DOI: 10.5061/dryad.cfxpnvx2p
title: Early-life exposures to diet soda and aspartame are associated with autism in males

description: 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, DSearly odds were tripled among autism (OR = 3.1; 95% CI: 1.02,
 9.7) and non-regressive autism (OR = 3.5; 95% CI: 1.1, 11.1)
 cases; ASPearly odds were even higher: OR = 3.4 (95% CI: 1.1, 10.4) and
 3.7 (95% CI: 1.2, 11.8), respectively, among autism and non-regressive
 autism cases (p &lt; 0.05 for each). 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.
description: Participants  Recruitment and data collection for the Autism Tooth Fairy
 Study (ATFS) were performed through The University of Texas Health Science
 Center at San Antonio (UTHSCSA) from May 2011, through June 2014. The
 largest recruitment source (n=255) was the Interactive Autism Network
 (IAN), an Internet-based U.S. registry (1) which included &gt;21,600
 individuals with ASD, and &gt;22,000 parents of individuals with ASD
 (2). Most families recruited through IAN had &gt;1
 child diagnosed with ASD (cases; n=178).  Neurotypically-developing
 children (controls; n=77) identified through IAN recruitment included
 siblings of cases (n=61), and offspring of friends/associates of IAN
 parents (n=16) (3). 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 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 (4).  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).
 In early data collection, all parents provided written informed 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. Materials 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. 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<sub>other</sub>) 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<sub>other</sub> 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<sub>other</sub>/day was calculated and summed to estimate
 total maternal intake of diet drinks/day
 (DD<sub>total</sub>/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.  Procedure 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 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. 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<sub>early</sub>). The secondary, dichotomized exposure
 variable – daily early-life exposure to aspartame
 (ASP<sub>early</sub>) – was defined as maternal intake of ≥177
 mg/day of aspartame during pregnancy/breastfeeding, from the sum of
 aspartame dosages in DS + DD<sub>other</sub> + packets
 consumed by the mother. 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 had previously been published
 (5); for comparable products from other brands, NNS dosages
 were imputed according to the NNS types specified on product labels.
 Leading DS brands sweetened exclusively with aspartame contained ≥177 mg
 of aspartame/can (5); this dosage was therefore selected as the cut-point
 for ASP<sub>early </sub>to identify offspring with daily
 early-life aspartame exposure comparable to that in 1 can of the leading
 diet colas (5).  For DD<sub>other</sub>, ingredient-labeling
 specified types of NNS used, but not dosages, which are proprietary. Based
 on limited available data, a 12-ounce serving of DD<sub>other
 </sub>sweetened exclusively with aspartame was estimated to contain
 approximately 100 mg of aspartame. A tabletop packet contains 37 mg of
 aspartame (6). 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 offspring with
 DS<sub>early</sub> and ASP<sub>early</sub>
 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 Χ<sup>2</sup> statistic in
 Stata/IC 14.2 for Windows. To assess associations between diagnostic status and the
 primary and secondary exposures under study –
 DS<sub>early</sub> and ASP<sub>early</sub> –
 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 <em>meglm</em> command
 in Stata/IC 14.2 for Windows.  Unadjusted odds ratios (ORs) for the primary and secondary
 exposures of interest – DS<sub>early</sub> and
 ASP<sub>early</sub> – 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 (≥ $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 of any NNS, from
 the combination of DS, DD<sub>other</sub>, 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, for example.
 We also performed subgroup analyses for non-regressive ASD cases and
 non-regressive autism cases, using these same exposure variables and
 covariates. These models were all fit using the <em>meglm</em>
 command in Stata/IC 14.2 for Windows. Sensitivity Analyses To examine influences on maternal intake of these products,
 we compared the proportions 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, educational
 level, and ethnicity. Among these six demographic subsets – mothers of
 offspring from households with incomes ≥ $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 in 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 Χ<sup>2</sup> statistic in Stata/IC
 14.2 for Windows. References The
 Interactive Autism Network: Research Overview: Kennedy Krieger Institute;
 2023 [updated 2023. Available
 from: https://iancommunity.org/cs/ian_research/overview. Interactive Autism Network: Patient-Centered Outcomes Research Institute; 2023 [2023]. Available from: https://www.pcori.org/research-results/2015/interactive-autism-network#section_professional_abstract. Heilbrun LP, Palmer RF, Jaen CR, Svoboda MD, Miller CS, Perkins J. Maternal Chemical and Drug Intolerances: Potential Risk Factors for Autism and Attention Deficit Hyperactivity Disorder (ADHD). J Am Board Fam Med. 2015;28(4):461-70. Palmer RF, Heilbrun L, Camann D, Yau A, Schultz S, Elisco V, et al. Organic Compounds Detected in Deciduous Teeth: A Replication Study from Children with Autism in Two Samples. J Environ Public Health. 2015;2015:862414. Franz M. Diet Soft Drinks.  Diabetes Self-Management: Rapaport Publishing, Inc.; 2010. Stein PJ. The Sweetness of Aspartame: A Biochemistry Lab for Health Science Chemistry Courses. Journal of Chemical Education. 1997;74(9):1112.
description: Please note that the second spreadsheet contain a data
 dictionary, which defines the meanings of numeric values assigned to
 categorical variables, and provides further explanatory notes, including
 the meanings of dichotomous variables.
description: # Early-life exposures to diet soda and aspartame are associated with
 autism in males.
 [https://doi.org/10.5061/dryad.cfxpnvx2p](https://doi.org/10.5061/dryad.cfxpnvx2p) Please see the attached READ ME file, which contains both a detailed data dictionary and also an 'exposure-variables map' worksheet, to describe which exposure variables were included in each of the tables and figures in our manuscript. ## Description of the data and file structure We have organized the variables in our dataset into the following categories, and ordered them in this sequence: 1\. numeric study identifiers 2\. demographic variables and other covariates 3\. diagnosis\- and regression\-related variables 4\. early\-life exposures through maternal diet during pregnancy or breastfeeding: a. computed daily exposure variables, dichotomized b. computed minimum mean-daily-equivalent exposure variables, dichotomized c. original retrospective dietary recall data recorded by biological mothers, for their pregnancy or breastfeeding periods <br> This READ ME file describes the meanings of values of numeric variables; missing data codes; and abbreviations used. ## Sharing/Access information This dataset stored and hosted by Dryad is the sole way of accessing the data used in all analyses in our published manuscript. All the data used in our published manuscript were derived from the Autism Tooth Fairy Study, led by Raymond F. Palmer, Ph.D, Principal Investigator, and Lynne ## Code/Software This is an optional, freeform section for describing any code in your submission and the software used to run it. Describe any scripts, code, or notebooks (e.g., R, Python, Mathematica, MatLab) as well as the software versions (including loaded packages) that you used to run those files. If your repository contains more than one file whose relationship to other scripts is not obvious, provide information about the workflow that you used to run those scripts and notebooks.