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. Author manuscript; available in PMC: 2026 Jan 9.
Published in final edited form as: Int J Hyg Environ Health. 2025 Dec 15;272:114733. doi: 10.1016/j.ijheh.2025.114733

Associations of glyphosate and aminomethylphosphonic acid (AMPA) concentrations with birth outcomes in pregnant women from the Midwestern U.S.

Kelsi A Morris 1,2,3, Maria E Cinzori 1,2, Brad A Ryva 2,4,5, Nicole Talge 3, Khyatiben V Pathak 6,9, Brooke Lovell 6,9, Patrick Pirrotte 6,9, Susan L Schantz 7,8, Rita S Strakovsky 1,2,*
PMCID: PMC12781968  NIHMSID: NIHMS2132196  PMID: 41401588

Abstract

Background:

Pregnant women are ubiquitously exposed to the herbicide glyphosate and its major metabolite aminomethylphosphonic acid (AMPA). Therefore, we examined associations of glyphosate and AMPA with birth outcomes.

Methods:

Pregnant women from the Illinois Kids Development Study (I-KIDS) participating in this pilot study (n=300) provided first-morning urine samples at median 13-weeks gestation for glyphosate and AMPA measurement. We calculated gestational-age-at-birth using the last menstrual period after ultrasound confirmation and abstracted birthweight from electronic medical records and calculated sex-specific birthweight-for-gestational-age z-scores (BWz). We measured birth length (cm) and anogenital distance (AGD) within 24-hours of birth and calculated the anogenital index (AGI, mm/m) by dividing each AGD value (in mm) by the body length (in m). Multivariable linear regression models evaluated associations of glyphosate or AMPA with birth outcomes and differences in associations by newborn sex.

Results:

In all newborns, only 2-fold higher AMPA was associated with −0.07cm shorter birth length (95%CI: −0.16, 0.02). In males, each 2-fold higher glyphosate was associated with −0.06 lower BWz (95%CI: −0.13, 0.01), −0.14cm shorter birth length (95%CI: −0.31, 0.04), and 0.63 longer AGIlong (95%CI: −0.19, 1.44). In females, each two-fold higher glyphosate was associated with 0.54 longer AGIlong (95%CI: −0.16, 1.25). AMPA was associated with 0.61 (95%CI: −0.01, 1.24) and 0.48 (95%CI: 0.02, 0.94) longer AGIlong in males and females, respectively. Some associations gained precision in sensitivity analyses removing infants born preterm or after controlling for gestational-age-at-birth.

Conclusions:

Glyphosate or AMPA were associated with smaller birth size and longer AGIlong, with evidence of sexual-dimorphism, although most findings were imprecise. Larger studies are needed, including those that consider implications of these findings for child lifelong health.

Keywords: aminomethylphosphonic acid, birth outcomes, glyphosate, pregnancy

INTRODUCTION

Anthropometric measures at birth and gestational age at birth are important indicators of fetal development and are associated with short- and long-term child health. For example, both small and large birth size are associated with increased cardiovascular and metabolic disease risk (Smith et al. 2016; Whincup et al. 2008), and being born early- or pre-term is linked to poor health outcomes in infancy and adulthood (Crump et al. 2013; Crump et al. 2021; Sipola-Leppanen et al. 2015). Additionally, anogenital distance (AGD) at birth is a proposed androgen-mediated marker of reproductive development (Liu et al. 2014) with links to long-term reproductive health. Fetal development is driven by highly-regulated changes in maternal hormones across pregnancy. Many environmental contaminants are endocrine disrupting chemicals (EDCs), and exposure to various EDCs has been associated with changes in maternal hormones (Pacyga et al. 2024; Ryva et al. 2024), birth size (Puche-Juarez et al. 2023), gestational age at birth (Puche-Juarez et al. 2023), and AGD at birth (Nelson et al. 2020). Given the importance of the in utero environment in child long-term health, and the potential for EDCs to disrupt developmental processes, it is critical to understand how other environmental contaminants impact fetal growth and development.

Glyphosate-based herbicides are the most commonly used class of domestic and commercial broad-spectrum herbicides globally due to their low cost, wide range of growth control, and effectiveness when compared to other herbicides. Reflective of these attributes, glyphosate-based herbicides have had over a 200-fold increase in usage since 1974 (Maddalon et al. 2021; Mesnage et al. 2015). Due to the widespread usage of glyphosate-based herbicides, women are ubiquitously exposed to glyphosate and its major environmental metabolite, aminomethylphosphonic acid (AMPA) (Koskinen et al. 2016), as up to 50% of glyphosate can degrade into AMPA after application to soil (von Merey et al. 2016). Exposure to glyphosate and AMPA occurs through consumption of contaminated food or liquid and dermal contact in the general population, and inhalation of contaminated air in those occupationally exposed or living in-or-near agricultural communities (Connolly et al. 2019). Reflective of its widespread applications, greater than 70% of the women from the National Health and Nutrition Examination Survey (NHANES, 2013-2018) had measurable urinary levels of glyphosate (National Health and Nutrition Examination Survey 2013-2014, 2015-2016, 2017-2018). Importantly, there is some limited evidence from experimental rodent models that glyphosate is an EDC with potentially androgenic effects during pregnancy, including increased AGD in both male and female offspring, delayed first estrous for female offspring, as well as increased testosterone in female offspring, which are considered endpoints for androgen-mediated activity (Manservisi et al. 2019).

In humans, extensive epidemiologic research has focused on occupational glyphosate exposures and cancer-related endpoints (Andreotti et al. 2018; Meftaul et al. 2020; Weisenburger 2021) but comparatively little is known about non-occupational glyphosate or AMPA exposure in pregnant individuals. Only three prior studies examined associations of glyphosate exposure with birthweight and reported either null (Sathyanarayana et al. 2010) or inverse associations (Gerona et al. 2022; Reynier and Rubin 2025). Similarly, only five prior studies considered associations of glyphosate or AMPA with gestational age at birth. In three studies, glyphosate and AMPA were not associated with preterm birth (Ashley-Martin et al. 2024; Lesseur et al. 2022; Varde et al. 2023), whereas one study reported that glyphosate and AMPA were associated with higher odds of preterm birth (Silver et al. 2021) and another reported that higher county-level glyphosate usage was associated with shorter gestation length (Reynier and Rubin 2025). Consistently, to our knowledge, only one small epidemiologic study examined associations of glyphosate with newborn AGD, reporting that glyphosate was marginally associated with longer AGD in female newborns (Lesseur et al. 2021). Given the ubiquitous exposure of pregnant women to glyphosate and AMPA and potential consequences for fetal health, it is critical to continue exploring risks associated with exposure in pregnancy. Therefore, our primary objective was to examine associations of gestational glyphosate and AMPA exposures with birth anthropometrics (including birth size and AGD) and gestational age at birth. Given the sexually-dimorphic nature of these outcomes, we additionally considered differences in associations by fetal sex.

MATERIALS AND METHODS

Illinois Kids Development Study (I-KIDS) recruitment and enrollment

This is a pilot study that utilized data from pregnant women enrolled in the Illinois Kids Development Study (I-KIDS) – a prospective pregnancy cohort designed to evaluate the impacts of prenatal chemical exposures on infant neurodevelopment. I-KIDS participants were recruited at their first prenatal care appointment from two local obstetric clinics in Champaign-Urbana, IL. Details about I-KIDS recruitment have been extensively described (Pacyga et al. 2021; Pacyga et al. 2022; Pacyga et al. 2023). Briefly, eligible women were ≤ 15 weeks pregnant, 18-40 years old, fluent in English, carrying a low-risk (as determined by their physician) singleton pregnancy, and able/willing to complete most study visits. All women provided written informed consent to participate, and the study was approved by the Institutional Review Board at the University of Illinois, Urbana-Champaign.

Collection of maternal sociodemographic, lifestyle, and health information at enrollment

At the first home study visit (median 13 weeks gestation), women completed an interviewer-administered questionnaire to ascertain their sociodemographic, lifestyle, and health characteristics. Briefly, women reported their age, race, ethnicity, employment status, parity, and pre-pregnancy weight and height. We calculated pre-pregnancy BMI (kg/m2) using self-reported weight and height, and conception season using due dates based on the first day of the last menstrual period (confirmed via first trimester ultrasound). Women also completed a semi-quantitative food frequency questionnaire (FFQ) adapted for pregnancy from the full-length Block 98 FFQ (NutritionQuest, Berkeley, CA) (Bodnar and Siega-Riz 2002; Laraia et al. 2007). The FFQ queried dietary intake during the previous three months, therefore information collected at median 13 weeks gestation reflects maternal diet from conception through the first trimester.

Urine collection and measurement of urinary glyphosate and AMPA

At median 13 weeks gestation, women also collected a first-morning urine sample in polypropylene cups. Immediately following the study visit, trained researchers transported the urine sample to the laboratory to measure specific gravity (SG) using a hand-held refractometer and aliquoted urine into polypropylene cryovial tubes for long-term storage at −80°C. The samples were sent to City of Hope’s Integrated Mass Spectrometry Shared Resource (IMS-SR) (TGen, Phoenix, AZ) for glyphosate and AMPA measurement using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on a Vanquish UHPLC coupled to a TSQ Altis triple quadrupole mass spectrometer (ThermoFisher, San Jose, CA, USA) over a concentration range of 0-5 ng/mL (Lesseur et al. 2021). Urinary creatinine amount was used as a determinant for injection volume as described previously. The established assay limits for glyphosate and AMPA were as follows: the limits of detection (LOD) were 0.014 ng/mL and 0.013 ng/mL, respectively, while the limits of quantitation (LOQ) were 0.041 ng/mL and 0.04 ng/mL, respectively (Lesseur et al. 2021).

Collection of newborn anthropometrics and gestational age at birth

Body length and head circumference were measured in triplicate at the hospital within 24 hours of birth by trained researchers. If we were unable to visit or measure these outcomes at the hospital (n=19 body length; n=17 head circumference), we extracted these data from electronic medical records. Birthweight was abstracted from electronic medical records (n=269) or from hospital crib cards (n=31). I-KIDS researchers measured newborn AGD in triplicate using a digital caliper at the hospital within 24 hours of birth. AGD is measured from distinct anatomical landmarks in males and females, which results in four measures of AGD: long and short in both males and females. AGDshort is measured from the anus to the fourchette in females, and from the anus to scrotum in males. AGDlong is measured from the anus to the clitoris in females, and from the anus to the base of the penis in males, representing sexually-dimorphic development of fetal genitalia (Barrett et al. 2018; Kloboves et al. 2022; Schwartz et al. 2019). The range of % coefficients of variation (CVs) for all outcomes measured in triplicate was 0.8%-3.5%. We calculated gestational age at birth using ultrasound-confirmed due date and delivery date obtained from participants at the hospital visit within 24 hours of birth or at follow-up child visits.

Statistical analysis

Derivation of the analytic sample and presentation of descriptive statistics

The current pilot study includes 300 women selected to have characteristics representative of the full cohort (Supplemental Figure 1). These women were selected to participate in the study because they had an early-pregnancy urine sample to measure glyphosate and AMPA, at least one outcome of interest, and all relevant covariates. We described the analytic sample using n (%) or median (25th, 75th percentiles), and described chemical concentrations using min, max, and 25th, 50th (median), and 75th percentiles (Tables 1–2). We described the outcomes using 25th, 50th (median), and 75th percentiles and additionally evaluated differences in outcomes by fetal sex using an unpaired t-test, except for gestational age at birth (Wilcoxon’s rank-sum test) (Table 3).

Table 1.

Characteristics of Illinois Kids Development Study (I-KIDS) participants.

Women who remained in study through delivery (n = 578) Analytic sample (n = 300) Analytic sample, women carrying females (n = 150) Analytic sample, women carrying males (n = 150)
Characteristic n (%)4 or median (25th, 75th percentile)
Age (years) 1 30.0 (28.0, 33.0) 31.0 (28.0, 33.0) 31.0 (28.0, 34.0) 31.0 (29.0. 33.0)
Race/ethnicity 1
Non-Hispanic White (ref) 464 (80.3) 243 (81.0) 124 (82.7) 119 (79.3)
Other2 113 (19.6) 57 (19.0) 26 (17.3) 31 (20.7)
Employment status 1
Unemployed 81 (14.0) 39 (13) 19 (12.7) 20 (13.3)
Employed (ref) 497 (86.0) 261 (80) 131 (87.3) 130 (86.7)
Educational attainment
Some college or less 115 (19.9) 43 (14.3) 17 (11.3) 26 (17.3)
College graduate or higher 463 (80.1) 257 (85.7) 133 (88.7) 124 (82.7)
Annual household income
< $60,000 171 (29.6) 83 (27.7) 43 (28.7) 40 (26.7)
≥ $60,000 403 (69.7) 217 (72.3) 107 (81.3) 110 (73.3)
Parity1
No children (ref) 302 (52.2) 163 (54.3) 83 (55.3) 80 (53.3)
≥ 1 child 275 (47.6) 137 (45.7) 67 (44.7) 70 (46.7)
Pregnancy intention
Trying 383 (66.3) 210 (70.0) 103 (68.7) 107 (71.3)
Not trying 194 (33.6) 90 (30.0) 47 (31.3) 43 (28.7)
Smoking in the 1st trimester
No 515 (89.1) 268 (89.3) 135 (90.0) 133 (88.7)
Yes 28 (4.8) 11 (3.7) 3 (2.0) 8 (5.33)
Unknown 35 (6.1) 21 (7.0) 12 (8.0) 9 (6.0)
Early pregnancy diet quality (max = 100) 51.5 (43.9, 59.8) 52.2 (45.4, 59.9) 52.3 (45.3, 60.1) 51.6 (45.4, 59.9)
Early pregnancy whole grains score (max=10) 1 1.8 (0.9, 2.9) 1.9 (1.0, 3.1) 1.9 (1.0, 3.1) 1.9 (1.0, 3.2)
Pre-pregnancy BMI (kg/m2) 24.6 (21.9, 29.9) 24.7 (22.0, 29.3) 24.6 (21.9, 28.7) 24.7 (22.0, 29.5)
Pre-pregnancy BMI 1,3
Under-/healthy weight 307 (53.1) 160 (53.3) 82 (54.7) 78 (52.0)
Overweight 128 (22.2) 70 (23.3) 35 (23.3) 35 (23.3)
Obese 143 (24.7) 70 (23.3) 33 (22.0) 37 (24.7)
Conception Season 1
Winter (ref) 142 (24.6) 66 (22.0) 35 (23.3) 31 (20.7)
Spring 150 (26.0) 76 (25.3) 34 (22.7) 42 (28.0)
Summer 138 (23.9) 72 (24.0) 35 (23.3) 37 (24.7)
Fall 142 (24.6) 86 (28.7) 46 (30.7) 40 (26.7)
Newborn sex 1
Female 296 (51.2) 150 (50.0) -- --
Male (ref) 279 (48.3) 150 (50.0) -- --
Gestational age at urine collection (weeks) 13.4 (12.4, 14.1) 13.3 (12.4, 14.1) 13.3 (12.6, 14.1) 13.4 (12.3, 14.1)
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1

Included as covariates in models

2

Includes Hispanic, Black, Asian, American Indian or Alaska Native, Native Hawaiian or Other Pacific Islander, Multiracial, Other.

3

Modeled as two categories in all statistical analyses (women with under-/healthy weight and overweight were combined (ref) and compared to women with obesity).

4

In women who remained in study through delivery, values may not add up to 100% due to missing data:

Race/ethnicity missing = 1, Annual household income missing = 4, Parity missing = 1, Pregnancy intention missing = 1, Newborn sex missing = 3, Early pregnancy diet quality missing = 87, Early pregnancy whole grains score missing = 87, Gestational age at urine collection (weeks) missing = 89.

I-KIDS, Illinois Kids Development Study; BMI, body mass index; ref, reference.

Table 2.

Urinary concentration of glyphosate and aminomethylphosphonic acid (AMPA) at median 13 weeks gestation

All women (n=300) Women carrying females (n=150) Women carrying males (n=150)
Analyte LOD, ng/mL n (%) > LOD n (%) = 0 Median (25th, 75th) n (%) > LOD n (%) = 0 Median (25th, 75th) n (%) > LOD n (%) = 0 Median (25th, 75th)
Glyphosate (ng/ml) 0.014 277 (92.3) 0 (0.0) 0.13 (0.05, 0.29) 141 (94.0) 0 (0.0) 0.12 (0.06, 0.28) 136 (90.7) 0 (0.0) 0.16 (0.05, 0.29)
AMPA (ng/ml) 0.013 226 (75.3) 9 (3.0) 0.04 (0.01, 0.11) 112 (74.7) 5 (3.3) 0.04 (0.01, 0.12) 114 (76.0) 4 (2.7) 0.04 (0.01, 0.11)
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All chemical concentrations were specific gravity-corrected.

LOD; limit of detection; AMPA, aminomethylphosphonic acid.

Table 3.

Distribution of birth outcomes.

All newborns Female newborns Male newborns
Birth Outcome n Median (25th, 75th percentile) n Median (25th, 75th percentile) n Median (25th, 75th percentile) P-value1
Gestational age at birth (weeks) 300 39.43 (38.57, 40.29) 150 39.57 (38.86, 40.43) 150 39.43 (38.43, 40.14) 0.28
Birthweight (g) 296 3507.34 (3175.00, 3848.50) 149 3379.00 (3060.00, 3779.00) 147 3628.00 (3303.00, 3875.38) 0.0002
Birthweight z-score 296 0.32 (−0.38, 1.01) 149 0.23 (−0.47, 1.02) 147 0.37 (−0.20, 0.97) 0.14
Birth length (cm) 298 50.00 (48.50, 51.67) 150 49.50 (48.00, 51.00) 148 50.67 (49.28, 52.00) <0.0001
Head circumference (cm) 298 34.80 (34.00, 35.83) 150 34.55 (33.47, 35.47) 148 35.17 (34.40, 36.13) <0.0001
AGDshort (mm) - - 130 9.57 (7.13, 11.41) 128 18.83 (16.33, 22.67) <0.0001
AGDlong (mm) - - 128 34.34 (31.11, 35.87) 125 45.10 (41.95, 47.62) <0.0001
AGIshort2 (mm/m) - - 130 19.51 (14.35, 22.51) 128 37.83 (32.16, 44.34) <0.0001
AGIlong2 (mm/m) - - 128 68.10 (63.51, 73.23) 125 89.38 (82.74, 95.12) <0.0001
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1

P-values represent unpaired t-test, except gestational age at birth (Wilcoxon’s rank-sum test) comparing female versus male newborns.

2

Anogenital distance divided by infant body length at birth (m).

AGD, anogenital distance; AGI, anogenital index.

Modeling maternal urinary glyphosate concentrations

To avoid potential bias from imputing values <LOD, which may result in left censoring of data, we included all machine-read values of glyphosate (n=23) and AMPA (n=74) that were below the LOD (Succop et al. 2004). To account for urinary dilution, we used the following equation: SG-adj-Chem = Chem[1.015–1/(SG-1)], for which SG-adj-Chem is the SG-adjusted glyphosate or AMPA concentration (ng/mL), Chem is the observed glyphosate or AMPA concentration, 1.015 is the mean SG of urine samples in our analytic sample, and SG is each urine’s specific gravity at median 13 weeks gestation. We ln-transformed our exposures to improve model fit, to help with potential outliers, and to ease interpretation of results. To avoid undefined estimates during ln-transformation, we replaced 0.00 ng/mL AMPA values with 0.0001 in a small number of women (n=9).

Modeling birth outcomes

We used birthweight in grams (g) to calculate sex-specific birthweight-for-gestational-age z-scores (BWz) (Talge et al. 2014). We used the mean of the triplicate body length (cm) and head circumference (cm) measurements in statistical analyses. To ensure that our AGD-related findings modeled the potential roles of glyphosate and AMPA in reproductive development independent of body size, we divided AGDshort and AGDlong (mm) by each newborn’s body length (to which we will refer as anogenital index, AGI) based on prior recommendations (Loreto-Gomez et al. 2017; Thankamony et al. 2009); for this purpose, we modeled body length in meters (m) to improve interpretability. Outcomes were not ln-transformed and were all modeled as continuous variables.

Covariate selection and operationalization

To select the most appropriate set of covariates, we used a directed acyclic graph informed by prior literature to select variables predictive of our exposures and outcomes (Ashley-Martin et al. 2024; Lesseur et al. 2021; Lesseur et al. 2022; Manservisi et al. 2019) (Supplemental Figure 2), with a resulting minimal sufficient adjustment set (MSAS). We further evaluated Pearson, Spearman (continuous variables), and polychoric (categorical variables) correlation coefficients between all covariates to avoid multicollinearity, but these were not a concern (all r < 0.4). We additionally considered splines between continuous forms of covariates and each outcome to identify which variable form (e.g., continuous, categorical) of each covariate best suited our models. Our final statistical models adjusted for maternal age (continuous), race/ethnicity (reported separately as race and ethnicity, and operationalized as follows: Non-Hispanic White [ref] and Other (includes Hispanic White, Black, Asian, American Indian or Alaska Native, Native Hawaiian or Other Pacific Islander, Multiracial, Other)), employment status as a measure of socioeconomic position in our cohort (employed [ref], unemployed), pre-pregnancy BMI (under/healthy/overweight [ref], obese), early pregnancy whole grain consumption (continuous), season of conception (winter [ref], spring, summer, fall), and parity (no prior children [ref], any prior children) because parity is a known predictor of birth outcomes and because mothers of children may have unique lifestyle (e.g. eating) habits that may increase or decrease their exposure to pesticides.

Models evaluating associations of chemicals with head circumference did not additionally adjust for mode of delivery because mode of delivery could potentially be on the causal pathway and because additional adjustment for delivery mode had no effect on our findings (data not shown). We converted whole grain consumption in grams to a score (1-10, with 10 being the best/highest intake) using the Alternative Healthy Eating Index (AHEI) 2010 method (Chiuve et al. 2012), as the score was highly correlated with our exposures and outcomes of interest. In cases of missing early pregnancy whole grain intake (n=2), we used mid-to-late pregnancy whole grain intake (median 35 weeks gestation), as whole grain consumption did not differ across pregnancy (data not shown).

Analyses to address primary and secondary objectives

To address our primary objective, we evaluated associations of SG-adjusted, ln-transformed urinary glyphosate or AMPA with gestational age at birth (weeks), birthweight (g), BWz, body length (cm), head circumference (cm), AGIshort, and AGIlong using unadjusted and covariate-adjusted linear regression models.

Our secondary objective was to understand whether relationships between our exposures and outcomes differed by newborn sex. To obtain sex-specific effect estimates and 95% confidence intervals (CIs) in linear regression models, we included a multiplicative interaction term between glyphosate or AMPA and fetal sex. Given the exploratory nature of our study, we interpreted sex-specific findings regardless of the significance of the interaction P-value. All analyses evaluating associations of glyphosate or AMPA with AGI were a priori stratified by newborn sex given the anatomical differences in AGI (and measured landmarks) and were therefore included in primary analyses. We did not adjust for multiple comparisons due to the exploratory nature of our study (Rothman 1990).

Sensitivity analyses

We conducted sensitivity analyses to ensure the robustness of our findings. First, we excluded babies born preterm (<37 weeks gestation) (n=16) when evaluating each exposure-outcome relationship. Second, despite gestational age at birth potentially being on the causal pathway between glyphosate/AMPA and anthropometric measures at birth, we conducted analyses adjusting for gestational age at birth when evaluating each anthropometric outcome (except BWz).

Interpretation of findings

When reporting our findings, we back-transformed the β-estimate and 95% confidence intervals (CIs) to account for the ln-transformation of the exposure variables. the ln-transformation of the exposure variables. Based on recommendations from the American Statistical Association, rather than considering P-values for linear regression models, we assessed patterns in the direction and magnitude of associations, as well as the range of the 95% confidence interval to identify potentially meaningful results (Amrhein et al. 2019; Wasserstein and Lazar 2016). We described associations as being statistically significant (P ≤ 0.05) or trending (P ≤ 0.1). All findings were reported as a unit increase in each outcome for each two-fold increase in exposures. All analyses were performed in SAS version 9.4 (SAS Institute Inc, Cary, NC).

RESULTS

Characteristics of the analytic sample

We outline participant characteristics in Table 1. Most women were non-Hispanic White (81%), college educated (86%), and had annual household incomes > $60,000 (72%). Few women smoked in the first trimester of pregnancy (4%) and most perceived their early-pregnancy health as being excellent or good (95%). Women in the analytic sample had a median age of 31 years and pre-pregnancy BMI of 24.7 kg/m2 (with approximately half (53%) classified as having under- or normal weight before pregnancy). The median early pregnancy whole grain intake score was 1.9 points. By design, participant characteristics were similar to the full I-KIDS cohort.

Characteristics of the exposures

As outlined in Table 2, glyphosate levels were >LOD in 92% of women and >0.0 ng/mL in 100% of women. The median (25th, 75th percentile) SG-adjusted urinary glyphosate concentration in early pregnancy was 0.13 ng/mL (0.05, 0.29). These levels are approximately two-fold lower compared to unadjusted urinary glyphosate concentrations in 18-40 year old women from NHANES, who had median (25th, 75th percentile) urinary glyphosate concentrations of 0.29 ng/mL (0.14, 0.50) across cycle years 2013-2018 (National Health and Nutrition Examination Survey 2013-2014, 2015-2016, 2017-2018); however, our median concentrations include machine-read values below the LOD, and the LOD in our study was lower than in NHANES (0.014 ng/mL in our study vs. 0.2, 0.2, and 0.1 ng/mL in NHANES survey years 13-14, 15-16, and 17-18, respectively). AMPA levels were >LOD in 75% of women and >0.0 ng/mL in 97% of women. The median (25th, 75th percentile) SG-adjusted urinary concentration of AMPA in early pregnancy was 0.04 ng/ml (0.01, 0.11), with no available NHANES comparison. Concentrations of glyphosate and AMPA were comparable between women carrying female and male fetuses (Table 2).

Characteristics of the outcomes

Distributions of birth outcomes are reported in Table 3. Briefly, the median (25th, 75th percentile) birthweight and BWz were 3507.34 g (3175.00, 3848.50) and 0.32 (−0.38, 1.01), respectively. Additionally, the median (25th, 75th percentile) birth length and head circumference were 50.00 cm (48.50, 51.67) and 34.80 cm (34.00, 35.83), respectively, and median (25th, 75th percentile) gestational age at birth was 39.43 weeks (38.57, 40.29). In male newborns, median (25th, 75th percentile) short and long AGD were 18.83 mm (16.33, 22.67) and 45.10 mm (41.95, 47.62). In female newborns, median (25th, 75th percentile) short and long AGD were 9.57 mm (7.13, 11.41) and 34.34 mm (31.11, 35.87). Most anthropometric measurements at birth were larger in male newborns compared to female newborns; however, gestational age at birth did not differ by newborn sex.

Associations of maternal urinary glyphosate or AMPA with birth outcomes

In unadjusted and covariate-adjusted linear regression models, we did not observe overall associations between early pregnancy maternal urinary glyphosate levels and gestational age at birth, birthweight, BWz, birth length, head circumference, AGIshort, or AGIlong (Supplemental Table 1 and Table 4). There was a trending association of maternal AMPA concentrations with shorter newborn body length in adjusted models, where each 2-fold increase in maternal urinary AMPA levels was associated with −0.07 cm (95% CI: −0.16, 0.02) shorter newborn body length (Table 4).

Table 4.

Associations of glyphosate and aminomethylphosphonic acid (AMPA) with birth outcomes in all children, newborn females, and newborn males.

Exposure/birth outcome All newborns Female newborns Male newborns P int
Glyphosate n β (95%CI) n β (95%CI) n β (95%CI)
Gestational age (weeks) 300 0.05 (−0.04, 0.13) 150 0.09 (−0.04, 0.21) 150 0.01 (−0.1, 0.13) 0.40
Birthweight (g) 296 −6.77 (−33.96, 20.42) 149 13.8 (−25.43, 53.02) 147 −20.75 (−55.91, 14.41) 0.19
Birthweight z-score 296 −0.03 (−0.09, 0.02) 149 0.00 (−0.08, 0.08) 147 −0.06 (−0.13, 0.01)# 0.26
Birth length (cm) 298 −0.09 (−0.22, 0.05) 150 −0.01 (−0.20, 0.18) 148 −0.14 (−0.31, 0.04)# 0.33
Head circumference (cm) 298 −0.03 (−0.12, 0.06) 150 −0.06 (−0.18, 0.07) 148 −0.00 (−0.11, 0.11) 0.51
AGIshort (mm/m) - - 130 0.18 (−0.35, 0.71) 128 −0.01 (−0.90, 0.88)
AGIlong (mm/m) - - 128 0.54 (−0.16, 1.25)# 125 0.63 (−0.19, 1.44)#
AMPA n β (95%CI) n β (95%CI) n β (95%CI)
Gestational age (weeks) 300 0.00 (−0.05, 0.06) 150 −0.01 (−0.09, 0.07) 150 0.02 (−0.06, 0.11) 0.58
Birthweight (g) 296 0.23 (−17.89, 18.34) 149 −1.78 (−25.69, 22.13) 147 3.44 (−22.8, 29.67) 0.77
Birthweight z-score 296 0.00 (−0.03, 0.04) 149 −0.00 (−0.05, 0.05) 147 0.01 (−0.05, 0.06) 0.88
Birth length (cm) 298 −0.07 (−0.16, 0.02)# 150 −0.07 (−0.19, 0.05) 148 −0.07 (−0.20, 0.06) 0.99
Head circumference (cm) 298 0.00 (−0.06, 0.06) 150 −0.02 (−0.09, 0.06) 148 0.02 (−0.06, 0.11) 0.50
AGIshort (mm/m) - - 130 0.11 (−0.23, 0.46) 128 0.32 (−0.34, 0.97)
AGIlong (mm/m) - - 128 0.48 (0.02, 0.94)* 125 0.61 (−0.01, 1.24)*
Open in a new tab

Linear regression models evaluated associations of glyphosate and AMPA with birth outcomes (overall and by newborn sex). Models controlled for maternal age, race/ethnicity, employment status, parity, pre-pregnancy body mass index, whole grain consumption, season of conception. β-estimates and 95%CIs for associations of glyphosate or AMPA were back-transformed to represent a unit change in birth outcome for every two-fold increase in chemical concentrations. AGI, anogenital index; AMPA, aminomethylphosphonic acid; CI, confidence interval; Pint, interaction P-value (glyphosate or AMPA with fetal sex).

*

P≤0.05,

#

P≤0.1.

Sex-specific associations of maternal urinary glyphosate or AMPA with birth outcomes

In unadjusted sex-stratified models, neither glyphosate nor AMPA were associated with gestational age, birthweight, BWz, birth length, head circumference, or AGIshort in either males or females (Supplemental Table 1). In adjusted models, neither glyphosate nor AMPA were associated with gestational age at birth, birthweight, head circumference, or AGIshort in males or in females (Table 4). However, in adjusted models, maternal glyphosate was associated with lower BWz and shorter birth length in newborn males, in whom each two-fold increase in glyphosate was associated with a −0.06 (95% CI: −0.13, 0.01) lower BWz and −0.14 cm (95% CI: −0.31, 0.04) shorter body length. Sex-stratified associations of AMPA with birth length were non-significant, but retained the same effect estimates as in the primary model. In unadjusted and adjusted models, urinary glyphosate was associated with longer AGIlong in male newborns, and urinary AMPA was associated with longer AGIlong in both male and female newborns (Supplemental Table 1). Interestingly, after covariate adjustment, there was a trending association of urinary glyphosate with longer AGIlong in female newborns (Table 4). Specifically, in adjusted models, each two-fold increase in maternal glyphosate was associated with a 0.63 mm/m (95% CI: −0.19, 1.44) longer AGIlong in male newborns and 0.54 mm/m (95% CI: −0.16, 1.25) longer AGIlong in female newborns. Each two-fold increase in AMPA was associated with 0.61 mm/m (95% CI: −0.01, 1.24) longer AGIlong in male newborns and 0.48 (95% CI: 0.02, 0.94) longer AGIlong in female newborns.

Sensitivity analyses

Findings did not differ substantially after exclusion of infants born preterm, with small gains in precision for birth length overall (for glyphosate), as well as for male birthweight, but small losses in precision for male AGIlong (Supplemental Table 2). In models that adjusted for gestational age at birth, we observed small gains in precision for birth length (with the association of glyphosate with birth length becoming statistically significant overall and in males) and birthweight in males, and a loss of precision for AGIlong in females and males when considering glyphosate exposure (Supplemental Table 3). After adjusting for gestational age at birth, there was also a trending association of glyphosate (each two-fold increase) with smaller head circumference (β= −0.10 cm; 95% CI: −0.21, 0.02) (Supplemental Table 3).

DISCUSSION

Summary of findings

In a sample of relatively healthy pregnant women, maternal urinary AMPA concentrations were associated with shorter body length in all newborns, whereas glyphosate was associated with smaller birthweight in male newborns, but many associations were imprecise. Additionally, in utero exposures to both glyphosate and AMPA were associated with longer AGIlong in male and female newborns. Our findings were generally robust to several sensitivity analyses, although there was some evidence for the role of preterm birth in our findings, which will need to be explored in cohorts with greater prevalence of preterm birth. Overall, findings from this study suggest that prenatal glyphosate and AMPA exposures are associated with fetal growth and development, but larger studies are needed to corroborate these observations as many of our findings lacked precision.

Glyphosate was not associated with gestational age at birth

In the current study, we observed no evidence that glyphosate or AMPA were associated with gestational age at birth, which is generally supported by other studies, except for one study in women from Puerto Rico. Specifically, maternal urinary glyphosate or AMPA levels were not associated with preterm birth in Canadian women from the MIREC study, in U.S. women enrolled in TIDES (Ashley-Martin et al. 2024; Lesseur et al. 2022), or in women from Charleston, South Carolina (Varde et al. 2023), whereas maternal urinary glyphosate and AMPA concentrations in Puerto Rican women enrolled in the PROTECT cohort were associated with an higher odds of preterm birth (Silver et al. 2021). Concentrations of glyphosate and AMPA in I-KIDS are lower compared to those in the PROTECT, as is the prevalence of preterm birth. Overall, glyphosate and AMPA exposure do not appear to be associated with elevated risk of preterm birth, but additional studies are needed in sensitive populations that have broader distributions of glyphosate/AMPA exposure and gestational age at birth.

Higher glyphosate was associated with smaller body size in a newborn sex-specific manner

To our knowledge, no prior epidemiologic studies have considered the roles of prenatal exposure to glyphosate and AMPA in both birthweight and birth length. In the current study, higher maternal urinary glyphosate levels were associated with lower BWz and shorter body length in male newborns, whereas AMPA was associated with shorter body length in all newborns. While some of these associations were imprecise, they remained or gained precision following several sensitivity analyses. Prior studies investigating the relation between glyphosate and birthweight are limited, but studies generally report null or inverse associations, similar to those observed in the current study. The Pregnancy Environmental Exposures Study (PEES) in Indiana (n = 155) observed that maternal urinary glyphosate levels in early pregnancy were associated with smaller birthweight percentiles in male newborns, which corroborates our findings despite the comparatively higher levels of glyphosate in women from PEES (mean (SD) urinary glyphosate: 3.33 (1.67) ng/mL) compared to I-KIDS participants (Gerona et al. 2022). These findings may indicate a linear relationship between glyphosate and birthweight, but additional studies are needed to support this postulation. Despite some agreement with prior studies, our results do not corroborate findings from the Agricultural Health Study (AHS) conducted in Iowa and North Carolina (n = 2246), which reported null associations between prenatal glyphosate exposure and infant birthweight (Sathyanarayana et al. 2010). The AHS used questionnaires to approximate glyphosate exposure instead of urinary glyphosate concentrations, which may have led to exposure misclassification and potentially biased results towards the null. Additional studies may be needed to compare glyphosate biomarker data from those regions of the U.S. to levels in Illinois to understand if additional factors (e.g., different exposure distributions) led to these discrepant findings. Interestingly, our birthweight findings are in line with findings from the National Vital Statistics System (NVSS) that considered county-level variation in agriculture-related glyphosate applications rather than urinary exposure biomarkers and found that average birthweights were lower following the introduction of glyphosate to agricultural fields in that area (Reynier and Rubin 2025). Although additional exposure assessment studies are needed to understand the sources of glyphosate exposure in I-KIDS participants, these concordant findings could potentially suggest that herbicide field application is a major source of glyphosate exposure in participants from our study, as Urbana-Champaign is surrounded by corn and soybean farms.

Higher glyphosate and AMPA were associated with longer AGIlong

Anogenital distance is a sexually dimorphic marker of reproductive development (Fischer et al. 2020; Liu et al. 2014). Prior studies have shown that in utero exposure to some EDCs (e.g., phthalates, bisphenols) is associated with disrupted AGD in humans at birth (Barrett et al. 2017; Mammadov et al. 2018; Swan et al. 2005; Swan et al. 2015). In our study, higher maternal urinary glyphosate or AMPA in early pregnancy were associated with longer AGIlong in both male and female newborns. To our knowledge, the only prior study that examined this relationship in humans observed a similar association among U.S. children born to women enrolled in The Infant Development and the Environment Study (TIDES) cohort. In TIDES, the observed relationship became non-significant after adjustment for covariates (Lesseur et al. 2021), which may be due to their small sample size (n=94). Our findings are also supported by a prior study in rats, in which prenatal exposure to glyphosate at the EPA reference dose (1.75 mg/kg bw/day) caused longer AGD in male pups at postnatal day 4 (Manservisi et al. 2019). In male newborns, shorter, and not longer AGDlong, has been associated with infertility and sperm motility issues (Eisenberg et al. 2011; Eisenberg and Lipshultz 2015), whereas little is known about the potential implications of longer AGD for health in males. However, longer AGDlong in female newborns has been associated with higher adult serum testosterone levels and multi-follicular ovaries (Barrett et al. 2018). Our findings (and those from the limited prior studies) support the potential role of glyphosate in adverse reproductive development, particularly in females. Given these findings, additional epidemiologic and experimental studies are needed to explore the role of glyphosate in reproductive development and long-term implications for reproductive health.

Possible biological underpinnings

Glyphosate is an herbicide that disrupts the shikimate pathway that supports amino acid production in plants, but is not found in humans (Casida 2017). Because of this, glyphosate was previously thought to be inert in humans, but recent research suggests that bacteria present in the human digestive tract may contain the shikimate pathway, indicating that glyphosate may disrupt the human microbiome (Lehman et al. 2023). As maternal microbiome disruption is associated with altered fetal growth (Miko et al. 2022), future studies should consider whether changes to either maternal or fetal microbiome drive glyphosate-mediated pregnancy complications. There is also evidence that glyphosate activates oxidative stress and inflammatory responses in certain cell lines from mammals and fish (Peillex and Pelletier 2020); with additional limited evidence that AMPA functions as an herbicide by producing reactive oxygen species and disrupting chlorophyll biosynthesis in crops (Gomes et al. 2014). To that end, although less investigated than glyphosate, AMPA exposure caused cytotoxicity in human cells and increased biomarkers of inflammation and oxidative damage (Martinez et al. 2020). This aligns with research in adult rats, demonstrating that chronic glyphosate exposure increased oxidative stress and caused liver dysfunction (Mesnage et al. 2017); in humans, glyphosate and AMPA were also associated with markers of liver damage (Eskenazi et al. 2023). Both oxidative stress and inflammation in utero have been linked to altered birth size (Arogbokun et al. 2021; Ragsdale et al. 2019). Furthermore, according to the Developmental Origins of Health and Disease (DOHaD) hypothesis, birth size is a reliable predictor of future metabolic health (Calkins and Devaskar 2011). Therefore, substantially more research is needed to understand the long-term implications of glyphosate exposure in utero, particularly for cardiometabolic diseases. There is also some evidence that glyphosate is an endocrine disrupting chemical, but the literature is limited. In rats, glyphosate exposure in pregnancy resulted in higher testosterone in newborn pups (Manservisi et al. 2019), and elevated testosterone levels have been linked to longer AGD (Barrett et al. 2018). Because we and others observed that associations of glyphosate and AMPA with birth outcomes are sexually-dimorphic, and especially given our findings related to possible disruptions in reproductive development endpoints (AGD), additional research is needed to unravel the roles of hormonal mechanisms in glyphosate/AMPA-related adverse health outcomes.

Strengths and limitations

Our study has several limitations, but also important strengths. First, although urinary concentrations of glyphosate in I-KIDS are lower compared to a few cohorts, we contribute data related to the lower end of the exposure curve, which may be generalizable to many individuals without occupational exposure. Additionally, urinary glyphosate and AMPA were quantified from one first-morning urine sample in early-pregnancy instead of a pooled sample across pregnancy as we have done for other chemicals in our cohort. While one timepoint may not represent exposure across pregnancy, which may lead to exposure misclassification, first-morning urine samples have been shown to have better precision than spot urine samples for measuring various compounds in urine, including other non-persistent chemicals (Mok et al. 2022; Witte et al. 2009). Similarly, despite the low concentrations of AMPA in our cohort, our findings contribute to the currently limited literature related to AMPA distributions in pregnant U.S. women. Second, we were unable to consider preterm birth as an outcome due to the healthy nature of our cohort; however, several of our findings related to birth size agree with prior studies, many of which were more heterogeneous than our study. Third, there is always chance for unmeasured confounding. For example, we lack data about maternal physical activity and time spent outside, which are predictors of birth outcomes and may be associated with glyphosate exposure from outdoor air; however, we used a priori consideration as well as prior literature to inform final covariate selection. Additionally, we leveraged the rich data collection in I-KIDS to account for potential confounding by dietary sources of glyphosate, as diet is also an important driver of fetal growth and development. Lastly, I-KIDS is a relatively homogeneous cohort of primarily healthy, non-Hispanic White, and educated women, which limits the generalizability of our findings to other populations and also limits our ability to consider the roles of pregnancy complications (e.g. gestational diabetes, preeclampsia) in the relationships of glyphosate with birth outcomes. However, the homogenous nature of our sample may help reduce the impacts of unmeasured confounding when considering biological hypotheses and chemical exposures.

Conclusions

In a sample of mostly non-Hispanic White U.S. pregnant women from the Midwest with relatively high socioeconomic status, higher maternal urinary glyphosate levels were associated with lower BWz in male newborns, whereas higher maternal urinary glyphosate and AMPA were associated with longer AGIlong in both male and female newborns. While some associations lacked precision, given that these measures are known predictors of both the in utero environment and of long-term child health, additional studies are needed to recapitulate these findings in larger and more diverse populations, to investigate the underlying mechanisms, and to understand potential short- and long-term implications of these observations for both the mother and child.

Supplementary Material

1

ACKNOWLEDGEMENTS:

The biological specimens from the Carle Foundation Hospital were used in this study. We thank contributors, patients and their families whose help and participation made this work possible.

FUNDING SOURCES:

This publication was made possible by the National Institute of Health grants ES032227, ES022848, ES007255, UGOD023272, UHOD023272, HD087166, and the U.S. Environmental Protection Agency grant RD83543401. This publication includes work performed at the Integrated Mass Spectrometry Shared Resource, supported by the National Cancer Institute of the National Institutes of Health (Grant P30CA033572). Its contents are solely the responsibility of the grantee and do not necessarily represent the official views of the US EPA or NIH. Further, the US EPA does not endorse the purchase of any commercial products or services mentioned in the publication. This project was also supported by the USDA National Institute of Food and Agriculture, Michigan AgBioResearch.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ETHICAL APPROVAL: All women provided written informed consent to participate, and the study was approved by the Institutional Review Board at the University of Illinois, Urbana-Champaign.

COMPETING INTERESTS: Authors have no conflicts of interest to disclose.

DATA AVAILABILITY STATEMENT:

Authors do not have permission to share data.

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NIHMS2132196-supplement-1.docx (267.6KB, docx)

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