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

Abstract

Objective: [1] To determine whether apoptosis can be measured in ejaculated spermatozoa by flow cytometry using the Annexin V assay, which measures expression of phosphatidylserine on the outer leaflet of the cell membrane, or the TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP [deoxy-uridine triphosphate] nick end labeling) assay, which measures occurrence of DNA strand breaks and [2] to correlate the outcome with routine semen variables 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 for various reasons. Main Outcome Measure(s): Percentage of apoptotic spermatozoa by two different assays, percentage of necrotic spermatozoa, concentration and motility of 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 were apoptotic according to both assays. A significant inverse correlation was seen between phosphatidylserine expression (Annexin V assay) and sperm concentration ( r = −0.389; P<.05) and motility ( r = −0.289; P<.05). A highly significant inverse correlation was seen between DNA double-strand breaks (TUNEL assay) and sperm concentration ( r = −0.629; P<.0001). Conclusion(s): Flow cytometry can easily and reliably detect phosphatidylserine expression on the outer leaflet of the cell membrane and DNA strand breaks, both of which are hallmarks of apoptosis. About 20% of ejaculated spermatozoa are apoptotic, and the concentration of spermatozoa is lower in men with more apoptotic spermatozoa.