Semen samples were obtained from male participants between April 2013 and February 2014. Informed consent for enrollment into the study and for the use of semen in analysis was obtained from all participants. The institutional review board at Seoul National University Bundang Hospital reviewed and approved the study (B-1205-155-003). Subjects without a history of genital inflammation or genital surgery, subjective symptoms, self-reported medical risk factors and taking any prescription medications were included.

Semen samples were collected by masturbation after 3 days of sexual abstinence. After liquefaction for 30 minutes at room temperature, sperm quality was assessed using computer-assisted semen analysis (SAIS-PLUS 10.1, Medical Supply Co., Seoul, Korea) and classified according to World Health Organization guidelines published in 2010. Strict criteria for the definition of normal spermatozoa were used during morphological assessment. Baseline semen characteristics were as follows: volume, 2.9±1.4 mL (range, 1.0 to 6.0 mL), concentration, 99±82 million/mL (range, 18 to 460 million/mL), motility, 54.6±15.3% (range, 10.1% to 75.8%), total motile sperm, 177±238 million (range, 17 to 1,485 million); and normal forms by strict morphology, 11.3±5.6% (range, 2.3% to 25.0%). All semen samples contained motile sperm and no sample had significant numbers of round cells or leukocytospermia in accordance with World Health Organization guidelines (<1 million round cells/mL).

The semen was processed by the conventional swim-up method. After centrifuging the semen (300 xg for 5 minutes), a pellet was obtained via removal of seminal plasma. The pellet was suspended in fresh Ham's F10 (1.5 mL) supplemented with 10% serum substitute supplement (Irvine Scientific, Santa Ana, CA, USA). After centrifugation (300 xg for 5 minutes), the supernatant was discarded and Ham's F10 with 10% serum substitute supplement media (0.5 mL) was gently layered on the pellet and incubated at 37℃ in a 5% CO₂ atmosphere for 1 hour. The supernatant (0.5 mL) was then transferred to a conical tube. Computer-assisted semen analysis and normal forms by strict morphology were assessed in the processed samples.

Nuclear DNA integrity was measured by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as described previously [11]. The samples were smeared on a silane-coated slide (DAKO, Glostrup, Denmark) and air-dried. Sperm samples were fixed with 4% paraformaldehyde for 1 hour at 15℃ to 25℃, and then washed with phosphate-buffered saline (PBS). Sperm were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (Sigma-Aldrich, St. Louis, MO, USA). A commercial apoptosis detection kit was used (In Situ Cell Death Detection Kit, Roche Diagnostics GmbH, Mannheim, Germany). The remaining procedures were performed as per manufacturer's instructions. Counterstaining was performed using a mounting medium with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Sperm with fragmented DNA had nuclei stained green, whereas the nuclei of other cells were blue. Sperm head with >50% of the area stained green was considered positive. At least 500 sperm were counted per experimental set and the percentage of sperm with fragmented DNA was determined as DNA fragmentation index (DFI).

This method was used for the detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a known biomarker for oxidative stress. A specific antibody (Argutus Medical OxyDNA Test, BD Biosciences, Franklin Lakes, NJ, USA) conjugated to fluorescein isothiocyanate (FITC) was used. The intensity of FITC fluorescence was then assessed under fluorescent microscopic evaluation (Fig. 1). Briefly, sperm samples were fixed by 4% paraformaldehyde and permeabilized. The primary antibody was then added for 1 hour, according to the manufacturer's instructions. At least 500 sperm were counted in different areas of each slide. Sperm head with >50% of the area stained green was considered positive.

PLCζ was detected by immunofluorescent staining with a polyclonal anti-PLCζ antibody as described before [12]. Sperm samples were fixed with 4% paraformaldehyde/PBS, permeabilized with 0.5% (v/v) Triton X-100/PBS and stored at 4℃ until use. Sperm smears were created on pre-coated glass slides (Menzel-Gläser, Braunschweig, Germany), incubated in 3% bovine serum albumin-PBS, and labelled with a polyclonal anti-PLCζ antibody (25 µg/mL; Santa Cruz Biotechnology, Dallas, TX, USA) in 0.05% bovine serum albumin-PBS overnight at 4℃. After three washes with PBS, samples were labeled with Alexa Fluor 555 goat anti-rabbit IgG for 1 hour (1:400 dilution; Invitrogen, Merelbeke, Belgium), counterstained with 5 µg/mL Hoechst 33258 and 5 µg/mL FITC (Alexa 488, Invitrogen, Grand Island, NY, USA) for 15 minutes at 37℃ in the dark, washed with PBS and mounted in glycerol-DABCO. Sperm (n=400) were examined using a fluorescent microscopy (Axioskop40, Carl Zeiss Microscopy, Jena, Germany) (Fig. 2) [1]. Sperm head with >50% of the area stained green was considered positive regardless of the localization. PLCζ immunostaining was performed twice in each donor sample and the consistency was assessed by the Pearson's correlation test.

While several parameters were not normally distributed, the data are expressed as the mean±standard deviation. Nonparametric Spearman's correlation test was used to assess an association between different numerical parameters. Immunoreactivity of PLCζ was averaged from two measurements. A correlation between two measurements of PLCζ immunoreactivity was assessed by the Pearson's correlation test because this variable showed a normal distribution. Intergroup differences were assessed by nonparametric Wilcoxon test. A P-value of <0.05 (two-tailed) was considered statistically significant. Statistical analysis was performed using SPSS ver. 18.0 (SPSS Inc., Chicago, IL, USA).