1H NMR-based metabonomics for infertility diagnosis in men with varicocele

Abstract

Purpose: "Omics" techniques have been used to understand and to identify biomarkers of male infertility. We report on the first metabonomics models created to diagnose varicocele and infertility among men with varicocele.

Methods: We recruited 35 infertile men with varicocele (VI group), 21 fertile men with varicocele (VF group) and 24 fertile men without varicocele (C group). All men underwent standard semen analysis, scrotal duplex ultrasonography, and sexual hormone level measurement. Hydrogen-1 nuclear magnetic resonance (¹H NMR) spectra of seminal plasma were used to create metabonomics models to discriminate between men with and without varicocele, and between fertile and infertile men with varicocele.

Results: Using the statistical formalisms partial least square discriminants analysis and genetic algorithm-based linear discriminant analysis (GA-LDA), we created two models that discriminated the three groups from each other with accuracy of 92.17%. We also created metabonomics models using orthogonal partial least square discriminants analysis and GA-LDA that discriminated VF group from VI group, with an accuracy of 94.64% and 100% respectively. We identified 19 metabolites that were important in group segregation: caprate, 2-hydroxy-3-methylvalerate, leucine, valine, 3-hydroxybutyrate, lactate, alanine, 4-aminobutyrate, isoleucine, citrate, methanol, glucose, glycosides, glycerol-3-phosphocoline, n-acetyltyrosine, glutamine, tyrosine, arginine, and uridine.

Conclusions: ¹HNMR-based metabonomics of seminal plasma can be used to create metabonomics models to discriminate between men with varicocele from those without varicocele, and between fertile men with varicocele from those infertile with varicocele. Furthermore, the most important metabolites for group segregation are involved in the oxidative stress caused by varicocele.

Keywords: Fertility; Metabolome; Metabolomics; NMR spectroscopy; Semen.

Fig. 1

Typical ¹H NMR spectrum (400 MHz, D₂O) of seminal plasma. The areas under the peaks are associated with the concentration of metabolites weighted by the number of hydrogen nuclei in each chemical environment. Some peaks had their respective chemical compounds assigned in the spectrum

Fig. 2

Results of PLS-DA using 80 samples and three classes—control group (C), 24 samples (red dots); varicocele infertile group (VI), 35 samples (green dots); and varicocele fertile group (VF), 21 samples (blue dots). Score plot (left) and VIP score plot (right) showing the 20 most important variables for discrimination

Fig. 3

Results of the OPLS-DA model to discriminate the control group (C), 24 samples (red dots), from the varicocele group (V), 56 samples (green dots). Score plot (left) and permutation test (right)

Fig. 4

Results of OPLS-DA model to discriminate the varicocele infertile group (VI), 35 samples (red dots), from the varicocele fertile group (VF), 21 samples (green dots). Score plot (left) and permutation test (right)

Fig. 5

a Score plot of GA-LDA to discriminate varicocele infertile (blue), varicocele fertile (red), control (black) groups. Training samples (balls) and test samples (dots). The importance of each variable selected for the model are shown by the loading of discriminant functions 1 and 2 in b and c respectively

Fig. 6

a Score plot of GA-LDA to discriminate Varicocele group (blue) from control group (red); training samples (balls) and test samples (dots). The importance of each variable selected is shown by the loading of discriminant function 1 in b

Fig. 7

a Score plot of GA-LDA to discriminate the varicocele infertile group (blue) from the varicocele fertile group (red); training samples (balls) and test samples (dots). The importance of each variable selected is shown by the loading of discriminant function 1 in b