Detection of Cholesterol and its Oxidized Derivatives in Human Sperm Membranes through a Fast and Reliable LC-MS Method

The trafficking of membrane cholesterol (Chol) in mammals represents key processes in cells function, particularly in sperm physiology since Chol removal is strictly associated with motility gain. Moreover, the involvement of oxidized Chol derivatives, known as oxysterols, has been recently summoned in the regulation of sperm function. The study of sterols dynamic in human sperm is largely hampered by the low sample availability and the inadequate sensitivity of current methods based on chromatographic techniques. In this study we aimed to develop a robust and reliable method based on liquid chromatography-mass spectrometry (LC-MS) to quantify the sperm levels of Chol and major oxysterols 7β-OH-Cholesterol (7β -OHC) and 7-keto-Cholesterol (7-KC). In particular, by finely tuning the reverse-phase chromatography parameters, the unambiguous identification of Chol, 7β -OHC and 7-KC in biological samples was allowed on the bases of their different retention times, accurate m/z determination and natural isotope abundance pattern. Finally, by applying the method on real sperm samples from 12 semen donors, we documented that normozoospermic subjects (total sperm count >39 × 106 cells/ejaculate) showed an underrepresentation of all steroids compared to subjects with oligozoospermia (total sperm count ≤ 39 × 106 cells/ejaculate; respectively P=0.048 for Chol, P=0.006 for 7β-OHC and P=0.001 for 7-KC). These preliminary data suggest further investigation about the impact of disorders of the spermatogenic process on the composition and function of the sperm membrane.