The effect of age on the expression of apoptosis biomarkers in human spermatozoa  Alinne Colin, M.D., Gerardo Barroso, M.D., M.Sc., Nardhy Gómez-López, Ph.D., E. Hakan Duran, M.D., Sergio Oehninger, M.D., Ph.D.  Fertility and Sterility  Volume 94, Issue 7, Pages 2609-2614 (December 2010) DOI: 10.1016/j.fertnstert.2010.04.043 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Detection of sperm phosphatidylserine (PS) translocation (early apoptotic marker) according to man's age. (A) Representative plot and histogram showing the gating strategy and parameters used for the flow cytometry analysis. Annexin-V fluorescein isothiocyanate conjugate (FL1-H [530/30 filter - height of fluorescence intensity])/IP uptake (FL2-H [585/42 filter - height of fluorescence intensity]). (Right) 65.9% represents viable cells [Annexin-V(-)/IP(-)], 29.33% represents cells in early apoptosis [Annexin-V(+)/IP(-)], 4.67% represents necrotic cells [Annexin-V(+)/IP(+)] and 0.1% represents necrotic old cells [Annexin (-)/IP(+)]. (Left) Histogram represents all the positive cells by Annexin-V, necrotic and apoptotic cells. (B) Diagrammatic representation of the effect of age on PS translocation (Annexin-V(+)/IP (-) cells) according to stratified age groups (∗P = .01 by ANOVA). (C) Positive correlation was demonstrated between male age and PS translocation (Annexin-V(+)/IP (-) cells) (r = 0.50, P<.01). Fertility and Sterility 2010 94, 2609-2614DOI: (10.1016/j.fertnstert.2010.04.043) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Detection of sperm DNA fragmentation (late apoptotic marker) according to man's age. (A) Representative plot and histogram showing the gating strategy and parameters used for the flow cytometry analysis. (Right) 88.4% represents viable cells [DNA(+)/IP(-)], 8.4% represents cells in late apoptosis [DNA(+)/IP(+)]. (Left) 0.1% and 3.1% represent unstained cells. (B) Diagrammatic representation of the effect of age on DNA breaks [DNA(+)/IP(+)]. There were no statistical differences between groups, but a trend showing increased DNA fragmentation with age is evident. Fertility and Sterility 2010 94, 2609-2614DOI: (10.1016/j.fertnstert.2010.04.043) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions