Study groups

The research was conducted in a population of 198 men of reproductive age (median age: 32.50; range (20–41)). Between January 2017 and November 2020, the subjects were recruited from the Andrology Outpatient Clinics in Poznan and Szczecin and via traditional and social media advertisements using the same protocols and following the same exclusion criteria as previously established [20, 21]. Briefly, all the patients and volunteers underwent andrological and ultrasound examinations. A questionnaire about fertility status, genitourinary and systemic diseases, medications, occupation, and lifestyle was completed by all the participants. Individuals with active systemic or genitourinary inflammation, cryptorchidism, hypogonadism, testicular injury or cancer, body mass index > 32 kg/m2, a smoking habit, or double local temperature factor were excluded. The participants were divided into four groups: (A) men with proven fertility (at least one offspring over the past 2 years) not exposed to prolonged genital heat stress, serving as the controls, n = 29; (B) men who had worked at least 2 years as professional drivers, n = 54; (C) infertile men (no pregnancy after at least 1 year of regular unprotected sexual intercourse without any apparent reason in their partner(s)) with clinically diagnosed varicocele (dilatation of the vessels of the pampiniform plexus at least 3 mm in diameter), n = 78; and (D) infertile men not exposed to prolonged (clinical or environmental) genital heat stress, n = 37.

Manual semen analysis

Semen samples were collected before any medical intervention. Three to 5 days of sexual abstinence were asked of the participants before ejaculates were collected by masturbation. After liquefaction at room temperature, standard semen analysis was done according to the 5th WHO laboratory manual within 60 min of ejaculation by a trained technician [22]. After initial macroscopic assessment of the samples (color, viscosity, volume, and pH), microscopic assessment was performed under a bright-light microscope equipped with a contrast phase (DM 2000, Leica, Heerbrugg, Switzerland). The sperm concentration was estimated with an improved Neubauer hemocytometer (Paul Marienfeld, Lauda-Königshofen, Germany). Motility characteristics were evaluated using the standard grading system: progressive motility, nonprogressive motility, and immotility. Both eosin staining and hypo-osmotic swelling (HOS) tests were used to assess sperm viability. Sperm morphology was assessed according to Kruger’s strict criteria following Papanicolaou staining of the previously washed sperm smears. To distinguish peroxidase-positive leukocytes from peroxidase-negative round cells (other round cells), the LeucoScreen Kit (FertiPro N.V., Beernem, Belgium) was used.

Preparation of sperm suspensions

Collected semen samples were centrifuged at 1800 rpm for 7 min at room temperature. The sperm pellets were washed in warm phosphate-buffered saline (PBS), pH 7.4, by centrifugation at 1800 rpm for 7 min. The fresh sperm suspensions were immediately used for fluorescence and flow cytometry studies. To assess sperm DNA fragmentation, an aliquot of sperm suspension was fixed in 1% formaldehyde at 4 °C for 20 min. After washing two times in PBS, the fixed sperm cells were resuspended in ice-cold 75% (v/v) ethanol and stored at − 20 °C for the TUNEL assay for no less than 3 months [21].

Evaluation of membrane fluidity

The level of phospholipid scrambling in the sperm membrane lipid bilayer was evaluated by merocyanine 540 (M540) staining [23]. Sperm cells were incubated with 4.09 µmol merocyanine 540 (M540) dye (Ex/Em 563/607 nm, emission of red fluorescence) for 15 min in darkness at 37 °C in a CO2 atmosphere. Then, flow cytometry analysis was performed (see below). The subpopulation of sperm cells without membrane lipid disorders (M540-negative sperm) was calculated.

Evaluation of PS translocation

Phosphatidylserine (PS) externalization in sperm membranes was evaluated by using the Annexin V-FITC Apoptosis Detection Kit (Beckman Coulter, Fullerton, CA, USA) according to the manufacturer’s guidelines [23]. Sperm cells washed in PBS supplemented with Ca2+ and Mg2+ were incubated with Annexin V-FITC and propidium iodide (PI) for 15 min in darkness on ice. Then, flow cytometry analysis was performed (see below). The subpopulation of apoptotic sperm cells (Annexin V-positive and PI-negative sperm) was calculated.

Evaluation of mitochondrial membrane potential

The mitochondrial membrane potential of sperm was evaluated by using lipophilic cationic 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide JC-1 staining [24]. Sperm cells were incubated with 10 µg/ml JC-1 dye (Ex/Em490/529 nm, emission of green fluorescence of JC-1 monomers and Ex/Em 514/590 nm, emission of orange fluorescence of JC-1 aggregates) for 25 min in darkness at 37 °C in a CO2 atmosphere. After washing in warm PBS, sperm smears were evaluated under a fluorescence microscope (Olympus BX41, Tokyo, Japan) equipped with a triple emission filter (DAPI/FITC/Texas Red) at × 1000 magnification. The subpopulation of sperm cells with polarized mitochondria (JC-1–positive sperm) was calculated.

Detection of mitochondrial superoxide anion

Mitochondrial superoxide anion generation in sperm was evaluated by using MitoSOX Red staining [25]. Sperm cells were incubated with 2 µmol MitoSOX Red dye (Ex/Em 561/603, emission of red fluorescence) for 15 min in darkness at 37 °C in a CO2 atmosphere. After washing three times in warm PBS, flow cytometry analysis was performed (see below). The subpopulation of sperm cells that emitted red fluorescence (MitoSOX Red-positive sperm) was statistically calculated.

Evaluation of DNA fragmentation

Nuclear DNA strand breaks in sperm were evaluated by using the FlowTACS Apoptosis Detection Kit (Trevigen, Inc., Minneapolis, MN, USA) by the TUNEL/PI method [26]. The previously fixed (see above) and permeabilized (0.1% Triton X-100 in 0.1% sodium citrate) sperm cells were incubated with the labeling solution containing 1 × binding buffer, biotinylated dNTPs, Mn2+, and terminal deoxynucleotidyl transferase (TdT) enzyme (not in negative control) for 45 min at 37 °C. Then, the sperm pellet was incubated with FITC-labeled streptavidin solution for 20 min at room temperature. After the TUNEL labeling reaction, sperm cells were stained with PI to discriminate apoptotic cells from necrotic cells in the flow cytometry analysis. The subpopulation of sperm with DNA fragmentation (TUNEL-positive sperm) was calculated.

Flow cytometry analysis

Cytofluorometric evaluation of sperm suspensions was performed using a Beckman Coulter flow cytometer (Cell Lab Quanta SC MPL, Beckman Coulter, Fullerton, CA, USA) equipped with a 488-nm argon-ion laser for excitation. Samples were measured at a flow rate of 150–250 cells per second. For each analysis, at least 10,000 events were acquired. The sperm population was gated on the basis of the electronic volume (EV, parameter depending on the cell size) and side scatter signals (SS, parameter depending on cellular granules). The intensity of green (480–550 nm, for FITC) and/or red fluorescence (590–670 nm, for M540, PI) was detected using the FL1 and FL3 channels, respectively. MitoSOX Red fluorescence was detected in channel FL2 (561–550 nm). The fluorescence data were obtained at a fixed gain setting in logarithmic mode (FL1, FL2, FL3). For acquisition and data analysis, the Cell LabQuanta SC MPL Analysis software (Beckman Coulter) was used. Fluorescence reading was repeated two times with distinct samples. Binding specificity was checked with a fluorescence microscope (Olympus) (Fig. 2).

Statistical analysis

All statistical calculations were performed using the Python 3 with Pandas (https://pandas.pydata.org/ ver 0.24.2), Matplotlib (https://matplotlib.org/ ver 3.0.3), SciPy (https://www.scipy.org/ ver 1.2.1), Seaborn (https://seaborn.pydata.org/ ver 0.11.0) and scikit-posthoc (https://pypi.org/project/scikit-posthocs/ver 0.5.4) libraries as previously described (17, 18). The data distribution was verified using the Shapiro‒Wilk test. As the variables were not normally distributed, the nonparametric Kruskal‒Wallis test followed by the Dunn test with Holm correction was applied to compare the parameters among the studied groups. Correlations were assessed using the Spearman rank test. A p < value of 0.05 after the post hoc test was considered significant.