To evaluate the sperm chromatin decondensation (SCD) rates of the annexin-negative (nonapoptotic) sperm fraction of patients with infertility using hamster intracytoplasmic sperm injection (H-ICSI). In healthy donors, the depletion of apoptotic sperm using annexin V-based magnetic-activated cell separation (MACS) enhances hamster oocyte sperm penetration but does not increase SCD rates following H-ICSI.A prospective-controlled study.Male infertility clinic, European Academy of Andrology Center Leipzig.Twenty-one male infertility patients with subnormal spermiogram.Spermatozoa were separated by Annexin V-MACS.Apoptosis signaling (disruption of transmembrane mitochondrial potential, transmembrane mitochondrial potential [TMP], and activation of caspases-3 [CP3]) and SCD rates of human spermatozoa after hamster intracytoplasmic sperm injection.Infertility patients showed high levels of sperm with active CP3 and disrupted TMP, which correlated negatively with SCD rates. Annexin V-MACS resulted in a significant enrichment of spermatozoa with inactive CP3 and intact TMP in the annexin-negative fraction. Similarly, annexin-negative sperm had the highest SCD rates following H-ICSI compared with controls and annexin-positive sperm.These results suggest that nonapoptotic spermatozoa prepared by annexin V-MACS display higher early fertilization potential following ICSI. The technique should be evaluated in a clinical setting for its impact on ICSI outcomes in patients diagnosed with infertility.