Sperm DNA fragmentation assay kit was purchased from Zhejiang Xingbo Biotechnology Co., Ltd. (Ningbo, China); Acrosome integrity assay kit, membrane integrity assay kit, ROS assay kit, mitochondrial membrane potential (MMP) assay kit and apoptosis assay kit were purchased from Celula, Medical Technology Co., Ltd. (Chengdu, China); GSH, total superoxide dismutase (T-SOD), Catalase (CAT) activities kit and iron assay kit were purchased from Nanjing Jiancheng Institute of Bioengineering (Jiangsu, China); RNA and protein assay kit were obtained from TIANGEN Biochemical Technology Co. Ltd. (Beijing, China); BCA protein Assay kit (Solarbio, China); Rabbit antibodies against GPX4, RIPK1, RIPK3, and, pyroptosis markers of GSDME and HSP 90 were all obtained from Cell Signaling Technology Biological Reagent Co., Ltd. (Shanghai, China).

The study was approved by the Ethics Committee of the Peking University International Hospital. This study involving human subjects was performed in accordance with guidelines of the Declaration of Helsinki. Informed patient consent was obtained from the participants who visited the reproductive medicine center in the Peking University International Hospital for infertility treatment during January 2021 to June 2021.

Totally, 26 males with bilateral varicocele (grade 2 or grade 3) and 20 healthy males were enrolled. All participants were examined in the standing positions, and the diagnosis standards and methods of varicocele included was visible swelling of the scrotum, and palpation of the spermatic cord at rest and during the Valsalva maneuver. Moreover, the ultrasound criteria for diagnosing a varicocele was spermatic vein diameter ≥2 mm and retrograde blood flow. There three grades of varicocele by the clinical grading system: grade 0 (subclinical): non-palpable and visualized only by Color Doppler ultrasound (CDUS); grade 1: palpable only with Valsalva maneuver; grade 2: easily palpable but not visible; and grade 3: easily visible. According to abovementioned criteria, varicocele on either side of the spermatic vein was excluded in 20 control males (Wein, 2007).

Semen samples were collected in sterile containers from healthy males and bilateral varicocele patients by masturbation after 2–7 days of sexual abstinence. After liquefaction, the characteristics of sperm concentration, pH, sperm motility, and sperm morphology were examined using a computer-assisted semen analyzer (WL-9000 sperm analyzer, Beijing Weili New Century Technology Development Co., LTD., Beijing, China) according to WHO guidelines (Sun et al., 2018). All semen samples were centrifuged at 500 g for 5 min. Then, the seminal plasma and spermatozoa were separated and stored at −80°C until analysis.

Intracellular ROS was detected using propidium iodide (PI) and dichloro-dihydro-fluorescein diacetate (DCFH-DA) fluorescence staining (Sperm ROS Detect Kit™, Celula, China) (Yu et al., 2019). Briefly, the spermatozoa were added with DCFH-DA at a final concentration of 40 μM and PI at a final concentration of 10 μM. After incubation for 25 min at 37°C, the samples were centrifuged at 800 g for 3 min. All analyses were performed by flow cytometry (50 μL, 5000 particles and max 5 min, Sparrow, Celula, Chengdu, China).

The sperm chromatin structure assay required a minimum of 10 μL semen samples with a concentration of ≥0.5 million/mL. Firstly, the frozen specimens were thawed in a 37°C water bath and re-suspended in TNE (Tris, NaCl and EDTA) buffer at the ratio of 1:9 (μL). Subsequently, the samples were treated with 400 μL of acid (pH1.20) for 30 s to denature DNA at the sites of strand breaks. Thirdly, acridine orange staining solution was added to stain the single-strand DNA breaks (presenting with red fluorescence) and double-strand DNA breaks (presenting with green fluorescence). At least 5,000 sperm cells were analysed per sample within 5 min using flow cytometry. The sperm fragmentation index was calculated as red fluorescence/(red and green fluorescence).

Sperm acrosome integrity was determined as described in a previous study (Yániz et al., 2017). Briefly, spermatozoa were stained with the fluorochromes of acrosome integrity assay kit (The labelling mixture included PI, Hoechst 33342, and carboxyfluorescein diacetate). At least 5000 spermatozoa were examined using flow cytometry.

The integrity of human spermatozoa sperm membrane was stained with PI (Sperm Membrane Integrity Kit™, Celula, China). This fluorescent probe has a high affinity for sperm DNA but is non-permeable to the sperm plasma membrane, only staining damaged (non-viable) sperm plasma membranes. Sperm with membrane damage passes through the FL2 channel and emits red fluorescence. Briefly, 2.5 μl PI (1 mg/ml) was added to 10 μl sperm samples, and then 500 μl 1% (w/v) NaCl was added. After incubation in the dark for 5 min, the samples were detected by flow cytometry (50 μL, 5000 particles and max 5 min). Sperm plasma membrane integrity was defined as the percentage of PI-negative cells (membranes intact) per sample per treatment.

MMP was evaluated using the specific probe JC-1 (Sperm MMP Staining Kit™, Celula, China) according to the manufacturer’s instruction. The semen samples were centrifuged at 500× g for 20 min and the supernatant was removed. JC-1 is a lipid cationic fluorochrome that is widely used as a fluorescent probe for the detection of MMP. When the MMP value is high, JC-1 accumulates in the mitochondrial matrix and emits orange fluorescence (585 nm). Conversely, when the MMP value is low, JC-1 accumulates less in the mitochondrial matrix, and emits green fluorescence (525 nm) as a monomer. The MMP was calculated using fluorescence ratio of JC-1-aggregates (red) to JC-1-monomer (green).

The sperm apoptosis was evaluated using the fluorescent probes Annexin V-FITC (Sperm Apoptosis Staining Kit™, Celula, China) according to the manufacturer’s instruction. Briefly, 45 µL of human spermatozoa (>5 * 10⁶/mL) were incubated with Annexin-V, H-42, and Annexin V-FITC for 60 min at room temperature in the dark. A total of 5000 sperm cells were analyzed by flow cytometry (50 μL, 5000 particles and max 5 min). Apoptosis was calculated using the following formula: Annexin V-FITC positive/H-42 positive and PI negative.

The relative iron concentration in cell lysates was assessed using the Iron Assay Kit (Nanjing Jiancheng Institute of Bioengineering (Jiangsu, China) according to the manufacturer’s instructions (Sun et al., 2015).

Spermatozoa were prepared as 10% cell lysates in normal saline and centrifuged at 3000 rpm at 4°C for 15 min. The supernatant was collected. The relative concentrations of GSH, T-SOD, and CAT were assessed using the corresponding kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions (Deng et al., 2017).

Total RNA was extracted from human spermatozoa using a universal RNA extraction kit (TIANGEN Biochemical Technology Co. Ltd., Beijing, China) according to the manufacture’s protocol. Subsequently, cDNA was obtained by reverse transcription using a commercial kit (TIANGEN Biochemical Technology Co. LTD., Beijing, China). Abundance of mRNA transcripts encoding iPLA 2β, P53, Zinc finger E-box binding homeobox 1 (ZEB1), GPX4, and GAPDH were measured by quantitative real-time PCR. Primer sequences are shown in Table 1.

Total protein from human spermatozoa were extracted with RIPA lysis buffer on ice and Protein Extraction Assay kit (TIANGEN Biochemical Technology Co. Ltd., Beijing, China). BCA protein Assay kit (Solarbio, China) was used to quantitate concentration of protein in the supernatant. Equivalent amounts of protein were separated on 10%–15% SDS-polyacrylamide gels and blotted onto a nitrocellulose membrane. After blocking with 5% skimmed milk for 2 h at room temperature in TBS with 0.1% Tween-20, membranes were probed with GPX4 (1:5000 dilution), GSDME (1:5000 dilution), RIPK1 (1:5000 dilution), RIPK3 (1:5000 dilution), GAPDH (1:5000 dilution), and HRP conjugated IgG antibodies (1:10,000 dilution). Relative band intensity was then determined by ImageJ 1.53 software (National Institutes of Health, America).

All experiments were performed independently for at least three times. The independent sample t-test was used to analyze numerical data between two groups. Student t-test was used to analyze significant differences between two groups. Data analysis was performed by using Statistical Program for Social Sciences (SPSS) software, version 22.0 (IBM Corporation, Armonk, NY, United States), and a p-value <0.05 indicated statistical significance.

This study included 20 healthy males aged 29–34 years old (control group) and 26 patients with bilateral varicocele aged 28–40 years old (patient group) (Table 2). The duration of abstinence (3.77 d vs. 3.7 d), PH (7.35 vs. 7.3) and sperm counts (231.94 *10⁶ ells vs. 177.67 *10⁶ cells) between the two groups were not significantly different (p > 0.05). However, there were significant differences in other basic semen parameters, including semen volume (2.58 mL vs. 3.87 mL), sperm concentration (87.25 *10⁶/mL vs. 53.32 *10⁶/mL) and normal morphology (1.4% vs. 0.85%) (p < 0.05). Additionally, sperm progressive rate (47.68% vs. 20.83%), sperm motility (58.46% vs. 24.38%) and sperm DNA fragmentation index (10.78% vs. 22.07%) between the two groups were significantly different (p < 0.001) according to the 2010 WHO criteria (Murray et al., 2012).

Firstly, sperm acrosome integrity and membrane integrity were compared between controls and patients with bilateral varicocele by flow cytometry. There were decreases in the acrosome integrity in patients with bilateral varicocele (Figures 1A–C). Next, we analyzed membrane integrity. The results demonstrated that the membrane integrity also significantly decreased in patients with bilateral varicocele (Figures 1D–F).

In the next set of experiments, we investigated whether mitochondrial function and sperm motility are involved in MMP and apoptosis rate. Our results showed that there was ∼2-fold decrease in MMP level in patients with bilateral varicocele (Figures 2A–C). Moreover, apoptosis rate of human spermatozoa significantly (∼2.5-fold) increased in patients with bilateral varicocele (Figures 2D–F). Altogether, the results of these experiments demonstrate that MMP and apoptosis rate are involved in decreased sperm motility observed in these patients.

To investigate the ferroptosis level between control and patients with bilateral varicocele, ROS and iron level were detected in the seminal plasma. Comparing with control group, ROS level was increased in patients with bilateral varicocele (p < 0.05) (Figures 3A–C). However, the level of iron was reduced in patients with bilateral varicocele (p < 0.01) (Figure 3D). These results together suggest that men with varicocele increased ROS levels and decreased iron levels, which might suggest the activation of the ferroptosis pathway.

Next, we investigated whether ROS and iron level in human spermatozoa affects antioxidant enzyme activities. Quantification of GSH in sperm lysates showed that there was a ∼78% (p < 0.01) decrease of cellular GSH in patients with bilateral varicocele (Figure 4A). T-SOD levels in patients with bilateral varicocele were decreased by ∼33% (p < 0.01) (Figure 4B); CAT levels in patients with bilateral varicocele were decreased by ∼95% (p < 0.01) (Figure 4C). According to these results, antioxidant enzyme activities were also contributed by ferroptosis.

To examine whether ROS are directly involved in ferroptosis in response to bilateral varicocele, mRNA and protein of ferroptosis-related signal pathways were detected. Quantitative real-time PCR was used to detect the mRNA expression levels of P53, iPLA 2β, ZEB1 and GPX4 in the semen. These results showed that the mRNA expression levels of P53, iPLA 2β, and ZEB 1 in the patient group was significantly higher than that of the control group (p < 0.05) (Figures 5A–C). However, the mRNA expression level of GPX4 in the patient group was significantly lower than that of the control group (p < 0.05) (Figure 5D). Besides, the level of GPX4 protein was reduced in the patient group (Figure 6A). Therefore, the results of these data demonstrate that ROS exposure might be associated with activation of ferroptosis pathway in bilateral varicocele men.

Except ferroptosis, we also examined whether ROS are directly involved in pyroptosis in response to bilateral varicocele. Pyroptosis and necroptosis-related proteins of RIPK1, RIPK3, and pyroptosis markers of GSDME and HSP 90 were detected. There were obvious differences in the protein expression of RIPK1, RIPK3 GSDME, and HSP 90 between two groups (Figures 6B, C). In the patients group, expression of ferroptosis, pyroptosis and necroptosis-related proteins were increased than control group.