Isolation of spermatozoa with low levels of fragmented DNA with the use of flow cytometry and sorting

OBJECTIVE: To develop a method to select spermatozoa with low DNA fragmentation rates for assisted reproduction technologies (ART). DESIGN: Multistep prospective cohort study. SETTING: University hospital. PATIENT(S): Semen samples of 34 infertile men were prepared in parallel with swim-up and fluorescence-activated cell sorting (FACS), and 11 semen samples were used for testing the staining strategy. INTERVENTION(S): Flow cytometric sorting of YO-PRO-stained spermatozoa. MAIN OUTCOME MEASURE(S): Assessment of recovery of spermatozoa and purity after sorting, quantification of sperm DNA fragmentation and viability after sorting and after swim up preparation. RESULT(S): Staining with YO-PRO could be performed successfully in regular culture medium, both dead and apoptotic spermatozoa were labeled without the dye entering the viable spermatozoa. Compared with the conventional swim-up method, the sorted viable population showed a significantly reduced number of spermatozoa with fragmented DNA. CONCLUSION(S): A novel method has been developed, which not only might improve the outcome of ART, but can also help to clarify the ongoing controversy of the role of DNA fragmentation in male infertility.