Sequencing Matters: Comparative Effectiveness of Density-Gradient Centrifugation and MACS for Enhancing Sperm Quality and DNA Integrity

This systematic review of 25 original studies examined the efficacy of density gradient centrifugation (DGC) combined with magnetic-activated cell sorting using Annexin V (MACS) for improving sperm quality in infertile men undergoing ICSI. The evidence consistently demonstrates that DGC-MACS significantly reduces sperm DNA fragmentation, with reported reductions ranging from 2.82% to 21.9% in absolute terms, and relative reductions of 39%-83%. The combination of DGC followed MACS achieved superior outcomes compared to either technique alone, reducing DNA fragmentation index (DFI) 4.1 to ± 1.3% compared with 8.1 ± 4.1% for DGC alone and 7.4 ± 3.9% for MACS alone. The treatment improved sperm motility, membrane integrity and overall spermatozoa health by reducing protamine deficiency and chromosomal abnormalities. Clinical results in ICSI cycles showed that although fertilization rates were similar between the treated and control groups, DGC followed by subsequent MACS treatment significantly improved embryo quality (72.5% vs. 51.47% top-quality day-3 embryos), blastocyst formation rate (69.69% vs. 48%), pregnancy rates (60.7% vs. 51.5%, p=0.014), and live birth rates (47.4% vs. 31.2%, p=0.001) with a reduced miscarriage rate (14.7% vs. 20.6%, p=0.034). The technique proved most beneficial in patients with high baseline DNA fragmentation (≥30%) and in those with asthenozoospermia or asthenoteratozoospermia. Studies suggest, and consistently supported by our study results, that performing DGC before MACS (DGC-MACS) may yield superior results compared to the reverse sequence. This is because DGC leads to a primary separation of sperm based on density, motility, and morphology and thus producing a high-density fractions enriched in morphologically normal sperms. MACS by specifically binding to phosphatidylserine (PS) residues on sperm membranes selectively removes PS‑positive/apoptotic sperm in a subsequent purification step. Overall, the literature strongly supports the use of combined DGC-MACS as an effective sperm preparation technique for ICSI in infertile men with elevated sperm DNA fragmentation.