Lipid signature of motile human sperm: Characterization of sphingomyelin, ceramide, and phospholipids with a focus on very long polyunsaturated fatty acid

Abstract

Sperm membrane lipids are crucial in male fertility, influencing sperm motility, viability, and functional competence. This study comprehensively characterizes the phospholipid and sphingolipid composition in highly motile human spermatozoa obtained through the swim-up method, a widely used technique in assisted reproductive technology (ART). Using two-dimensional thin-layer chromatography and phosphorus analysis, we identified choline glycerophospholipids (CGP, 45%), ethanolamine glycerophospholipids (EGP, 26%), and sphingomyelin (SM, 17%) as predominant phospholipids, with minor components including cardiolipin, lysophospholipids, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and neutral lipids. Gas chromatography analysis of glycerophospholipids (GPL) revealed a high long chain (C20-C22) polyunsaturated fatty acid (PUFA) content (46.3%), particularly docosahexaenoic acid (DHA, 29 22:6n-3), which was more abundant in CGP (46%) than EGP (26%). Sphingolipid analysis 30 indicated that ceramide (Cer) and SM shared similar fatty acid profiles due to their metabolic relationship, with very-long-chain (VLC) PUFA (≥C26) being more prevalent in SM (10%) than in Cer (6%). Additionally, argentation chromatography allowed the identification of highly unsaturated VLCPUFA species in Cer, including 28:3n-6, 28:4n-6, and 30:4n-34 6, which had not been previously quantified in motile human spermatozoa. Given the essential function of sphingolipid metabolism in spermatogenesis, capacitation, and acrosomal exocytosis, our findings suggest that the balance of VLCPUFA-containing SM and Cer emerges as a key factor in sperm performance and fertilization potential. This study provides novel insights into the lipid signature of human sperm and highlights the relevance of membrane lipid remodeling for male fertility and ART outcomes.