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Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose

E Isachenko, Department of Obstetrics and Gynaecology, University Woman’s Hospital Ulm, Ulm, Germany
V Isachenko, Department of Obstetrics and Gynaecology, University Woman’s Hospital Ulm, Ulm, Germany
J Weiss, Department of Obstetrics and Gynaecology, University Woman’s Hospital Ulm, Ulm, Germany
R Kreienberg, Department of Obstetrics and Gynaecology, University Woman’s Hospital Ulm, Ulm, Germany
I Katkov, Cancer Center, University of California at San Diego, La Jolla, United States
M Schulz, Center of Biotechnology in Reproduction, Department of Basic Sciences, La Frontera University, Temuco, Chile
A Lulat, CReATe Cord Blood Bank, Toronto, Canada
J Risopatrón, Center of Biotechnology in Reproduction, Department of Basic Sciences, La Frontera University, Temuco, Chile
R Sanchez, Center of Biotechnology in Reproduction, Department of Basic Sciences, La Frontera University, Temuco, Chile

Correspondence: Evgenia Isachenko, Email: e.isachenko{at}yahoo.de

Abstract

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryo-induction of capacitation and acrosomal reaction. Spermatozoa were isolated using the swim up procedure performed using three different media: a) Human Tubal Fluid medium (HTF, control), b) HTF with 1% human serum albumin (HSA) and c) HTF with 1% HSA and 0.25 M sucrose. 30 mcl suspensions of cells from each group were dropped directly into liquid nitrogen and stored for at least 24 hrs. Cells were thawed by quickly submerging the spheres in HTF with 1% HSA at 37° C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosomal reaction were investigated. Sperm viability, acrosome reaction and capacitation were detected using double fluorescence CTC-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye JC-1. The number of progressive motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1 ± 3.2 %, P < 0.05) compared to controls (19.4 ± 1.9 %). The combination of HSA and sucrose (65.2 ± 7.9 %) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compare to HSA alone (28.6 ± 4.7 %). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as human serum albumin and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.