Intracellular sperm and seminal plasma Ca+2 concentrations affect cryodamage susceptibility after a cycle of freezing and thawing

Abstract

Objective: Sperm cells can be affected in viability, fertilizing ability and motility by freezing and subsequent thawing in assisted reproduction techniques. Ca+2 is an important cellular metabolism messenger. In other mammals it has been noted that high calcium concentrations in the external medium may decrease the motility and metabolism of sperm and “calcium intoxication” may be a factor in cryodamage. Subsequently, the intracellular and seminal plasma Ca+2 contents are potential predictors of sperm quality after freezing-thawing process. Our aim with this study is to increase the information of the parameters of the cell physiology that are affecting sperm survival after freezing/thawing process, and to determine the predictive value of plasma and cellular Ca+2 determinations. Materials and Methods: This study was performed in 94 human sperm samples after freezing-thawing process, studied according with WHO statements for concentration of motile and alive sperm, both fresh and after thawing. We measured intracellular Ca+2 by incubation with Fluo Ca+2 indicator Fluo-4 AM (Molecular Probes) (30′ 37°C) by fluorometry (Excitation: 485nm Emmision: 538nm) (Fluoroskan, Labsystems). Seminal plasma Ca+2 were measured with an Alcyon analyzer (Abbot). One way analysis of variance was used to detect differences between Ca+2 and sperm rate survival after thawing. Linear regression analysis was used to determine the extent of correlation between parameters compared. The diagnostic performance of the tests performed was evaluated using Receiver Operating Characteristic (ROC) curve analysis. Results: Intracellular sperm Ca+2 was expressed as fluorescence arbitrary units/50 million cells and Ca+2 in seminal plasma was expressed as mg/dl. AUCROC: area under the curve in a receiver operating characteristic analysis. CI: confidence interval. PPV: positive predictive value. NPV: negative predictive value. Tabled 1 A significant negative relationship between intracellular Ca+2 (r=−0.36) and seminal plasma Ca+2 (r=−0.30) concentrations and post-thaw motile sperm recovery rate was observed. Predictive values demonstrated that Ca+2 has good diagnostic accuracy. ∗ denotes statistically significant relationship, p<0.05. Conclusions: There was a negative correlation between the concentrations of intracellular and plasma seminal Ca+2 with the percentage of sperm survival after thawing. This findings strongly suggests that high intracellular sperm Ca+2 and seminal plasma Ca+2 negatively affect sperm metabolism and increase the possibilities of cryodamage after a freezing/thawing process. Research should be focused now in introducing Ca+2 quelators and phospholipids in the freezing media to prevent increased calcium flux into the sperm.