Introduction

Plastic pollution is one of the most urgent environmental problems in the current century. Over the past two decades, an increasing number of people have focused on small debris from the breakdown of plastic waste in the environment. Thompson et al. (2004) initially introduced the term "microplastics" to describe plastic debris. Microplastics are regarded as plastic debris smaller than 5 mm (Tang et al. 2024). Recently, nanoplastics, a class of microplastics within size ranging from 1 to 100 nm or 1000 nm, has attracted considerable interest (Atugoda et al. 2023). Microplastics have the potential to disperse widely throughout various ecosystems (Yuan et al. 2022). It has been reported that down to 20 nm nanoplastics were detected in water and seafood packaging samples (Ruan et al. 2024). Microplastics and nanoplastics can enter the food chain and ultimately accumulate in the human body, potentially causing health risks (Eze et al. 2024; Tang et al. 2024). Therefore, the United Nations Environment Programme has recognized microplastics as an emerging environmental issue (UNEP 2011).

Polystyrene is one of the most frequently used polymers in plastics, and thus, polystyrene microplastics are abundant in the environment (Atugoda et al. 2023; Osman et al. 2023). Toxicological evaluations on animal models indicate that polystyrene microplastics, especially those smaller than 10 μm, have the potential to induce male reproductive toxicity. These microplastics can disrupt the blood-testis barrier (Hu et al. 2022; Jin et al. 2021) and hinder spermatogenesis and steroidogenesis by inducing oxidative stress and inflammation in mice and rats (Hong et al. 2023). Our recent study demonstrated that polystyrene nanoplastics (25–100 nm) significantly reduced male fertility by causing sperm toxicity in mice (Xu et al. 2023). However, the toxicity of polystyrene microplastics and nanoplastics on male reproduction in humans is unknown. Recently, Zhao et al. (2023) detected microplastics (21.8–286.7 μm) in human testes and semens by infrared spectroscopy. They found that polystyrene microplastics are the most predominant microplastics in human testes (Zhao et al. 2023). Due to the limitations of infrared spectroscopy, microplastics smaller than 10 μm cannot be detected. Therefore, we aimed to investigate whether polystyrene microplastics smaller than 10 μm contaminate the male reproductive system and whether they truly cause male reproductive toxicity in humans.

Numerous methods have been proposed to control microplastic pollution (Padervand et al. 2020). Compared to other methods, magnetic iron oxide nanoparticles (nano-Fe3O4) effectively aggregate and coprecipitate microplastics in a quick, simple, and economical way (Shi et al. 2022). In addition, nano-Fe3O4 is promising for application in biomedicine due to its good biocompatibility and low toxicity (Liu et al. 2020). However, intervention with nano-Fe3O4 for toxicity of nanoplastics has not been reported. Therefore, we explored whether polystyrene nanoplastics could be removed by nano-Fe3O4 and evaluated their potential intervention effect on nanoplastic-induced sperm toxicity.

Experimental

The polystyrene microplastics smaller than 10 μm present in human semen samples and the microplastics released from the disposable paper cups were measured using pyrolysis–gas chromatography/mass spectrometry (Py-GCMS) as described previously (Leslie et al. 2022). To determine the toxicity of microplastics and nanoplatics and the intervention on their toxicity by nano-Fe3O4 in human sperm, we exposed human sperm to synthetic and environmental microplastics and nano-Fe3O4 treated nanoplastics. Sperm functions and physiological parameters were assessed as previously described (Luo et al. 2019; Xu et al. 2023). The semen samples used in these experiments are described in Table S1. Details about the sample collection, sample quality assurance and quality control, Py-GCMS analysis, sperm treatments, assessment of sperm functions and physiological parameters, statistical analysis, and other related procedures are described in Text S1.

Results and discussion

Polystyrene microplastics in human semen

Results of pyrolysis–gas chromatography/mass spectrometry (Py-GCMS) show polystyrene microplastics smaller than 10 μm in all three seminal pools, with a concentration of 3.57 ± 0.32 μg/mL, ranging from 3.05 to 4.15 μg/mL (Figs. 1a, S1 and S2). To analyze the small polystyrene microplastics in human semen, we used a 220 nm filter membrane because it is the smallest filter membrane that can purify the crude extract. Polystyrene microplastics smaller than 220 nm were detected in all three seminal pools with a concentration of 0.26 ± 0.06 μg/mL (0.15 to 0.33 μg/mL) (Figs. 1a and S3). The polystyrene microplastics detected in this study were approximately one-fifth of the total microplastics abundance in human semen reported by Zhao et al. (2023). Overall, these results suggest that microplastics smaller than 10 μm can penetrate the blood-testis barrier and accumulate in the male reproductive system in humans. Therefore, evaluating the potential male reproductive risk of polystyrene microplastics is necessary.

Fig. 1
figure 1

Polystyrene microplastics smaller than 10 μm were detected in human semen and could enter or bind to human sperm. a Polystyrene microplastics in human semen were detected using pyrolysis–gas chromatography/mass spectrometry. Data: the mean ± standard error of the mean (N = 3). b The ability of polystyrene microplastics to enter human sperm was assessed by exposing the sperm to green fluorescent polystyrene microplastics (0.5 mg/mL) for 4 h on 3 individuals (bar: 5 μm). The yellow and red arrows indicate the sperm head and tail, respectively

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Adverse effects of polystyrene microplastics on human sperm

The synthetic polystyrene microplastics used in the study exhibited a regular spherical morphology with uniform sizes near 25 nm, 50 nm, 100 nm, 500 nm, 4 μm, and 10 μm (Figs. S4 and S5). We demonstrated that 25–100 nm polystyrene nanoplastics can penetrate human sperm, while 0.5–10 μm polystyrene microplastics bind to the sperm surface (Fig. 1b). Although these six sizes of polystyrene microplastics have been shown to cause male reproductive toxicity in mice and rats (Hong et al. 2023), their effects on human sperm may differ and are worth testing.

To evaluate the male reproductive toxicity of polystyrene microplastics, we first exposed human sperm with polystyrene microplastics at 500 μg/mL, which is one hundred times greater than that detected in human semen (Fig. 1a). The results showed that 25 nm polystyrene nanoplastics had adverse impacts on human sperm functions and physiological parameters even after 1 min of incubation and lasted for 4 or 12 h (Figs. S6 and S7). In addition, 50 nm‒4 μm polystyrene microplastics affected human sperm functions only after 12 h of incubation (Fig. S6a–c), while 10 μm polystyrene microplastics had no significant impact on human sperm (Figs. S6 and S7). Sperm are the executors of male fertility, and their functional and physiological parameters are indicative of male reproductive capacity (Bertolla 2020). Since polystyrene microplastics affect sperm functions and physiological parameters, they may pose a potential risk to male reproduction in humans. Therefore, we next evaluated whether polystyrene microplastics are harmful to human sperm at semen-relevant concentrations.

Polystyrene nanoplastics (25–100 nm), but not polystyrene microplastics (0.5 and 4 μm), cause adverse effects on human sperm at semen-related concentrations (5 or 50 μg/mL) (Figs. 2 and S8–11). In addition, 25 nm polystyrene nanoplastics have synergistic effects with bisphenol A on human sperm at semen-related concentrations (Fig. S12). Bisphenol A is a commonly used plastic additive and a classical environmental endocrine-disrupting chemical that adversely affects the male reproductive system (Rahman et al. 2021). These data confirmed previous findings that nanoplastics can act as enhancers of other harmful substances in the environment (Zhang et al. 2023). Here, we demonstrated that polystyrene nanoplastics are more toxic to human sperm than polystyrene microplastics, which may be attributed to their different abilities to enter and accumulate in human sperm (Fig. 1b). In conclusion, these results indicate that attention should be given to the risk of male fertility caused by polystyrene nanoplastics. Since there are various types, sizes, and shapes of microplastics in the environment, we subsequently examined the impact of environmental microplastics on human sperm.

Fig. 2
figure 2

Adverse effects of polystyrene nanoplastics on human sperm functions. Human sperm were exposed to different concentrations of 25 nm polystyrene nanoplastics in high-saline solution for 12 h to examine sperm viability (a) and progressive motility (b). Human sperm were exposed to polystyrene nanoplastics in human tubal fluid medium (a capacitation buffer) for 4 h to measure penetration ability (c) and capacitation (d). Polystyrene nanoplastics cause adverse effects on viability, progressive motility, penetration ability, and progesterone-induced capacitation of human sperm at 5–500 μg/mL. Data: the mean ± standard error of the mean (N = 6). Different significance was assessed by the One-way ANOVA and Dunnett's test: **p < 0.01 and ***p < 0.001 (control vs treatment groups)

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Absence of impact of some environmental microplastics on human sperm

In addition to polystyrene microplastics, three other types of microplastics, polyethylene microplastics, polypropylene microplastics, and polyethylene terephthalate microplastics, were released from disposable paper cups (Fig. S13). The total concentrations of the environmental microplastics were approximately 360 μg/mL for sizes smaller than 10 μm and 18 μg/mL for sizes smaller than 220 nm (Fig. S13). However, these environmental microplastics did not significantly affect human sperm (Fig. S14–16). Overall, we assumed that the amount of nanoplastics released from a disposable paper cup did not reach the levels that cause toxicity to human sperm. However, there is a male reproductive health risk when nanoplastics accumulate in semen at concentrations harmful to human sperm. Therefore, eliminating nanoplastics in the environment is an important issue.

Removal of nanoplastics using magnetic iron oxide nanoparticles

In this study, we evaluated whether 25 nm polystyrene nanoplastics could be eliminated by nano-Fe3O4 (Fig. 3a). The mean diameter and zeta potential of the nano-Fe3O4 particles were 22.4 nm and − 31.3 mV, respectively (Fig. 3b). Upon mixing with 25-nm polystyrene nanoplastics (250 μg/mL) in either high-saline solution or human tubal fluid medium, the nano-Fe3O4 (1.3 mg/mL) aggregated with the 25 nm polystyrene nanoplastics (Fig. 3c). The mean removal rates of polystyrene nanoplastics in the high-saline solution and human tubal fluid medium were 98.5% and 98.7%, respectively (Fig. 3c). In addition, neither the residual polystyrene nanoplastics in the nano-Fe3O4 treated supernatant nor nano-Fe3O4 had a significant impact on sperm function or physiological parameters (Table 1). In conclusion, the aggregation and coprecipitation of polystyrene nanoplastics by nano-Fe3O4 is an effective intervention against the male reproductive toxicity induced by nanoplastics in human sperm.

Fig. 3
figure 3

Magnetic iron oxide nanoparticles can remove polystyrene nanoplastics. a The strategy for calculating the removal rate and assessing the alleviatory effect of magnetic iron oxide nanoparticles on 25 nm polystyrene nanoplastics. b The characteristics of the magnetic iron oxide nanoparticles were analyzed (bar: 50 nm). c Morphology of the magnetic iron oxide nanoparticles aggregates and the removal rates of polystyrene nanoplastics by magnetic iron oxide nanoparticles in different buffers (bar: 100 nm). Data: the mean ± standard error of the mean (N = 3)

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Table 1 Effect of magnetic iron oxide nanoparticles on human sperm toxicity induced by polystyrene nanoplastics
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Conclusion

Here, we demonstrated that human semen was contaminated by polystyrene microplastics smaller than 10 µm. Polystyrene nanoplastics of 25–100 nm can penetrate human sperm and damage them at semen-relevant concentrations, while polystyrene microplastics of 0.5–10 μm can bind to the sperm. In addition, the aggregation and coprecipitation of polystyrene nanoplastics by magnetic iron oxide nanoparticles is an effective intervention against the male reproductive toxicity induced by nanoplastics on human sperm. These findings provide new insights into the male reproductive toxicity of microplastics in humans.