Comparison of Methods for Rapid Determination of Cholesterol Concentration in Human Sperm Membrane in Clinical Laboratory Practice

Abstract

Objective: Cholesterol is an important structural component of the plasma membrane of mammalian cells. Cholesterol, among other important roles, plays a special role in sperm membranes. Change in the lipid composition of the sperm membrane, particularly the outflow of cholesterol, is an integral part of the process of capacitation and subsequent acrosomal reaction necessary for the sperm to fertilize an egg. Deviations in cholesterol concentration in sperm membrane may indicate a decrease in the fertilizing potential of sperm. To determine the optimal method for rapid analysis of the cholesterol content in human sperm membranes in the IVF laboratory, four methods of quantitative determination of cholesterol were compared in terms of practicality and effectiveness of their use to assess the concentration of cholesterol in human sperm membranes: the method of enzymatic colorimetric detection, the Lieberman–Burchard method, infrared spectroscopy and high-performance liquid chromatography. Methods: 101 ejaculates of patients with established normozoospermia (according to WHO criteria) were used in the work. Spermatozoa were separated from the semen by double centrifugation with the addition of DPBS medium. The resulting cellular pellet was used to determine the concentration of cholesterol in a sample by one of four methods: enzymatic colorimetric detection (FCD), HPLC, Lieberman-Burchard method, infrared spectroscopy. Results and Discussion: The following cholesterol concentrations were obtained by enzymatic colorimetric detection, Lieberman–Burchard, infrared spectroscopy and high-performance liquid chromatography: 1.0 ± 0.3, 1.32 ± 0.15, 5.1 ± 1.8 and 1.53 ± 0.18 nmol/10⁶ cells, respectively. The Lieberman–Burchard method, enzymatic colorimetric detection and HPLC showed similar results, the obtained average cholesterol concentrations coincide within the error. The mean cholesterol concentration in sperm membranes obtained using infrared spectroscopy method significantly exceeds the values presented in the literature and the values obtained using other methods. In addition, this method requires an amount of analyzed material that significantly exceeds the volume of one ejaculate. Conclusions: As a result of comparing four methods of quantitative cholesterol analysis, the method of enzymatic colorimetric detection is proposed as a method of rapid analysis of cholesterol in human sperm membranes suitable for routine use in a clinical laboratory. The advantages of this method include the low toxicity of the method, it’s cost-effectiveness and a significant reduction in the time of complete analysis: from sample preparation to obtaining the result.