Refining the seminal biomarker detection: metabolome profiles before and after the liquefaction procedure

Abstract

Research question: Should seminal metabolites be analysed from fresh ejaculate or after liquefaction to establish a protocol for biomarker discovery? Design: Semen samples were collected from 15 healthy donors, with two aliquots obtained for each donor, one before and one after the liquefaction process, resulting in total of 30 samples for analysis. Non-targeted metabolomics analysis was conducted using liquid chromatographyhigh-resolution mass spectrometry on these paired samples. Data quality was assessed using MarkerView software. Metabolites were identified using the 2021 NIST Mass Spectral Library, PeakView, CEU Mass Mediator and Sirius software. Results: A total of 1664 mass-to-charge ratio values were detected and 76 metabolites were identified, including amino acids, lipids, carbohydrates and compounds related to oxidative stress and sperm function. Principal component analysis did not reveal any statistically significant differences between the pre- and post-liquefaction samples. However, univariate statistical testing detected subtle changes in metabolite levels, most (1611) having similar or increased intensities in post-liquefaction samples, along with notable interindividual variability. Conclusions: The semen liquefaction process does not seem to affect the overall metabolic profile, allowing flexibility in sample analysis without compromising data integrity. This supports the robustness of metabolomics for semen analysis and its potential for identifying new fertility biomarkers.