Effect of the Environmental Protection Agency reference dose of glyphosate on the ovarian function and cycle during the postnatal period

Dear Editor, Glyphosate is one of the most widely used herbicides in agriculture worldwide. Several studies have shown that exposure to glyphosate and glyphosate-based herbicides (GBHs) can have adverse effects on female reproductive health, including hormonal alterations and ovarian dysfunction [1]. However, there are some controversies, and most studies focus on the prenatal period and at high exposure doses. In the present study, our general objective was to determine whether glyphosate behaves as an endocrine disruptor when administered chronically at the Environmental Protection Agency (EPA) reference dose, or the estimated daily exposure that would not cause adverse effects throughout a lifetime, during the postnatal period. Additionally, two specific aims were set: (i) to evaluate if the EPA dose of glyphosate during the puberal stage alters the estrous cycle and the ovarian morphology, and (ii) to evaluate if it has an impact on ovarian gene expression. For this purpose, female Sprague Dawley rats (Rattus norvegicus, n = 16; 1-month-old) were randomly assigned to one of the following groups: control (n = 8, receiving water) and treated (n = 8, receiving an oral dose of glyphosate at 1 mg/kg/day) (Supplemental file M&M). The animals were weighed and treated throughout the eight-week treatment period, and their estrous cycle was monitored daily. On the day of sacrifice (in the estrus stage), blood samples were collected via intracardiac puncture to evaluate serum levels of E2 and P4; the animals were weighed, and the ovaries were extracted and weighed. The extracted ovaries were stored either in 10% formalin (for H&E staining, morphological analysis), in RNA later (for RNA-Seq), or in empty cryotubes (for RT-qPCR analysis of 12 genes) and stored in a −80°C freezer. After following the estrous cycle of each animal, we observed an alteration in the estrous cycle from the treated animals, noting a significant decrease in the days the treated animals stayed in the diestrus stage (Figure 1A, P < .05), and alterations in the pattern of the estrous cycle of the treated animals compared to the control group were noted (Figure 1B and Supplemental Figure 1), as well as, a decrease in serum levels of E2 (Figure 1C; P < .05). In contrast, although an increase was noted in the serum levels of P4, it did not reach significance (Figure 1C; P > .05). Morphological analysis of the whole ovary revealed no significant differences in the percentage of follicles at the different stages of development (Figure 1D; P > .05), even though there was a trend of a reduction of secondary and antral follicles, by contrast, with an increase in primordial and atretic follicles. Regarding the corpus luteum (CL), we observed a significant increase in the number of CL in the treated animals compared to the control group (Figure 1D; P < .05), both from previous cycles and in total CL. But no significant differences in the number of newly formed CLs from the cycle current were observed (Figure 1D; P < .05), suggesting no alterations in ovulation. No significant differences were observed in the animals’ weight curve either during or at the end of the treatment, nor in the ovarian weight (Supplemental Figure 2A and B; P > .05). RNAseq was performed in 5 ovaries from each group (Supplemental Figure 4). Eight over-represented genes (Ccnb1, Ccnd2, Dhh, Ihh, Inhbb, Angpt2, Amh, and Plk1) as well as some steroidogenesis enzymes (Hsd3b2, Cyp19a1, Cyp11a1 and Star) were selected to be validated by RT-qPCR and normalized with 18 s and Actb. Interestingly, Ccnb1, Ccnd2, Inhbb, and Plk1 showed a significant decrease in expression in the treatment group (Figure 1E; P < .05), showing that chronic oral exposure to low glyphosate (EPA reference dose) compromises ovarian and endocrine function during the pubertal period. However, no significant differences were observed in the other assessed genes (Supplemental Figure 3A and B; P > .05).