Seminal Plasma Protein N-Glycan Peaks Are Potential Predictors of Semen Pathology and Sperm Chromatin Maturity in Men

Abstract

Background: Male infertility is increasingly becoming a health and demographic problem. While it may originate from congenital or acquired diseases, it can also result from environmental exposure. Hence, the complexity of involved molecular mechanisms often requires a multiparametric approach. This study aimed to associate semen parameters with sperm DNA fragmentation, chromatin maturity and seminal plasma protein N-glycosylation.

Methods: The study was conducted with 166 participants, 20-55 y old, 82 normozoospermic and 84 with pathological diagnosis. Sperm was analyzed by Halosperm assay and aniline blue staining, while seminal plasma total protein N-glycans were analyzed by ultra-high-performance liquid chromatography.

Results: Sperm DNA fragmentation was significantly increased in the pathological group and was inversely correlated with sperm motility and viability. Seminal plasma total protein N-glycans were chromatographically separated in 37 individual peaks. The pattern of seminal plasma N-glycan peaks (SPGP) showed that SPGP14 significantly differs between men with normal and pathological semen parameters (p < 0.001). The multivariate analysis showed that when sperm chromatin maturity increases by 10%, SPGP17 decreases by 14% while SPGP25 increases by 25%.

Conclusion: DNA integrity and seminal plasma N-glycans are associated with pathological sperm parameters. Specific N-glycans are also associated with sperm chromatin maturity and have a potential in future fertility research and clinical diagnostics.

Keywords: DNA fragmentation; Halosperm; N-glycosylation; aniline blue; male infertility; seminal plasma; sperm; sperm chromatin maturity.

Figure 1

Representative microscopic image of sperm after Halosperm assay for evaluation of DNA fragmentation. (A,B) Non-fragmented sperm and (C,D) Severely fragmented sperm. Magnification 400×, scale bar shows 20 µm.

Figure 2

Representative microscopic image of sperm after aniline blue staining for assessment of sperm chromatin maturity of (A) normozoospermic men and (B) men with pathological semen parameters. Arrows indicate immature dark-blue stained sperm. Magnification 1000×, scale bar shows 50 µm.

Figure 3

(A) Representative chromatogram of N-glycans isolated from seminal plasma total proteins determined by ultra-high-performance liquid chromatography based on hydrophilic interactions (HILIC-UHPLC). EU = emission units. Structures of glycans identified in the glycan peak SPGP14 are biantennary digalactosylated disialylated glycan with (B) and (C) without core fucose.