Measuring apoptosis in human spermatozoa: a biological assay for semen quality?

Objective: [1] To determine whether apoptosis can be measured in ejaculated spermatozoa by flow cytometry using the Annexin V assay, which measures ex pression of phosphatidylserine on the outer leaflet of the cell membrane, o r the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP [deoxy-uri dine triphosphate] nick end labeling) assay, which measures occurrence of D NA strand breaks and [2] to correlate the outcome with routine semen variab les and the hypoosmotic swelling (HOS) test. Design: Pilot study and clinical trial. Setting: Large teaching hospital and fertility center. Patient(s): Men whose semen was studied fur various reasons. Main Outcome Measure(s): Percentage of apoptotic spermatozoa by two differe nt assays, percentage of necrotic spermatozoa, concentration and motility o f spermatozoa, and outcome of the HOS test. Result(s): Apoptosis can be measured in spermatozoa by flow cytometry using the Annexin V assay and the TUNEL assay. Twenty percent of spermatozoa wer e apoptotic according to both assays. A significant inverse correlation was seen between phosphatidylserine expression (Annexin V assay) and sperm con centration (r = -0.389; P<.05) and motility (r = -0.289; P<.05). A highly s ignificant inverse correlation was seen between DNA double-strand breaks (T UNEL assay) and sperm concentration (r = -0.629; P<.0001). Conclusion(s): Flow cytometry can easily and reliably detect phosphatidylse rine expression on the outer leaflet of the cell membrane and DNA strand br eaks, both of which are hallmarks of apoptosis. About 20% of ejaculated spe rmatozoa are apoptotic, and the concentration of spermatozoa is lower in me n with more apoptotic spermatozoa. (C) 2000 by American Society for Reprodu ctive Medicine.