Polystyrene microplastics and di-2-ethylhexyl phthalate co-exposure: Implications for female reproductive health

Abstract

Microplastics and phthalates are prevalent and emerging pollutants that pose a potential impact on human health. Previous studies suggest that both microplastics and phthalates can adversely affect the reproductive systems of humans and mammals. However, the combined impact of these pollutants on the female reproductive system remains unclear. Here we show the impacts of exposure to polystyrene microplastics (PS-MPs) and di-2-ethylhexyl phthalate (DEHP) on female Sprague-Dawley rats' reproductive systems. We find that co-exposure to PS-MPs and DEHP results in a marked increase in cystic and atretic follicles, oxidative stress, fibrosis, and dysregulation of serum sex hormone homeostasis in the ovaries of the rats. Proteomic analysis identified differentially expressed proteins that were predominantly enriched in signaling pathways related to fatty acid metabolism and tight junctions, regulated by transforming growth factor β1 (TGF-β1). We further confirm that co-exposure to DEHP and PS-MPs activates the TGF-β1/Smad3 signaling pathway, and inhibiting this pathway alleviates oxidative stress, hormonal dysregulation, and ovarian fibrosis. These results indicate that exposure to the combination of microplastics and phthalates leads to a significant increase in atretic follicles and may increase the risk of polycystic ovary syndrome (PCOS). Our study provides new insights into the reproductive toxicity effects of microplastics and DEHP exposure on female mammals, highlighting the potential link between environmental pollutants and the occurrence of PCOS. These findings highlight the need for comprehensive assessments of the reproductive health risks posed by microplastic pollution to women and contribute to the scientific basis for evaluating such risks.

Keywords: DEHP; Microplastics; PCOS; Reproductive toxicity; TGF-β1/Smad3.

Graphical abstract

Fig. 1

Pathological lesions in ovarian tissue and follicle counts. a, H&E staining of ovaries. b, The number of follicles at each stage. ∗: p < 0.05. The asterisks (∗) indicate the comparison with the control group.

Fig. 2

Fibrosis of ovarian tissue. a, Masson-trichrome staining of ovaries. b, Collagen volume fraction. c, The mRNA level of Col1a1. d, The mRNA level of Col3a1. e, ELISA determination of COL1A1 concentration in ovaries. f, ELISA determination of COL3A1 concentration in ovaries. ∗ or ^: p < 0.05; ∗∗ or ^^: p < 0.01; ∗∗∗ or ^^^: p < 0.001. ^, ^^, or ^^^ on the bar indicates the comparison with the control group; ∗, ∗∗, or ∗∗∗ on the short lines indicate the comparison between the two groups indicated.

Fig. 3

Dyshomeostasis of sex hormones. a, E2 levels in serum. b, T levels in serum. c, E2/T ratio. ∗ or ^: p < 0.05; ∗∗ or ^^: p < 0.01; ^^^: p < 0.001. ^, ^^, or ^^^ on the bar indicate the comparison with the control group; ∗, ∗∗, or ∗∗∗ on the short lines indicate the comparison between the two groups indicated.

Fig. 4

Identification of differentially expressed proteins. a, The Venn diagram of co-expressed proteins in the ovarian. The columns indicate the number of proteins in each group. The bottom table shows the number of specific or shared proteins. b, The volcano plots (from top to bottom and left to right): Saline group vs. MP group, Saline group vs. DEHP group, Saline group vs. MP + L-DEHP group, Saline group vs. MP + H-DEHP group. Red points: up-regulated protein, fold change >2 and p < 0.05; Blue points: down-regulated protein, fold change <0.5 and p < 0.05; Gray points: protein with non-significantly different proteins, absolute log₂ (fold change) < 1 or p > 0.05.

Fig. 5

KEGG enrichment of differentially expressed proteins.

Fig. 6

Co-exposure activates the TGF-β1/Smad3 signaling pathway. a, Immunohistochemistry for p-Smad3. b, The integral optical density of p-Smad3. c, TGF-β1 concentration in ovaries. d, The mRNA levels of p-Smad3 in ovaries. e, The mRNA levels of TGF-β1 in ovaries. ∗ or ^: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001. ^ or ^^^ on the bar indicate the comparison with the control group; ∗ or ∗∗ on the short lines indicate the comparison between the two groups indicated.

Fig. 7

SIS3 alleviated ovarian fibrosis and sex hormone disorders. a, Masson-trichrome staining of ovaries. b, ELISA determination of COL1A1 concentration in ovaries. c, ELISA determination of COL3A1 concentration in ovaries. d, The mRNA levels of Col1a1. e, The mRNA levels of Col3a1. f, Collagen volume fraction. g, E2 concentration in serum. h, T concentration in serum. i, E2/T ratio. Panels g–i show the restoration of sex hormone homeostasis with SIS3 treatment. ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001; ns: no signification. ∗, ∗∗, or ∗∗∗ on the short lines indicate the comparison between the two groups indicated.