Label-Free Enrichment of Live Sperm Using Contactless Dielectrophoresis in Human and Bovine Samples

Abstract

Efficient selection of live sperm is critical for the success of assisted reproductive technologies (ART) in both livestock and human reproduction, yet conventional preparation methods such as centrifugation and density gradients can introduce mechanical stress and reduce the number of intact cells. This study evaluated contactless dielectrophoresis (cDEP), a label-free microfluidic technique, for enrichment of live sperm in frozen-thawed bovine and human semen. For bovine samples, semen from six ejaculates was processed in synthetic oviductal fluid fertilization medium (SOF-FERT) and CytoBuffer™ on a Cyto R1™ platform equipped with a HOAPES microfluidic chip known as CytoChip D (CytoRecovery, Blacksburg, VA). Human semen samples from three donors, with multiple sorting runs per donor, were processed in InVitroCare Sperm Wash (InVitroCare Inc., Frederick, MD) and CytoBuffer™ and sorted on the same platform under identical operating conditions. Sorting for both species was performed at 578 Vpp and 500 kHz for 30 min at a flow rate of 2 μL/min, maintaining buffer conductivity below 100 μS/cm. Post-sorting outlet fractions (collected, control, and waste) were stained with Calcein-AM and SYTOXTM Deep Red (Invitrogen, Thermo Fisher Scientific, Waltham, MA) to assess membrane integrity using fluorescence microscopy, followed by manual quantification using a hemocytometer. In bovine samples, the cDEP-collected fraction (COL) contained a greater proportion of live sperm than the unsorted control (CON; mean = 62% vs. 27% respectively; P = 0.029). Differences among outlet types were evaluated using one-way analysis of variance (ANOVA), followed by Tukey's test for pairwise comparisons. In human samples, the cDEP-collected fraction (COL) contained a greater proportion of live sperm than the unsorted control (CON; mean = 73.6% vs 27.2% respectively, P < 0.001). Differences among outlet types were evaluated using a linear mixed-effects model accounting for repeated measures within donor. These findings indicate that cDEP can effectively enrich live sperm samples and support its potential as a gentle and reproducible front-end selection step for ART.