The role of glyphosate in infertility: the mechanistic link

Abstract

In light of the recent Roundup lawsuit, glyphosate has been widely accepted as a significant environmental toxin that may affect humans in various ways including cancer and infertility. Exposure to even low doses of glyphosate-based herbicides during pregnancy has been found to impair fertility, cause intrauterine growth restriciton and induce fetal malformations. In this study we sought to determine the underlying mechanism by which glyphosate negatively impacts oocyte quality, fertilization rates as well as embryo development. Experimental case-control study of mouse oocytes and pronuclear embryos, exposed in vitro to increasing glyphosate concentrations and followed through day 5 of development. We utilized multiple assays including reactive oxygen species (ROS) generation and zinc depletion to examine the possible underlying detrimental mechanisms. Metaphase II mouse oocytes (n=200) were retrieved from 8-10 week female mice and a subset (n=100) were fertilized using IVF. The oocytes as well as the fertilized mouse embryos were then exposed to increasing concentrations of glyphosate (0-200 μM) for 2h - 4h as per protocol. The oocytes were divided into four groups that were treated as follows: Group A: ROS detection assay, Group B: Zinquin ethyl ester assay, Group C: fixed, stained and scored based on the spindle structure (microtubule morphology -MT and chromosomal alignment - CH) as indicators of the oocyte's capacity to sustain exposure. All groups were compared to Group 4: untreated controls. Exposed embryos were incubated for up to 120 hours post fertilization and evaluated for full and hatching blast rate conversion. They were photographed and graded daily based on their appearance and development using published embryo grading protocols. A subset of the treated embryos (n = 10 for each concentration) were treated, in a similar fashion as the oocytes, in order to evaluate for ROS overproduction and zinc depletion. Confocal microscopy was used to assess the embryos. Statistical analysis was performed using t-test, ANOVA and chi-square. A p-value < 0.05 was considered statistically significant. Oocytes treated with increasing glyphosate concentrations > 50 μΜ were found to have poor scores for MT and CH and that difference was statistically significant as compared to controls (p< 0.05). Embryos followed to 96 hours post fertilization (early blastocyst) and 120 hours (full and hatching blastocyst) after glyphosate exposure (0-200 μM) were assessed and those exposed to glyphosate concentrations > 100 μM showed significantly increased arrest rates and poor morphology scores compared to controls. ROS overproduction as well as zinc depletion was evident in embryos treated with high glyphosate concentrations. These observations were statistically significant compared to untreated controls (p<0.05). This work suggests the possible underlying mechanisms by which glyphosate negatively affects reproductive health in the mouse model. Possible fertility implications in humans will require further research.