ORIGINAL ABSTRACT

BACKGROUND: Glyphosate and its primary metabolite, aminomethylphosphonic acid (AMPA), are widespread environmental contaminants with potential endocrine-disrupting properties. Despite near-universal glyphosate and AMPA exposure, human studies evaluating their association with maternal hormonal regulation during gestation remain scarce. OBJECTIVE: To assess associations between urinary glyphosate and AMPA concentrations and repeated measures of maternal hormone levels during pregnancy and explore effect modification by gestational timing and fetal sex. METHODS: We analyzed data from 752 pregnancies in the PROTECT birth cohort in Puerto Rico. Glyphosate and AMPA were measured in up to three urine samples per participant collected at 18 ± 2-, 22 ± 2-, and 26 ± 2-weeks' gestation. Serum concentrations of sex steroid (estriol, progesterone, testosterone), thyroid (T3, T4, fT4, TSH), and stress-related (CRH) hormones were assessed at two periods. Linear mixed effects models estimated differences in serum hormone levels associated with interquartile range (IQR) increases in urinary glyphosate/AMPA, adjusting for demographic, biological, and technical covariates. RESULTS: Glyphosate and AMPA were associated with lower estriol concentrations across pregnancy. Each IQR increase in AMPA was associated with 10.6% lower estriol (95% CI: -18.1%, -2.4%), while glyphosate was associated with 8.3% lower estriol (95% CI: -14.6%, -1.4%). AMPA was associated with 4.0% higher T3 (95% CI: 0.9%, 7.0%). In sensitivity analyses, AMPA was linked to 7.9% higher TSH at 26 ± 2-weeks' gestation (95% CI: 0.5%, 15.9%), while glyphosate was associated with 9.5% higher CRH at this time point (95% CI: 1.5%, 18.2%). Stratified models revealed stronger associations among participants carrying female versus male fetuses. SIGNIFICANCE: This study provides first evidence of associations between prenatal glyphosate and AMPA exposure and disruption of multiple maternal hormonal pathways during pregnancy. Findings suggest these contaminants may interfere with estrogenic, thyroid, and stress hormone systems critical for pregnancy maintenance and fetal development, with implications for understanding mechanisms underlying adverse birth outcomes. IMPACT: This study provides the first human evidence that glyphosate and AMPA, ubiquitous environmental contaminants detected in 75-95% of pregnant women, disrupt multiple maternal hormonal pathways during pregnancy. Among 752 participants in a Puerto Rico birth cohort, we demonstrate significant reductions in estriol (8-11% decreases) and alterations in thyroid and stress hormones associated with glyphosate exposure. These findings illuminate potential biological mechanisms underlying previously observed associations between glyphosate exposure and adverse birth outcomes, including preterm birth and altered fetal growth. Given the widespread use of glyphosate-based herbicides globally, our results have significant implications for public health policy and regulatory decision-making. The hormonal disruptions we identified suggest that pregnant populations may be particularly vulnerable to glyphosate exposure, supporting the need for enhanced protective measures and exposure reduction strategies during critical windows of fetal development.