Differences in generation of reactive oxygen species (ROS) in highly motile fractions of spermatozoa as a discriminating tool for semen samples from infertility patients

Eyup Hakan Duran; Mahmood Morshedi; Steven Taylor; Sergio Oehninger

doi:10.1016/S0015-0282(02)04159-6

Objective: To assess the differentiating ability of ROS generated by highly motile fractions of spermatozoa in samples of donors and patients, and to determine interrelation among semen parameters, ROS, sperm DNA denaturation, fragmentation and phosphatidylserine externalization. Design: Prospective study. Materials/Methods: Ten fertile donors and 66 infertility patients were evaluated. Following computer assisted evaluation of basic semen parameters, each sample was subjected to a density gradient separation using 90% and 45% Percoll layers. The 90% fraction containing motile sperm was further mixed with paramagnetic beads coated with anti CD45 Ab to remove possible contaminating white blood cells. Generation of ROS in this fraction was determined by chemiluminescence as previously described (Biol Reprod, 1989;40:183–97), using luminol as the probe. Briefly, suspensions of spermatozoa at 10 × 106/mL in a volume of 400L were exposed to 4L of luminol and 12.4 U horseradish peroxidase. Chemiluminescence was monitored as counts integrated over a 10-min period and results were expressed as the difference between post-luminol and pre-luminol conditions. A second chemiluminescence reading was performed after the addition of N-formyl-L-methionyl-L-leucyl-L-phenylalanine, to verify that the samples were free of contamination (Fertil Steril, 1992; 57:1317–25). Motile sperm fractions from donors as well as those of patients with high ROS levels (>20 × 104 cpm, n=24) were evaluated further for phosphatidylserine externalization (annexin V staining with annexin V and 6-CF), DNA denaturation (acridine orange) and fragmentation (In Situ Cell Death Detection Kit, TUNEL) as previously described (Fertil Steril,1984; 42:87–91, Hum Reprod,2000; 15:1338–44). Statistical analysis was done to compare various groups by one way ANOVA with post hoc Bonferroni correction and Mann-Whitney U test. Results: Samples from patients could be grouped into high and low ROS levels. Patients in the high ROS group had significantly higher levels of ROS when compared to both donors and the low ROS group (67,000/20,000/8,700 cpm respectively, p <0.001). ROS levels in donors were similar to those of low ROS patients. Semen parameters from patient samples with high or low ROS were not significantly different. Various semen parameters of donor samples were significantly higher than those of the patients (p <0.01). Nonetheless, the motile fraction of donor semen samples had ROS levels similar to the patient group with low ROS. Patients with elevated ROS levels had higher magnitudes of DNA denaturation (54% vs 33%, p <0.05), DNA fragmentation (14% vs 3%, p <0.05) and phosphatidylserine externalization (21% vs 9%, p <0.01) than those of donors. Conclusions: ROS determination could distinguish a group of semen samples from infertility patients that is not identifiable by routine means of semen analysis. A single underlying mechanism may exist to increase ROS production, DNA denaturation/fragmentation and phosphatidylserine externalization.

Differences in generation of reactive oxygen species (ROS) in highly motile fractions of spermatozoa as a discriminating tool for semen samples from infertility patients

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