This study was declared exempt by the ethics committee of the Guangzhou Women and Children’s Medical Center. All studies involving human subjects were conducted in accordance with guidelines laid down in the Declaration of Helsinki. Written informed consents were obtained by the participants, and the subjects’ data on medical history, lifestyle, and smoking status with a structured questionnaire were collected. Individuals who had never smoked were defined as non-smokers. Current smokers with smoking dose more than 1 pack per day over 10 years or smoking dose more than 2 packs per day over 5 years were defined as heavy smokers. They were men who visited the reproductive medicine center in the Guangzhou Women and Children’s Medical Center for infertility treatment during March 2017 to December 2017. Non-smokers were randomly selected from patients who were age-matched to heavy smokers. All subjects underwent physical examinations and at least two semen analyses. Men who were unhealthy or had a known cause of defective spermatogenesis, such as varicocele, infection, obstruction of the vas deferens, chromosomal abnormalities, or microdeletions of the azoospermia factor region on the Y chromosome were excluded. Patients who were diagnosed with azoospermia, severe oligozoospermia (sperm concentration <5*106 cells/mL), hemospermia, leukospermia, and necrozoospermia were also excluded. Finally, 70 heavy smokers and 75 non-smokers were recruited.
Semen collection and analysis
Semen samples were collected in sterile containers from patients by masturbation after 2-7 days of sexual abstinence. Samples were allowed to liquefy for at least 30 min at room temperature. Analysis of semen volume, pH, sperm concentration, motility, vitality, sperm morphology, and computer-assisted semen analysis were carried out according to WHO guidelines . Samples were centrifuged at 1000 *3 g for 10 min, and seminal plasma and cell pellets were separated and stored at -80°C until analysis.
Cell culture and cigarette smoke condensate treatment
GC-2spd cell lines were purchased from the American Tissue Culture Collection (ATCC) and maintained in Dulbecco modified Eagles media (DMEM, Gibco, USA) supplemented with heat-inactivated 10% fetal bovine serum (Biological Industries, Israel) in a 95% air-humidified incubator with 5% CO2 at 37°C as our previous study .
Cigarette smoke condensate (CSC) was prepared according to the study  and resuspended at a concentration of 50 mg/mL in dimethyl sulfoxide (DMSO) as stock solution. For smoke-exposure experiments, cells were cultured in medium with 400 μg/mL of CSC. After exposure to CSC for 24 hours, cells were used for further study.
Cell viability assay
Cell viability was determined with a Cell Counting Kit-8 (CCK-8) assay. Briefly, cells were plated at a density of 5×104 cells/well in a 96-well plate with 100 μl of medium. Following treatment, cell viability was evaluated with CCK-8 according to manufacturer’s instructions, and absorbance was measured at 450 nm with a microplate reader. Cell viability was expressed as the percentage of live cells vs. controls (set at 100%).
The relative iron concentration in cell lysates was assessed using the Iron Assay Kit (no. ab83366; Abcam) according to the manufacturer’s instructions and our previous study .
Lipid peroxidation assay
The relative malondialdehyde (MDA) concentration in cell lysates was assessed using a Lipid Peroxidation (MDA) Assay Kit (no. ab118970; Abcam) according to the manufacturer’s instructions.
The relative glutathione (GSH) concentration in cell lysates was assessed using the Glutathione Assay Kit (no. CS0260; Sigma) according to the manufacturer’s instructions.
Western blot analysis
Following treatment, cells were washed twice with cold PBS and extracted with RIPA lysis buffer on ice. Extracted cells were then sonicated and centrifuged at 15,000 g for five minutes. Protein extraction was carried out in accordance with existing protocols (Beyotime, China). Protein content was determined using a BCA protein assay kit, according to manufacturer’s instructions. Equivalent amounts of protein were separated on 10-15% SDS-polyacrylamide gels and blotted onto a nitrocellulose membrane. After blocking at room temperature for two hours with 5% non-fat milk in TBS with 0.1% Tween-20, membranes were probed with GPX4 (1:5,000 dilution), GAPDH (1:5,000 dilution) and HRP conjugated IgG antibodies (1:10,000 dilution); and visualized by exposure to Gel Doc XR (BioRad, USA). Relative band intensity was then determined by standard scanning densitometry normalized with GAPDH.
The chi-square test was used to compare groups of categorical variables, and an independent sample t-test was used to analyze numerical data in two groups. The nonparametric Mann-Whitney test was used to analyze differences between nonhomogeneous variances. One-way ANOVA with Tukey or Dunnett posttest was used to analyze data in multiple groups (SPSS version 17.0 for Windows). A two-sided P value of 0.05 was considered statistically significant.