Density Gradient Centrifugation Alone or the Combination of DGC with Annexin V Magnetic-Activated Cell Sorting Prior to Cryopreservation Enhances the Postthaw Quality of Sperm from Infertile Male Patients with Poor Sperm Quality

Objective. To examine whether density gradient centrifugation (DGC) alone or its combination with annexin V magnetic-activated cell sorting (DGC-MACS) can be used to process semen samples from infertile male patients with poor sperm quality prior to subjecting these to freeze/thaw process in order to optimize the outcomes of sperm freezing. Methods. This study enrolled sixteen patients with sperm $\text{concentration}\ge 20\times {10}^6/\text{mL}$ , sperm $\text{motility}<30$ %, and/or <4% normal sperm morphology. Sperms were processed by DGC or DGC-MACS prior to the freeze/thaw process. Sperm motility, hyperosmotic swelling test (HOS), TUNEL test, and morphological analysis were performed before and after the freeze/thaw process. Results. The freeze/thaw process had a detrimental effect on sperm motility, viability, morphology, and DNA integrity in all three groups (RAW, DGC, and DGC + MACS groups). The DGC and DGC + MACS groups showed increased sperm motility, viability, and normal morphology following freeze/thaw than untreated frozen controls. The motility and viability were not significantly different between DGC-MACS-CPT (cryopreservation-thawing) and DGC-CPT groups. Moreover, almost no grade A or grade B sperm was observed in the DGC-MACS-CPT groups. The sperm selected by DGC or DGC + MACS showed decreased levels of sperm DNA fragmentation than RAW samples following freeze/thaw. Moreover, the sperm DNA fragmentation following freeze/thaw in the DGC-MACS-CPT group was significantly lower than that in the DGC-CPT group. Conclusions. Sperm preparation by DGC before cryopreservation improved the quality of sperm postthaw in infertile males with poor sperm quality. If the sperm quality following freeze/thaw is foreseen to be insufficient for artificial insemination with husband’s sperm or in vitro fertilization, or if there is high DNA fragmentation in RAW sperm, DGC + MACS should be used prior to cryopreservation to reduce sperm DNA fragmentation and improve the quality of sperm available for intracytoplasmic sperm injection.