Biological properties of mouse spermatozoa separated by using the microfluidic sperm sorter

Abstract

The microfluidic sperm sorter (MFSS) is a novel device that is able to separate spermatozoa with high motility and low DNA fragmentation from raw semen without washing and centrifugation process. However, some biological properties and effect on embryonic development of spermatozoa separated by the MFSS are as yet unexplained. The purpose of this study was to compare and investigate the biological properties and the developmental ability between spermatozoa separated by the MFSS and the swim-up (SU) procedure. Motility parameters and mitochondrial membrane potential (MMP) of spermatozoa prepared by MFSS and SU were studied. Cleavage and blastocyst rate were compared between embryos with spermatozoa separated by MFSS and those with spermatozoa processed by SU. Spermatozoa derived from C57BL/6J mouse were divided into two aliquots. One aliquot was processed by the MFSS and separated into high motility (HM) and low motility (LM) spermatozoa; the second aliquot was centrifuged (1800rpm, 10min) and processed by the swim-up procedure for 20-30 min (SU). Sperm motility parameters of three groups were analyzed by using the sperm motility analyzing system (SMAS). MMP was evaluated with the flow cytometer after JC-1 staining. In addition, spermatozoa of each group were injected into ovulated MII oocytes. After 3.5 hours from ICSI, a part of fertilized oocytes derived from each group was stained immunohistochemically with gamma H2AX antibody to detect DNA damage, and the others were cultured until to the blastocyst stage. By the SMAS analysis, the motile sperm rate was 90.5%, 47.8%, and 89.5% for HM, LM, and SU, respectively. The average speed of HM was significantly higher than that of LM and SU (HM: 116.8±8.6 μm/sec, LM: 68.2±8.2, SU: 83.1±4.1, p<0.05). MMP of spermatozoa in HM and SU were significantly higher than that of LM. After ICSI, there was no significant difference in the rate of pronuclear formation between each group. By immunohistochemical staining, high positive rate of gamma H2AX was observed in male pronucleus of embryos with LM spermatozoa. In addition, the blastocyst rate of embryos with HM was significantly higher than that of LM and was comparable to that of SU (HM: 84.3%, LM: 56.1%, SU: 79.7%, p<0.05). In this study, we demonstrated that spermatozoa with high sperm motility and developmental ability were able to separate by using the MFSS. By optimizing the MFSS system, the most appropriate spermatozoa for IVF/ICSI would be provided efficiently.