Daily Early-Life Exposures to Diet Soda and Aspartame Are Associated with Autism in Males: A Case-Control Study

Abstract

Since its introduction, aspartame—the leading sweetener in U.S. diet sodas (DS)—has been reported to cause neurological problems in some users. In prospective studies, the offspring of mothers who consumed diet sodas/beverages (DSB) daily during pregnancy experienced increased health problems. We hypothesized that gestational/early-life exposure to ≥1 DS/day (DSearly) or equivalent aspartame (ASPearly: ≥177 mg/day) increases autism risk. The case-control Autism Tooth Fairy Study obtained retrospective dietary recalls for DSB and aspartame consumption during pregnancy/breastfeeding from the mothers of 235 offspring with autism spectrum disorder (ASD: cases) and 121 neurotypically developing offspring (controls). The exposure odds ratios (ORs) for DSearly and ASPearly were computed for autism, ASD, and the non-regressive conditions of each. Among males, the DSearly odds were tripled for autism (OR = 3.1; 95% CI: 1.02, 9.7) and non-regressive autism (OR = 3.5; 95% CI: 1.1, 11.1); the ASPearly odds were even higher: OR = 3.4 (95% CI: 1.1, 10.4) and 3.7 (95% CI: 1.2, 11.8), respectively (p < 0.05 for each). The ORs for non-regressive ASD in males were almost tripled but were not statistically significant: DSearly OR = 2.7 (95% CI: 0.9, 8.4); ASPearly OR = 2.9 (95% CI: 0.9, 8.8). No statistically significant associations were found in females. Our findings contribute to the growing literature raising concerns about potential offspring harm from maternal DSB/aspartame intake in pregnancy.

Full text

Citation: Fowler, S.P.; Gimeno Ruiz
de Porras, D.; Swartz, M.D.; Stigler
Granados, P.; Heilbrun, L.P.; Palmer,
R.F. Daily Early-Life Exposures to
Diet Soda and Aspartame Are
Associated with Autism in Males: A
Case-Control Study. Nutrients 2023,
15, 3772. https://doi.org/10.3390/
nu15173772
Academic Editor: Ruggiero
Francavilla
Received: 7 August 2023
Revised: 22 August 2023
Accepted: 25 August 2023
Published: 29 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
nutrients
Article
Daily Early-Life Exposures to Diet Soda and Aspartame Are
Associated with Autism in Males: A Case-Control Study
Sharon Parten Fowler 1, *, David Gimeno Ruiz de Porras 2,3,4 , Michael D. Swartz 5, Paula Stigler Granados 6,
Lynne Parsons Heilbrun 7and Raymond F. Palmer 8
1Department of Medicine, Joe R. & Teresa Lozano Long School of Medicine, The University of Texas Health
Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA
2Southwest Center for Occupational and Environmental Health, Department of Epidemiology, Human
Genetics and Environmental Sciences, School of Public Health in San Antonio, The University of Texas Health
Science Center at Houston, 7411 John Smith Drive, San Antonio, TX 78229, USA; david.gimeno@uth.tmc.edu
3Center for Research in Occupational Health, Universitat Pompeu Fabra, 08003 Barcelona, Spain
4CIBER of Epidemiology and Public Health, 28029 Madrid, Spain
5
Department of Biostatistics and Data Science, School of Public Health, The University of Texas Health Science
Center at Houston, 1200 Pressler Street, Houston, TX 77030, USA; michael.d.swartz@uth.tmc.edu
6Divisions of Environmental Health and Global Health, School of Public Health, San Diego State University,
5500 Campanile Drive, San Diego, CA 92182, USA; pstiglergranados@sdsu.edu
7Department of Epidemiology, Human Genetics, and Environmental Sciences, School of Public Health in San
Antonio, The University of Texas Health Science Center at Houston, 7411 John Smith Drive,
San Antonio, TX 78229, USA; lynne.heilbrun@uth.tmc.edu
8Department of Family Practice and Community Medicine, Joe R. & Teresa Lozano Long School of Medicine,
The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive,
San Antonio, TX 78229, USA; palmerr@uthscsa.edu
*Correspondence: fowlers@uthscsa.edu
Abstract:
Since its introduction, aspartame—the leading sweetener in U.S. diet sodas (DS)—has
been reported to cause neurological problems in some users. In prospective studies, the offspring of
mothers who consumed diet sodas/beverages (DSB) daily during pregnancy experienced increased
health problems. We hypothesized that gestational/early-life exposure to
≥
1 DS/day (DS
early
) or
equivalent aspartame (ASP
early
:
≥
177 mg/day) increases autism risk. The case-control Autism
Tooth Fairy Study obtained retrospective dietary recalls for DSB and aspartame consumption during
pregnancy/breastfeeding from the mothers of 235 offspring with autism spectrum disorder (ASD:
cases) and 121 neurotypically developing offspring (controls). The exposure odds ratios (ORs) for
DS
early
and ASP
early
were computed for autism, ASD, and the non-regressive conditions of each.
Among males, the DS
early
odds were tripled for autism (OR = 3.1; 95% CI: 1.02, 9.7) and non-regressive
autism (OR = 3.5; 95% CI: 1.1, 11.1); the ASP
early
odds were even higher: OR = 3.4 (95% CI: 1.1,
10.4) and 3.7 (95% CI: 1.2, 11.8), respectively (p< 0.05 for each). The ORs for non-regressive ASD in
males were almost tripled but were not statistically significant: DS
early
OR = 2.7 (95% CI: 0.9, 8.4);
ASP
early
OR = 2.9 (95% CI: 0.9, 8.8). No statistically significant associations were found in females.
Our findings contribute to the growing literature raising concerns about potential offspring harm
from maternal DSB/aspartame intake in pregnancy.
Keywords:
autism; autism spectrum disorder; pregnancy; diet; diet soda; diet beverages; aspartame;
non-nutritive sweeteners; artificial sweeteners; high-intensity sweeteners
1. Introduction
Over the past 40 years, the prevalence of diagnosed autism spectrum disorder (ASD)
in the U.S. has dramatically risen [
1
], from fewer than 0.3 per 1000 children diagnosed
with autism before 1980 [
2
] to 27.6 per 1000 children diagnosed with ASD in 2020 [
3
].
Changes in diagnostic definitions and guidelines and increased testing availability and
Nutrients 2023,15, 3772. https://doi.org/10.3390/nu15173772 https://www.mdpi.com/journal/nutrients

Nutrients 2023,15, 3772 2 of 22
funding have made major contributions to this increase in diagnosed cases; under the
added impacts of changes in dietary, environmental, and other exposures affecting the
intrauterine environment, ASD prevalence has reached unprecedented proportions. Males
have been disproportionately affected: autism prevalence among boys is almost quadruple
that among girls, and a recent study estimated that approximately 1 in 23 U.S. boys aged
8 years or older in 2020 had been diagnosed with ASD [
3
]. The degree to which ASD
diagnoses have risen during this time highlights the potential role of non-genetic influences,
including early prenatal exposures to heavy metals, organophosphate pesticides, and other
environmental toxins, in offspring autism risk [4].
Maternal diet during pregnancy and breastfeeding represents an important additional
non-genetic influence on offspring autism risk and has been studied increasingly closely
over the past 15 years. Zhong et al. [
5
], in their systematic review, reported evidence of
the protective role of maternal intake of prenatal vitamins, folic acid, and vitamin D in
reducing offspring autism risk. Peretti et al. [
6
] also reported decreased offspring autism
risk associated with a higher maternal intake of folic acid, omega 6 fatty acids, and vitamin
D during pregnancy and an increased autism risk among the offspring of mothers deficient
in omega 3 fatty acids and polyunsaturated fatty acids. Although the results from different
individual studies varied, the available data suggest that these selected nutrients may
indeed exert neuroprotective effects during development.
In contrast, the maternal dietary intake of methanol during pregnancy was identified
as a potential risk-increasing exposure for offspring. Peretti et al. [
6
] called attention to
the work of Walton and Monte [
7
], who found that the dietary intake of methanol during
pregnancy was approximately twice as high among biological mothers of children with
ASD as among mothers of neurotypically developing children. Aspartame, a leading
non-nutritive sweetener (NNS) in the U.S., is a ubiquitous source of dietary methanol in
this country, and aspartame-sweetened products constituted the major sources of dietary
methanol included in Walton and Monte’s calculations of maternal methanol intake. If their
preliminary findings are correct, the possibility that pregnant women might unknowingly
be exposing their unborn children to an increased autism risk through their consumption
of aspartame-sweetened diet products during pregnancy is of particular concern.
The safety of aspartame, a leading sweetener used in diet sodas, other diet beverages,
and over 6000 dietary, pharmacologic, and other products over the past 40 years [
8
], has
been the subject of intense debate since before it entered the marketplace. In 1981, the
U.S. Food and Drug Administration (FDA) approved the use of aspartame as a tabletop
sweetener and, in 1983, as an ingredient in diet sodas (DS) and other products [
8
]. Aspar-
tame rapidly became the leading DS sweetener used in the U.S; DS consumption doubled,
and aspartame consumption increased 17-fold, over the next 8 years [
9
]. By the end of
1983, however, the FDA had received numerous complaints of adverse reactions among
aspartame consumers. Prominent complaints included headache, anxiety, and depres-
sion [
10
,
11
]. These and other neurological problems—including irritability, mood disorders,
cognitive problems, and seizures—were subsequently reported to be increased among users
of diet sodas/beverages (DSB) and other aspartame-sweetened products [
10
–
22
], which are
leading vehicles of aspartame intake in the U.S. [
23
]. Impaired cognitive performance and
increased anxiety-related behaviors also developed in aspartame-fed animals [
24
–
30
]. Such
reports contributed to the prolonged controversy over the safety of aspartame [
29
,
31
–
37
].
While a review of this debate is beyond the scope of this report, we highlighted results with
potential neurophysiologic relevance to our investigation.
Each of the three first-phase metabolites of aspartame has been studied with regard to
its impacts on neurologic function. Aspartame is metabolized in the intestine into aspartic
acid, an excitatory neurotransmitter; phenylalanine, which is involved in neurotransmitter
regulation; and methanol, the metabolites of which include formaldehyde, formate, and
other toxins [
38
]. These three first-phase metabolites represent approximately 40%, 50%,
and 10% of aspartame by weight, respectively [
38
]. The adverse neurological impacts
following the consumption of aspartic acid, phenylalanine, and/or aspartame include

Nutrients 2023,15, 3772 3 of 22
changes in neurotransmitter levels [
25
,
38
,
39
] and excitotoxicity, with adverse impacts on
neuron function/survival [
40
–
42
]. Primates, including humans, are uniquely vulnerable to
methanol [
43
,
44
], the blood levels of which rise following aspartame consumption
[44–46].
Exposures to methanol and formaldehyde resulted in increased neuronal apoptosis, neu-
rodegeneration, and cognitive problems [43,47,48].
The potential mechanisms that might underlie the associations between aspartame in-
take and long-term health problems in humans and their offspring were reported in animal
studies. Prominent among these is a recurring finding of the markedly decreased availabil-
ity of the reduced form of glutathione—glutamine sulfhydryl (GSH)—in aspartame-fed
animals [
25
,
44
–
46
,
49
–
52
]. This finding is crucial because GSH protects the developing
brain by providing antioxidant defense against oxidative stress [
53
,
54
], by scavenging
toxins [
53
,
55
], and by supporting methylation processes [
56
]. Animals exposed to as-
partame and its metabolites exhibited a range of problems, including increased levels
of free radicals, oxidative stress, lipid peroxidation, inflammation, and mitochondrial
dysfunction; increased permeability of the blood–brain barrier (BBB); excitotoxicity and
neuronal apoptosis; and decreased brain serotonin, noradrenaline, and dopamine lev-
els
[25–27,29,38,41,42,44–46,50,51,57–63].
Several investigators attributed these adverse
impacts to methanol and its metabolites [44–46,59,64].
Adverse impacts on the gut microbiota (GM) were also detected in animals exposed
to aspartame [
29
,
61
,
65
–
73
] and two of its metabolites: formaldehyde [
67
] and pheny-
lalanine [
66
]. Among other changes, aspartame-fed rats exhibited a doubling of serum
propionate [
65
]. Propionic acid, a short-chain fatty acid produced by gut bacteria, was
hypothesized to increase ASD risk through multiple pathways, including increased gut
and BBB permeability [
74
,
75
]; decreased GSH and neurotransmitter levels; increased oxida-
tive stress, excitotoxicity, and neuroinflammation [
76
,
77
]; and altered mitochondrial and
immunological function [78].
Taken together, as summarized in Figure 1, such physiological impacts could create
a perfect storm: increasing the access of toxins to the developing brain, decreasing the
developing brain’s antioxidant and detoxification capacities, and adversely impacting
the microbiome–gut–brain axis, which is critical in neurodevelopment [
79
,
80
]. Further
discussion of the processes summarized in Figure 1is included in Section 4.
In order to address the question of whether maternal aspartame consumption in-
creases autism risk in offspring exposed to aspartame in early life—in utero and during
breastfeeding—we aimed to retrospectively assess the maternal intake during pregnancy
and breastfeeding of diet soda and of aspartame from multiple sources, among biological
mothers of offspring with either autism specifically or any ASD (cases) and among mothers
of neurotypically developing offspring (controls), to evaluate the associations between
daily early-life exposures to these products and current autism and ASD status in the
case-control Autism Tooth Fairy Study (ATFS).
The primary aim of this study was to examine whether the odds of daily exposure
during pregnancy/breastfeeding to
≥
1 DS or an equivalent amount of aspartame, through
the maternal diet, were significantly higher among offspring diagnosed with autism (autism
cases), compared with neurotypically developing offspring (controls). The secondary aim
of the study was to assess whether these exposure odds were higher among offspring with
any ASD (ASD cases), compared with controls. Since males have increased developmental
vulnerability to adverse early-life exposures and since the risk of autism is four times
higher in boys than in girls, we performed both sex-specific and pooled analyses for both
diagnostic categories.

Nutrients 2023,15, 3772 4 of 22
Nutrients 2023, 15, x FOR PEER REVIEW 4 of 22
times higher in boys than in girls, we performed both sex-specific and pooled analyses for
both diagnostic categories.
Figure 1. Potential impacts and interactions of aspartame and its metabolites on known autism risk
factors. (OCM: one-carbon metabolism, including the transsulfuration pathway, methionine cycle,
and folate cycle; GSH: reduced glutathione, a major antioxidant involved in defense against oxida-
tive stress; detoxification; maintenance of methylation capacity; SAM: S-adenosylmethionine, the
major methyl donor for cellular methylation processes; BBB: blood–brain barrier).
2. Materials and Methods
2.1. Participants
Recruitment and data collection for the Autism Tooth Fairy Study were performed
through the University of Texas Health Science Center at San Antonio (UTHSCSA) from
May 2011 to June 2014. The largest recruitment source (n = 255) was the Interactive Autism
Network (IAN), an Internet-based U.S. registry [81] that included >21,600 individuals with
ASD and >22,000 parents of individuals with ASD [82]. Most families recruited through
IAN had ≥1 child diagnosed with ASD (cases; n = 178). Neurotypically developing chil-
dren (controls) identified through IAN recruitment included siblings of cases (n = 61), and
offspring of friends/associates of IAN parents (n = 16) [83]. Additional recruitment (n =
101) was conducted through media outreach in San Antonio and South Texas (SA/STX),
through which 57 cases, including 7 referred from local autism services, and 44 controls
were enrolled. The ATFS, thus, included a total of 356 offspring: 235 ASD cases and 121
controls. In addition to completing questionnaires regarding their children’s early-life
Figure 1.
Potential impacts and interactions of aspartame and its metabolites on known autism risk
factors. (OCM: one-carbon metabolism, including the transsulfuration pathway, methionine cycle,
and folate cycle; GSH: reduced glutathione, a major antioxidant involved in defense against oxidative
stress; detoxification; maintenance of methylation capacity; SAM: S-adenosylmethionine, the major
methyl donor for cellular methylation processes; BBB: blood–brain barrier).
2. Materials and Methods
2.1. Participants
Recruitment and data collection for the Autism Tooth Fairy Study were performed
through the University of Texas Health Science Center at San Antonio (UTHSCSA) from
May 2011 to June 2014. The largest recruitment source (n= 255) was the Interactive Autism
Network (IAN), an Internet-based U.S. registry [
81
] that included > 21,600 individuals with
ASD and >22,000 parents of individuals with ASD [
82
]. Most families recruited through
IAN had
≥
1 child diagnosed with ASD (cases; n= 178). Neurotypically developing
children (controls) identified through IAN recruitment included siblings of cases (n= 61),
and offspring of friends/associates of IAN parents (n= 16) [
83
]. Additional recruitment
(
n= 101
) was conducted through media outreach in San Antonio and South Texas (SA/STX),
through which 57 cases, including 7 referred from local autism services, and 44 controls
were enrolled. The ATFS, thus, included a total of 356 offspring: 235 ASD cases and
121 controls. In addition to completing questionnaires regarding their children’s early-life
exposures, ATFS parents provided one or more of their children’s shed deciduous teeth for
compositional analyses, results of which are reported elsewhere [84].
The study was conducted according to the guidelines of the Declaration of Helsinki
and was approved by the Institutional Review Board (IRB) of the University of Texas
Health Science Center at San Antonio (UTHSCSA) (UTHSCSA IRB protocol
number 11-313,

Nutrients 2023,15, 3772 5 of 22
approved on 12 May 2011). In early data collection, all parents provided written in-
formed consent before completing self-administered hard-copy questionnaires. During
later Internet-based enrollment and data collection, all parents reviewed an IRB-approved
online study information sheet prior to completing online questionnaires through secure
websites, in accordance with the protocol approved by the IRB of the UTHSCSA.
2.2. Materials
2.2.1. Demographic and Neurodevelopmental Data Collected
Parents provided demographic data about themselves and their households; the birth
year and sex of each child; and whether each child had been diagnosed with either autism
or autistic disorder, Asperger’s disorder, pervasive developmental disorder—not otherwise
specified, or childhood disintegrative disorder. Children with any of these diagnoses were
included as ASD cases. Parents were also asked whether each child had been diagnosed
with any additional behavioral, developmental, and/or learning disabilities/disorders;
non-cases with none of these diagnoses were included as controls.
Parents were asked, “Was there ever a time when your child used at least three words
you could understand (besides mama or dada) on a daily basis for at least a month, and
then seemed to stop talking for a while (at least a month) where he/she used no words that
you could understand?” Cases where parents responded “no” were considered unlikely
to have experienced a regressive form of autism or ASD and were, thus, categorized as
“non-regressive” cases.
2.2.2. Early-Life Exposures to Diet Sodas, Other Diet Drinks, Aspartame, and Other NNSs
Biological mothers completed retrospective questionnaires on their intake of diet sodas
(DS) and other diet drinks (DD
other
) during pregnancy/breastfeeding. For each child they
were asked, “While you were pregnant or breastfeeding your child, how often did you
drink diet drinks containing artificial sweeteners? Please count diet sodas first, such as Diet
Coke, Diet Dr. Pepper, and Diet Sprite, and then other diet drinks, such as Crystal Light,
sugar-free Kool-Aid, Slim-Fast, and other ‘lite’ drinks”. Within each of these two beverage
subcategories, mothers recorded the number of cans or bottles of DS and the number of
glasses, cans, or bottles of DD
other
that they had consumed per time unit; whether this time
unit was daily, weekly, monthly, annually, or never; and the specific brand(s) consumed.
Mean intake of DS/day and DD
other
/day was calculated and summed to estimate total
maternal intake of diet drinks/day (DDtotal/day) during pregnancy/breastfeeding.
Each mother was also asked, “While you were pregnant or breastfeeding your child,
how many little packets of low-calorie sweeteners (such as Sweet ‘N Low, Equal or Splenda)
did you use in your coffee, tea, or other foods and beverages? In answering the question,
please keep in mind the number of drinks you had each day, and how many packets of
sweetener you added to each drink. Also, please include the number of packets you used
in cereal or other food”. Intake of the following three leading NNS packets was specifically
requested: “Equal/Nutrasweet (blue)”, “Splenda (yellow)”, and “Sweet’N Low (pink)”;
space was left for recording intake of other NNS packet brands. Maternal estimates of
intake of these products during pregnancy and breastfeeding were part of an extensive
questionnaire that gathered data on maternal and child exposures to a number of household
products and other environmental exposures throughout pregnancy and the early life of
the child.
2.3. Procedure
2.3.1. Case Definitions
Two case definitions were specified for analyses. The primary case definition of
interest was autism disorder (autism); the secondary case definition was any ASD. In
addition, subgroup analyses focused on offspring with non-regressive versions of each case
definition, among whom the relative etiologic influence of intrauterine/neonatal exposures
was expected to be maximized. In all analyses, controls were neurotypically developing

Nutrients 2023,15, 3772 6 of 22
offspring and—for sex-specific analyses—of the same sex as cases. Both sex-specific and
pooled analyses were performed for each of the case definitions and exposures.
2.3.2. Exposure Variables
Maternal intake of
≥
12 ounces (1 can) of DS/day during pregnancy/breastfeeding
defined the primary, dichotomized exposure variable: daily early-life exposure to DS
(DS
early
). The secondary, dichotomized exposure variable—daily early-life exposure to as-
partame (ASP
early
)—was defined as maternal intake of
≥
177 mg/day of aspartame during
pregnancy/breastfeeding, from the sum of aspartame dosages in DS + DD
other
+ packets
consumed by the mother. This cut-point was selected because it equals the minimum aspar-
tame content per can of the leading diet colas sweetened with aspartame alone. Estimates
of total maternal aspartame intake from these three sources were possible because mothers
were asked to report the specific brands they had consumed. Dosages of aspartame and
other NNSs used in each of the leading brands of DS were previously published [
85
];
for comparable products by other brands, NNS dosages were imputed according to the
NNS types specified on product labels. Leading DS brands exclusively sweetened with
aspartame contained
≥
177 mg of aspartame/can [
85
]; this dosage was, therefore, chosen
as the cut-point for ASP
early
to identify offspring with daily early-life aspartame exposure
comparable to that in 1 can of the leading diet colas [85].
For DD
other
, ingredient-labeling-specified types of NNS used, but not dosages, which
are proprietary. Based on limited available data, a 12-ounce serving of DD
other
exclusively
sweetened with aspartame was estimated to contain approximately 100 mg of aspartame.
A tabletop packet contains 37 mg of aspartame [86].
2.4. Statistical Analyses
Offspring born in 1984 or thereafter, with complete early-life DS exposure data and
neurodevelopmental diagnostic data, were included in analyses. The proportions of off-
spring with DS
early
and ASP
early
exposures were separately calculated, by sex, for the
following diagnostic groups: controls, all ASD cases, ASD cases without autism, autism
cases, and the non-regressive version of each case definition. Differences in the proportions
of exposed controls and cases, in each category, were calculated using the Pearson
χ2
statistic in Stata/IC 14.2 for Windows.
To assess associations among diagnostic status and the primary and secondary ex-
posures under study—DS
early
and ASP
early
—multilevel mixed-effects generalized linear
models (MEGLMs) were used to consider potential influences at two levels: the individual
child (level one) and shared intrauterine and familial influences, through the mother (level
two). Recruitment source was treated as a level-one predictor. A total of 356 children,
including children from 79 sibships, were included in analyses. Models were fit using the
meglm command in Stata/IC 14.2 for Windows.
Unadjusted odds ratios (ORs) for the primary and secondary exposures of interest—
DS
early
and ASP
early
—were separately computed for each case definition, by sex, and for
all participants, pooled. Adjusted models included the following covariates: recruitment
source (IAN vs. SA/STX); year of birth (range, 1985 to 2011), to adjust for secular trends
in autism diagnosis; and three dichotomous demographic variables: ethnicity of the child
(non-Hispanic white (NHW) vs. other), maternal education (college graduate vs. not),
and household income (
≥
USD 100,000/year vs. less). In pooled analyses, we additionally
adjusted for sex of the child. For comparison purposes, we also calculated separate ORs
for early-life exposures to
≥
1 serving/day of aspartame specifically or to any NNS, from
the combination of DS, DD
other
, and/or tabletop packets, with minimum serving size
defined as one tabletop sweetener packet. The minimum daily dosage for these analyses
would, thus, be 37 mg for aspartame, 36 mg for saccharin, and 12 mg for sucralose. We also
performed subgroup analyses for non-regressive ASD and non-regressive autism, using
these same exposure variables and covariates. These models were all fit using the meglm
command in Stata/IC 14.2 for Windows.

Nutrients 2023,15, 3772 7 of 22
2.5. Sensitivity Analyses
To examine influences on maternal intake of these products, we compared the pro-
portions of biological mothers of males who reported consuming DS, aspartame, or any
other NNSs during pregnancy and/or breastfeeding, within dichotomized substrata of
three demographic factors previously associated with cardiometabolic risk: income, educa-
tional level, and ethnicity. Among these six demographic subsets—mothers of offspring
from households with incomes
≥
USD 100,000/year vs. less; mothers with
≥
4 years of
college vs. less education; and mothers of NHW children vs. children of other or mixed
ethnicities—we compared the proportions of mothers whose mean total reported intake
of diet products equaled or exceeded the dose of 1 packet/day of any NNS, 1 packet/day
of aspartame, 1 DS/day, and 177 mg/day of aspartame. The significance of differences
between sociodemographic substrata in the proportions of mothers who reported daily
consumption of each of these diet products was assessed using the Pearson
χ2
statistic in
Stata/IC 14.2 for Windows.
3. Results
Table 1displays characteristics of controls, ASD cases, autism cases, and non-regressive
autism cases, by sex, for the 257 boys (203 ASD cases + 54 controls) and 99 girls (32 ASD
cases + 67 controls) included in the analyses. Maternal education was high overall:
≥
60%
of mothers in each diagnostic/sex subgroup were college graduates. Over 60% of families
had incomes above the 2010 U.S. median of USD 50,000 [
87
]; incomes were slightly higher
for controls and non-regressive autism cases. Among boys, roughly 70% of both cases and
controls were from IAN, and 70% of cases were NHW, although the proportion of controls
who were NHW was slightly lower: 64%. Among girls, approximately 90% of cases were
from IAN and were NHW, whereas approximately 60% of controls were from IAN and
were NHW.
Among males, the proportion of offspring with daily DS
early
and ASP
early
exposures
dramatically increased with the severity of the diagnostic category and further increased
with the non-regressive condition (Table 2). Nearly one in four males with non-regressive
autism (23.3%) had ASP
early
exposures, and 22.1% had DS
early
exposures, compared with
7.4% of controls. Among girls, no comparable trends emerged between their diagnostic
status and either DSearly or ASPearly proportions.
Similarly, the exposure odds for DS
early
progressively increased with diagnosis sever-
ity and non-regressive condition among males (Table 3). In the adjusted multivariable
model, the exposure odds for DS
early
were tripled among males with autism: OR = 3.14
(95% confidence interval (CI): 1.02–9.65) and were even higher for males with non-regressive
autism: OR = 3.49 (95% CI: 1.10–11.1). No statistically significant associations were detected
between DS
early
and any of the following: total ASD in either sex; any ASD diagnostic
category in pooled analyses; or any ASD diagnostic category in girls, among whom ORs
were consistently <1. Thus, the association between DS
early
and diagnostic status was
specific to autism in males in our analyses.

Nutrients 2023,15, 3772 8 of 22
Table 1. Demographic characteristics, by sex and diagnostic status, among 356 offspring in the Autism Tooth Fairy Study.
Characteristic
Boys (n= 257) Girls (n= 99)
Cases Cases
Controls
(n= 54)
Any ASD
(n= 203)
Autism
(n= 140)
Non-Regressive Autism
(n= 86)
Controls
(n= 67)
Any ASD
(n= 32)
Autism
(n= 28)
Non-Regressive Autism
(n= 19)
Birth year of child
(Mean (SD))
2001.3
(4.1)
2001.4
(4.3)
2001.5
(4.2)
2001.5
(4.1)
2002.2
(3.8)
2001.8
(4.3)
2001.9
(4.3)
2001.4
(4.6)
Education of mother (n) 54 202 139 86 67 32 28 19
<High school (%) 0 4 3 3 3 0 0 0
High school graduate (%) 13 5 8 9 4 9 7 5
<4 yrs college (%) 20 31 29 19 28 25 29 21
≥4 yrs college (%) 67 60 60 69 64 66 64 74
Family income (n) 50 198 135 83 66 32 28 19
<USD 25K (%) 12 18 19 16 14 9 7 5
USD 25K to <USD 50K (%) 10 16 14 12 17 28 29 16
USD 50K to <USD 100K (%) 32 35 36 30 29 34 39 53
USD 100K to <USD 200K (%)
32 25 25 34 29 19 18 21
≥USD 200K (%) 14 7 7 8 12 9 7 5
Ethnicity of child (n) 53 202 139 85 66 32 28 19
Hispanic (%) 25 16 15 13 32 9 7 5
Non-Hispanic white (%) 64 70 68 74 58 88 93 95
Other/mixed (%) 11 14 17 13 11 3 0 0
Recruitment source (n) 54 203 140 86 67 32 28 19
IAN (%) 69 74 73 72 60 87 89 89
SA/STX (%) 31 26 27 28 40 13 11 11
ASD = autism spectrum disorder; SD = standard deviation; IAN = Interactive Autism Network; SA/STX = San Antonio/South Texas; K = 1000.

Nutrients 2023,15, 3772 9 of 22
Table 2. Percentage of cases and controls exposed to ≥1 diet soda or ≥177 mg of aspartame daily a.
Boys (n= 257) Girls (n= 99)
n
≥1 Diet Soda/Day ≥177 mg/Day of Aspartame
n
≥1 Diet Soda/Day ≥177 mg/Day of Aspartame
% % % %
Controls 54 7.4 7.4 67 17.9 19.4
ASD cases, excluding autism 63 9.5 9.5 4 25.0 25.0
Non-regressive ASD cases, excluding autism 45 11.1 11.1 4 25.0 25.0
All ASD cases, combined 203 16.3 17.2 32 12.5 12.5
Non-regressive ASD cases, combined 131 18.3 19.1 * 23 17.4 17.4
Autism cases 140 19.3 * 20.7 * 28 10.7 10.7
Non-regressive autism cases 86 22.1 * 23.3 * 19 15.8 15.8
a
In early life and during gestation and/or breastfeeding; based on dietary intakes retrospectively reported by biological mothers. Bold-highlighted results indicate statistically
significantly increased exposure proportions among cases, compared with controls. * p< 0.05 for difference from proportion of male controls with daily early-life exposures to these
products.

Nutrients 2023,15, 3772 10 of 22
Table 3. Odds ratios a(OR) for early-life exposure bto ≥1 diet soda/day, by autism spectrum disorder (ASD) category.
Condition Boys Girls All Participants Combined c
nOR 95% CI nOR 95% CI nOR 95% CI
ASD: all cases
Unadjusted c257 2.4 0.8 to 7.2 99 0.7 0.2 to 2.2 356 1.4 0.7 to 2.8
Adjusted d246 2.4 0.8 to 7.3 98 0.5 0.1 to 1.8 344 1.4 0.7 to 2.9
Non-regressive ASD
Unadjusted c185 2.8 0.9 to 8.5 90 1.0 0.3 to 3.4 275 1.8 0.8 to 3.8
Adjusted d177 2.7 0.9 to 8.4 89 0.7 0.2 to 3.0 266 1.8 0.8 to 3.8
Autism
Unadjusted c194 3.0 0.99 to 9.0 95 0.6 0.1 to 2.1 289 1.5 0.7 to 3.1
Adjusted d183 3.1 * 1.02 to 9.7 94 0.3 0.1 to 1.3 277 1.5 0.7 to 3.2
Non-regressive autism
Unadjusted c140 3.5 * 1.1 to 11.1 86 0.9 0.2 to 3.4 226 2.0 0.9 to 4.3
Adjusted d132 3.5 * 1.1 to 11.1 85 0.5 0.1 to 2.3 217 2.0 0.9 to 4.4
a
ORs were derived using multilevel mixed-effects generalized linear model analyses. Bold-highlighted results indicate statistically significantly increased exposure ORs (p< 0.05).
b
Early-life exposure: through maternal diet during pregnancy and breastfeeding. Exposures were calculated based on diet soda intake during this period, retrospectively recalled by
biological mothers, for 356 offspring in the Autism Tooth Fairy Study.
c
Sex was included as a covariate in both “unadjusted” and adjusted analyses for all participants combined.
d
Adjusted for mother’s identity; recruitment source; child’s ethnicity (non-Hispanic white vs. other); year of birth; mother ’s education (
≥
4 years of college vs. less), and household
income (≥USD 100,000/year vs. less). OR = odds ratio; CI = confidence interval; * p< 0.05 for difference from odds of daily early-life exposure to ≥1 diet soda, among controls.

Nutrients 2023,15, 3772 11 of 22
Likewise, adjusted ORs for ASP
early
progressively increased with diagnosis severity
and non-regressive condition in males (Table 4), among whom ORs were more than tripled
among all autism cases and even higher among non-regressive autism cases: OR = 3.42
(95% CI: 1.12–10.4), and OR = 3.72 (95% CI: 1.18–11.8), respectively. As in the case of
DS
early
, no statistically significant associations were found between ASP
early
and total ASD
in boys, between ASP
early
and any ASD-related diagnoses in girls (Table S1a), or among
all participants combined (Table S1b). ORs for early-life exposure to
≥
1 serving/day of
any NNS—from either DS, DD
other
, or tabletop packets—increased with the severity of
the diagnosis and ranged from 1.60 (95% CI: 0.69–3.70) for total ASD to 2.09 (95% CI:
0.84–5.18) for non-regressive autism, though were not statistically significant. ORs for daily
early-life exposure to
≥
1 serving/day of aspartame were higher and ranged from 1.94
(95% CI: 0.76–4.95) for total ASD to 2.63 (95% CI: 0.97–7.19) for non-regressive autism, but
none of these was statistically significant. Taken together, these results suggest a possible
threshold effect, with autism diagnosis in boys being associated with more-than-tripled
odds of early-life exposure to either
≥
1 DS/day or comparable daily aspartame exposure
(
≥
177 mg/day) in this study. Lower daily dosages, however—for either NNS in general or
aspartame specifically—were not associated with autism status in this study sample. Nor
were statistically significant associations found between either DS
early
or ASP
early
exposure
and total ASD in boys, between either exposure and any ASD diagnosis in girls (Table S1a),
or among all participants combined (Table S1b).
Table 4.
Adjusted odds ratios
a
among males for daily early-life
b
exposures to any NNS
c
or to
aspartame specifically d.
Condition in Males nDaily Exposure ORs 95% CI
ASD: all cases 246
≥1 serving/day of any NNS 1.6 0.7 to 3.7
≥1 serving/day of aspartame 1.9 0.8 to 5.0
≥177 mg/day of aspartame 2.6 0.9 to 7.8
Non-regressive ASD 177
≥1 serving/day of any NNS 1.7 0.7 to 4.1
≥1 serving/day of aspartame 2.1 0.8 to 5.7
≥177 mg/day of aspartame 2.9 0.9 to 8.8
Autism 183
≥1 serving/day of any NNS 2.0 0.8 to 4.7
≥1 serving/day of aspartame 2.5 0.95 to 6.5
≥177 mg/day of aspartame 3.4 * 1.1 to 10.4
Non-regressive autism 132
≥1 serving/day of any NNS 2.1 0.8 to 5.2
≥1 serving/day of aspartame 2.6 0.97 to 7.2
≥177 mg/day of aspartame 3.7 * 1.2 to 11.8
a
ORs were derived using multilevel mixed-effects generalized linear model analyses for 246 male offspring
in the Autism Tooth Fairy Study: adjusted for mother ’s identity; recruitment source; child’s ethnicity (non-
Hispanic white vs. other); year of birth; mother ’s education (
≥
4 years of college vs. less), and household income
(
≥
USD 100,000/year vs. less). Bold-highlighted results indicate statistically significantly increased exposure
ORs (
p< 0.05
).
b
Early-life exposures: exposures that occurred during gestation and/or breastfeeding, through
maternal diet during these times. These were calculated based on aspartame and other NNS intake during this
period, from the sum of diet sodas, other diet drinks, and sweetener packets, retrospectively recalled by biological
mothers.
c
NNS: non-nutritive sweetener:
≥
1 serving/day of any NNS denotes either
≥
1 packet/day of any NNS,
≥
1 DS/day, or
≥
1 other diet drink/day. Minimum daily dosage for this category, thus, varies by NNS: 1 tabletop
packet contains 36 mg of saccharin, 12 mg of sucralose, or 37 mg of aspartame, for example.
d
For aspartame
specifically:
≥
1 serving/day of aspartame denotes either
≥
1 packet/day of aspartame,
≥
1 aspartame-sweetened
DS/day, or
≥
1 other aspartame-sweetened diet drink/day. Minimum daily dosage for this category is, thus,
37 mg, the dosage in 1 tabletop packet.
≥
177 mg/day of aspartame denotes total daily aspartame intake, from the
sum of packets + DS + other diet drinks, equivalent to
≥
177 mg, the dosage of aspartame in 1 can of a leading diet
cola sweetened only with aspartame. * p< 0.05 for difference from odds of daily early-life exposure to
≥
177 mg of
aspartame, among male controls.

Nutrients 2023,15, 3772 12 of 22
Sensitivity Analyses
The results from the sensitivity analyses for male offspring, displayed in Supplemen-
tary Figure S1a–c, demonstrate that the proportion of biological mothers consuming either
≥
1 DS/day or
≥
177 mg of aspartame/day were comparable across the substrata of house-
hold income, maternal education, and offspring ethnicity. However, point estimates for the
proportions of mothers who reported consuming the mean equivalent of
≥
1 packet/day of
any NNS were slightly higher for offspring from more affluent vs. less affluent households,
32% vs. 25%, respectively (Figure S1a); and for offspring of mothers with college degrees vs.
fewer years of education: 30% vs. 22%, respectively (Figure S1b). These proportions were
comparable for NHWs and other ethnic groups: 27% vs. 26%, respectively (Figure S1c).
None of the differences between the subgroups shown in Figure S1a–c were statistically
significant. Among the mothers of all males in our study, 28% reported mean daily NNS
consumption during pregnancy equivalent to
≥
1 tabletop packet of NNS. These results are
comparable to those from earlier studies, in which 24%–30% of pregnant women reported
using DS/DSB or other NNSs during pregnancy [88–90].
4. Discussion
To our knowledge, this is the first study to specifically examine the associations
between offspring autism status and the daily maternal intake of diet soda and aspartame
during pregnancy/breastfeeding. Among boys, we found more than tripled odds of
exposure to either DS
early
or ASP
early
among autism cases, compared with controls. These
associations were both diagnosis- and sex-specific: no statistically significant associations
were found for total ASD in boys nor for either diagnostic category in girls, who represented
only 17% (n= 28) of total autism cases in our study.
Several possible explanations exist for the lack of associations among girls in our
study; these include insufficient statistical power, inherent sex dimorphism in response
to DS/aspartame exposures, and possibly even the recruitment strategy itself, which, by
including as controls neurotypically developing female siblings of male cases, increased the
likelihood that any early-life exposures found to be risk-enhancing among their brothers
with ASD might appear to be negatively associated with ASD in the analyses for females.
Further research with larger sample sizes for both sexes and prospectively gathered data
would be important for investigating this association further in females and in all partici-
pants combined.
4.1. Non-Regressive Cases
Boterberg et al., in their overview of publications related to regression in ASD, noted
that there is no single published definition for regression in ASD [
91
]. Along with Ozonoff
et al. [
92
], however, they delineated language loss as a core component of regression
in ASD [
91
,
92
] and noted that this component was included in the earliest descriptions
of regression in autism [
91
]. Regression was historically observed to occur somewhere
during the second year after birth or thereafter, although data from newer, prospective
studies indicated that regression may occur at earlier ages, even within the first year after
birth [
91
,
92
]. In an attempt to maximize our ability to detect associations between exposures
in early life—in utero and/or through maternal milk—and current autism status and to
minimize the influence of later-childhood exposures and influences on autism risk, we
attempted to identify and focus on earlier-onset, non-regressive cases by designating a
subset of cases whose parents had reported no loss of speech for them, at any point, as
“non-regressive”. We then performed additional analyses for this subgroup, including only
offspring without such reported language loss. Since, as Ozonoff et al. noted, regressive
events may in fact also occur earlier in life without being detected [
92
], our “non-regressive”
category may include some offspring with very-early-life regression; thus, the cases in
this subgroup might best be broadly interpreted as “early-onset” and likely include both
offspring whose differences were noted very early after birth and some for whom regression
occurred somewhat later, though still earlier than the onset of speech acquisition.

Nutrients 2023,15, 3772 13 of 22
We had hypothesized that, among these earlier-onset, “non-regressive” cases, the
relative influence of prenatal exposures—compared with exposures later in infancy and
early childhood—would be higher than that among all cases combined. Our findings
supported this hypothesis: among males, the exposure ORs for non-regressive cases of
ASD and autism routinely exceeded those for total ASD and autism cases.
4.2. Results from Earlier Studies in Adult Consumers and Gestationally Exposed Offspring
As previously noted, diet sodas and other diet beverages (DSB) are leading vehicles of
aspartame intake in the U.S. [
23
]. Three clinical trials and two prospective studies previ-
ously reported adverse neurological reactions to aspartame and to the daily intake of diet
beverages. Increased headache [
18
] and problems with nervousness/irritability, depres-
sion, memory, and spatial orientation [
17
,
20
] were reported among aspartame-consuming
trial participants, with neurological symptomatology increased among individuals with
a history of major depression [
17
]. Among 318,257 participants in the National Institutes
of Health-American Association of Retired Persons (NIH-AARP) Diet and Health study
followed for >10 years, incident depression was significantly higher among consumers
of aspartame-sweetened tea and coffee, compared with non-consumers, which monoton-
ically increased with DS consumption [
21
]. Similarly, among 1484 older members of the
Framingham Heart Study Offspring cohort followed over a decade, the hazards of incident
Alzheimer’s disease were nearly tripled among participants with cumulative DS consump-
tion
≥
1/day [
22
]. The increased incidence of other major cardiometabolic and related
problems was also reported among daily adult DSB consumers [93–95].
Despite the adverse impacts from NNS and DSB consumption reported in studies of
animals and human adults, respectively, the potential neurodevelopmental impacts of early-
life exposure to NNSs in general and to DS/DSB in particular in humans remain largely
unexplored. This is a question of particular concern because between 24% and 30% of
pregnant women reported using either NNSs in general [
88
] or DS/DSB specifically [
89
,
90
]
during their pregnancies. Furthermore, a recent Australian study reported detecting one or
more NNSs in 100% of 15 cord blood samples analyzed and in 77% of 13 amniotic fluid
samples analyzed [
96
]. Their study is the first to provide direct evidence of transplacental
passage of NNSs in humans. Its authors also raised the troubling concern of the possible
dose accumulation of NNSs within the fetus [
96
]. If early-life exposures to aspartame and
one of its leading vehicles, DS, do indeed significantly increase autism risk in the unborn
child, then it is critical for women of reproductive age to be informed of this association.
Health impacts among the very young—a particularly vulnerable population—remained
largely unstudied in humans until 2010, when a publication from the first of three prospec-
tive studies described an increased risk of adverse health outcomes among offspring
exposed daily in utero to DSB [
64
,
89
,
97
,
98
] or to the combination of DS + aspartame pack-
ets [
99
]. These studies were the Danish National Birth Cohort (DNBC; n= 60,466 pregnant
women and their infants) [
64
,
97
]; the Canadian Healthy Infant Longitudinal Development
Study (CHILD; n= 2686 pregnant women and their infants) [
89
,
98
]; and Project Viva (PV;
n= 1683 children from a prospective pre-birth cohort) [
99
]. In the DNBC, daily maternal
DSB intake during pregnancy was associated with a 38% increase in total preterm birth
(<37 weeks’ gestation); a 67% increase in early-preterm birth (<32 weeks’ gestation) [
64
];
and—among mothers with gestational diabetes—almost doubled risk of offspring over-
weight/obesity by age 7 years [
97
]. Similarly, offspring overweight/obesity by age 1 year
was doubled among CHILD offspring exposed to DSB daily in utero [
89
]. In Project Viva,
the children of mothers in the highest quartile of DS + aspartame consumption during
pregnancy had significantly higher BMI z-scores and sum of skinfolds throughout early
and middle childhood, compared with unexposed offspring [
99
]. Furthermore, the as-
sociation between maternal NNS intake during pregnancy and subsequent offspring’s
BMI z-scores increased from age 3 to 18 years [
99
]. Thus, in each of these prospective
studies, daily gestational exposure to these products was associated with increased car-
diometabolic risk among offspring, even after adjustment for such important risk factors as

Nutrients 2023,15, 3772 14 of 22
pre-pregnancy maternal body mass index, caloric intake, physical activity, diabetes status,
and demographic characteristics.
4.3. Sex Differences in Developmental Responses to Early-Life Exposures from Earlier Studies
In the results from both CHILD [
89
] and the DNBC [
97
], it is notable that the doubling
of the overweight/obesity risk observed among DSB-exposed offspring occurred in male,
but not in female, offspring [
89
,
97
]. This is of particular relevance to the current investi-
gation because increased neurodevelopmental vulnerability among males was previously
reported for both intrauterine and other early-life exposures, including environmental
neurotoxicants [
100
], maternal stress [
101
], and dysbiosis of the gut microbiota [
79
]. This
heightened vulnerability among males contributes to the four- to eight-fold increased risk
of neurodevelopmental problems in human males [100,101].
4.4. Is Dietary Methanol from Aspartame Contributing to These Associations?
As noted earlier, the DNBC previously reported a 38% increased risk of total preterm
birth (<37 weeks’ gestation), and a 67% increased risk of early-preterm birth (<32 weeks’
gestation) among offspring whose mothers had consumed DSB daily during their ges-
tation [
64
]; the authors suggested that the aspartame metabolite methanol may have
contributed to these impacts. Gestational exposures to dietary methanol were estimated in
an earlier study by Walton and Monte [
7
] for 161 ASD cases and 550 controls, by means of
retrospective dietary questionnaires completed by their biological mothers. The mothers
reported their intake of key dietary sources of methanol—including DS, other diet products,
and processed fruit and vegetable juices—during pregnancy. Based on their estimates, the
mean calculated dietary methanol exposure in utero for ASD cases was more than double
that among controls: 142 vs. 67 mg/day, respectively, for both sexes combined (p< 0.001
for difference) [
7
]. Although the key exposure of interest in their study and the range
and specificity of the dietary products examined differed from ours, Walton and Monte
were the first to report an association between estimated gestational dietary exposure to an
aspartame metabolite—methanol—and subsequent ASD status. The congruence of their
findings with our own offers support for the need to further investigate the influence of
these early-life dietary exposures on autism risk.
4.5. Metabolic Impacts of Aspartame, Methanol, and Their Metabolites on Availability of Reduced
Glutathione (GSH) and Other Products of One-Carbon Metabolism
One of the most frequently reported impacts of aspartame consumption in animal
studies is a decrease in the availability of GSH, as well as the adverse metabolic impacts
that follow from it. GSH and S-adenosyl methionine (SAM), the major methyl donor for
cellular methylation processes [
52
], are among a number of neuroprotective molecules
maintained through folate-dependent one-carbon metabolism (OCM), a system of interre-
lated processes that include the folate cycle, the methionine cycle, and the transsulfuration
pathway, through which GSH is synthesized, and redox balance is maintained [
102
]. Dys-
regulation of OCM was observed in both aspartame-fed animals and individuals with
autism
[49,50,52,103–105].
In mice, the aspartame-triggered blockade of the transsulfura-
tion pathway and the methionine cycle within OCM has led to the reduced availability of
GSH, SAM, and other OCM metabolites [
52
]. It is notable that decreased GSH and increased
oxidative stress, mitochondrial dysfunction, inflammation, dysbiosis of the gut microbiota,
and leaky gut were also observed in both animals fed aspartame and individuals with
autism [75,103,106–108].
4.6. Sensitivity Analyses
Since maternal overweight and obesity (OW/OB), diabetes, and related conditions
are associated with an increased risk of ASD among offspring [
109
–
114
] and might also
influence a woman’s decision to use diet products, these represent potential unmeasured
confounders in our results. Although we have no data on maternal cardiometabolic risk

Nutrients 2023,15, 3772 15 of 22
factors in our study sample, we have data for three key demographic measures—household
income, educational attainment, and ethnicity—which were previously associated with
cardiometabolic risk in population-based studies. In earlier studies, a higher prevalence
of both OW/OB and diabetes was found among individuals with lower education and
income [
115
–
117
] and in some ethnic groups other than NHWs [
118
,
119
]. We, therefore,
included these three sociodemographic measures in all adjusted calculations of ORs for
DS
early
and ASP
early
(reported in Table 3, Table 4and Table S1a,b). In addition, for males,
we compared the maternal consumption of different diet products within demographic
subgroups defined by these variables.
A higher consumption of diet products within demographic subgroups historically at
a higher risk of OW/OB and diabetes would be expected if maternal consumption of diet
products in our study population was primarily driven by pre-existing cardiometabolic
problems. Our sensitivity analyses (Figure S1a–c), however, revealed that the proportion
of mothers with average NNS intakes
≥
1 packet/day of NNS were similar across ethnic
subgroups (Figure S1c) and were in fact higher among mothers with college degrees
(Figure S1b) and from more affluent households (Figure S1a), although none of these
differences were statistically significant. These results were congruent with those from the
National Health and Nutrition Examination Survey (NHANES) data from 1999–2000 to
2013–2014, in finding NNS consumption to be higher among NHWs and among participants
with higher education and income [
88
]. The NHANES data, like our own, suggest the
possibility that NNS consumption may have been primarily adopted as a preventive, health-
promotion strategy, rather than simply in response to existing cardiometabolic problems,
in each of these study populations.
4.7. Future Directions
While our findings do not establish a causal relationship between daily early-life
exposure to diet sodas/aspartame and autism risk in males, they nonetheless raise concerns
that justify further research, especially given the current widespread use of diet products
among pregnant women. It would be important to study the association between maternal
DBS/NNS intake during pregnancy and subsequent offspring autism risk within the
context of a larger population in which both the maternal intake of these products and
additional maternal characteristics, including cardiometabolic and other risk factors, could
be prospectively measured. In the meantime, the congruence between our results and
those of earlier studies in finding associations between early-life exposures to DSB and/or
aspartame and subsequent chronic health problems in offspring suggests a pattern of
increased risk that requires further study.
Autism typically results from a perfect storm of adverse impacts, with toxicant expo-
sures overlaid upon genetic polymorphisms that confer decreased neuroprotection [
102
].
As previously summarized, early-life aspartame exposure could potentially contribute to
this storm by increasing toxicant load while simultaneously decreasing neuroprotective
potential, as indicated in Figure 1. Although daily early-life exposure to DS/aspartame
represents only one potential risk-increasing exposure within the total exposome of the
developing child, nonetheless—within the context of male sex and other risk factors—it
might contribute to overwhelming the delicate neurodevelopmental susceptibility tipping
point of a child already at increased risk.
4.8. Strengths and Limitations
Our dietary questionnaires were retrospectively completed and focused on maternal
intake several years earlier, during pregnancy/breastfeeding. Diagnostic status, elapsed
time, and/or changes in product formulations may have led to recall bias and/or other
information bias in the maternal dietary recalls. As noted earlier, no covariate data were
available for maternal overweight/obesity and diabetes, maternal mental health, and other
potential confounders in our study. In addition, no dietary recall data were available for
the paternal intake of these products during the preconceptual period. Our estimates of

Nutrients 2023,15, 3772 16 of 22
offspring aspartame exposure were based upon the maternal intake of three product cate-
gories: diet sodas, other diet beverages, and sweetener packets. While these are prominent
vehicles for aspartame consumption, aspartame itself is included as an ingredient in over
6000 dietary, pharmacologic, and cosmetic products [
8
]; thus, our intake estimates only
represented the minimum maternal intake of aspartame: both cases and controls were
likely exposed to aspartame from additional sources. The overall study sample was small
and had low statistical power. In addition, the number of controls relative to cases was
small, especially among males, and included both unrelated controls and siblings of cases.
Further research is needed to determine whether the apparent associations observed in our
study are artifacts of these limitations or in fact arise from the deleterious consequences
of early-life exposures to these products. In support of a causal relationship between
these early-life exposures and autism risk in males is the striking congruence between
our results and those from animal studies as well as from large-scale prospective studies
that—even after adjusting for both maternal body mass index, diabetes status, and other
key potential confounders—found striking associations between daily gestational exposure
to non-nutritive sweeteners and/or diet beverages and subsequent major health problems
among offspring, especially among males.
5. Conclusions
Compared with male controls, males with autism in our study had more than tripled
odds of having been exposed daily—gestationally and/or through breastfeeding—to either
diet soda itself or comparable doses of aspartame from multiple sources. These exposure
odds were the highest among cases with non-regressive autism. These associations do not
prove causality. Taken in concert, however, with previous findings of increased prema-
turity and cardiometabolic health impacts among infants and children exposed daily to
diet beverages and/or aspartame during pregnancy, they raise new concerns about the
potential neurological impacts, which need to be addressed. Further research—including
larger sample sizes of both sexes and prospective measurement of exposures, additional
potential confounders, and outcomes—is needed to evaluate these associations in other
study populations and to assess whether they extend to overall ASD risk in boys and to
autism and/or ASD risk in girls.
In the meantime, however, the possibility that early-life exposures to these products
through maternal diet might increase offspring neurodevelopmental risk—at least among
boys—is of particular concern. Between 24% and 30% of pregnant women reported using
either non-nutritive sweeteners in general [
88
] or diet sodas and other diet beverages specif-
ically [
89
,
90
] during their pregnancies. Furthermore, a recent study provided evidence
of the transplacental passage of these substances in humans [
96
] and raised troubling
questions regarding the dose accumulation of non-nutritive sweeteners within the fetus.
Taken together with widespread gestational exposure to these products and previously
reported associations between gestational exposure to these products and adverse health
impacts in offspring, these findings suggest that, in the spirit of the Precautionary Prin-
ciple [
120
], women should exercise caution when considering the use of these products
during pregnancy and breastfeeding, until further assessments are available. The maternal
consumption of these products during periods of heightened offspring vulnerability rep-
resents a modifiable potential risk factor, the elimination of which might help to protect
susceptible offspring in the next generation.
Supplementary Materials:
The following supporting information can be downloaded at https:
//www.mdpi.com/article/10.3390/nu15173772/s1. Table S1a: Adjusted ORs among female offspring
for daily early-life exposures to any NNS or to aspartame specifically; Table S1b: Adjusted ORs
among all offspring for daily early-life exposures to any NNS or to aspartame specifically; Figure S1a:
Among mothers of all males, percentage who consumed
≥
1 DS/day,
≥
177 mg/day of aspartame,
≥
1 packet/day of aspartame (mean daily equivalent), or
≥
1 packet/day of any NNS (mean daily
equivalent) during pregnancy and/or breastfeeding, by substratum of household income; Figure S1b:
Among mothers of all males, percentage who consumed
≥
1 DS/day,
≥
177 mg/day of aspartame,

Nutrients 2023,15, 3772 17 of 22
≥
1 packet/day of aspartame (mean daily equivalent), or
≥
1 packet/day of any NNS (mean daily
equivalent) during pregnancy and/or breastfeeding, by substratum of maternal education; Figure S1c:
Among mothers of all males, percentage who consumed
≥
1 DS/day,
≥
177 mg/day of aspartame,
≥
1 packet/day of aspartame (mean daily equivalent), or
≥
1 packet/day of any NNS (mean daily
equivalent) during pregnancy and/or breastfeeding, by ethnic group.
Author Contributions:
Conceptualization, S.P.F., L.P.H. and R.F.P.; methodology, R.F.P., L.P.H. and
S.P.F.; software, R.F.P., L.P.H. and S.P.F.; validation, L.P.H. and S.P.F.; formal analysis, M.D.S., S.P.F. and
R.F.P.; investigation, R.F.P., L.P.H. and S.P.F.; resources, R.F.P.; data curation, L.P.H., R.F.P. and S.P.F.;
writing—original draft preparation, S.P.F.; writing—review and editing, R.F.P., D.G.R.d.P., P.S.G.,
L.P.H., M.D.S. and S.P.F.; visualization, S.P.F.; supervision, R.F.P., D.G.R.d.P., P.S.G. and M.D.S.; project
administration, R.F.P. and L.P.H.; funding acquisition, R.F.P. All authors have read and agreed to the
published version of the manuscript.
Funding:
The ATFS was funded by Autism Speaks, through a Suzanne and Bob Wright Trailblazer
Award to R.F.P. and through grant number 1R21ES023604-01A1, from the National Institute of Envi-
ronmental Health Sciences of the National Institutes of Health, also awarded to R.F.P. D.G.R.d.P. was
partially funded by the Southwest Center for Occupational and Environmental Health (SWCOEH),
the Centers for Disease Control and Prevention, and the National Institute for Occupational Safety
and Health (NIOSH)’s Education and Research Center (grant number T42OH008421) at The Univer-
sity of Texas Health Science Center at the Houston School of Public Health. No external funding was
received for the data analyses presented here.
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki and was approved by the Institutional Review Board (IRB) of the University
of Texas Health Science Center at San Antonio (UTHSCSA) (UTHSCSA IRB protocol number 11-313,
approved on 12 May 2011.
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement:
The data presented in this study are openly available in the Dryad
Digital Repository at https://doi.org/10.5061/dryad.cfxpnvx2p [121].
Acknowledgments:
The authors are deeply grateful to the following individuals and institutions,
who helped make this research possible: the Department of Family and Community Medicine at the
Joe R. and Teresa Lozano Long School of Medicine, UTHSCSA, for its support of the Autism Tooth
Fairy Study (ATFS); the South Texas Autism Research group, whose members—including Claudia
S. Miller, Stephen T. Schultz, Georgianna G. Gould, and David E. Camann—provided insights and
support for this undertaking; and to the hundreds of parents who participated in the ATFS, who
together—through their great generosity in sharing their experiences with study investigators and
team members—made this research possible.
Conflicts of Interest:
The authors declare no conflict of interest. The funding institutions had no role
in either the design or the execution of the study; in the collection, analysis, and interpretation of the
data; or in the writing or reviewing of the manuscript.
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