From failing fertility to faulty fatty acids: GC reveals all

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  • Published: Mar 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: From failing fertility to faulty fatty acids: GC reveals all

The mystery of poor sperm motility may be related to fatty acids

Male infertility is a common problem, one cause of which is asthenozoospermia (poor sperm motility). Around 1 in 5,000 men have complete sperm immotility, although many more suffer from the problem to a lesser extent. This condition is little understood, despite numerous studies of affected sperm. A 1999 paper by Conquer et al. suggested a link between low levels of polyunsaturated fatty acids, in particular docosahexaenoic acid (DEA), and asthenozoospermia. There also seemed to be a link with high levels of mono-unsaturated fatty acids, such as oleic acid. No differences in fatty acids were noted in the blood plasma, suggesting a metabolic problem rather than poor dietary intake.

The Nanjing researchers aimed to take a complete metabolic profile of fatty acids and other metabolites in seminal plasma. This was a metabonomic study, quantitatively evaluating the response of an entire range of metabolites to genetic changes or a pathological condition. The study was carried out using 60 volunteers, 30 suffering from asthenozoospermia and 30 healthy controls.

Fatty acid derivatisation and GC-MS reveal seminal metabolites

Seminal plasma samples were dissolved in chloroform/methanol, centrifuged, and the supernatant solution was treated with methoxyamine/pyridine, then derivatised with a mixture of MSTFA (N-methyl-N-(trimethylsilyl)trifluoroacetamide) and TMCS (trimethylchlorosilane), converting the fatty acids to their more volatile trimethylsilyl esters for GC-MS.

The GC was an Agilent 7890A fitted with an HP-5ms column, coated with 3% phenylmethylsiloxane. The temperature programme ran from 70-300 °C. The carrier gas was helium, and the flow rate was 1 ml/min. The mass spectrometer was an Agilent 5975C with an electron ionisation source.

In all, 89 compounds were identified, including fatty acids, sugars, cholesterol, amino acids and various smaller species, such as acetamide and lactic acid; 25 of the compounds were chosen for method validation. The method was shown to give good linearity and sensitivity for the 25 compounds, with a limit of detection (LOD) ratio of 0.004 (v/v, samples/(samples + water)) and a limit of quantification ratio (LOQ) of 0.05 (v/v, samples / (samples + water)).

A statistical comparison was carried out for all 89 identified compounds, looking at any differences between the asthenozoospermic samples and the control samples, using PCA (principal component analysis) and PLS-DA (partial least squares discriminant analysis). The PCA plots showed that the healthy and asthenozoospermic groups tended to separate from each other. Significantly increased levels of the mono-unsaturated acid, oleic acid, were found in asthenozoospermic samples, in line with previous work. Significantly higher levels of palmitic acid (a saturated fatty acid) were also noted, along with low levels of the amino acid valine. These have also been previously suggested as factors that may affect sperm motility. Four other compounds, benzoic acid, cholecalciferol, D-pinitol and nonadecanoic acid, were also found at significantly different levels in the two groups of samples, although the authors implied that the effects of oleic acid, palmitic acid and valine were likely to be more important.

Abnormal levels of fatty acids and valine linked to poor sperm motility

The researchers examined a large number of compounds, although some well-known seminal molecules that are difficult to analyse by GC, such as prostaglandins, were not detected.

The paper seemed to show a link between abnormal levels of oleic acid, palmitic acid and valine and poor sperm motility. However, some caution needs to be observed, since the number of participants was relatively small and a large number of compounds were examined. The results will need to be confirmed to ensure that they are not simply due to statistical noise. However, if confirmed these results do offer genuine new insights into a little-understood condition.

Related Links

Andrologia, 2017, Early View paper. Tang et al. Metabonomic analysis of fatty acids in seminal plasma between healthy and asthenozoospermic men based on gas chromatography mass spectrometry.

Lipids, 1999, 34, 793-799. Conquer et al. Fatty acid analysis of blood serum, seminal plasma, and spermatozoa of normozoospermic vs. asthenozoospermic males.

Wikipedia, Asthenozoospermia

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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