A comparative study was performed during 2015. Informed consent was obtained from all subjects, and the use of human semen for this study was approved by the Institutional Review Board of the Seoul National University Bundang Hospital (IRB No. B-1412-277-006). Semen samples were obtained from 18 healthy male partners who attended our infertility clinics for infertility investigations (a single sample per person). None of the subjects had a history of genital inflammation or surgery, no subjective symptoms or self-reported medical risk factors were identified, and none of the subjects had taken any prescribed medications. One subject was a smoker, and fertility was proven in seven subjects. The median age of the subjects was 35.5 years (range, 31–47 years).

Semen samples were collected by masturbation after 3 days of sexual abstinence. After liquefaction for 30 minutes at room temperature, semen analysis was performed using motion analysis equipment (SAIS-PLUS 10.1; Medical Supply Co., Seoul, Korea), as described previously [13]. Strict criteria for the definition of normal sperm were used during the morphological assessment. In accordance with the World Health Organization guidelines (fifth edition, 2010), normal samples were defined by semen volume ≥1.5 mL, sperm count ≥15 million/mL, motility ≥40%, and a percentage of normal form of ≥4%. Round cells were counted (without peroxidase staining), and all the studied samples were found to contain <1 million/mL round cells. The basic semen characteristics were as follows (median [range]): semen volume, 3.0 mL (1.5–9.5 mL); sperm count, 75.5 million/mL (12.8–152 million/mL); total motility, 45.9% (25.1%–75%); total motile sperm count, 81 million (7.2–552 million); and percentage of normal form, 5.0% (1.5%–8.1%).

After a routine semen examination, the remainder of each semen sample was divided into three aliquots. One aliquot was processed by the swim-up method as previously described [13] (the swim-up fraction), and the other aliquot was processed by the DGC method (the DGC fraction). The third aliquot was not processed and served as the control (raw semen).

In the swim-up method, after initial centrifugation of the semen (300×g for 5 minutes) for removal of the seminal plasma, the pellet was suspended in fresh Ham's F10 medium (1.5 mL) supplemented with 10% serum substitute supplement (Irvine Scientific, Santa Ana, CA, USA) [13]. The mixture was again centrifuged (300×g for 5 minutes); the supernatant was discarded; and Ham's F10 media with 10% serum substitute supplement (0.5 mL) was gently layered on the pellet, followed by incubation at 37℃ in a 5% CO₂ atmosphere for 1 hour. The supernatant (0.5 mL) was then transferred to a conical tube and later used for further experiments.

In the DGC method, the semen samples were processed by discontinuous silane-coated silica gradient (Sydney IVF Sperm Gradient; COOK Medical, Brisbane, Australia) as described in the kit instructions. After initial centrifugation of the semen sample (300×g for 5 minutes) for removal of the seminal plasma, the pellet was suspended in fresh Ham's F10 medium (3 mL) supplemented with 10% serum substitute supplement. The prewashed semen (3 mL) was then layered on top of a discontinuous silane-coated silica gradient in a 15-mL conical tube (40%/80%). Next, the conical tube was centrifuged at 300×g for 5 minutes, and the sperm collected from the bottom layer (80% layer) was washed twice by resuspension in 4 mL of Ham's F10 medium, followed by centrifugation (300×g for 5 minutes). After double centrifugation, the supernatant was removed and the pellet was resuspended in 1 mL of Ham's F10 medium supplemented with 10% serum substitute supplement. The resuspended pellet was later used for further experiments.

SDF levels were measured using the Halosperm assay (Halotech DNA, Madrid, Spain) as described previously [14,15]. Semen samples were diluted with Ham's F-10 medium (Sage, Trumbull, CT, USA) to adjust the concentration to 5 to 10 million/mL. Agarose gel from the kit was incubated for 5 minutes at 90℃ to 100℃ and then for 5 minutes at 37℃; this was followed by adding and mixing 25 µL of semen in a marked Eppendorf test tube. The mixtures (15 µL) were then placed on a super-coated slide and covered with a coverslip. The slides were kept for 5 minutes at 4℃ in a refrigerator to create a microgel with the implanted sperm. The coverslips were then carefully removed, followed by immersion of the slides into the prepared acid denaturation solution (80 µL in 10 mL of distilled water) for 7 minutes. The slides were then transferred to a tray with a lysis solution from the kit and incubated for 25 minutes. Next, the slides were rinsed with distilled water, followed by dehydration for 2 minutes in increasing concentrations of ethanol (70%, 90%, and 100%). After drying, the slides were stained with Diff-quick solution I and II (each for 7 minutes), rinsed with distilled water, and allowed to dry at room temperature.

Finally, each slide was examined under a light microscope at ×100 magnification, and at least 300 sperm cells were scored. Each sperm cell was categorized as having a large halo, medium halo, small halo, no halo, or degraded. A large halo meant that the sperm cell produced a halo with a thickness equal to or greater than the length of the minor diameter of the core, a medium halo meant that the sperm produced a halo with a thickness smaller than the length of the minor diameter of the core and greater than one-third of the minor diameter of the core, and a small halo meant that the sperm produced a halo with a thickness equal to or smaller than one-third of the minor diameter of the core. Sperm cells with a small halo or no halo, and those that were degraded, were classified as sperm cells with fragmented DNA.

Simultaneous two-probe two-color fluorescence in situ hybridization was performed using a probe set for the X and Y chromosomes. The probes used for fluorescence in situ hybridization were from loci DXZ1 and DYZ3. All the probes were directly labeled with fluorescent dyes; the X chromosome was labeled with Spectrum Orange, and the Y chromosome was labeled with Spectrum Green (Vysis Inc., Abbott Park, IL, USA). The detailed processes for the denaturation, hybridization, and 4’,6-diamidino-2-phenylindole (DAPI) counterstaining were performed as previously described [16,17]. At least 300 sperm cells per slide were analyzed under a Nikon E600 epifluorescent microscope (Tokyo, Japan).

Chromatin structure was assessed by toluidine blue staining, as described previously [13]. Based on a visual assessment, the stained sperms were categorized as having the following toluidine blue colors: dark purple, intermediate, and light blue. Only dark purple sperm cells were considered to have an abnormal chromatin structure. At least 300 sperm cells were counted per slide, and the percentage of sperm cells with a dark-purple color was determined.

Statistical analysis was performed using MedCalc ver. 10.4 (MedCalc Software, Mariakerke, Belgium). The SDF level, the percentage of normal X or Y sperm cells, and the percentage of sperm cells with an abnormal chromatin structure were compared between the raw and processed semen samples using the Wilcoxon signed-rank test. The Spearman correlation test was used to assess associations between different semen parameters. A p-value of <0.05 was considered to indicate statistical significance.