Plasma Membrane Translocation of Phosphatidylserine and Determination of Reactive Oxygen Species: A Study of Their Association With the Cryopreservation-Thawing Survival of Fractionated Human Spermatozoa

Abstract

Objectives: (1) To evaluate sub-lethal plasma membrane damage of sperm during cryopreservation-thawing by the assessment of phosphatidylserine (PS) translocation, (2) to examine the relationship between generation of reactive oxygen species (ROS) and cryopreservation-related membrane alterations, and (3) to study the value of membrane PS translocation for prediction of the ability to survive cryopreservation.Design: Prospective cohort study.Materials and Methods: Sub-fertile semen samples (n=10) were studied. Membrane PS translocation was evaluated with annexin V binding and generation of ROS was measured by chemiluminescence using luminol. Motility parameters were assessed by computer analysis. All measurements were performed in purified semen fractions with high and low sperm motility (Percoll density gradient separation) independently, before and after cryopreservation-thawing using TEST-Yolk buffer and glycerol.Results: Freezing-thawing induced significant levels of PS externalization in both sperm fractions. However, the induction of PS translocation in the fractions with high sperm motility was significantly higher than that of the fractions with low sperm motility. Significantly higher ROS levels were detected in pre-freeze samples of the fractions with low sperm motility compared to the fractions with superior motility. After freezing-thawing, ROS levels were significantly reduced in both fractions. There was a significant and negative correlation between annexin V staining before freezing and the ability to survive cryopreservation in the fractions with high sperm motility (r=−0.45, p=0.006). Furthermore, levels of annexin V staining <10% in a given fraction with high sperm motility predicted a >45% cryosurvival rate, with a sensitivity of 83% and a specificity of 80%.Conclusions: (1) Cryopreservation-thawing induced membrane PS translocation; (2) the effects of cryopreservation on sperm plasma membrane integrity were not associated with an increase in ROS generation as detected by chemiluminescence; and (3) the degree of annexin V binding before freezing may be used to predict the ability to survive cryopreservation-thawing. Objectives: (1) To evaluate sub-lethal plasma membrane damage of sperm during cryopreservation-thawing by the assessment of phosphatidylserine (PS) translocation, (2) to examine the relationship between generation of reactive oxygen species (ROS) and cryopreservation-related membrane alterations, and (3) to study the value of membrane PS translocation for prediction of the ability to survive cryopreservation. Design: Prospective cohort study. Materials and Methods: Sub-fertile semen samples (n=10) were studied. Membrane PS translocation was evaluated with annexin V binding and generation of ROS was measured by chemiluminescence using luminol. Motility parameters were assessed by computer analysis. All measurements were performed in purified semen fractions with high and low sperm motility (Percoll density gradient separation) independently, before and after cryopreservation-thawing using TEST-Yolk buffer and glycerol. Results: Freezing-thawing induced significant levels of PS externalization in both sperm fractions. However, the induction of PS translocation in the fractions with high sperm motility was significantly higher than that of the fractions with low sperm motility. Significantly higher ROS levels were detected in pre-freeze samples of the fractions with low sperm motility compared to the fractions with superior motility. After freezing-thawing, ROS levels were significantly reduced in both fractions. There was a significant and negative correlation between annexin V staining before freezing and the ability to survive cryopreservation in the fractions with high sperm motility (r=−0.45, p=0.006). Furthermore, levels of annexin V staining <10% in a given fraction with high sperm motility predicted a >45% cryosurvival rate, with a sensitivity of 83% and a specificity of 80%. Conclusions: (1) Cryopreservation-thawing induced membrane PS translocation; (2) the effects of cryopreservation on sperm plasma membrane integrity were not associated with an increase in ROS generation as detected by chemiluminescence; and (3) the degree of annexin V binding before freezing may be used to predict the ability to survive cryopreservation-thawing.