NMR-based plasma metabolomic discrimination for male fertility assessment of rats treated with Eurycoma longifolia extracts

Male infertility is one of the leading causes of infertility which affects many couples worldwide. Semen analysis is a routine examination of male fertility status which is usually performed on semen samples obtained through masturbation that may be inconvenient to patients. Eurycoma longifolia (Tongkat Ali, TA), native to Malaysia, has been traditionally used as a remedy to boost male fertility. In our recent studies in rats, upon the administration of high-quassinoid content extracts of TA including TA water (TAW), quassinoid-rich TA (TAQR) extracts, and a low-quassinoid content extract including quassinoid-poor TA (TAQP) extract, sperm count (SC) increased in TAW- and TAQR-treated rats when compared to the TAQP-treated and control groups. Consequently, the rats were divided into normal- (control and TAQP-treated) and high- (TAW- and TAQR-treated) SC groups [Ebrahimi et al. 2016]. Post-treatment rat plasma was collected. An optimized plasma sample preparation method was developed with respect to the internal standards sodium 3- (trimethylsilyl) propionate- 2,2,3,3- d4 (TSP) and deuterated 4-dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA). Carr-Purcell-Meibum-Gill (CPMG) experiments combined with orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to evaluate plasma metabolomic changes in normal- and high-SC rats. The potential biomarkers associated with SC increase were investigated to assess fertility by capturing the metabolomic profile of plasma. DSA was selected as the optimized internal standard for plasma analysis due to its significantly smaller half-height line width (W h/2) compared to that of TSP. The validated OPLS-DA model clearly discriminated the CPMG profiles in regard to the SC level. Plasma profiles of the high-SC group contained higher levels of alanine, lactate, and histidine, while ethanol concentration was significantly higher in the normal-SC group. This approach might be a new alternative applicable to the fertility assessment in humans through the quantitative metabolomic analysis of plasma without requiring semen.

ABBREVIATIONS: TA: Tongkat Ali; LOD: limit of detection; LOQ: limit of quantification; HPLC-UV: high performance liquid chromatography-ultrviolet; PDA: photodiode array; NMR: nuclear magnetic resonance; FID: free induction decay; LC-MS: liquid chromatography-mass spectrometry; GC-MS: gas chromatography-mass spectrometry; HSQC: heteronuclear single quantum coherence; CPMG: Carr-Purcell-Meibum-Gill; VLDL: very low density lipoprotein; HDL: high density lipoprotein; EDTA: ethylenediaminetetraacetic acid; ANOVA: analysis of variance; AMIX: analysis of mixtures; SIMCA: soft independent modeling of class analogy; PCA: principal components analysis; OPLS-DA: orthogonal partial least-squares discriminant analysis; VIP: variable importance plot; AUROC: area under the receiver operating characteristic; TSP: sodium 3-(trimethylsilyl) propionate- 2,2,3,3- d4; DSA: deuterated 4-dimethyl-4-silapentane-1-ammonium trifluoroacetate; ESI: electrospray ionization; TCA: trichloroacetic acid; ACN: acetonitrile; dd H2O: distilled deionized water; FSH: follicle-stimulating hormone; LH: luteinizing hormone; OECD: Organisation for Economic Co-operation and Development.