To gain a better understanding of male infertility, the EHU’s Human Reproduction group is examining the molecular characteristics of sperm with greater precision

Researchers from the EHU-University of the Basque Country have developed a robust protocol to detect the molecules involved in the metabolic reactions of sperm. The new method enables a detailed analysis to be made even with small samples, and the largest number of potential biomarkers identified to date to be detected; this advance could help to diagnose male infertility and develop future clinical solutions.

Human infertility is a significant health and social problem. It affects approximately 17.5% of the world's population, and almost half of all the cases are linked to male factors. Currently, male fertility is assessed by means of a semen analysis, in which several general parameters are measured under a microscope: sample volume, sperm concentration, motility, morphology, etc. “While this is interesting, it is not enough. A semen analysis lacks diagnostic and prognostic capabilities. Having normal values does not mean that a person is fertile,” explained Irune Calzado, a researcher in the Human Reproduction group. So finding reliable biomarkers to better evaluate male infertility is urgently needed.

The EHU researchers focused on the set of molecules involved in sperm metabolism. To do this, they used metabolomics, a technique that analyses metabolites. Metabolites are the products and intermediates of the metabolic reactions that occur in the body: lipids, amino acids, proteins, nucleic acids, carbohydrates, and so on. They provide a great deal of information: “For example, by analysing metabolites, you can find out whether a metabolic pathway is altered in a disease,” the researcher explained.

Most of the research carried out in this field so far has been based on seminal plasma, but not on sperm. In fact, Human Reproduction researchers have developed a specific methodology to enable this research to be carried out on sperm. EHU researchers had to address various methodological challenges, because analysing sperm presents certain difficulties: “Given their toughness, sperm are very difficult to break. So we have had to optimise a high-frequency ultrasound technique (sonication) to ensure that the sperm can be broken down properly and that we can extract all the metabolites from them,” said Calzado.

What is more, some patients ejaculate very few sperm, and it is even more difficult to detect metabolites in these samples. So the researchers wanted to find out the minimum quantity of sperm from which the highest quantity of metabolites can be detected. They verified that “a sample of 1.25 million sperm is the smallest sample that can provide reliable results. That was another of the challenges posed by the study: to take steps so that in the future a better diagnosis can be made using samples with a low sperm count”, she added.

Potential biomarkers: 955 metabolites and 473 lipids

So thanks to the robust methodology developed by the EHU’s Human Reproduction group, a comprehensive analysis of lipids and metabolites can be performed from a minimum quantity of cells. Using mass spectrometry, the researchers determined the potential biomarkers that sperm could have. For the first time, they identified 955 metabolites and 473 lipids that could be present in sperm. “We obtained an overview of sperm metabolites. From now on, we will need to start to explore what happens to these metabolites in each disorder. We now need to find out what alterations patients with infertility have. For example, in some disorders it is likely that certain lipids, or certain proteins, will not be found, or that there will be a larger or smaller quantity, and so on. All of that remains to be explored,” explained the EHU researcher.

The EHU's Human Reproduction group achieved a robust platform for discovering biomarkers to diagnose male infertility in the future, and to find clinical applications. “This opens the door to providing these patients with a better diagnosis and treatment. We have taken an important step forward; the aim now is to search for the biomarkers for each disorder,” she concluded.

Research into male infertility is in its infancy; in fact, “historically most research has been carried out on women. The biggest burden of infertility has always been placed on women. But it has been proven that infertility is the couple's problem”, said Calzado. 30% of cases are related to female factors; another 30% have a male origin, and the rest involve combined causes or are linked to unexplained infertility. So women and men bear a similar burden in this problem, but historically the issue has not been explored in men. “That is why, in our opinion, there is still a great deal of research to be done in this area, and our aim is to focus on that.”