Sequential Sperm Population Enrichment for ICSI: DGC-MACS versus MACS-DGC- A Hypothesis-Generating Perspective

Abstract Intracytoplasmic sperm injection (ICSI) requires selecting a suitable spermatozoon from an adequate pool of candidates. The order of density gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) may influence the characteristics of the recovered sperm population, but its clinical significance remains uncertain. This commentary hypothesizes that DGC followed by MACS (DGC-MACS) may offer practical advantages over the reverse sequence (MACS-DGC) when sperm availability is sufficient. DGC enriches motile, apparently morphologically normal spermatozoa while reducing debris and other cells; subsequent MACS enriches the population for spermatozoa without detectable externalized phosphatidylserine (PS). However, PS negativity does not establish complete functional competence, and DGC has not been validated as a measure of intrinsic sperm stress resistance. We therefore discuss membrane sialidase activity and mitochondrial membrane potential (MMP) as illustrative, exploratory biomarkers of complementary functional domains. Their assessment is proposed in parallel research aliquots, not for real-time selection of individual spermatozoa for ICSI. We also introduce a provisional PS-negative Enrichment Ratio (PSNER) based on the relative proportions of PS-negative spermatozoa before and after DGC-MACS, while acknowledging its dependence on processing conditions, baseline semen quality, and selective recovery. Direct randomized comparisons of DGC-MACS and MACS-DGC, with live birth as the primary outcome, alongside validation of the proposed biomarkers and index, are needed before clinical implementation. Lay Summary In some fertility treatments, a specialist helps fertilization by injecting a single sperm directly into an egg. Before this step, laboratories use different methods to prepare sperm samples and increase the chances of selecting a suitable sperm. Two methods can be used together. One separates sperm from unwanted cells and other material, while the other helps remove sperm showing certain changes on their surface. However, it is not yet clear whether the order in which these methods are performed affects the chances of having a baby. In this paper, we suggest that using the first method before the second may offer practical advantages when enough sperm are available. We also propose studying additional signs of sperm health and developing a new way to describe changes in sperm samples during preparation. These ideas have not yet been proven to improve fertility treatment outcomes. Further studies are needed to compare the two methods in different orders and determine whether either approach increases the chances of having a baby.