Effects of Semen Storage and Separation Techniques on Sperm DNA Fragmentation

Objective: To determine the effect of semen storage and separation techniques on sperm DNA fragmentation.

Design: Controlled clinical study.

Setting: An assisted reproductive technology laboratory.

Patient(s): Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation.

Intervention(s): One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD.

Main Outcome Measure(s): DNA fragmentation as measured by SCD.

Result(s): There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels.

Conclusion(s): The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use.