A total of 41 male patients (age range, 27–45 years) were enrolled in this study, which was performed at the Andrology Centre in Coimbatore City, Tamil Nadu, India. Semen samples were collected from each patient after informed consent was obtained. All procedures were performed according to Institutional Review Board policy. All patients underwent serology assessment for viral or bacterial infections, including tests for human immunodeficiency virus (HIV)-1 and 2, hepatitis B surface antigen, hepatitis C virus, and other standard laboratory tests for sexually transmitted infections, before undergoing the SCSA or SCD tests. Prior to sample collection, patients were asked to adhere to an ejaculatory abstinence regime spanning 24–48 hours. The semen samples were collected via masturbation into a sterile wide-mouthed calibrated container. After liquefaction for 1 hour at room temperature, 200–500 µL of the raw semen was aliquoted into cryovials without cryoprotectant and flash-frozen in liquid nitrogen. Samples were analyzed fresh or frozen/thawed. Two SCD kits (CANfrag and Halosperm) and SCSA were chosen for the DFI analyses. A CANfrag kit was utilized on 36 patient samples and a Halosperm kit on 31 samples, and all 41 patients were assessed via SCSA (Figure 1). The DFI value, the correlation of DFI between the different assays, the coefficient of variation (CV) between technical replicates, and the impact on the choice of ART procedure were compared among SCSA and the specific kits (Figure 1). All reagents were purchased from Sigma-Aldrich (Millipore-Sigma, St. Louis, MO, USA) unless otherwise noted.

Individual semen samples, stored in liquid nitrogen tanks (−196°C), were thawed in a 37°C water bath and then immediately placed on crushed ice. An aliquot of raw semen was transferred to a solution of TNE buffer (0.01 M Tris-HCl, 0.15 M NaCl, and 1 mM EDTA; pH 7.4) at 4°C to yield a final concentration of approximately 1–2×10⁶ sperm/mL. A total of 200 µL of this sperm suspension was admixed with 400 µL of a solution containing 0.08 N HCl, 0.15 M NaCl, and 0.1% (v:v) Triton X-100 at 4°C. Importantly, the HCl was diluted from a commercial solution of 2.0 N HCl. After 30 seconds, sperm were stained by adding 1.2 mL of staining solution containing 6 µg/mL acridine orange (AO, chromatographically purified; Polysciences, Warrington, PA, USA), 0.2 M Na2PO4, 0.1 M citric acid (pH 6.0), 1 mM EDTA, and 0.15 M NaCl to yield an AO:DNA-P molar ratio of ≥2 [23]. The acid-/AO-stained sample was placed in a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) sample chamber, and sample flow was initiated to bring the sheath flow and sample flow to equilibrium within 2 minutes. Subsequently, 5,000 sperm were analyzed at an event rate of 100–250 events/sec. If the event rate exceeded 250 events/sec, a new sample was prepared to ensure full equilibrium between the AO dye and the sperm. The flow cytometer was calibrated with a reference sample at the start of sample analysis, and the same reference sample was analyzed after every five test samples to calibrate the instrument. Each test sample was analyzed in duplicate, and replicates of the data were utilized to determine the percentage of sperm with measurably increased red fluorescence (sperm with fragmented DNA as determined using SCSAsoft). If a >10% difference in DFI was observed between the raw X and Y means in the replicates, the sample was repeated. The standard deviations between the replicates were calculated.

Low-melting-point agarose, which was pre-provided in a microcentrifuge tube, was placed in a float in boiling water (90°C–100°C) for 5 minutes and then transferred to a 37°C water bath for equilibration. An aliquot of fresh or flash-frozen semen sample was added to this tube of melted agarose in order to achieve a final sperm concentration of 15–20 million/mL. A volume of 150 µL of this agarose-semen sample mixture was pipetted onto the provided slide, covered with a coverslip (25 mm×50 mm), and allowed to solidify at 4°C for 5 minutes. The coverslip was carefully removed, and the slide was kept on a horizontal staining tray and immersed in 1 mL of acid denaturant solution for 7 minutes. After this period, the excess acid solution was drained, 1 mL of lysis buffer was added, and the slide was incubated at room temperature for 10 minutes. The slide was washed gently with 20 mL of distilled water to remove the lysis buffer, and sequential dehydration steps were performed using dehydrating solutions 1, 2, and 3 (provided in the kit). The slides were air-dried and stained using the staining solution provided with the kit (working stain prepared: 300 µL of stain+100 µL of dilution buffer), 250 µL of which was used to stain each slide. The slides were incubated for 5 minutes, air-dried, and examined under ×400 magnification using a light microscope (CH20i; Olympus, Tokyo, Japan). Each slide was scored for 200 spermatozoa by three lab technicians. Sperm cells with absent or small halos (≤1/3 of the head width) were counted as sperm with fragmented DNA; otherwise, they were considered normal sperm. The means and standard deviations were calculated.

Low-melting-point agarose (1%), which was pre-provided in a microcentrifuge tube, was placed in a float in boiling water (90°C–100°C) for 5 minutes and then transferred to a 37°C water bath for equilibration. An aliquot of fresh or flash-frozen semen sample was added to this tube of melted agarose in order to achieve a final sperm concentration of 5–10 million/mL. A drop of 10–15 µL of this agarose-semen sample mixture was pipetted onto the pre-treated slide and covered with a coverslip (18×18 mm or 22×22 mm), the edges of which were pressed gently to obtain an uniform distribution of the gel on the slide. The slide was then incubated at 4°C for 5 minutes. The coverslips were carefully removed, and the slides were immediately placed on a horizontal staining tray. An acid denaturant solution was freshly prepared (80 µL of the acid denaturant+10 mL of distilled water, provided in the kit), added to the gel, and allowed to react for 7 minutes. On completion, the slides were placed on another tray of lysis buffer and incubated at room temperature for 25 minutes. The slides were washed with abundant distilled water to completely remove the lysis solution and were then incubated for 5 minutes. Following this, the slides were sequentially dehydrated using 70%, 90%, and 100% ethanol (Changshu Hongsheng Fine Chemical, Jiangsu, China) for 2 minutes each, respectively. The slides were air-dried and stained using Diff-Quik Stain (Cell Life Ref: CL06; Cell Life, Visakhapatnam, India). First, azure A (eosin-red) stain was added and incubated for 7 minutes, and azure B (nigrosin-blue) stain was added and incubated for 7 minutes. The slides were incubated for an additional 5 minutes, air-dried, and examined under ×400 magnification using a light microscope (CH20i, Olympus). Each slide was scored for 500 spermatozoa by three lab technicians. Sperm cells with absent or small halos (≤1/3 of the head width) were counted as sperm with fragmented DNA; otherwise, they were considered normal sperm. The means and standard deviations were calculated.

The CV was calculated for the triplicate readings from SCD and the duplicate readings from SCSA using the formula ([standard deviation/mean]×100). The DFI value was used as an indicator of sperm quality, namely excellent (<15%), good to fair (15%–25%), fair to poor (>25%–50%) and very poor (>50%). The specific statistical tests used to determine significant differences (p<0.05) are mentioned in the respective parts of the Results section.

The DFI derived from SCSA was significantly lower than the DFI derived using the CANfrag SCD kit (Wilcoxon test, p<0.001) (Table 1). Additionally, in the correlation analysis between SCSA and the SCD kits, both SCD kits had a strong significant correlation with the SCSA kit (Spearman rank tests, p-values in Table 1). Of the patients tested, 29 had their sperm analyzed using all three tests. Two-way analysis of variance (ANOVA) revealed that DFI was the lowest when determined using SCSA and was slightly greater when determined using either Halosperm or CANfrag (Figure 2). The median DFI levels were 19.3%, 24.7%, and 29% for SCSA, Halosperm and CANfrag, respectively, and the level associated with CANfrag was significantly higher than those for SCSA or Halosperm (two-way ANOVA, p<0.0001) (Figure 2).

We calculated the CVs of the replicates for each patient to determine the technical repeatability of the tests. The CVs ranged from 0%–18% (mean, 4.7%) for SCSA, 2%–54% (mean, 24.2%) for CANfrag and 7%–62% (mean, 27.2%) for Halosperm. The CV associated with SCSA was significantly lower than the CVs associated with CANfrag or Halosperm (repeated-measures ANOVA and Bonferroni post-hoc test, p<0.001). When an arbitrary cut-off value of 10% was set for CV, we observed that 30 of 36 patients (83.3%) for CANfrag, 29 of 31 (93.5%) for Halosperm, and 4 of 41 (9.7%) for SCSA would require repetition of the test. The mean DFI, along with its 95% confidence interval, was calculated for each test (Figure 3). Outside lines closer to the mean indicate higher repeatability of the test.

Since DFI informs the choice of ART procedure, we compared DFI and the subsequent ART procedure chosen in 29 patients when SCSA, Halosperm or CANfrag was used. A heatmap representing the classification of the sperm into four categories of sperm potential showed a striking difference in the number of patients presenting with a higher DFI and lower fertility potential when an SCD kit was used (Figure 4A). The clinical management plan for each of these patients was further analyzed. This comparison revealed that if the SCSA testing method had been used for assessing sperm damage, and 25% DFI was set as the cutoff above which IVF/ICSI was selected instead of in vivo or intrauterine insemination, clinicians would have selected only nine of 29 patients for IVF/ICSI when using SCSA as opposed to 14 of 29 and 22 of 29 when using Halosperm and CANfrag, respectively (chi-square test, p=0.003) (Figure 4B).