Polystyrene microplastics (PS-MPs), as an emerging environmental pollutant, have attracted considerable attention regarding their toxic effects on the male reproductive system and underlying mechanisms. This study aims to investigate whether PS-MPs promote the expression and translocation of thioredoxin interacting protein (Txnip) by inducing oxidative stress, and thereby activate the apoptosis signal-regulating kinase 1 (ASK1)-cysteinyl aspartate specific proteinase9/3 (Caspase9/3) signaling pathway, ultimately mediating the apoptosis of mouse spermatocytes.

Mouse testicular spermatocyte line GC-2 was used as a model and treated with 5 μm PS-MPs (0, 10, 20, 40 μg/mL). Cell morphology and viability were analyzed after 48 and 72 h of exposure, and cell morphology was observed by optical microscopy, and cell viability was detected by CCK-8 assay (n=3). The remaining mechanistic studies were based on PS-MPs exposure for 48 h. Flow cytometry was performed to determine the levels of reactive oxygen species (ROS) (n=7, 9) and apoptosis rates (n=11, 13). Transcriptome sequencing and GO function enrichment analysis were conducted to screen differentially expressed genes (DEGs) and signaling pathways. RT-qPCR and Western blotting were utilized to detect Txnip expression at mRNA and protein levels (n=3), and immunofluorescence assay was adopted to observe Txnip subcellular translocation. The protein levels of downstream apoptosis-related molecules, such as ASK1, p-ASK1, pro-cysteinyl aspartate specific proteinase 3 (pro-Caspase3), cleaved-cysteinyl aspartate specific proteinase 3 (Cleaved-Caspase3) and Caspase9 were detected by Western blotting. Small interfering RNA was used to knock down Txnip expression; RT-qPCR and Western blotting were employed to verify the knockdown efficiency, and flow cytometry and Western blotting were utilized to detect apoptosis rates (n=12, 13) and the expression of downstream apoptosis-related molecules. CCK-8 assay was conducted to screen the appropriate intervention concentration of N-Acetylcysteine (NAC), and the expression of Txnip was detected by RT-qPCR and Western blotting (n=3). The apoptosis rate (n=7, 8), Txnip translocation and expression of downstream apoptosis-related molecules were detected by flow cytometry and Western blotting.

After 48 h of PS-MPs exposure, the number of cells was decreased and altered morphology was observed in all exposure groups. Compared with 0 μg/mL group, Except for the 10 μg/mL group, with no significant difference in cell viability, Compared with 0 μg/mL group, all other exposure groups demonstrated significantly decreased viability (P<0.0001). After 72 h of PS-MPs exposure, all exposure groups showed reduced cell numbers, altered morphology, and significantly decreased cell viability (P<0.001). Compared with 0 μg/mL group, exposure to PS-MPs for 48 h significantly increased ROS levels (P<0.001) and promoted cell apoptosis (P<0.01). Transcriptome analysis showed that DEGs were significantly enriched in oxidative stress and apoptosis regulatory pathways, with Txnip showing the most significant expression. After 48 h of PS-MPs exposure, Compared with 0 μg/mL group, the mRNA and protein levels of Txnip were mildly increased in the 10 μg/mL group, while the levels in all other exposure groups were increased significantly (P<0.05). Furthermore, knockdown of Txnip significantly inhibited cell apoptosis (P<0.0001). PS-MPs exposure induced the translocation of Txnip from the nucleus to mitochondria and accumulate in the cytoplasm, thereby promoting ASK1 phosphorylation, Caspase9 activation, and Caspase3 cleavage. Knockdown of Txnip significantly inhibited the activation of the ASK1-Caspase9/3 pathway. Co-exposure of PS-MPs with 1 mmol/L NAC for 48 h also inhibited Txnip expression (P<0.01) and translocation, thereby blocking downstream pathway activation and ultimately reducing apoptosis (P<0.0001).

PS-MPs can upregulate Txnip expression and promote its translocation through oxidative stress, thereby activating the ASK1-Caspase9/3 signaling pathway and finally inducing apoptosis in mouse GC-2 cells.

Current evidence on whether exposure to fine particulate matter (PM_2.5) affects male semen quality exhibits considerable heterogeneity, and a systematic evaluation of PM_2.5 spermatotoxicity is lacking. This study aims to provide new evidence on the association between PM_2.5 exposure during the spermatogenic cycle and semen parameters in a representative population of reproductive age men, and to conduct a systematic evaluation integrating existing evidence from population, animal, and cellular studies.

Based on the Chongqing Preconception Reproductive Health and Birth Outcome Cohort, a questionnaire survey was conducted on male volunteers with pregnancy plans recruited in Chongqing Maternal and Child Health Hospital from November 2018 to September 2023. Their semen samples were collected and analyzed following standardized procedures, including semen volume, total sperm count, sperm concentration, progressive motility, total motility, and normal sperm morphology. PM_2.5 exposure data were obtained from the Tracking Air Pollution in China (TAP) dataset. Individual-level exposure during different stages of the spermatogenic cycle was estimated based on the residential addresses from the recovered questionnaires. Multiple linear regression analysis was employed to estimate the dose-response relationships between PM_2.5 exposure and semen parameters. Targeted Risk Assessment of Environmental Chemicals (TRAEC) strategy was adopted for systematic evaluation. Eligible studies were retrieved from PubMed, Web of Science, and ScienceDirect databases. The findings from the cohort analysis were also incorporated to jointly assess the reproductive toxicity of PM_2.5 exposure, yielding a comprehensive quantitative score, and determine the risk level according to predefined criteria.

The cohort study enrolled 5735 male volunteers subjected from January 2018 to September 2023, with a mean age of 31.42±4.58 years. Each interquartile range increase in PM_2.5 exposure during the spermatogenic cycle was associated with a 12.30% (95%CI: -16.21 to -8.22, P<0.0001) decrease in total sperm count and a 10.25% (95%CI: -13.64 to -6.72, P<0.0001) decrease in sperm concentration. The TRAEC-based evaluation incorporated 41 studies, revealing that PM_2.5 exposure during the spermatogenic cycle posed a moderate risk for total sperm count and total motility, and a low risk for sperm concentration, progressive motility, semen volume, and normal sperm morphology.

Both the cohort study and the TRAEC-based systematic evaluation consistently demonstrate that PM_2.5 exposure primarily causes decreases in total sperm count and sperm concentration, suggesting potential spermatogenic toxicity of PM_2.5 exposure.

It is recommended that health authorities should leverage the existing maternal and child healthcare networks and preconception screening systems to simultaneously issue reproductive health protection alerts during air pollution episodes, and to provide targeted health education and risk communication for men planning pregnancy.

Infertility represents a major global reproductive health challenge. Metabolite levels in both partners are highly correlated and may serve as potential confounders for each other. However, how the overall preconception plasma metabolic profiles of both partners jointly influence natural conception outcomes remains unclear. Based on the Chongqing Preconception Reproductive Health and Birth Outcomes Cohort (PREBIC), this study aims to explore the association between preconception plasma metabolite levels in couples of reproductive age and conception outcomes.

The Chongqing PREBIC was launched in January 2019 at the Chongqing Health Center for Women and Children, recruiting couples of reproductive age undergoing preconception health examinations to establish baseline cohorts and collect blood samples. Follow-up was then performed every 3 months for clinical pregnancy outcomes following 24 months of unprotected intercourse. The study ultimately included 1292 couples of reproductive age for analysis. According to natural conception status, participants were assigned into a pregnant group (n=967) and a non-pregnant group (n=325). Baseline plasma samples were subjected to standardized metabolomic testing using the Bruker IVDR NMR platform, yielding 167 metabolic parameters. Elastic network regression was employed to screen key metabolites, and logistic regression models were constructed to evaluate the association between metabolites and 2-year pregnancy outcomes, and confounding factors such as age, body mass index (BMI), smoking and alcohol consumption were adjusted.

Among the 1292 couples of reproductive age, the 2-year pregnancy rate was 74.85%. Eighty-two metabolites showed positive correlations between partners, with a mean correlation coefficient of 0.15. After controlling for inter-variable correlations using elastic network model, 14 male metabolites and 20 female metabolites were found to be associated with conception outcomes. Logistic regression revealed gender-specific associations between plasma metabolite levels in both partners and conception outcomes: in males, triglycerides in low-density lipoprotein were positively associated with conception outcomes (Q4 vs Q1: RR=1.12, 95%CI: 1.03 to 1.19, P adjust=0.047). In females, low-density lipoprotein, apolipoprotein A in high-density lipoprotein, apolipoprotein B in low-density lipoprotein, creatinine, and leucine were negatively associated with conception outcomes. Acetic acid in in both partners was positively correlated with conception outcomes (Q4 vs Q1: male RR=1.13, 95%CI: 1.04 to 1.21, P adjust=0.047; female RR=1.18, 95%CI: 1.05 to 1.28, P adjust=0.007).

Preconception metabolite levels in couples of reproductive age may act on future reproductive potential in a gender-specific manner, with common key metabolites identified.

Based on these findings, it is recommended that the health policy-making departments accelerate the transition of preconception care services from focusing solely on women toward a dual-partner co-management model. Incorporate the pre-pregnancy metabolism of both males and females of reproductive age into routine preconception eugenics health services, with priority monitoring of metabolic indicators, including lipid homeostasis and energy metabolism; develop precision intervention measures according to metabolic differences between genders; improve the urban-rural integrated preconception health management system covering both partners; and enhance reproductive health at the public health source level, providing policy basis and technical support for optimizing birth population quality.