Comparative Efficacy of Swim-Up, Density-Gradient Centrifugation, and Microfluidic Sorting in Sperm Preparation, and the Impact on Motility, Morphology, and DNA Integrity.

 Reactive oxygen species (ROS) are considered a major factor contributing to sperm DNA damage during sperm preparation for assisted reproductive technologies (ART). This study aimed to investigate whether microfluidic sorting can select sperm with a low DNA fragmentation index (DFI) and to explore the underlying mechanisms. We compared the effects of three sperm preparation methods-swim-up, density-gradient centrifugation, and microfluidic sorting-on sperm quality and DNA integrity.  Semen samples from 12 patients were divided into three equal portions and processed using swim-up, density-gradient centrifugation, and microfluidic sorting techniques. Sperm concentration, motility, morphology, DFI, intracellular H2O2 levels, and mitochondrial O2⁻ levels were measured and compared across the three methods. Additionally, DFI was assessed in both fresh and frozen-thawed sperm samples.  Sperm prepared using microfluidic sorting exhibited significantly higher total motility (85.3 ± 3.2%) and progressive forward motility (72.5 ± 2.8%) compared to density-gradient centrifugation (total motility: 70.1 ± 3.5%; progressive motility: 58.4 ± 3.1%). Microfluidic sorting also resulted in a significantly lower DFI (8.2 ± 1.5%) compared to density-gradient centrifugation (25.6 ± 2.3%) and swim-up (15.4 ± 1.8%). Intracellular H2O2 levels were similar across all methods, but mitochondrial O2⁻ levels were significantly lower in microfluidic-sorted sperm (12.3 ± 1.2%) compared to fresh semen (20.5 ± 1.8%). After cryopreservation, sperm prepared by microfluidic sorting and swim-up maintained lower DFI levels (10.5 ± 1.6% and 14.8 ± 1.9%, respectively) compared to density-gradient centrifugation (28.3 ± 2.5%).  Microfluidic sorting is an effective method for selecting sperm with higher motility, normal morphology, and lower DFI, while also reducing mitochondrial O2⁻ levels. This method shows promise for improving sperm quality and DNA integrity, particularly in the context of ART and cryopreservation. Further clinical studies are needed to validate these findings and explore the long-term implications of microfluidic sorting in ART procedures.