R21-ES021330; T32-
MH073124; Grant sponsor: Eunice Kennedy Shriver National Institute of Child Health and Human Development; Grant number: U54-HD079125;
Grant sponsor: U.S. Environmental Protection Agency; Grant numbers: R829388; R833292; RD-83543201; Grant sponsor: U.S. Department of
Defense; Grant number: AR110194; Grant sponsor: The Allen Foundation.
Received May 24, 2017; accepted for publication October 19, 2017
Address for correspondence and reprints: Irva Hertz-Picciotto, Department of Public Health Sciences, MIND Institute (Medical Investigations of
Neurodevelopmental Disorders), University of California, Davis, Med Sci 1C, Davis, CA, 95616. E-mail: iher@ucdavis.edu
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com)
DOI: 10.1002/aur.1938
C 2018 International Society for Autism Research, Wiley Periodicals, Inc.
V

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Folstein & Piven, 1991; Smalley, Tanguay, Smith, &
Gutierrez, 1992; Wassink & Piven, 2000; Wassink,
Piven, & Patil, 2001] had demonstrated aberrant brain
development, establishing a pathobiologic basis for
autism and replacing the psychogenic explanation. By
early 2000s, an intense focus on single gene mutations
operating alone or in combination dominated ASD etio-
logic research [Abrahams & Geschwind, 2008; Bartlett,
Gharani, Millonig, & Brzustowicz, 2005; Bespalova &
Buxbaum, 2003; Wassink, Brzustowicz, Bartlett, & Szat-
mari, 2004]. Overwhelming evidence supports a role, in
a subset of persons with ASD, for numerous gene var-
iants, with rare ones contributing a small proportion
and common variants, as yet largely unidentified, com-
prising the bulk of the heritability [Gaugler et al.,
2014]. Nonetheless, underlying the literature on genet-
ics is an implicit assumption that genes act indepen-
dently of more mutable factors such as epigenetic
marks or environmental exposures, or at least, the latter
can be ignored while the search to understand ASD
genetics advances.
Yet the complexity of neurodevelopment, the rapid-
ity of early neurogenesis, and the wide complement of
increasing idiopathic developmental disorders with
challenging disabilities provide a backdrop for a broader
perspective on causal factors in ASD. Furthermore, a
long history of environmental disasters is a compelling
rationale for interrogating the complex ecology sur-
rounding human development. This tale traces back
more than a century, to use of leaded paints resulting
in severe, diverse impairments in young children [Gib-
son, 1904]. Early studies of exceedingly high exposures
prompted regulations to reduce lead exposures, and
later to the landmark investigation by Needleman
which established, for the first time, that alterations in
behavior and intellectual proficiency occurred at levels
previously considered safe [Needleman et al., 1979]. As
cognitive deficits from lead exposure are not necessarily
accompanied by clinical symptoms, they have been
characterized as a ‘silent’ epidemic. High exposure to
another metal, mercury, from industrial waste in Mini-
mata Bay, Japan [Harada, 1978, 1995] and from epi-
sodes of grain contamination in Iraq [Harada, 1978,
1995] caused severe physical and mental disabilities. As
with lead, subsequent research identified milder impair-
ments associated with low-level mercury exposures usu-
ally only after adjustment for fish consumption (a
major source of both low-level mercury and fatty acids
beneficial for brain development) [Karagas et al., 2012;
Oken et al., 2008; Sagiv, Thurston, Bellinger, Amarasiri-
wardena, & Korrick, 2012]. A further example is that of
(polychlorinated biphenyls (PCB’s), an industrial chemi-
cal), which mistakenly contaminated cooking oil in two
separate incidents, one in Japan and one in Taiwan
[Hsu et al., 1985; Kuratsune, Yoshimura, Matsuzaka, &
2 Hertz-Picciotto et al./Environmental contributions to ASD
Yamaguchi, 1972]. Those exposed in utero were the
most severely affected (by PCBs or mercury) and
because PCBs are persistent (half-lives of years in
humans), children born to exposed mothers, even years
after the incident, suffered similarly [Chen & Hsu,
1994]. At far lower prenatal levels, PCBs are now known
for their ability to disrupt thyroid hormones, immune
function, hearing, and neurobehavioral outcomes
[Jacobson & Jacobson, 1996; Winneke et al., 1998].
Defining Environment
Although the word ‘environment’ is often used to refer
to exogenous chemicals in air, water, soil, food, and
household products, our biology and our health are
influenced by a much wider array of exposures: nutri-
tion, family resources (financial, educational, social),
household structure, neighborhood attributes (urban/
suburban/rural, walkability, housing stock, social capi-
tal), workplaces, pets, microbiome, and for the fetus,
the intrauterine environment with maternal health and
physiology playing a dominant role. What these expo-
sures share—distinct from inherited nuclear DNA—is
the capacity to be modified, and this malleability opens
the door to interventions at various levels. The poten-
tial to reduce disability by altering these factors is the
premise and foundation of environmental research on
autism.
This paper is divided into four parts. First, we delin-
eate critical concepts in causation: root causes, suscepti-
ble time windows, and multifactorial causation. With
this foundation, the second section starts with a histori-
cal overview of environmental etiologic research, then
summarizes the literature on specific prenatal factors
studied by at least three research groups. Although nei-
ther comprehensive nor systematic, this review briefly
but critically evaluates both positive and null findings,
and where available, meta-analyses. The third section
selects three examples from the CHildhood Autism
Risks from Genes and the Environment (CHARGE)
Study, a large case-control investigation of causes and
contributing factors to ASD, to illustrate methodologic
approaches to central challenges in observational (non-
randomized) research: accurate exposure assessment,
inference from association to causation, and integration
of molecular (genetic, epigenetic), with environmental
influences on health. The fourth and final section out-
lines future directions for exposomics and gene-
environment interaction.
Concepts of Causation
Root Causes
A useful classification of causes for any health condi-
tion distinguishes root or initiating factors from those
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that occur after the disorder or disease is apparent or
has been diagnosed. These latter exposures may alter
course or severity of the disabilities, whereas the root
causes foster the initial pathogenic changes ultimately
leading to a diagnosable phenotype. Here, we focus on
exposures, events, or conditions that occur prior to the
full onset of symptoms, and hence may be causally rele-
vant to the emergence of ASD. Symptoms, however,
often go unnoticed, delaying diagnoses for years. Some
root causes for ASD may also exacerbate symptoms after
the behavioral syndrome has developed. However, only
those factors present before symptoms are sufficient to
warrant a diagnosis—even if the symptoms went unno-
ticed—would be considered potentially etiologic. Inter-
ventions that reduce or eliminate the root causes would
be expected to mitigate the disabling symptoms and
such actions are preventative, in that they lower the
number of individuals meeting diagnostic criteria.
Throughout life, however, multiple molecular, physio-
logic, microbiologic, chemical, nutritional, family, psy-
chologic, and other factors may influence autism-
related behaviors. These also might serve as targets for
interventions to improve lives of those affected with
ASD.
Time Trends in Autism
In 2003, the California Department of Developmental
Services (DDS), which coordinates services for persons
with developmental disabilities, including autism,
reported a steep and steady rise in cases of autistic dis-
order from the start of its electronic records 1990.
Notably, the Diagnostic and Statistical Manual (DSM)
of mental health conditions was revised to broaden the
defining symptoms, a change consolidated in the DSM
IV [American Psychiatric Association, 1994]. A later
analysis of age-specific incidence showed the California
rise to be unrelenting for more than a decade, reaching
a 7-fold increase in cumulative incidence to age five
between 1990 and 2001 birth cohorts [Hertz-Picciotto &
Delwiche, 2009b]. The DSM changes, a trend towards
earlier age at diagnosis, and a shift in clinical practice
favoring inclusion of milder cases together accounted
for only one-third of the observed rise [Hertz-Picciotto
& Delwiche, 2009b]. Another team reported that about
a quarter could be attributed to ‘diagnostic substitution’
wherein the same behavioral manifestation may have
prompted a different diagnosis in earlier years [King &
Bearman, 2009]. Given some overlap between diagnos-
tic substitution and younger ages at diagnosis, about
half the rise likely reflected changing clinical practice
rather than a true rise. The California DDS has excluded
cases without major impairments, and beginning in
2003, cases with fewer than three functional limita-
tions. Data collected by the Centers for Disease Control
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(CDC) indicate a greater rise in ASD without intellec-
tual disability nationally [National Academies of Sci-
ence, 2015]; these data include more recent years than
the analyses of DDS data, and CDC data sources are
inconsistent over time, that is, cover different regions
with widely different prevalence proportions. Unlike
other data sources, the California DDS system, with its
consistent source of case ascertainment throughout sev-
eral decades, provides a robust vehicle for understand-
ing time trends. Thus, based on two independent
quantitative analyses [Hertz-Picciotto & Delwiche,
2009a; King & Bearman, 2009] the rapid, steady
increase in diagnoses in California during the 1990s
and early 2000s was only partially attributable to diag-
nostic artifacts. An additional analysis showed only 4%
of the rise could be attributed to trends toward later
maternal age at childbearing [Shelton, Tancredi, &
Hertz-Picciotto, 2010]. Paternal age would be expected
to have a similar impact.
What explains the rest of the trend? De novo gene
mutations and copy number errors of replication occur
too slowly to be plausible explanations. On the other
hand, rapid recent changes in the environment include:
increased consumption of sweetened beverages [Bleich
et al., 2009]; rise in intake of high fructose corn syrup
from <10g to nearly 100g per person per day [Bray,
Nielsen, & Popkin, 2004], and new ultrasound equip-
ment introduced for fetal imaging with 8-fold higher
thermal doses [Webb et al., 2017], both beginning by
early 1980’s; increasing C-section deliveries [Osterman
& Martin, 2014]; rising use of brominated flame retard-
ants in household products [Lorber, 2008]; globalization
of commerce and human travel bringing many new
viruses across borders; and phthalates becoming perva-
sive in scented products, to name just a few.
Parallel trends offer possible clues but, in a society,
marked by rapid changes in all sectors of life, any single
change is likely confounded by a multiplicity of others.
Most parallel trends turn out to be coincidences, as
data mining readily shows (Spurious Correlations
http://tylervigen.com/page?page52). Thus, parallel
trends, by themselves, cannot be construed as evidence
of causality, and are generally far more likely to be
chance occurrences.
Moreover, causes for autism need not exhibit a rise
parallel to that of ASD incidence. Exposures exhibiting
a downward, nonmonotonic, or no trend may still play
a causal role. This apparent paradox could occur if, for
instance, the chemical of concern was replaced by
another with similar or greater neurodevelopmental
toxicity. Plasticizers are often replaced with chemicals
having very similar structures. More generally, the net
impact of multiple risk factors with changing prevalen-
ce’s may negate the impact of trends in any particular
causal factor.
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Finally, the other ‘half’ of the rise in autism, that is,
the previously unrecognized cases now being diag-
nosed, as well as the background of cases that have
been diagnosed all along, also require etiologic explana-
tion. Both genetic susceptibility and environmental fac-
tors may be involved.
Critical Time Windows
Another key concept for understanding causation is
that of the ‘critical time window’. Autistic symptoms
commonly appear in the second and sometimes the
first year of life, indicating a strong likelihood of origins
in the prenatal and early postnatal periods. Neurogene-
sis takes place at an astonishing rate, averaging 250,000
new neurons per minute during gestation; the result is
100 billion neurons at birth [Davis & Pfaff, 2014]. Most
of the growth is in the third trimester, when 40,000
synapses are formed per minute. Moreover, brain devel-
opment comprises an array of qualitatively different
processes with overlapping timing: neural tube forma-
tion, cell proliferation and differentiation, migration,
dendritic arborization, synaptogenesis, apoptosis, for-
mation, and connectivity of cortical mini-columns, and
myelination. Synaptogenesis, considered to play a
major role in ASD, accelerates in mid-2nd trimester and
continues through adolescence, whereas neural migra-
tion is completed by birth [Tau & Peterson, 2010].
Evidence from ASD genetics implicates disruption of
these processes. Variants enriched in ASD are found in
genes that regulate synaptogenesis [De Rubeis et al.,
2014; Patel, Ioannidis, Cullen, & Rehkopf, 2015], cell
migration [Boitard et al., 2015; Reiner, Karzbrun, Kshir-
sagar, & Kaibuchi, 2016; Wang et al., 2014; Wong
et al., 2014], dendritic growth [De Rubeis et al., 2014]
as well as more basic functions such as chromatin
remodeling, cell signaling and cell cycle regulation
[Anney et al., 2011; De Rubeis et al., 2014]. Transcrip-
tomics along with whole exome sequencing of histone
modifying genes similarly suggest altered expression of
genes involved in synapse formation, neuronal connec-
tivity, and apoptosis [De Rubeis et al., 2014; Mahfouz,
Ziats, Rennert, Lelieveldt, & Reinders, 2015; Zeidan-
Chulia et al., 2014; Zhang et al., 2016]. Gene expression
is regulated by epigenetic changes, which in turn may
have environmental origins.
Brain development unfolds with precise timing and
spatial attributes, and several epidemiologic studies sug-
gest specificity of susceptible windows for environmen-
tal influences on ASD, summarized in [Schmidt, Lyall,
& Hertz-Picciotto, 2014a]. Empirical research on thalid-
omide traced the exposures to an early insult on days
20–24 days post-conception [Rodier, Ingram, Tisdale,
Nelson, & Romano, 1996]. Maternal intake of prenatal
vitamin supplements appeared protective when taken
4 Hertz-Picciotto et al./Environmental contributions to ASD
close to conception, but not in later time periods,
according to two separate studies [Schmidt et al., 2011,
2012; Suren et al., 2013]. Studies on maternal fever sug-
gest first and second trimesters as being influential
[Atladottir et al., 2010], whereas air pollution results
indicate the strongest associations with ASD following
third trimester or early postnatal exposures. A broad
survey of parents regarding stressors during pregnancy
implicated weeks 21–32 in autism [Beversdorf et al.,
2005]. Thus, time interval(s) for brain vulnerability may
vary by the agent, its mechanism, and the target of its
action. A critical window may be identifiable for expo-
sures that are uncorrelated across time periods (e.g.,
fever) or for associations not observed in other time
windows (prenatal vitamins, thalidomide), but difficult
to discern when exposures are highly correlated over
time.
Often the critical, hence relevant, window for a par-
ticular exposure is unknown. Or, the exposure informa-
tion is only available in broad intervals enveloping
periods outside the susceptible window. In these situa-
tions, relevant exposure may be measured with error,
and if exposure is categorized, will result in misclassifi-
cation [Hertz-Picciotto, Pastore, & Beaumont, 1996].
Errors in capturing exposure in the susceptible window
will usually be independent of the diagnosis, that is,
just as likely in those with, as in those without, ASD
(‘nondifferential misclassification’). These errors bias
the association towards the null, that is, towards find-
ing no effect, when both exposure and outcome are
dichotomous variables [Rothman, Greenland, & Lash,
2008]. Consequently, lack of knowledge about the criti-
cal window for a given exposure can lead to Type 2
errors in statistical tests (null hypothesis not rejected
when the alternative hypothesis is true), and underesti-
mation of effect sizes.
Multifactorial Causation
Much of biomedical science proceeds by tackling single
hypothesized causes of medical conditions, even when
multiple risk factors are established. Although the con-
cept that a single or small group of genes would explain
the vast majority of ASD cases had considerable traction
not long ago, today, it is broadly acknowledged that
hundreds of genes may contribute to ASD risk. Thus, a
prevailing consensus accepts multifactorial causation of
ASD across the population.
Less appreciated is the multifactorial causation within
an individual. The impacts of causal factors—both
genetic and environmental—are often interdependent,
and a person’s inherent ability to adapt will determine
their ‘tipping point.’ Whereas each of us inherits
genetic susceptibility, along with components of our
parental epigenetic profiles, our environmental ‘toxic
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Figure 1. Causal ‘pies’. This pie represents a hypothetical set
of causes that all together is minimally sufficient to induce the
disease or condition of concern in some members of the popula-
tion. Represented here is one possible pie, or set of sufficient
causes, but there may be (usually are) many different sufficient
sets across the population. For any such set, removal of any
one factor will make the set insufficient, and therefore the indi-
vidual would not meet criteria for the diagnosis, though lesser
impairments might still occur.
burden’ or ‘load’ also influences risk. As described by
Herbert [Herbert & Weintraub, 2012] the toxic load
includes chemical, infectious, psychologic, nutritional,
and other stressors, and can erode an individual’s resil-
ience through effects on the epigenome, endocrine
homeostasis, cell signaling and a range of physiologic
and molecular adaptive mechanisms, ultimately over-
whelming adaptive capacity. From the perspective of a
multifactorial model encompassing both genetic and
environmental factors, genetic inheritance determines
baseline susceptibility, and epigenetics—both inherited
and acquired via interaction with the environment—
likely acts by setting or resetting that threshold for
adaptation.
One conceptualization of multifactorial causation is
presented in Figure 1. This ‘pie’ represents a minimally
sufficient set of causes, that is, together, the factors in a
single pie will result in ASD in at least some segment of
the population [Rothman et al., 2008]. These multiple
causes need not occur simultaneously. For instance,
vitamins might be influential around conception,
maternal illnesses in mid- to late gestation, and certain
exogenous chemicals even later or earlier. Across the
population, there may be dozens or hundreds of such
pies.
Under this conceptual model of multifactorial causa-
tion, removal of one piece of the pie results in a set
that is no longer sufficient. The resulting phenotype
might range from virtually free of symptoms to having
clear impairments but not meeting diagnostic criteria.
Thus, despite thousands of stressors in the human
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experience, even in the earliest stages of life, it is not neces-
sary to identify and remove or reduce them all in order to
lessen the risk! Instead, large gains in improving the lives
for those at risk can be achieved by focusing on com-
monly encountered factors that are disabling for large
proportions of people. In other words, some of the
greatest impacts will occur when interventions success-
fully target the most prevalent causal factors.
Etiologic Research on Environmental
Contributions to ASD: A Developmental
Perspective
The 20th Century
Pre-conception of environmental etiologic research.
Figure 2 provides a timeline of ASD etiologic research.
At the turn of the 21st century, evidence for genetic
contributions to ASD comprised high family recurrence,
twin concordance, chromosomal abnormalities and
some identified candidate genes [Bailey et al., 1995; Fol-
stein & Rutter, 1977b,; Ritvo et al., 1989; Wassink &
Piven, 2000; Wassink et al., 2001]. At the same time,
understanding of nongenetic root causes for ASD
amounted to a few clues about infectious origins and
pharmacologic agents. These included an unusually
high prevalence of autistic symptoms in children
exposed to congenital rubella [Chess, 1977; Desmond
et al., 1967], prenatal thalidomide, a medication pre-
scribed as a sedative for morning sickness in pregnancy
[Rodier et al., 1996; Stromland, Nordin, Miller, Aker-
strom, & Gillberg, 1994], or prenatal valproate expo-
sures [Rodier et al., 1996]. Another epidemiologic study
suggested that maternal gestational or infant (first 18
postnatal months) influenza, ear infection, or fever of
unknown origin, occurred more often in children with
autism than in their unaffected siblings [Deykin & Mac-
Mahon, 1979]. Other studies indicated links between
ASD and suboptimal birth outcomes (preterm delivery,
APGAR score) [Nelson, 1991], which may represent
causal agents, mediating factors, or markers of little eti-
ologic significance. Overall, research on risk factors or
causes of autism was scattered: as a field of inquiry, this
was the pre-conception period.
The 21st Century
Conception & gestation of environmental etiologic
research. Continuing on our timeline, despite a
largely barren landscape of nongenetic research at the
turn of the 21st century, a new wave of environmental
studies began to appear. Initially, many were ‘ecologic’
studies, denoting a particularly weak epidemiologic
design that correlates group-level outcomes such as
prevalences or rates, with a group-level statistic for
exposure (median levels, proportion exposed, etc.).
Such studies are prone to various biases, including
5
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Hertz-Picciotto et al./Environmental contributions to ASD
Figure 2. Timeline of etiologic research on autism. Shown is a broad sweep of research on autism etiology, highlighting major framing publications on autism incidence, prev-
alence and causation (white background), along with genetics (black background), and emphasizing prenatal modifiable factors: medical (medium blue background, environmen-
tal chemical (grey background), and gene-by-environment interactions (light blue background).
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confounding at the group-level, reverse causation
(when exposure and outcome are measured in the same
time interval, the outcome could be influencing the
exposure), and cross-level confounding (the latter
referred to as “ecologic” bias). [Morgenstern, 1995].
Birth. In 2006–2007, high quality individual-level
studies emerged. Within a decade, scores of reports pro-
duced results on environmental chemical and physical
agents, lifestyle, infectious agents, trauma, and medical
conditions or interventions. We review major classes of
risk or protective factors occurring in the prenatal
period in Tables 1, 2, and 3. This literature, represents
the Infancy & Childhood of the field. A separate sec-
tion probes, more deeply, methodologic issues relevant
to causal inference using three instructive examples of
environmental chemicals, nutrition, and maternal med-
ical conditions, with special reference to work con-
ducted at the UC Davis MIND Institute.
Chemicals and Pollutants (Table 1)
Air pollution. Over a dozen full-scale epidemiologic
studies have been published, most indicating that pre-
natal exposure to air pollutants increased risks for ASD
[Becerra, Wilhelm, Olsen, Cockburn, & Ritz, 2013;
Hartz, Heinonen, Shapiro, Siskind, & Slone, 1975; Kalk-
brenner et al., 2010; Kalkbrenner, Schmidt, & Penlesky,
2014; Raz et al., 2015; Talbott et al., 2015; Volk, Hertz-
Picciotto, Delwiche, Lurmann, & McConnell, 2011;
Volk, Lurmann, Penfold, Hertz-Picciotto, & McConnell,
2012; Windham, Zhang, Gunier, Croen, & Grether,
2006]. However, different pollutants were evaluated,
including coarse and fine particles, nitrogen dioxide,
ozone, metals, solvents, and diesel particles. Metrics of
exposure varied from proximity to freeways to model-
based estimates of concentrations, sometimes based on
annual averages, and in other studies based on more
temporally sensitive models. The models are con-
structed from air monitoring, source information, dis-
persion models, and sometimes also land topography
and meteorologic variables; model validation was per-
formed for many, though for limited time periods and
geographic areas. Outcomes also varied from maternal
response to a single question about whether they have
a child with autism to a full clinical evaluation using
established standardized instruments (e.g., ADOS and
ADI-R).
The pattern of findings is generally consistent across
U.S. studies, though not necessarily for the same pollu-
tants. Although virtually all studies adjusted for at least
one socioeconomic variable, concerns about residual
confounding remain: Air pollution is highest in com-
munities with the lowest socioeconomic levels in most
of the U.S., and, a recent analysis of correlates for SES
INSAR Hertz-Picciotto et al./Environmental contributions to ASD
using the nationally representative NHANES (National
Health and Nutrition Examination Survey) provided
unequivocal evidence that household income is posi-
tively correlated with numerous serum nutrients, and
negatively correlated with blood and urine measure-
ments of metals, hydrocarbons and volatile organics
that are typical constituents of ambient air pollution,
along with a wide range of viral and inflammatory
markers [Patel et al., 2015]. Notably, nutritional, viral
and inflammatory exposures have each been associated
with ASD. Moreover, two large rigorous studies con-
ducted in Europe, where there is less of a link between
wealth and clean air, showed no association with ASD
[Gong et al., 2017; Guxens et al., 2016]. Additionally, a
troubling discrepancy is the inconclusive literature on
cigarette smoking in relation to ASD, even though
tobacco smoke and ambient air pollution contain thou-
sands of chemicals in common, some associated with
ASD in epidemiologic studies and many with biologic
plausibility. Moreover, many studies on tobacco smoke
failed to control for confounding or inappropriately
controlled for potential intermediates (such as birth-
weight). In short, unanswered questions about con-
founding in studies of air pollution and about the true
association of smoking with ASD remain.
Pesticides. Studies of pesticides and ASD are dis-
cussed under “Methodologic Challenges” below.
Phthalates. Phthalates are a ubiquitous class of
chemicals used as plasticizers, stabilizers, emulsifiers,
dispersants, solvents, and lubricants and as enteric coat-
ings on pharmaceuticals and nutritional supplements.
They are in vinyl flooring, adhesives, pesticides, food
packaging, children’s toys, medical tubing, air fresh-
eners, toiletries (soaps, shampoos, hair products), and
cosmetics, [Centers for Disease Control and Prevention,
2016; Kim, Hong, Bong, & Cho, 2015; U.S. Environ-
mental Protection Agency Office of Prevention, 2006].
In short, they are ubiquitous. Four studies have
addressed phthalates in relation to ASD, autistic symp-
toms, or common co-occurring behaviors. In a Scandi-
navian population-based sample, maternal report of a
child with ASD was associated with vinyl flooring in
the bedrooms [Larsson, Weiss, Janson, Sundell, & Bor-
nehag, 2009]. Phthalates are a major component in
vinyl flooring and off-gas continuously. Two cohort
studies measured phthalate metabolites in maternal 3rd
trimester urine samples: one found poorer scores on all
domains of the Social Responsiveness Scales (SRS) [Mio-
dovnik et al., 2011], whereas the other measured differ-
ent phthalates, and showed no association with the
composite SRS [Braun et al., 2014b]. In a fourth study,
phthalates measured in house dust samples collected
7
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Table 1. Evidence on Environmental Chemicals and Pollutants in Relation to Autism Spectrum Disorder or Autism Symptoms
Environmental chemical
or pollutant class: State of the evidence: Reviews or influential references:

Air pollution Associated with increased risk of autism in close to a dozen studies in the Windham et al. [2006]; Kalkbrenner et al.
U.S. and Asia, but not in several European populations. Different exposure [2010]; Volk et al. [2011, 2012]; Roberts
measures have included: (a) modeled estimates by Census tract; (b) prox- et al. [2013]; von Ehrenstein et al.
imity of geocoded home address to freeway; (c) model-based estimates of [2014]; Raz et al. [2015]; Guxens et al.
NO2, ozone, PM2.5, and PM10 at geocoded home address; (d) other air con- [2016]; Gong et al. [2017]
taminant monitoring and land use regression. Results most consistent for
PM2.5; other pollutants not entirely consistent. Two European studies do
not confirm air pollution associations with ASD. Because of strong correla-
tions between air pollution and socioeconomic status, residual confound-
ing may be affecting multiple studies. Apparent discrepancy with cigarette
smoke (see below) also raises questions. Air pollutants induce oxidative
stress and have been associated with cognitive and neurobehavioral
deficits.
Pesticides: Organophos- Four studies provide evidence, all used objective measures of exposure. Two Rauh et al. [2011]; Eskenazi et al. [2007];
phates (OP’s) cohort studies reported prenatal OP exposures associated with PDD symp- Roberts et al. [2007]; Shelton et al.
toms: one measured chlorpyrifos in plasma (cord or maternal); the other [2014]
measured metabolites (nonspecific for organophosphate pesticides) in
urine. Two case-control studies linked proximity to agricultural applica-
tions of OP’s with autism risk. Third trimester exposures to chorpyrifos,
the most commonly used OP, showed OR>3.0. Pesticide drift measured in
air samplers at distances several kilometers away from agricultural applica-
tions correlates with amounts applied [Wofford et al., 2014]. Organophos-
phate exposures during gestation have also been associated with
cognitive impairment, volumetric differences in brain regions, attention
deficits, and tremor (see Table 4).
Pesticides: Pyrethroids Association observed with applications of pyrethroids in two studies, includ- Roberts et al. [2007]; Shelton et al. [2014]
ing two large case-control studies. Both geocoded residential addresses
and assessed proximity to agricultural applications during the pregnancy.
One observed association with bifenthrin, which degrades most slowly of
all pyrethroids. The other study found total pyrethroids or Type II pyreth-
roids associated primarily when exposures were in the pre-conception or
3rd trimester, after adjustment for confounders. Numerous mechanisms
have been proposed for neurodevelopmental toxicity [Shelton et al.,
2012].
Phthalates Three of four studies suggest an association with neurobehavior; one of two Larsson et al. [2009]; Miodovnik et al.
reported an association with ASD diagnosis, and one of two found mater- [2011]; Braun et al. [2014a]; Philippat
nal prenatal urinary phthalate metabolites associated with higher SRS et al. [2015]
scores. One observed greater hyperactivity/impulsivity and inattention.
Studies used: (a) presence of vinyl flooring; (b) phthalate metabolites
measured in maternal prenatal urine; and (c) house dust phthalates. Given
the continuous nature of most phthalate exposure sources, urinary meas-
urements may provide reasonable approximations to chronic long-term
exposures. Phthalates disrupt androgen activity [Main et al., 2006;
National Research Council, 2008; Miodovnik et al., 2014].
Persistent pollutants: Three studies of PCBs in association with ASD or SRS scores, one with very Cheslack-Postova et al. [2013]; Braun et al.
PCBs low power. Two others (one using SRS, one using ASD diagnosis) found [2014a]; Lyall et al. [2017]; Jolous-
associations of higher scores or risk for the same congener peak (IUPAC Jamshidi et al. [2010]
#138/#158), while one found lower SRS and the other found higher ASD
risk with #153. Nondioxin-like PCBs can influence neuronal dendritic
development. Toxicologic studies demonstrate alterations in social behav-
iors in rodents. Disruption of thyroid hormone homeostasis is a mecha-
nism of neurodevelopmental toxicity in rodents.

ASD, autism spectrum disorder; PCBs, polychlorinated biphenyls; PDD, pervasive developmental delay.

after the child was diagnosed were associated with
other developmental delay but not autism; however, in
both groups, higher phthalate levels were associated
with hyperactivity-impulsivity and inattention [Philip-
pat et al., 2015]. The house dust protocol was designed
to obtain deep dust deposited over a period of years
that included, but was not specific to, prenatal expo-
sures. Thus, three of four studies in different popula-
tions, using three different methods to assess early life
exposure suggest potential impact on neurobehavior,

8 Hertz-Picciotto et al./Environmental contributions to ASD INSAR

Licensed to Mariana Wollinger Maciel Fassheber - marianawmaciel@gmail.com - 046.925.309-60
but not necessarily ASD. Phthalates have well-known
anti-androgenic properties [National Research Council,
2008; Main et al., 2006; Miodovnik, Edwards, Bellinger,
& Hauser, 2014] and sexual dimorphism is commonly
observed in epidemiologic studies of phthalates and cer-
tain behavioral and cognitive outcomes [Engel et al.,
2010; Whyatt et al., 2012]. With a high male:female
ratio in autism, an ‘extreme male brain’ resulting from
excessive androgens during gestation has been proposed
[Baron-Cohen et al., 2015]. The literature, however, is
contradictory [e.g., Kung et al., 2016]. Alternative more
biologically focused frameworks emphasize sexually
dimorphic fetal programming of stress responses by the
HPA-placental axis [Davis & Pfaff, 2014] and brain mas-
culinization in context of immunoregulatory processes
[McCarthy & Wright, 2017].
Persistent pollutants. PCBs are a class of chemicals
formerly used in industrial applications but now
banned; by 1985, production ended worldwide. They
are persistent in the environment bioaccumulate
through the food chain, have been associated with neu-
rodevelopmental deficits in young children [Vreugden-
hil, Lanting, Mulder, Boersma, & Weisglas-Kuperus,
2002; Winneke et al., 1998] and as recently as 2003–
2004 were still detected in 100% of the U.S. population
[Megson et al., 2013]. Three studies have investigated
associations of PCBs with ASD or with SRS scores. One
small study reported elevated OR’s but confidence inter-
vals (CI) were very wide [Cheslack-Postava et al., 2013].
In a cohort from Cincinnati, one specific PCB peak
(138/158) was associated with higher composite SRS
scores in a semi-Bayesian analysis with 52 pollutants,
but this may have been driven by a very high correla-
tion with another PCB (153) showing the opposite asso-
ciation [Braun et al., 2014b]. However, a large nested
case-control study that measured maternal PCBs in 2nd
trimester serum and confirmed diagnoses based on an
expert review of medical records, found that these same
two PCB peaks (138/158 and 153) were both associated
with significantly elevated risk for ASD [Lyall et al.,
2017b]. PCBs can disrupt thyroid hormones, though
effects appear small at today’s population levels.
Other persistent pollutants that are pervasive in
homes and detectable in the vast majority of U.S. resi-
dents and in other populations have yet to be ade-
quately studied as root causes of autism. Two examples
are polybrominated diphenyl ethers (PBDEs), a class of
compounds used as flame retardants in a wide variety
of household and building products [Wu et al., 2015]
and perfluoroalkyl and polyfluoroalkyl substances
(PFAs), which are ubiquitous due to their use as stain
resistant coatings on fabrics and carpets, as nonstick
coatings on cookware, and in firefighting foam, the
INSAR Hertz-Picciotto et al./Environmental contributions to ASD
latter now contaminating drinking water in the U.S.
[Hoffman et al., 2011; Post, Louis, Lippincott, & Proco-
pio, 2013]. Both PBDEs and PFASs are endocrine-
disrupting and have been linked with neurologic and
neurodevelopmental outcomes in a small number of
human studies [Braun et al., 2017; Herbstman et al.,
2010; Hoffman, Webster, Weisskopf, Weinberg, &
Vieira, 2010; Lopez-Espinosa, Mondal, Armstrong, Eske-
nazi, & Fletcher, 2016; Sagiv et al., 2015; Stein & Savitz,
2011; Zhao et al., 2015].
Proxy Exposures
Maternal and paternal age, inter-pregnancy interval (see
below) and season are all factors that can serve as surro-
gates for more proximal risk or protective factors. For
example, season of conception or birth can be a proxy
for cold/warm weather, air pollution, influenza preva-
lence, use of pesticides, or availability of fruits and veg-
etables. Winter conceptions have been associated with
higher incidence of ASD [Gadow, Perlman, Ramdhany,
& de Ruiter, 2016; Mackay et al., 2016; Zerbo, Iosif, Del-
wiche, Walker, & Hertz-Picciotto, 2011], though other
studies found different seasons [Atladottir et al., 2007;
Hebert et al., 2010; Hultman et al., 2002; Lee et al.,
2008]. As the net effect of season is likely not strong,
only large studies would have sufficient power.
Lifestyle Factors (Table 2)
General nutrition. Evidence is accumulating for a
potentially large role in autism etiology for gestational
nutrition. Several studies examining maternal nutrients
or diet support an association with folic acid and other
nutrients [Lyall, Schmidt, & Hertz-Picciotto, 2014;
Schmidt et al., 2011, 2012; Suren et al., 2013]; folic acid
studies are reviewed in detail below (‘Methodologic
Challenges’). Here we discuss iron, fatty acids, and fish
consumption, and short inter-pregnancy interval (IPI)
as a proxy. We omit postnatal factors (e.g., breastfeed-
ing) due to space constraints.
IPI. As recently systematically reviewed, a short IPI
has been consistently associated with ASD, conferring
about 2-fold higher risk for children born after an IPI
less than 12 months compared with those born after 36
or more months [Cheslack-Postava, Liu, & Bearman,
2011; Cheslack-Postava et al., 2014; Conde-Agudelo,
Rosas-Bermudez, & Norton, 2016; Coo et al., 2015; Dur-
kin, DuBois, & Maenner, 2015; Gunnes et al., 2013;
Zerbo, Yoshida, Gunderson, Dorward, & Croen, 2015].
Short IPI may indicate maternal depletion of nutrients,
such as folate, as essential nutrients are preferentially
distributed to the developing fetus during pregnancy,
and can remain low for up to a year postpartum
[O’Rourke, Redlinger, & Waller, 2000; Smits & Essed
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Table 2. Evidence on Lifestyle Factors in Relation to Autism Spectrum Disorder or Autism Symptoms
Lifestyle & Diet State of Evidence Reviews and Influential References2

Interpregnancy interval Shorter intervals (<12 months) from end of the previous pregnancy to the Cheslak-Postava et al. [2011]; Dodds et al.
start of pregnancy associated with about two-fold higher autism risk in [2011]; Gunnes et al. [2013]; Cheslack-
8 studies. Some evidence for dose-response, however three studies also Postava et al. [2014]; Coo et al. [2015];
showed higher risk with longer intervals. Associations could result from Durkin et al. [2015]; Zerbo et al. [2015];
maternal nutrient depletion or cultural, family planning, or lifestyle Conde-Agudelo et al. [2016]
factors.
Maternal folic acid and Higher folic acid intake and folic acid supplements associated with reduced Schmidt et al. [2011, 2012, 2017]; Suren
prenatal vitamins risk for ASD or autistic traits in several population-based studies; one et al. [2013]; Braun et al. [2014a];
study had null findings. Association with ASD diagnosis specific to expo- Steenweg-de Graaff et al. [2015]; Virk
sure near conception in two studies. Evidence for a stronger association et al. [2016]
when mother or child has genetic or environmental susceptibility. Two
studies observed no association of autistic traits with maternal serum
folate collected after the first trimester of pregnancy, similar to reported
lack of association with folic acid intake or supplements after first two
months. Mechanisms for folic acid protection may involve methylation of
DNA and altered gene expression. One study of interactions showed folic
acid protection against pesticide-related increased risk of ASD.
Maternal iron In a large case-control study, mothers of children with ASD were less likely Schmidt et al. [2014b]; Suren et al. [2013]
to report taking an iron supplement; lowest quintile of supplemental iron
associated with ASD, especially in older mothers and mothers with meta-
bolic conditions. A large birth cohort found no association with minerals.
Maternal fatty acids and Literature is inconsistent. Exposures have included (a) prenatal fish oil sup- Suren et al. [2013]; Lyall et al. [2013];
fish consumption plements, (b) intake of polyunsaturated fatty acids, omega-6 and omega-3 Steenweg-de Graaff et al. [2016]; Julvez
fatty acids and/or linoleic acid, and (c) fish consumption. Self-reported et al. [2016]
intake of polyunsaturated fatty acids, omega-6 fatty acids and linoleic
acid associated with lower ASD risk in one study, but in another, total
omega-6 and linoleic acid associated with more ASD traits. Another
observed increased ASD risk with very low omega-3 intake. Fish intake not
associated with ASD or traits in two studies, while another found fewer
autistic traits in association with high fatty fish consumption; this latter
was the only study to adjust for mercury.
Maternal smoking Evidence is inconsistent. Two recent meta-analyses, each based on fifteen Rosen et al. [2015]; Tang et al. [2015].
studies, reported the same OR of 1.02 (95% CI 5 0.93, 1.12 or 1.13).
However, two-thirds of original analyses either did not adjust for any con-
founders, or incorrectly adjusted for birthweight or gestational age,
potential intermediates, for which adjustment can induce bias. All relied
on self-report of smoking, known to be prone to underascertainment. No
studies examined genetic susceptibility, nor have objective exposure
markers been utilized in smoking assessment.

2001; van Eijsden, Smits, van der Wal, & Bonsel, 2008].
Short IPI could also be a marker for cultural, family
planning or lifestyle factors [Cheslack-Postava et al.,
2011; Dodds et al., 2011], and evidence for a U-shaped
relationship between IPI and ASD supports a contribu-
tion from such influences [Cheslack-Postava et al.,
2014; Durkin et al., 2015; Zerbo et al., 2015].
Iron. Iron is critical for fetal and placental growth
and brain development and functioning. During preg-
nancy, iron deficiency, which is common [O’Brien,
Zavaleta, Abrams, & Caulfield, 2003; Stoltzfus, 2001]
can induce fetal and infant iron-deficiency [Allen, 2000;
Colomer et al., 1990; Millard, Frazer, Wilkins, & Ander-
son, 2004; Tchernia, Archambeaud, Yvart, & Diallo,
1996]. Low iron has been associated with developmen-
tal delays and behavioral disturbances including
decreased social interaction [Pollitt, 1993]. Iron contrib-
utes to neurotransmitter production, myelination, and
immune function [Beard, 2000], processes shown to be
dysregulated in autism [Beard, 2000].
Results from two large studies of maternal iron during
pregnancy and ASD risk are inconsistent. In the
California-based CHARGE case-control study (520 cases,
346 typically developing controls), children of mothers
with the highest versus lowest iron intake from supple-
ments and cereals had half the odds of developing ASD,
especially when mothers were of advanced age, obese,
diabetic, or hypertensive [Schmidt, Tancredi, Krako-
wiak, Hansen, & Ozonoff, 2014b]. In contrast, in the
Norwegian birth cohort study [Suren et al., 2013], ASD
risk did not differ for those taking vitamin and mineral
supplements likely to contain iron, compared with
those taking no vitamins or minerals. The studies dif-
fered on obesity prevalence; potential for recall bias;

10 Hertz-Picciotto et al./Environmental contributions to ASD INSAR

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and measure of supplemental iron intake. Neither study
accounted for total dietary iron intake or maternal iron
status, concerns that future studies should heed.
Fatty acids and fish. Polyunsaturated fatty acids
(PUFAs), especially omega-3, play significant roles in
the structure, function, and development of human
brains [Freeman et al., 2006; McNamara & Carlson,
2006; Rombaldi Bernardi, de Souza Escobar, Ferreira, &
Pelufo Silveira, 2012]. Differential PUFA status in indi-
viduals with versus without ASD [Brigandi et al., 2015;
Jory, 2016; Wiest, German, Harvey, Watkins, & Hertz-
Picciotto, 2009; Yui, Imataka, Kawasaki, & Yamada,
2016], and small studies of fatty acid supplementation
to improve ASD symptoms were recently reviewed
[Mazahery et al., 2017]. Omega-3 consumption during
pregnancy has been associated with higher IQ [Richard-
son, Easton, & Puri, 2000b] and fewer social problems
[Richardson & Ross, 2000]. Similarly, lower omega-
3:omega-6 ratio in maternal blood was associated with
poor communicative development [Strain et al., 2008]
Maternal seafood and fish intake, the primary source of
PUFAs [Peet, Laugharne, Mellor, & Ramchand, 1996;
Richardson et al., 2000a], improves child neurodevelop-
mental outcomes (reviewed in [Avella-Garcia & Julvez
2014; Starling, Charlton, McMahon, & Lucas, 2015]),
however, positive impacts may be attenuated by con-
taminants such as mercury and PCBs, which bioaccu-
mulate in these foods [Avella-Garcia & Julvez, 2014].
The few studies that examined maternal fatty acids
and ASD risk are not concordant. The Norwegian study
found no association of prenatal fish oil supplements
with ASD risk [Suren et al., 2013]. In contrast, mothers
in the Nurses’ Health Study II who reported having a
child with autism also reported diets lower in omega-6,
linoleic acid, and total PUFAs, and very low intake (the
lowest 5%) of omega-3 [Lyall, Munger, O’Reilly, Santan-
gelo, & Ascherio, 2013], but similar intake of fish to
mothers not reporting a child with ASD. However, die-
tary information was not collected for the pregnancy
period for most participants, PUFA from supplements
was not available, and contaminants in fish were not
considered.
Two studies assessed autistic traits. The Generation R
Cohort of the Netherlands obtained the Social Respon-
siveness Scale on >4,600 children, and measured PUFAs
at midpregnancy: lower maternal plasma omega-
3:omega-6 ratio, and higher total omega-6 and linoleic
acid were associated with more autistic traits at 6 years
[Steenweg-de Graaff, Ghassabian, Jaddoe, Tiemeier, &
Roza, 2016]. Maternal omega-3 status and prenatal die-
tary fish intake were not associated with autistic traits.
This study did not account for mercury or PCB expo-
sure. A study that administered the Childhood Asperger
INSAR Hertz-Picciotto et al./Environmental contributions to ASD
Syndrome Test examined fish intake collected prospec-
tively; the researchers reported consumption of fatty
fish above the recommended limit during pregnancy
(>340g/week) was associated with fewer autistic traits
along with higher cognitive scores [Julvez et al., 2016].
This was the first study to control for confounding by
mercury (measured in umbilical cord blood). Additional
strengths were the large sample, inclusion of both die-
tary and supplement intake, and analyses by timing,
which showed stronger associations for prenatal as
compared with childhood intake. Conclusions on fish
and PUFAs will require more studies that include sup-
plements and diet, adjust for fish contaminants, and
consider exposure windows.
Smoking. The literature on maternal smoking and
ASD has been contradictory, and two meta-analyses
that employed random effects models on fifteen studies
reported the same estimated OR 5 1.02 and almost
identical CI (0.93, 1.12) [Rosen, Lee, Lee, Yang, & Bur-
styn, 2015] and (0.93, 1.13) [Tang, Wang, Gong, &
Wang, 2015]. Sensitivity analyses from both teams
found the results to be robust with no evidence of pub-
lication bias. Is this sufficient evidence of no effect to
be definitive? Two concerns detract from that conclu-
sion. First, of the 15 studies included in the analysis by
Tang et al. [2015], six did not adjust for any confound-
ers, and four adjusted for either birthweight or gesta-
tional age, both of which are strongly associated with
smoking and ASD and could be intermediates, for
which a standard adjustment as a confounder can intro-
duce bias. Second, virtually all studies ascertained
smoking by self-report, whether to a mid-wife, on a
standardized questionnaire, or for a birth certificate;
birth certificates have long been known to provide
underestimates of smoking prevalence, and more gener-
ally, self-report of smoking during pregnancy is subject
to significant under-reporting. Gold-standard biochemi-
cal measures of active smoking indicate about 25%
underascertainment using self-report. [Dietz et al.,
2011; Kang et al., 2013; Shipton et al., 2009; Swamy,
Reddick, Brouwer, Pollak, & Myers, 2011]. This high
level of misclassification could bias results towards a
no-effect finding.
Medical Factors (Table 3)
Medications. Described above, prenatal valproic acid
and thalidomide exposures predicted strongly elevated
risk for autism. More recently, some reports have sug-
gested that use of SSRI’s may, in certain subsets, be
associated with ASD [Croen, Grether, Yoshida, Odouli,
& Hendrick, 2011; El Marroun et al., 2014; Harrington
et al., 2014; Rai et al., 2013], with two studies suggesting
greatest risk with first trimester exposures [Croen et al.,
11
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Table 3. Evidence on Medical Conditions, Procedures, or Medications in Relation to Autism Spectrum Disorder or Autism
Symptoms
Medical Factors Summary of evidence Influential References or Reviews

Thalidomide Case reports of increased autism prevalence in children of mothers given
Valproic acid/anti- these drugs during pregnancy. With low prevalence of use, these factors
epileptic drugs can account for very few cases of autism today.
Selective Serotonin Most studies found an association of SSRI use during pregnancy and
Reuptake Inhibitors increased risk in either a specific time period or a specific subset. Meta-
analysis of four case-control studies produced and adjusted OR51.8 (95%
CI 5 1.5, 2.2). As with individual studies, confounding by indication could
not be excluded. Studies attempting to assess such confounding yielded
conflicting results.
Pre-eclampsia Several large studies provide evidence of increased risk for ASD. Studies
relied on (a) administrative medical databases (e.g., Medicaid), or (b)
detailed abstraction of medical records including placental insufficiency.
The latter produced a larger effect size than administrative databases,
possibly because detailed abstraction of notes in medical charts beyond
standard codes, is more complete. RR’s ranged from 1.2 to over 2.
Gestational diabetes High degree of consistency in associations with ASD across studies, includ-
ing two meta-analyses, several large cohorts, and a few moderate-sized
studies. Meta-analysis, two large cohorts and one moderate-sized case-
control study found RRs/OR of 1.3–1.5; two studies with <30 cases
observed RRs of 3–4 (with wide CI’s). Strong biologic plausibility; co-
occurring obesity may exacerbate effect. Whether controlled versus uncon-
trolled diabetes alters association has not been examined.
Maternal infections/fever Congenital rubella, maternal fever and influenza, and other viruses during
during pregnancy pregnancy are each associated with autism in multiple studies and in
meta-analysis. In two studies, the impactful timing of maternal fever was
2nd trimester, and limited to fevers not treated with antipyretics. Addi-
tionally, copy number variants and viral infection combined appear to
increase severity of autism symptoms.
Stromland et al. [1994]
Moore et al. [2000]; Rodier et al. [1996]
Croen et al. [2011]; Hviid et al. [2013]; El
Marroun et al. [2014]; Harrington et al.
[2014]; Rai et al. [2013]; Malm et al.
[2016]; Man et al. [2015]
Walker et al. [2015]; Mann et al. [2010];
Burstyn et al. [2010]; Buchmayer et al.
[2009]; Dodds et al. [2011]
Krakowiak et al. [2012]; Lyall et al. [2012];
Xiang et al. [2015]; Connolly et al. [2016];
Li et al. [2016]; Nahum Sacks et al. [2016]
Meta-analysis: Xu et al. [2014]
Chess et al. [1977]; Desmond et al. [1967];
Deykin and MacMahon [1979]; Zerbo
et al. [2012]; Atladottir [2010, 2012];
Webb et al. [2017]; Hornig et al. [2017]

2011; Harrington et al., 2014]. A meta-analysis of nine
studies produced pooled ORs for case-control studies and
cohort studies, respectively, of 2.1 (95% CI, 1.7, 2.7) and
1.8 (1.5, 2.2). As with many of the individual studies,
this analysis was subject to confounding by indication.
However, larger studies that did take such confounding
into account by comparing depressed mothers who did
vs. did not medicate (or did not medicate with SSRIs)
produced conflicting results [Malm et al., 2016; Rai et al.,
2013]; neither study examined timing of exposure. Nev-
ertheless, mother’s mental state can alter the intra-
uterine (and postnatal) environment for the child. At
this stage, no conclusion can yet be drawn as to whether
SSRIs influence risk of ASD.
Medical interventions. Use of ultrasonography for
imaging the fetus is now nearly universal. Both fre-
quency of use and thermal intensity have increased, the
latter by 8-fold during the 1990s as a result of FDA lifting
prior restrictions [Webb et al., 2017]. A few studies
addressed ultrasound in relation to ASD. One found no
association in analyses of the number of ultrasounds
received, but the study period for these exposures (1994–
1999) covered the changes in equipment [Grether, Li,
Yoshida, & Croen, 2010]. That problem, combined with
no estimate of duration or intensity of the thermal
index, suggest that the exposure metric may have done a
poor job of rank ordering children by their true doses. A
second null study randomized pregnancies to different
ultrasound and Doppler imaging protocols, but took
place before introduction of higher dose equipment
[Stoch et al., 2012]. The third investigation evaluated the
triple-hit hypothesis, which proposed ASD phenotypes
to be dependent on three insults combined: (a) environ-
mental exposures; (b) their specific timing; and (c)
genetic susceptibility [Williams & Casanova, 2010]. This
concept was operationalized for exposures to 1st trimes-
ter ultrasound and presence of CNV in an ASD popula-
tion (n 5 1749) from the Simons Simplex Collection
[Webb et al., 2017]. Those with vs. without a 1st trimes-
ter ultrasound scored significantly lower in social affect,
and significantly higher in repetitive/restricted behav-
iors. Limiting to males with CNV, the 1st trimester ultra-
sound associated with significantly lower nonverbal IQs,
and higher repetitive behavior scores. Without a control
group, ASD risk could not be assessed, but results suggest
1st trimester ultrasound may influence the severity of
impairments, particularly in those with underlying
genetic susceptibility.
Infertility treatments have also received attention.
Because both ASD and each type of treatment are both

12 Hertz-Picciotto et al./Environmental contributions to ASD INSAR

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rare, few if any studies have been sufficiently powered
[Lyall et al., 2017a].
Acute illnesses. Decades ago, two independent stud-
ies demonstrated a strong association of ASD with con-
genital rubella [Chess, 1977; Desmond et al., 1967]. In
another early epidemiologic study, prenatal influenza
or other viral infection appeared to predispose the off-
spring to develop autism [Deykin & MacMahon, 1979]
and maternal influenza was recently implicated in an
investigation from Denmark [Atladottir, Henriksen,
Schendel, & Parner, 2012], with a doubling of risk. In
another report, these researchers described 3-fold
increased autism risks after viral infection in the 1st tri-
mester, or 40% higher bacterial infection in the 2nd tri-
mester [Atladottir et al., 2010]. Two studies, including
one prospective cohort, observed an association of ASD
with fever in the 2nd trimester, primarily when not
treated with an anti-pyretic [Hornig et al., 2017; Zerbo
et al., 2012]. Prolonged fever (a week or longer)
appeared to confer a 3-fold higher risk of autism in the
Danish study. Supporting a role for acute infectious ill-
nesses or fever in ASD is a growing literature on inflam-
mation or immune dysregulation in relation to autism
[Braunschweig et al., 2012, 2013; Gesundheit et al.,
2013; Hsiao, 2013; Vargas, Nascimbene, Krishnan, Zim-
merman, & Pardo, 2005] and on animal models involv-
ing infection or immunological perturbations similar to
those induced by infection and resulting neurobehavio-
ral aberrations (see review emphasizing timing [Meyer,
Yee, & Feldon, 2007; Patterson, 2011]. Studies of sea-
sonality of birth or conception add circumstantial evi-
dence insofar as influenza and other infectious diseases
also follow seasonal patterns. Norwegian mothers of
male children with ASD versus male controls were
observed to have higher mid-pregnancy serum anti-
bodies to Herpes simplex virus-2 [Mahic et al., 2017],
however, the proportion who were seropositive did not
differ and several potential confounders were not con-
trolled. A previous study found no association with
HSV-2 [Atladottir et al., 2012].
Chronic conditions, inflammation. A literature on
complications of pregnancy and delivery has emerged.
Hypertensive disorders of pregnancy including pre-
eclampsia have been associated with ASD, consistently
in large, well-conducted studies [Buchmayer et al.,
2009; Burstyn, Sithole, & Zwaigenbaum, 2010; Mann,
McDermott, Bao, Hardin, & Gregg, 2010; Walker et al.,
2015]. Preeclampsia explained much of the association
between preterm delivery and ASD [Buchmayer et al.,
2009] and remained strongly associated even after
adjustment for intermediates, gestational age, or birth-
weight [Mann et al., 2010].
INSAR Hertz-Picciotto et al./Environmental contributions to ASD
Much work has examined maternal diabetes during
pregnancy as a risk factor for ASD with or without obe-
sity [Connolly et al., 2016; Krakowiak et al., 2012; Li
et al., 2016; Xiang et al., 2015], explored in greater
depth below.
Methodologic Challenges
To highlight methodological challenges and illustrate
conceptual issues surrounding environmental epidemio-
logic studies, three investigations from the CHARGE
Study are described. One of the linchpins of this field is
the difficulty capturing exposures accurately, which is
especially acute for case-control studies. For the exam-
ple of pesticides, we demonstrate one approach. A sec-
ond challenge is that of causal inference in the context
of risk factors for which a randomized controlled clini-
cal trial is not an option for either ethical reasons or
feasibility. The field of epidemiology has developed sev-
eral frameworks to ensure that inferences are based on
sound science, logical reasoning and transparency; we
illustrate with the case of maternal diabetes. From the
third example, maternal folic acid intake, we explore
issues of gene-environment interaction and epigenetics.
Briefly, the CHARGE Study was funded in 2002 to
identify causes and contributing factors for ASD, along
with other developmental delays, with a focus on envi-
ronmental chemicals and their interactions with
genetic susceptibility. The case-control design is well-
suited for outcomes with poorly understood etiology, as
this approach allows for multiple exposures to be inves-
tigated, and is more efficient for rare conditions (e.g.,
<5% risk) and those with a potential latency (time
between exposure and diagnosis) of years, than a pro-
spective cohort. With prenatal exposures of special con-
cern and diagnoses frequently made at ages 3 or
beyond, the case-control design also promised statisti-
cally powerful results within a shorter time frame.
Three groups of children were enrolled (ASD, other
developmental delays without ASD symptoms (DD),
and controls from the general population), and final
diagnoses of ASD, DD or typical development were
based on a full clinical assessment (for further protocol
details, see [Hertz-Picciotto et al., 2006]). To address the
primary limitation of case-control studies—retrospective
assessment of exposure—CHARGE obtained, whenever
feasible, objective measures. Two sources are described
in the examples below: a database of environmental
exposures, and medical records. Both carry information
collected in real-time for specific time windows. Such
historical data constitute strengths, essentially obviat-
ing this limitation of the case-control design. With
these types of exposure data, a case-control study can
13
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Table 4. Prospective Studies of Prenatal Measures of Pesticide Exposures and Child Neurodevelopmental Outcomes Other
than ASD
Outcome Study Population Reference

Memory and IQ New York inner-city children age 3 and age 7 years Rauh et al. [2006, 2011]
Brain volumetric measurements Same cohort, age 8–9 years PNAS, Rauh et al. [2012]
Tremor Same cohort, age 11 years Neurotoxicology, Rauh et al. [2015]
Mental & psychomotor development California farmworker children, age 24 months Eskenazi et al. [2007]
Memory and IQ Same cohort, age 7 years Bouchard et al. [2011]
Attention/ADHD Children ages 36 months and 5 years Marks et al. [2010]
Mental and psychomotor development and reasoning Urban child population, ages 12 months and 6–9 years Engel et al. [2011]

provide exposure assignments equal in quality to those
obtained in prospective studies.
Example #1-Pesticides: The challenge of exposure assessment
Humans exposures to pesticides occur from: (a) insect
or other pest control products (sprays, bombs, etc.) in
homes, malls, restaurants, or on pets and gardens, even
when used according to instructions; (b) drift from
applications on orchards or agricultural fields, well-
documented at distances of several kilometers from the
application site [Wofford et al., 2014]; and (c) residues
in fruits, vegetables, and, through bioaccumulation,
meats, fish, and dairy products [Schafer & Kegley, 2002;
Vogt et al., 2012].
Most pesticides are designed to be neurotoxic. Orga-
nophosphates, currently among the most abundantly
used insecticides, are acutely toxic due to inhibition of
the enzyme acetylcholinesterase (AChE), which breaks
down neurotransmitters at the post-synaptic mem-
brane. However, neurodevelopmental and neurologic
dysfunction occur at subacute levels, when AChE inhi-
bition is absent, [Voorhees, Rohlman, Lein, & Pieper,
2016]. Hence other toxic mechanisms are at play and
potentially relevant for gestational exposures in early
brain development, including inflammation, GABA sig-
naling, thyroid disruption, oxidative stress and mito-
chondrial dysregulation [Banks & Lein, 2012; Schuh,
Lein, Beckles, & Jett, 2002; Shelton, Hertz-Picciotto, &
Pessah, 2012; Voorhees et al., 2016]. Prenatal organo-
phosphate exposures have been associated with cogni-
tive impairments, attention deficits, and tremor in
children (Table 4) [Bouchard et al., 2011; Engel et al.,
2011; Rauh et al., 2011]. Some deficits persist into
school ages.
Three studies evaluated prenatal organophosphate
exposures and either an ASD diagnosis or symptoms.
Urine specimens collected from pregnant women in a
farmworker community were analyzed for organophos-
phate pesticide metabolites. When the children were 24
months of age, pervasive developmental delay (PDD)
symptoms were assessed. The proportion scoring
>97.5th percentile was unexpectedly high (possibly
related to administration of the scale in Spanish) and
doubled with a ten-fold increase in metabolites [Eske-
nazi et al., 2007].
Two case-control studies examined ASD in association
with distance of home to agricultural pesticide applica-
tions. All commercial pesticide applications in Califor-
nia agriculture are reportable by law to the Department
of Pesticide Regulation, including specific chemicals
used, amounts, dates of applications, and locations.
Both studies used Geographic Information Systems to
overlay maternal addresses with locations of the pesti-
cide applications from this database. Amounts of pesti-
cide applied within pre-defined distances of the
residences were compared for cases vs. controls. One
study showed increased risks for residential proximity
to two organochlorine pesticides in the first trimester,
and for applications of organophosphates and a specific
pyrethroid (bifenthrin) at any time during pregnancy
[Roberts et al., 2007].
The second case-control study used CHARGE partici-
pants [Shelton et al., 2014], for whom all diagnoses
were clinically confirmed. The three chemical classes
investigated were: organophosphate, pyrethroid, and
carbamate pesticides. Figure 3 demonstrates linkage of
pesticide use reports to the residences. All addresses
from 3 months before conception through pregnancy
were geocoded and buffers of radii 1.25, 1.5, or 1.75
kilometers were created. Overlaying on this map were
the locations of pesticide applications, with each child
assigned exposures within the buffer zone in the win-
dows of interest: the 3 months before conception, each
trimester, or the entire pregnancy. One salient finding
was the significant association between organophos-
phate exposures at any point during pregnancy and
60% higher risk for ASD (Fig. 4, top two panels). The
strongest association was for 2nd and 3rd trimester
chlorpyrifos (an organophosphate), with a 2.5- to 3-fold
elevation in ASD risk. Chlorpyrifos was banned in 2001
by the U.S. EPA for household products, and the safety
of its continued use in agriculture has been contested
based on both toxicologic and epidemiologic research.
In fall 2016, U.S. EPA determined that residues in food
and water exceeded standards for safe levels. In March
2017, the U.S. EPA decided not to follow through on its

14 Hertz-Picciotto et al./Environmental contributions to ASD INSAR

Licensed to Mariana Wollinger Maciel Fassheber - marianawmaciel@gmail.com - 046.925.309-60
Figure 3. Household linkage to Pesticide Use Report (PUR, a
database of pesticide applications based on mandated reporting
by commercial applicators). The dot represents a residence,
each square on the grid is 1 3 1 mile, representing the
township-range-section for which pesticide application loca-
tions are geocoded, and the circle is a buffer around the house,
used for assigning exposures. Shelton et al. [2014], online sup-
plementary material. (This figure was originally posted in the
online supplementary material of this Open Access journal.).
own proposed rule to ban use of chlorpyrifos in agricul-
ture [U.S. Environmental Protection Agency, 2017].
Additionally, preconception pyrethroid exposures
(Fig. 4, lower two panels) showed an 80% increased risk
for ASD. Marketed as a natural product (pyrethrins are
produced by the chrysanthemum plant), pyrethroids are
synthetic compounds chemically engineered to be
more toxic and to last longer. Pyrethrins rapidly
degrade in sunlight whereas bifenthrin has a half-life in
the outdoors of about a year.
In environmental health studies, assessment of expo-
sure stands out as the critical component. Objections
that pesticide applications do not reflect exposure [Burns,
Cohen, & Lunchick, 2015] were not supported by a
recent year-long environmental monitoring study con-
ducted in the California central valley. Air measurements
of chlorpyrifos in a town located near agricultural fields
exhibited a direct variation with the amount of this pesti-
cide applied within an 8 km buffer (Fig. 5) [Wofford et al.,
2014]. Similar high correlations were observed for air
concentrations of numerous other pesticides compared
with amounts known to have been applied, establishing
drift at distances similar to those used as buffers in
research studies on child development. Wofford et al.
thus demonstrate that pesticide applications serve as a
valid measure of exposures to nearby residents. Presence
INSAR Hertz-Picciotto et al./Environmental contributions to ASD
of the pesticides in air samples is sufficient to presume
human exposures, since even indoor air reflects outdoor
air pollution and only those who leave the area for
extended time periods would avoid breathing the air.
Although for some chemicals biologic measures may be
preferable, the organophosphate and pyrethroids com-
pounds are quickly metabolized and excreted. Hence,
even multiple specimens can easily fail to capture an
exposure that may have damaged the developing brain.
Residential proximity to applications recorded in real-
time not only is reasonable and now established as valid,
but may be an optimal exposure metric as it accounts for
exposures throughout preconception and pregnancy.
Further support for long-lasting effects from prenatal
pesticide exposures is a small MRI study in the Colum-
bia Children’s Cohort. Chlorpyrifos was measured in
3rd trimester blood samples. Twenty children, whose
mothers were nonsmokers with low exposure to air pol-
lution, underwent MRI at ages 8–9 years. Among those
with high (vs. low) chlorpyrifos, cortical regions of the
brain responsible for attention, receptive language proc-
essing, social cognition and inhibitory control were
enlarged [Rauh et al., 2012]. These represent core ASD
symptoms and common comorbid deficits, adding bio-
logic plausibility to the pesticide-ASD link.
Thus, two studies used an objective validated measure
of exposure with accurate timing. A third obtained a
biomarker prospectively. In total, three studies impli-
cate prenatal pesticides in ASD: each had a sizable sam-
ple, adjusted for confounding, and used an objective
measure of exposure specific to a period in pregnancy.
The two that examined pyrethroids both found associa-
tions, and all three observed associations of ASD with
organophosphate pesticides or their metabolites.
Example #2-Maternal diabetes: Causal inference in
non-randomized studies
Obesity prevalence has increased at all ages, and by
2010, over one-third of U.S. adults were obese [Flegal,
Carroll, Kit, & Ogden, 2012]. Over 20% aged 20–39
have metabolic syndrome [Ervin, 2009; Flegal, Carroll,
Ogden, & Curtin, 2010], diabetes prevalence doubled
from 1980 to 2012 [Geiss et al., 2014], and among preg-
nant women in California, 9% were diagnosed with
type 2 or gestational diabetes [Lawrence, Contreras,
Chen, & Sacks, 2008], while 5% of U.S. pregnancies in
2009 were complicated by hypertensive disorders [Mar-
tin et al., 2011]. Against this backdrop, a growing num-
ber of studies has observed an association between
maternal prenatal diabetes and ASD [Connolly et al.,
2016; Krakowiak et al., 2012; Li et al., 2016; Nahum
Sacks et al., 2016; Xiang et al., 2015; Xu, Jing, Bowers,
Liu, & Bao, 2014].
15
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Figure 4. Associations of agricultural pesticide applications with risk of autism spectrum disorder (ASD). Graphs present the odds
ratios (OR’s) comparing ASD cases to typically developing controls with respect to their exposures to organophosphate (upper two
panels) and pyrethroid (lower two panels) pesticides. Each column represents a different time window and results for the three dif-
ferent buffers (1.25, 1.5, and 1.75 km) are within each box, with a solid square representing the OR and a bar for the 95% confi-
dence interval. OR’s are plotted on the logarithmic scale.

whose mothers’ pregnancies were complicated by one

Figure 5. Comparison of average weekly concentrations of
chlorpyrifos detected at three monitoring sites in a rural Califor-
nia town over a one-year period, and total reported use of
chlorpyrifos by week in an 8 km study area surrounding the
monitoring sites. Reproduced from Wofford et al. [2014].
(Reprinted with permission from Springer.).
In the CHARGE Study, compared with mothers of
typically developing children, those of children with
ASD or other DD were disproportionately afflicted with
metabolic conditions during pregnancy, including obe-
sity, diabetes (type 2, gestational), and underlying
hypertension or preeclampsia [Krakowiak et al., 2012;
Walker et al., 2015]. Among nearly 1,000 children clini-
cally assessed using standardized instruments, those
or more metabolic conditions were 60% more likely to
have ASD and over twice as likely to have DD compared
with children whose mothers had healthy weight and
no metabolic conditions. These results were adjusted
for sociodemographic characteristics, calendar time,
and the child’s age and sex [Krakowiak et al., 2012].
Obesity, the most prevalent metabolic condition, drove
these associations. Metabolic conditions involve dysre-
gulation marked by elevated glucose, triglycerides, cho-
lesterol, leptin, and proinflammatory immune markers.
Maternal diabetes, primarily gestational, was associ-
ated with a doubled risk for DD and a 50% increased
likelihood of ASD although the latter did not reach sta-
tistical significance. An earlier meta-analysis had sug-
gested an ASD-maternal diabetes association [Gardener,
Spiegelman, & Buka, 2009], and recently, a meta-
analysis of 12 studies (9 case-control, including the
CHARGE Study; 3 cohort) also demonstrated signifi-
cantly elevated risks (cohort: pooled RR 5 1.5, 95% CI
1.3, 1.8; case-control: pooled OR 5 1.7, 95% CI 1.2, 2.4)
[Xu et al., 2014]. Newer large epidemiologic studies also
confirm the association between maternal diabetes
(alone or in combination with obesity) and risk for ASD
[Connolly et al., 2016; Li et al., 2016; Xiang et al.,
2015]. In a retrospective cohort of 322,000 births at an
HMO [Xiang et al., 2015], both pregestational and

16 Hertz-Picciotto et al./Environmental contributions to ASD INSAR

Licensed to Mariana Wollinger Maciel Fassheber - marianawmaciel@gmail.com - 046.925.309-60
Figure 6. Causal model linking maternal metabolic conditions
to environmental exposures, biomarkers in the newborn, bio-
markers in childhood, gene expression, and diagnosis. Bold
arrows represent associations that have been published [Krako-
wiak et al., 2012; Krakowiak et al., 2017a; Krakowiak et al.,
2017b].
gestational diabetes diagnosed at 26 weeks gestation
were significantly associated with 33% to 42% greater
ASD risk. Major strengths of the study were: adjustment
for critical confounders; medical records and glucose
values to confirm diabetes diagnoses; child follow-up
for a median 5.5 years; and diagnoses by pediatric
developmental specialists. Results were similar in a large
cohort of over 40,000 children [Connolly et al., 2016],
which showed diabetes and obesity associated with,
respectively, 56% and 50% greater risks for ASD; more-
over, offspring from pregnancies complicated by both
gestational diabetes and obesity had a 2.5-fold increased
likelihood of ASD. A large sample size and meticulous
protocol for case ascertainment from EMR were major
strengths of this study. The reliance on birth certificates
for maternal diabetes and height and weight (to calcu-
late obesity) is not optimal, though prevalences of these
conditions were comparable to other studies, suggesting
underascertainment was not major. In a smaller low-
income urban cohort from Boston, children of mothers
with gestational or pregestational diabetes accompanied
by obesity had a 3 to 4-fold increased risk for ASD,
adjusting for confounders [Li et al., 2016]. The
strengths were 6 years follow-up and medical records of
both maternal and child diagnoses. A fourth study
involved a cohort of over 230,000 births in which out-
comes were hospitalizations with one or more neuro-
psychiatric diagnosis, of which ASD was one [Nahum
Sacks et al., 2016]. A 4-fold increased risk was associated
with maternal gestational diabetes, but the number of
hospitalized cases of ASD was small.
Underlying biological mechanisms. Several mecha-
nisms have been hypothesized to explain the link of
maternal diabetes with offspring ASD. Metabolic condi-
tions induce persistent low-grade inflammation and
insulin resistance [Makki, Froguel, & Wolowczuk, 2013;
Pantham, Aye, & Powell, 2015]. Such inflammation
leads to abundant immune cells in adipose tissue,
INSAR Hertz-Picciotto et al./Environmental contributions to ASD
which maintain a proinflammatory environment and
reduce insulin sensitivity. If compensatory mechanisms
fail, maternal hyperglycemia may ensue resulting in an
oxygen-deprived fetal brain at a critical time [Burstyn,
Wang, Yasui, Sithole, & Zwaigenbaum, 2011; Eidelman
& Samueloff, 2002]. Other pathways may involve meta-
bolic and immune derangements, stimulating placental
proinflammatory responses and inducing oxidative
stress. Oxidative stress has been linked to ASD [Chau-
han, Chauhan, Brown, & Cohen, 2004; Yao, Walsh,
McGinnis, & Pratico, 2006], and skewed immune
marker profiles have been described in newborns and
children with ASD [Krakowiak et al., 2017a; Onore,
Careaga, & Ashwood, 2012]. Additionally, mothers may
experience a breach in immune tolerance from meta-
bolic dysfunction. Approximately 23% of mothers of
children with ASD produce anti-fetal brain autoantibod-
ies compared with 1% of mothers of typically develop-
ing children [Braunschweig et al., 2012, 2013]. Among
mothers whose child had ASD, we found gestational
diabetes to be associated with a 3-fold increased preva-
lence of having these autoantibodies [Krakowiak,
Walker, Tancredi, Hertz-Picciotto, & Van de Water,
2017b]. Both ASD and maternal anti-fetal brain autoan-
tibodies have been independently associated with the c-
MET “C” allele polymorphism, which regulates immune
response [Campbell et al., 2008; Heuer et al., 2011;
Jackson et al., 2009]. In animal models, impaired MET
signaling is associated with diabetes [Araujo et al.,
2012; Demirci et al., 2012]. Additionally, many ASD
genes involve insulin signaling pathways (e.g., PI3K/Tor
(PI3K and mTOR)) [Stern, 2011]. Finally, the maternal
diabetic environment could alter the placental epige-
nome and ultimately its regulation of fetal develop-
ment [Marsit, 2016]. Overall, numerous mechanisms
can plausibly link maternal diabetes with disrupted
brain development; moreover, biomarkers for these per-
turbations have been found, in some cases prior to the
ASD diagnosis.
Inferring causation when randomized trials are
inappropriate, unethical, or unfeasible. Conducting
randomized trials for studying environmental risk fac-
tors is often impossible. An intervention that increases
exposures suspected to cause harm would be unethical.
When exposures are present in diverse sources, not
revealed in product labels (e.g., cosmetics, air fresh-
eners, etc.), and/or have outcomes that take years to
manifest, even an intervention to reduce exposures
may be infeasible. For these reasons, epidemiologists
rely on observational studies, which makes inference of
causation from challenging, though not impossible.
Indeed, many etiologic factors have been established
17
Licensed to Mariana Wollinger Maciel Fassheber - marianawmaciel@gmail.com - 046.925.309-60
from nonrandomized investigations (e.g., cigarette
smoking and lung cancer).
When does evidence from observational associations
permit causal inference? Since the purpose of randomi-
zation is to control for systematic differences (con-
founders) between treated and untreated groups,
identification of confounders and their control is at the
core of the epidemiologic approach. When epidemio-
logic studies are referred to as ‘correlational’, this is usu-
ally an inaccurate characterization since a correlational
study by definition does not take into account con-
founders, and hence would fail the most elementary
test of rigor. More generally, methodologic soundness
of individual studies depends on: reliable, valid expo-
sure measures, high quality diagnostic or dimensional
outcome measures, a systematic approach to con-
founder control, minimization of selection bias, and
statistical analyses appropriate to the nature of the
data. Additionally, the body of literature should be
assessed in context of the Hill ‘viewpoints’ (first
described for evaluating epidemiologic evidence that
smoking caused lung cancer [Hill, 1965]), including:
temporality (the exposure precedes onset of the out-
come); strength of the association, consistency across
studies in different populations, biologic plausibility,
and coherence with known scientific facts.
Applying the above approach to the major studies
described above on maternal diabetes during preg-
nancy: (a) For exposure, all but one used medically
reported diagnoses of diabetes, or self-report validated
by medical reports [Krakowiak, Walker, Tancredi, &
Hertz-Picciotto, 2015], confirming both temporality and
validity of the exposure; the one exception used birth
certificates which appeared to show face validity based
on the prevalences of these conditions [Connolly et al.,
2016]. (b) For outcomes: most used physician-diagnosed
cases from medical records, one used gold standard
research-reliable instruments, and one used neuropsy-
chiatric hospitalization with ASD. Lacking a biologic
test for ASD, clinical diagnoses vary in how stringently
DSM or ICD criteria are evaluated, which might lead to
some misclassification (in both directions), and, if non-
differential relative to diabetes diagnosis, possibly may
explain substantially smaller associations in the larger
Kaiser and Ohio studies. (c) All studies used appropriate
multivariable models and (d) adjusted for maternal age,
a candidate confounder; most also included year of
birth, gender, and race/ethnicity. (e) Although the
strength of the associations varied, the two studies with
large relative risks each had fewer than 30 exposed
cases [Li et al., 2016; Nahum Sacks et al., 2016], result-
ing in CI consistent with the smaller, more precise esti-
mates of the other studies; hence, along with earlier
meta-analyses, these results therefore demonstrate con-
sistency. (f) Biologic plausibility was described above,
18 Hertz-Picciotto et al./Environmental contributions to ASD
and (g) genetic pathways related to both ASD and dia-
betes or insulin regulation offer further evidence for
coherence with known facts. The literature provides
substantial support for prenatal maternal diabetes as a
causative factor in ASD, based on the Hill viewpoints.
Example #3-Folic Acid: Integration of environment, genetics
& epigenetics
Periconceptional folic acid (FA) supplements can pre-
vent up to 70% of neural tube defects (NTDs). From
large scale RCTs [MRC Vitamin Study Research Group,
1991], first trimester folic acid supplementation is rec-
ommended worldwide to prevent neural tube defects
(NTDs): 4–5 (0.4–1) mg/day for women with (without)
a history of NTDs [Centers for Disease Control, 1991].
Cohort studies have also found that maternal FA sup-
plement intake before/during early pregnancy was asso-
ciated with fewer childhood behavioral problems [Roza
et al., 2010] or hyperactivity and peer problems [Schlotz
et al., 2010], improved verbal, verbal-executive func-
tion, attention, and social competence [Julvez et al.,
2009], and decreased risk for severe language delays
[Roth et al., 2011].
Previously, we found that maternal report of prenatal
vitamin intake near conception was associated with
40% reduced risk of ASD in 707 California-born
CHARGE Study children with autism or clinically-
confirmed typical development. Given that mothers
recalled their vitamin intake retrospectively, recall bias
could not be excluded. However, this result was repli-
cated in a prospective birth cohort study with 85,176
Norwegian children [Suren et al., 2013]. Prenatal vita-
mins contain high levels of FA, which is converted to
folate under genetic regulation by MTHFR677A. Addi-
tional population-based cohort studies [Braun et al.,
2014a; Steenweg-de Graaff et al., 2015] also found pre-
natal vitamin or FA supplements associated with
reduced risk for autistic traits. However, when these
studies examined maternal folate in serum collected after
the first trimester, their results were null [Braun et al.,
2014a; Steenweg-de Graaff et al., 2015], consistent with
the literature on timing of folic acid protection against
ASD risk [Schmidt et al., 2011, 2012; Suren et al., 2013].
The only study not consistent with an early prenatal
protective effect of FA was a large population-based
birth cohort study in Denmark [Virk et al., 2016]. This
study also failed to replicate one of the most established
risk factors for ASD, parental age, appearing to be an
outlier.
To date, only the CHARGE Study evaluated ASD and
FA intake by maternal and child genetics. We found
periconceptional maternal supplemental FA was associ-
ated with reduced autism risk only when mothers and/
or children had genotypes conferring inefficient folate
INSAR
Licensed to Mariana Wollinger Maciel Fassheber - marianawmaciel@gmail.com - 046.925.309-60
metabolism [Schmidt et al., 2012]. Thus, it was the
combined impact of not taking a prenatal vitamin near
conception and the mother or child having gene var-
iants producing less efficient folate-dependent one-car-
bon metabolism that put the child at particularly high
risk for developing autism [Schmidt et al., 2011].
The observed gene-environment interaction strength-
ens the evidence that the observed protection is not
due to biased reporting of folic acid intake. First of all,
because the finding was time-limited to the period
around conception, mothers who had typical children
would have had to systematically report an earlier con-
sumption of prenatal vitamins than when they actually
did initiate it, or mothers of affected children would
have had to systematically report a later than actual
time for initiating supplementation. However, this
hypothesized biased reporting would have to occur only
among those genetically endowed with inefficient met-
abolic profiles, an unlikely proposition.
Given the strength of the interaction between genes
and nutrition, supplemental folic acid could help over-
come ASD in susceptible subsets of the population; if
so, folic acid intake at levels higher than current recom-
mendations could be useful for mothers with genetic or
other susceptibility factors, including environmental
exposures, to reduce ASD risk. For example, our latest
findings indicate risk for ASD from prenatal pesticide
exposures was reduced in children whose mothers took
folic acid in the periconception, as compared to those
whose mothers did not [Schmidt et al., 2017]. Thus,
higher maternal folic acid intake in the periconception
could not only compensate for genetic risk, but also
help protect against other environmental chemicals.
The protective mechanisms of periconceptional FA
for NTDs [Blom, Shaw, den Heijer, & Finnell, 2006],
ASD [Schaevitz & Berger-Sweeney, 2012], and other
developmental conditions remain unknown. Folate’s
antioxidant properties [Joshi, Adhikari, Patro, Chatto-
padhyay, & Mukherjee, 2001], DNA methylating capac-
ity [James et al., 2004, 2009] and role in DNA repair
[Duthie, 1999] are all candidates. Folate is a major
methyl donor, suggesting epigenetic regulation near
conception when the DNA methylome is largely
stripped and then reestablished [Reik & Walter, 2001].
This timing is congruent with the protective associa-
tions observed in epidemiologic ASD studies [Blom,
2009; Schmidt et al., 2011]. Alterations of maternal die-
tary methyl-donor concentrations during methylome
establishment near conception has long-term epigenetic
and health outcomes implications [Cooney, Dave, &
Wolff, 2002; Heijmans et al., 2008; Steegers-Theunissen
et al., 2009; Wolff, Kodell, Moore, & Cooney, 1998].
Studies examining folic acid attenuation of effects of
environmental hazards on other outcomes, including
bisphenol A in mice [Dolinoy, Huang, & Jirtle, 2007],
INSAR Hertz-Picciotto et al./Environmental contributions to ASD
arsenic in humans [Howe et al., 2014; Lambrou et al.,
2012] and PM2.5 in a clinical trial [Zhong et al., 2017]
have also implicated DNA methylation mechanisms.
Future Directions
The first decade of concerted efforts to identify environ-
mental/modifiable causes of autism has produced a
plethora of clues about risk and protective factors, with
increasingly compelling evidence for a few: short inter-
pregnancy intervals, maternal diabetes. The field has
begun to mature, moving into its mid- to late child-
hood stage, where research is building and consolidat-
ing gains through replication of results and refinement
of methods, even while still uncovering new connec-
tions. Also arising from this literature is the recognition
of complexity in ASD etiology. Thus, we are poised to
approach adolescence, where new developments in
molecular biology, electronic connectedness and big
data create possibilities and opportunities to address
some of the major perplexing challenges.
The Exposome
Taking lessons from genome-wide studies, environmen-
tal health scientists have begun to explore moving from
candidate exposures to more comprehensive analyses of
the universe of human exposures. Although genetics is
exceedingly complex, the DNA code is, for the most
part, fixed for life. In contrast, environment has the
additional feature (curse, for researchers!) of being in
constant change. Environmental health investigators
are currently innovating ‘exposomic’ approaches.
One of the first environment-wide analyses assessed
266 unique environmental factors in relation to type 2
diabetes defined by a fasting blood glucose test. Similar
to GWAS, adjustment for multiplicity testing was
accomplished using false discovery rate, and results
were validated in four cohorts [Patel, Bhattacharya, &
Butte, 2010]. Novel potential risk factors were discov-
ered and confirmed, including two organochlorines (a
PCB and a pesticide), interesting in light of diabetes-
ASD associations described above. For ASD research, the
principal obstacle to applying similar statistical meth-
odologies is the lack of large amounts of environmental
data collected during relevant time windows in robust
samples sizes.
New technologies in exposure sciences are providing
solutions for such collection. Nontargeted chemical
analyses, for example, via gas (or liquid) chromatogra-
phy with quantitative time-of-flight mass spectrometry,
are identifying an increasingly large array of chemicals
in various media: food packaging [Cherta et al., 2015],
river water for pharmaceuticals [Martinez Bueno, Ulas-
zewska, Gomez, Hernando, & Fernandez-Alba, 2012],
19
Licensed to Mariana Wollinger Maciel Fassheber - marianawmaciel@gmail.com - 046.925.309-60
and organics in breast milk [Baduel, Mueller, Tsai, &
Gomez Ramos, 2015]. Wearable sensors are being
deployed for a variety of applications, such as: detec-
tion of infant movements [Chen, Xue, Mei, Bambang
Oetomo, & Chen, 2016; Zhu et al., 2015], physiologic
measurements in aging populations or athletes [Patel,
Chen, & Butte, 2012], electrolyte and metabolite moni-
toring in sweat, tears and saliva [Bandodkar & Wang,
2014] among others [Zheng et al., 2014]. Sensors for
exogenous chemicals include silicone wristbands [Ham-
mel, Hoffman, Webster, Anderson, & Stapleton, 2016],
skin patches [Romanyuk et al., 2014], or hand-held
breathalyzers [Gouma et al., 2017] for children or adults
[Hammel et al., 2016]. Sensor technology offers high-
dimensional data collection that can dramatically
advance the field of environmental epidemiology.
Another component of the exposome is the micro-
biome: the >90% of our cells that are not human, but
symbiotically inhabit our bodies. Research on micro-
biome is revealing different intestinal microbiota in
children with ASD versus those with typical develop-
ment, such as high prevalence of Clostridium [De
Angelis, Francavilla, Piccolo, De Giacomo, & Gobbetti,
2015; Finegold et al., 2010; Parracho, Bingham, Gibson,
& McCartney, 2005]. These findings comport with
greater prevalence of GI symptoms in ASD [Chaidez,
Hansen, & Hertz-Picciotto, 2014]. Relevance for etiology
remains unclear, although, as stated in our introduction
to causal concepts, interventions that alter the trajec-
tory after a diagnosis has been made may alleviate
symptoms and in some cases result in markedly
improved behavior. Numerous research groups are seek-
ing to develop probiotics to alter the species present
and their relative abundance in hopes these will ulti-
mately help children with autism lead more productive
and socially connected lives.
As in the field of genomics, the twin problems of
nonindependence (of genes, or of environmental fac-
tors) and mixtures present major statistical challenges.
Models that assume independence continue to be used,
despite evidence to the contrary (for both genes and
environmental chemicals). Hierarchical regression and
Bayesian methods have addressed nonindependence
across a large set of explanatory variables [Witte, Green-
land, Haile, & Bird, 1994], machine learning algorithms
accounting for nonindependence are being developed
[Zou & Hastie, 2005], as are corrections for nonindepen-
dence of alleles [Li et al., 2012]. For real-world scenarios
of simultaneous exposures, various tools address differ-
ent questions: principal components can identify clus-
ters of correlated items, reducing to a few principal
components that maximize orthogonality; weighted
quantile regression can estimate the effect of the whole
mixture on an outcome and apportion the relative con-
tributions of the parts [Czarnota, Gennings, & Wheeler,
20 Hertz-Picciotto et al./Environmental contributions to ASD
2015] and elastic net addresses correlated covariates,
and allows the number of predictors to be larger than
the number of observations. Network and pathway
models can characterize overlapping and/or inter-
changeable functionality, and also suggest potential
synergistic effects produced by chemicals acting on dif-
ferent steps along the same or converging pathways. An
NIEHS workshop recently emphasized the need for toxi-
cologists, statisticians and epidemiologists to work col-
laboratively, bringing their respective knowledge bases
to the mixture problem [Taylor et al., 2016].
Gene-Environment Interactions
Interestingly, although one-carbon metabolism genes
that influence levels and activity of folate appear to act
synergistically with folic acid intake to increase ASD
risk, these same genes have been missed in the vast
number of autism GWAS reports, likely because their
high-risk variants are too common. This raises a conun-
drum—how can we systematically identify the joint
contributions of genetic and environment to ASD? If
the scenario seen for folic acid is not unique—and
chances are that common genetic variants are largely
undiscovered—then potentially vast numbers of yet-
unidentified genes may increase ASD risk only in com-
bination with exposures that have gone unmeasured in
genetic studies. Exploration of genetic susceptibility in
combination with environmental exposures promises to
be fruitful, particularly for common variants.
To date, few studies have addressed gene-
environment interaction in ASD by examining synergis-
tic combined effects for a specific environmental expo-
sure (or a small set of exposures) and a genetic
susceptibility. Two used the CHARGE Study to examine
whether risks for ASD from nutrition or air pollution
differed by genotypes with known biologic relevance to
the selected exposure, comparing children with ASD to
typically developing controls [Schmidt et al., 2011,
2012; Volk et al., 2014]. Two others used the Simons
Simplex cohort of affected individuals to investigate
associations of a medical factor, in the presence of
CNVs, with severity scores [Mazina et al., 2015; Webb
et al., 2017]. All four of these studies found evidence
supporting the hypothesized interactions, and three of
the four either focused on a pre-specified time period or
examined several in which associations were stronger
for exposures in specific windows.
Big-data approaches can harness genome-wide data in
combination with environmental factors for discovery-
type investigation. Alternatively, an ‘exposomic’ screen,
evaluating a large universe of environmental factors, can
be used in combination with a single or small set of
genes. Both approaches require large samples—optimally,
thousands to tens of thousands of participants. The
INSAR
Licensed to Mariana Wollinger Maciel Fassheber - marianawmaciel@gmail.com - 046.925.309-60
primary limitation in either of these statistically equiva-
lent strategies is the sample size needs for reasonably
powered analysis of interactions involving combined
effects, when either genes or environmental exposures
are of high dimension (thousands to hundreds of thou-
sands of variables), even when the other has only a few
categories or even one binary or continuous factor.
Examining two high-dimensional sets of data– both
genomic and exposomic—in combination is a feasibility
challenge. Practical limits for sample sizes of such omic-
by-omic analysis have been described [Patel, 2016]:
under a wide range of assumptions, statistical power for
detecting GxE interactions based on as few as 10 to
over 10,000 environmental exposures, even with sam-
ple sizes of 10,000 to 60,000, is low. Moreover, interac-
tions with SNPs will magnify the nonindependence
problem discussed above [Rao & Province, 2016].
Although greater power may be achieved, for example,
for exposures more prevalent than the 20% maximum
assumed by Patel, or associated with greater interaction
relative risks [as was observed by Schmidt et al., 2011],
alternative strategies for gene-by-environment analyses
are needed.
Several alternatives to omic-omic analyses are feasible,
and lie within reach even for current sample sizes,
including: biologically-based pathways relevant to spe-
cific exposures [Schmidt et al., 2011, 2012; Volk et al.,
2014]; expanded gene pathways, for example, based on
gene ontology and/or toxicogenomic databases, to
restrict the number of genes in combination with a
mechanistically defined limited set of candidate expo-
sures; genome-wide searches in combination with candi-
date or known autism risk factors; and use of two-stage
methods for GWAS in combination with various new
exposomic analyses. Variations on these approaches are
discussed in [Patel et al., 2012; Patel, Chen, Kodama,
Ioannidis, & Butte, 2013; Patel, 2016]. The recently
launched ECHO (Environmental Influences on Child
Health Outcomes) program encompassing 50,000 chil-
dren can be an excellent platform for advancing Big Data
analysis of gene-environment interactions.
Finally, the type of gene-environment interaction dis-
cussed above—investigating the combination of inher-
ited genes and environmental exposures for synergistic
effects that are greater than the sum of the individual
effects—is only one of several ways in which the two can
interact. A second is the ability of environmental factors
to alter DNA through either (a) de novo mutational
events or (b) de novo replication defects resulting in
copy number variants, either deletions or duplications,
sometimes numbering in hundreds or more repeats of
genetic code. A third type of gene-environment interac-
tion involves epigenetic alterations of the genetic struc-
ture (see below) as a result of environmental factors,
both endogenous and exogenous, acting upon the
INSAR Hertz-Picciotto et al./Environmental contributions to ASD
epigenome to up- or down-regulate gene expression.
Thus, as epidemiologists move into gene-environment
interaction, epigenetics opens the door to understand-
ing the truly complex interplay between inherited and
noninherited molecules within our bodies and our eco-
logic niche.
Environmental Epigenetics
DNA methylation, histone modifications, micro-RNA,
noncoding RNAs, and chromatin remodeling all consti-
tute changes in the architecture and expression of
genes, without affecting the DNA sequence itself. A bur-
geoning field of environmental epigenetics has identi-
fied epigenetic changes or profiles associated with a
wide range of exposures. Environmentally-induced epi-
genetic changes, especially during periods of erasure
and reestablishment of the methylome during preg-
nancy [Reik & Walter, 2001], have potential as both
markers and mediators of environmental influences on
later developmental outcomes [Baccarelli & Bollati,
2009]. Human epidemiology and animal models indi-
cate that prenatal and postnatal environmental expo-
sures influence developmental trajectories, leading to
persistent changes in phenotypes and in chronic disease
susceptibility. Evidence is accumulating for epigenetic
effects, primarily DNA methylation in offspring, from
environmental exposures including stress, socioeco-
nomic status, parental body mass index, gestational dia-
betes, maternal antibiotic use, lead, arsenic, mercury,
bisphenol A, cigarette smoke, pesticides, and nutritional
factors [Breton et al., 2017; Finer et al., 2015; Ladd-
Acosta et al., 2014; Schmidt et al., 2016]. Similarly,
numerous studies have provided evidence for DNA
methylation or other epigenetic alterations in ASD
[Feinberg et al., 2015; Schroeder, Lott, Korf, & LaSalle,
2011; Schroeder et al., 2016] and other neurodevelop-
mental disorders as reviewed previously [Flashner,
Russo, Boileau, Leong, & Gallicano, 2013; Kubota &
Mochizuki, 2016; Loke, Hannan, & Craig, 2015; Vogel
Ciernia & LaSalle, 2016; Zhubi, Cook, Guidotti, & Gray-
son, 2014]. These studies implicate large epigenetic con-
tributions to ASD etiology, and suggest epigenetic
mechanisms as mediators of effects from environmental
factors. Nevertheless, to date, direct causal evidence
linking environmental factors with ASD through DNA
methylation or other epigenetic mechanisms remains
limited.
Most environmental epigenetics currently focuses on
DNA methylation, due to greater feasibility in large-scale
epidemiologic studies. Even so, numerous methodologi-
cal issues complicate measurement and interpretation of
DNA methylation marks, especially in relation to a brain
disorder like autism, including: species specificity, tissue
specificity, and cell heterogeneity [Bakulski & Fallin,
21
Licensed to Mariana Wollinger Maciel Fassheber - marianawmaciel@gmail.com - 046.925.309-60
2014]. For instance, different cell types have distinct epi-
genetic marks, and the specific composition of blood
cells shift in the presence of, for example, inflammation.
Other issues concern laboratory and statistical methods
[Breton et al., 2017]. Nevertheless, epigenetics has
opened up the big black box of how genes and environ-
ment play complementary roles in development and
health. The complex genetic architecture of ASD alters
susceptibility to environmental agents, while noninher-
ited factors influence gene expression in tissue specific
contexts, affecting not only the exposed organism, but
potentially also future generations.
Developing and applying new technologies for mea-
suring the exposome, generating mega-studies with
broad and high-quality genome and exposome data to
be analyzed using robust strategies, and finally, tying it
together with deep characterization of the epigenome
will be heavy challenges – yet not as far-flung as they
seem today. We are poised to dramatically advance our
understanding of the etiology of ASD as never before.
Conclusion
As noted, many factors reviewed here have associations
with other neurodevelopmental or psychiatric condi-
tions, and therefore may lack specificity for autism.
Genetic factors or critical time periods may influence
how these xenobiotics or noninherited conditions alter
brain connectivity and whether they lead to autism ver-
sus different deficits. Thus, the causal concepts of tim-
ing and susceptibility loom as keys to unlocking the
mysteries of autism etiology. Realistic strategies for pur-
suit of gene-by-environment synergies have been out-
lined and many of the tools are now available, but
require large enough studies. New developments in epi-
genetics offer yet further opportunities to capture the
complexity of environmental influences on this disor-
der. Major research gaps include determination of criti-
cal etiologic windows for environmental exposures,
how these vary by type of exposure, and whether epige-
netic vulnerabilities feature in defining those windows.
Parent-of-origin effects and hormonal hypotheses may
enable disentangling the maternal and paternal contri-
butions and the sex ratio. Additional work on how the
gut microbiotal populations influence behavior,
whether probiotics can induce desired changes, and if
so, in what time periods, is also needed. Continuing
targeted investigations into environmental chemicals,
particularly endocrine disrupting compounds, appears
to be another promising avenue with realistic opportu-
nities for interventions. Similarly, deeper dives into
maternal nutritional, obstetric, metabolic and other fac-
tors during the pre-conception, prenatal and perinatal
periods are likely to enhance understanding and
22 Hertz-Picciotto et al./Environmental contributions to ASD
uncover additional preventive measures. Although the
preconception and prenatal periods may be the most
influential, continued plasticity of the central nervous
system postnatally implies further opportunity during
the first year or two, to mitigate the debilitating fea-
tures and enhance the special giftedness of ASD. Our
goal is simple: by studying environmental—hence mal-
leable—factors that contribute to ASD, we amplify the
potential for a productive and fulfilling life for all
children.
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