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Sperm plasma membrane integrity measurement: a combined method
A Carreras1, J P Ramirez, C Mendoza
1Departamento de Bioquímica y Biologia Molecular, Facultad de Ciencias, Universidad de Granada, Spain.
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.
Insights
The hypo-osmotic swelling (HOS) test combined with eosin Y or propidium iodide staining effectively assesses sperm membrane integrity. High percentages of HOS-negative, stained sperm (types 2 and 4) indicate poor outcomes in in vitro fertilization (IVF).
Area of Science:
- Reproductive Biology
- Sperm Physiology
- In Vitro Fertilization
Background:
- Sperm plasma membrane integrity is crucial for male fertility.
- Assessing sperm viability and function aids in diagnosing infertility and optimizing assisted reproductive technologies.
- The hypo-osmotic swelling (HOS) test is a functional assay for sperm membrane status.
Purpose of the Study:
- To evaluate a combined hypo-osmotic swelling (HOS) test with eosin Y or propidium iodide staining for assessing sperm plasma membrane characteristics.
- To correlate sperm membrane integrity, assessed by the HOS-dye tests, with lipid peroxidation levels.
- To determine the prognostic value of the HOS-dye tests in predicting fertilization success in an in vitro fertilization (IVF) program.
Main Methods:
- Sperm samples were fractionated using a swim-up technique to isolate motile spermatozoa.
- The hypo-osmotic swelling (HOS) test was performed in conjunction with eosin Y (HOS-eosine) or propidium iodide (HOS-propidium) staining.
- Lipid peroxidation levels were measured in different sperm fractions.
- Spermatozoa were classified into four types based on HOS response and dye uptake.
Main Results:
- The HOS-dye tests differentiated sperm based on membrane integrity, categorizing them into four types.
- Fractionated motile sperm (Fraction II) exhibited higher percentages of intact membranes (Type 1) and lower lipid peroxidation compared to unselected sperm (Fraction I).
- Non-motile sperm fractions (Fraction III) showed compromised membrane integrity (higher percentages of Types 2, 3, and 4) and elevated lipid peroxidation.
- Preliminary IVF data indicated that a high incidence of Types 2 and 4 spermatozoa was associated with fertilization failure.
Conclusions:
- The combined HOS-dye test is a valuable method for assessing sperm plasma membrane integrity.
- Sperm membrane integrity, as indicated by HOS-dye test results, correlates with lipid peroxidation levels.
- The HOS-eosine test shows prognostic potential for predicting fertilization outcomes in IVF, with high rates of specific sperm types suggesting potential failure.