Isolation of spermatozoa with low levels of fragmented DNA with the use of flow cytometry and sorting  Sofia C. Ribeiro, Ph.D., Gideon Sartorius, M.D., Flurina Pletscher, M.Sc., Maria de Geyter, Ph.D., Hong Zhang, Ph.D., Christian de Geyter, M.D.  Fertility and Sterility  Volume 100, Issue 3, Pages 686-694.e4 (September 2013) DOI: 10.1016/j.fertnstert.2013.05.030 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Typical distribution of the YO-PRO–Hoechst–stained spermatozoa in FACS, as exemplified by sample no. 31. The upper plots (A and B) show the washed ejaculate sample before the sorting. (A) 355 nm fluorescence dot plot produced by the Hoechst dye; (B) 530 nm fluorescence dot plot produced by YO-PRO. The selection of the spermatozoa to sort was made by first gating the cells in P1 and then P2 (nonvital) or P3 (vital). (C) Vital spermatozoa population after sorting; (D) YO-PRO–Hoechst staining of the swim-up population. For the analysis, 10,000 events were acquired. P4 represents the nonviable population caused by the sorting procedure. Fertility and Sterility 2013 100, 686-694.e4DOI: (10.1016/j.fertnstert.2013.05.030) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Comparison of the number of spermatozoa with fragmented DNA, as determined by terminal deoxynucleotide transferase–mediated dUTP nick-end labeling (TUNEL), in the viable population sorted with fluorescence-activated cell sorting (FACS) and in the population after swim-up. In four samples the swim-up preparation failed to provide sufficient numbers of spermatozoa for TUNEL. The number of spermatozoa with fragmented DNA after swim-up was significantly higher than after sorting with FACS (P<.0001). Fertility and Sterility 2013 100, 686-694.e4DOI: (10.1016/j.fertnstert.2013.05.030) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Comparison of the relative number of spermatozoa susceptible to death, as determined by the Live/Dead Fixable Far Red kit in the viable population sorted with fluorescence-activated cell sorting (FACS) and the population after swim-up. The number of spermatozoa susceptible to death after swim-up was significantly higher than after sorting with FACS (P<.0001). Fertility and Sterility 2013 100, 686-694.e4DOI: (10.1016/j.fertnstert.2013.05.030) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Overview of experimental steps: (A) Evaluation of different staining strategies to distinguish between apoptotic/dead and viable spermatozoa. PI = propidium iodide. (B) Comparison between fluorescence-activated cell sorting (FACS) and swim-up for the preparation of semen samples. Hoechst+/YOPRO+ = spermatozoa population positively stained with Hoechst and YO-PRO (apoptotic and dead); Hoechst+/YOPRO− = spermatozoa population positively stained with Hoechst and unstained by YO-PRO (viable). Fertility and Sterility 2013 100, 686-694.e4DOI: (10.1016/j.fertnstert.2013.05.030) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Correlation between the results obtained by conventional semen analysis or sperm computer analysis regarding the spermatozoa concentration. Fertility and Sterility 2013 100, 686-694.e4DOI: (10.1016/j.fertnstert.2013.05.030) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 (A) With the use of simple regression analysis, the number of swim-up spermatozoa with fragmented DNA, as determined by terminal deoxynucleotide transferase–mediated dUTP nick-end labeling (TUNEL), correlated significantly with those susceptible to apoptosis, as given by the Live/Dead Fixable Far Red kit (correlation coefficient 0.762; P<.0001). (B) Furthermore, the number of spermatozoa susceptible to death significantly exceeded those with fragmented DNA (P<.0001). Fertility and Sterility 2013 100, 686-694.e4DOI: (10.1016/j.fertnstert.2013.05.030) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions