Sperm plasma membrane integrity measurement: a combined method

Abstract

Sperm plasma membrane characteristics were measured by a combined method consisting of the hypo-osmotic swelling test and staining with either the eosine Y (HOS-eosine test) or propidium iodide dye (HOS-propidium test). Sperm samples were washed and resuspended in BWW medium (fraction I). Aliquots of the washed spermatozoa were treated by a swim-up technique to select motile spermatozoa (fraction II). After separation of motile cells, residual sperm pellets were treated separately (fraction III). These three fractions were subjected to the hypo-osmotic swelling test, lipid peroxidation measurement, and the HOS-eosine and HOS-propidium tests. The HOS-eosine test makes it possible to distinguish 4 types of spermatozoa: type 1: HOS+/eosine-; type 2: HOS-/eosine-; type 3: HOS-/eosine+ and type 4: HOS+/eosine+ (Fig. 1). HOS-propidium test shows equal results as HOS-eosine test. Fraction I spermatozoa showed 55.2 +/- 3.6% type 1; 12.6 +/- 1.0 type 2; 28.0 +/- 2.9 type 3; and 4.2 +/- 0.6 type 4 cells. Fraction II spermatozoa were characterized by high percentages of type 1 cells, low percentages of types 3 and 4, and very low values of lipid peroxidation (5 times smaller than fraction I). Fraction III showed a low percentage of type 1, a high percentages of the other types, and an enhanced value of lipid peroxidation (2 times higher than fraction I). The prognostic value of the HOS-eosine test was evaluated in an IVF programme. Preliminary results show that a high incidence of types 2 and 4 spermatozoa is often associated with fertilization failure.