Improvement in sperm dna fragmentation index is observed at 15 minutes incubation time but not with longer incubation times using the microfluidics sperm sorting system

To evaluate the effect of incubation times on Sperm DNA Fragmentation Index (DFI) using the Microfluidics Sperm Sorting (MFSS) system. A total of 180 semen samples which were being processed by MFSS for Intra Cytoplasmic Sperm Injection (ICSI) underwent DFI testing on the raw sample (n=180) and post incubation at 15 minutes (n=145) and 30 minutes (n=63) in the the ZyMōt Multi (850μL) Sperm Separation Device). A total of 100 sperm were analyzed per sample. DFI testing was performed by the Terminal deoxynucleotidyl transferase (TdT) mediated dUTP Nick End Labelling (TUNEL) method with the in-situ cell death detection kit (Roche) using fluorescein and DAPI stains, pre and post treatment. The DFI showed a significant improvement after sperm sorting for 15 mins (presorting mean = 11.86%, post sperm sorting mean = 5.08% (p<0.001). When sperm from the same incubated samples were analysed at 30 minutes (n=.63), the DFI showed no significant improvement after sperm sorting for 30 mins, presorting mean = 13.5% normal, post sperm sorting mean = 14.9% normal (p>0.05). In fact it showed a slight rise in the number of Sperm with higher DFI after incubating for 30 mins. This study suggests that sperm sorting using MFSS may be helpful in obtaining sperm with less DNA fragmentation after 15 mins of incubation. It appears that incubating sperm for greater than 15 minutes does not yield sperm with low DFI. In fact increasing the time interval allows sperm with higher DFI to enter the outlet. More studies are needed to understand the time of incubation to allow for the optimal yield of sperm with low DFI.