Twenty-six male partners of candidate couples for intracytoplasmic sperm injection (ICSI) were involved in this prospective study at the Ibn Rushd Center of Reproductive Medicine of Constantine, Eastern Algeria. The necessary precautions were taken to protect the participants, according to the principles of the Declaration of Helsinki. Informed written consent was obtained from all of the patients.

The men's average age was 37.50±0.88 years and infertility dated back to at least one year prior (6.04±0.54 years). It was primary in 88.46% of cases and secondary in the remaining 11.54% cases. The etiology of infertility varied among oligospermia, asthenospermia, and teratospermia. The patients had no history of radiotherapy, chemotherapy, chronic illness, or varicocele. Only four smokers were recorded.

Semen samples were collected in sterile containers by masturbation after 3 to 5 days of sexual abstinence. After semen liquefaction at room temperature for at least one hour, density gradient centrifugation (DGC) was carried out. Briefly, 100 µL of PureSperm™ buffer (Nidacon International AB, Molndal, Sweden) were added to 900 µL of 100% PureSperm™ medium to obtain 1,000 µL of 90% PureSperm™. Five hundred fifty microliters of PureSperm™ buffer were added to 450 µL of 100% PureSperm™ medium to obtain 1,000 µL of 45% PureSperm™. One milliliter of the liquefied semen was then added on top of two layers of gradients (90% and 45%), the whole was centrifuged at 300 g for 25 minutes, and the supernatant was removed to add 200 µL of washing solution: FertiCult™ Flushing medium (FertiPro N.V, Beernem, Belgium). After centrifugation at 500 g for 10 minutes, the washing solution was aspirated and a culture medium (FertiCult™ IVF medium) was added to the pellet. An aliquot of unprocessed semen was kept in order to be freshly used for SCD assessment in a subsequent stage.

Standard semen parameters (volume, concentration, motility, and morphology) were evaluated according to the WHO guidelines [11].

The concentration was assessed by examining samples using phase-contrast microscopy at a final magnification of 200× or 400× in a Makler counting chamber. A drop of 10 or 20 µL of diluted semen was introduced into the Makler chamber and covered with a coverslip. The patient was considered oligospermic if the sperm count was below 20 million/mL.

Mobility was evaluated using a simple grading system. At least five microscopic fields were assessed systematically to classify 200 SPZ. The motility of each SPZ was graded according to whether it showed rapid progressive motility (denoted as a); slow or sluggish progressive motility (b); non progressive motility (c), or immobility (d). The percentage of a+b was calculated, and asthenozoospermia was diagnosed if this percentage was less than 50%.

Smears were prepared for morphological evaluation, Papanicolaou stained, and finally assessed according to David's classification [12]. We used the cut-off of 30% to assess normality of sperm morphology [12].

DNA fragmentation was assessed by SCD test [13]. In the absence of massive sperm DNA breakage and following acid denaturation and removal of nuclear proteins, dispersed DNA loops produce a characteristic halo. Sperm with fragmented DNA does not develop such a halo, or it is small. A Halosperm® kit (Halotech DNA SL, Madrid, Spain) was used following these steps: Semen was diluted in culture medium to obtain a maximum concentration of 20 million SPZ per milliliter. Agarose (100 µL) was fluidized for 5 minutes in water at 95℃ to -100℃; then the temperature was equilibrated at 37℃ for 5 minutes. Fifty microliters of prepared sperm were added to agarose and gently mixed at 37℃. A drop of 8 µL of this cell suspension was then deposited on the treated side of a slide and covered with a glass coverslip. The slide was placed on a cold surface and put in the fridge at 4℃ for 5 minutes. After removing the coverslip, it was immersed in the acid denaturing solution for 7 minutes, then incubated in a lysis solution for 25 minutes. Next, the slide was rinsed in abundant distilled water for 5 minutes and fixed with 70% ethanol, then with 100% ethanol, each time for 2 minutes. After drying, the slide was stained by the Diff-Quik reagent. The visualization was done under a bright field light microscope (MOTIC B1 Series) with ×20 and ×40 objectives. We counted 300 to 500 SPZ while identifying those with DNA fragmentation according to manufacturer instructions [14] and then calculated the DNA fragmentation index (DFI) as: We chose to fix the DFI threshold at 18% to distinguish between two groups of patients: a high fragmentation group (HFG: DFI>18%) and a low fragmentation group (LFG: DFI ≤18%). This threshold was used by other authors, who have indicated that SDF levels above 18%, as measured by SCD, are not compatible with the initiation and maintenance of a term pregnancy [15]. The mean age of patients in HFG and LFG were 37.84±1.13 years and 36.57±1.17 years, respectively, without a significant difference.

GraphPad Prism ver. 6.00 for Windows (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. To assess the relationships between DNA fragmentation and semen parameters, we calculated the Spearman's correlation coefficient. The Mann-Whitney test was used to compare quantitative parameters between HFG and LFG. Fisher's exact test was used to detect the difference in the morphologically normal SPZ frequency between the two study groups. The data are presented as mean±standard error in a 95% confidence interval (95% CI), and statistical significance was set at 0.05. All reported p-values are from two-sided tests.

The mean sperm concentration was 15.46±15.02× 10⁶/mL (median, 4.5×10⁶/mL) and was distributed as follows: <5×10⁶/mL in 50%; between 5 and 20×10⁶/mL in 27%; and ≥20×10⁶/mL in 23% of the men. The mean sperm motility was 46.14%±3.27%, and 27% of the men had a sperm progressive motility ≥50%. We used the cutoff of 30% (David's classification) to assess the normality of sperm morphology. In our series, 50% of the patients had 30% or more SPZ with normal morphology in their semen. Finally, the mean semen volume in all patients was 2.85±0.26 mL.

The mean DFI in our sample was 25.55%±2.49%. Among the 26 men, 19 (i.e., 73.07%) had a DFI>18%; they represent the HFG. In this group, the mean DFI was 31.22%±2.13%, while it was 10.14%±2.28% in the LFG (DFI ≤18%).

We did not find any correlation between sperm volume and DFI (r=0.24; p=0.25) (Table 1), and when comparing mean sperm volume between LFG and HFG, we did not find a significant difference (2.37±0.51 mL vs. 3.03± 0.29 mL, respectively; p=0.22; Table 2).

We found a significant negative correlation between DFI and sperm concentration (r=-0.65; p=0.0003) (Table 1, Fig. 1A). This is akin to the significant difference between mean sperm concentration of LFG and HFG (37.57± 13.16×10⁶/mL vs. 7.32±3.59×10⁶/mL, respectively; p=0.007; Table 2).

Sperm motility was negatively correlated with DFI (r= -0.45; p=0.03) (Table 1, Fig. 1B). However, even if mean sperm motility of the LFG seemed to be more important than in the HFG, the difference did not reach a significant level (54.17%±7.90% vs. 43.13%±3.26%; respectively, p=0.19; Table 2).

Plotting DFI with the percentage of normal SPZ in each sample did not illustrate any correlation between the two parameters (r=-0.18; p=0.68) (Table 1, Fig. 1C). According to this, there is also no significant difference in typical SPZ forms between LFG and HFG (29.50±4.50 vs. 38.14±7.00, respectively; p=0.78; Table 2). It is worth noting that 63% of patients in HFG had at least 30% morphologically normal SPZ. Even if this proportion was inferior to that of LFG (i.e., 85.71%), belonging to HFG or LFG did not correlate with the frequency of morphologically normal SPZ, distinguished as either ≥30% or <30% (odds ratio=2.77, p=0.62, 95% CI [0.27~28.41]).