Superparamagnetic extraction of ejaculated human spermatozoa with early apoptotic changes

Abstract

Introduction and Objective: Externalization of phosphatidylserine (EPS) on the outer leaflet of the plasma membrane is one of the earliest events of apoptosis. There is an inverse correlation between the proportion of EPS+ sperm and the semen quality. The aim of our study was to separate the spermatozoa with and without EPS and to evaluate the caspase activity (aCp), mitochondrial membrane potential (Δ_upper;ψm), and DNA fragmentation in both the groups.Design: A prospective study.Methods: Semen samples from healthy donors were pooled to create 18 pools of semen. After separation by 2-layer density gradient centrifugation, the mature and the immature fractions were collected as the 90% pellet and at the interface between the 47% and 90% layers, respectively. Each of these fractions were further divided into sub fractions of spermatozoa depending on EPS, using Annexin V labeled superparamagnetic beads, that bind specifically to EPS. The passage through a magnetic field separated out the EPS+ sperm from the EPS- ones. Using the carboxyfluorescein derivatives the levels of aCp 9, 8, and 3 were estimated in both the fractions. Sperm with intact Δ_upper;ψm were estimated in all the subsets using a lipophilic cationic dye. Spermatozoa with intact Δ_upper;ψm excite an intense red fluorescence while those with disrupted Δ_upper;ψm excite green. The proportion of cells with DNA fragmentation (TU+) was measured in the same fractions using the TUNEL assay. All fluorescence signals were analyzed using flow cytometry.Results: The EPS+ sperm had significantly higher levels of aCp, disrupted Δ_upper;ψm, and TU+ than the EPS- ones (p < 0.01). Conclusions: The EPS− spermatozoa isolated by superparamagnetic separation have significantly lower levels of aCp, disrupted Δ_upper;ψm, and TU+ than the EPS+ ones. We speculate that the removal of EPS+ spermatozoa may improve the outcome of assisted reproduction failures, attributed to high levels of sperm DNA damage. Introduction and Objective: Externalization of phosphatidylserine (EPS) on the outer leaflet of the plasma membrane is one of the earliest events of apoptosis. There is an inverse correlation between the proportion of EPS+ sperm and the semen quality. The aim of our study was to separate the spermatozoa with and without EPS and to evaluate the caspase activity (aCp), mitochondrial membrane potential (Δ_upper;ψm), and DNA fragmentation in both the groups. Design: A prospective study. Methods: Semen samples from healthy donors were pooled to create 18 pools of semen. After separation by 2-layer density gradient centrifugation, the mature and the immature fractions were collected as the 90% pellet and at the interface between the 47% and 90% layers, respectively. Each of these fractions were further divided into sub fractions of spermatozoa depending on EPS, using Annexin V labeled superparamagnetic beads, that bind specifically to EPS. The passage through a magnetic field separated out the EPS+ sperm from the EPS- ones. Using the carboxyfluorescein derivatives the levels of aCp 9, 8, and 3 were estimated in both the fractions. Sperm with intact Δ_upper;ψm were estimated in all the subsets using a lipophilic cationic dye. Spermatozoa with intact Δ_upper;ψm excite an intense red fluorescence while those with disrupted Δ_upper;ψm excite green. The proportion of cells with DNA fragmentation (TU+) was measured in the same fractions using the TUNEL assay. All fluorescence signals were analyzed using flow cytometry. Results: The EPS+ sperm had significantly higher levels of aCp, disrupted Δ_upper;ψm, and TU+ than the EPS- ones (p < 0.01). Conclusions: The EPS− spermatozoa isolated by superparamagnetic separation have significantly lower levels of aCp, disrupted Δ_upper;ψm, and TU+ than the EPS+ ones. We speculate that the removal of EPS+ spermatozoa may improve the outcome of assisted reproduction failures, attributed to high levels of sperm DNA damage.