EHU Researchers Develop Novel Metabolomics Protocol to Identify Sperm Biomarkers for Male Infertility Diagnosis
Key Insights
Researchers at the University of the Basque Country (search) developed a robust sonication-based methodology to extract and analyze metabolites directly from sperm cells for the first time.
The protocol enables reliable metabolomic analysis from as few as 1.25 million sperm, addressing the challenge of low-sperm-count samples.
Using mass spectrometry, the team identified 955 metabolites and 473 lipids as potential biomarkers, creating the largest sperm metabolite catalog to date.
Human infertility represents a significant global health and social challenge, affecting approximately 17.5% of the world's population, with male factors contributing to nearly half of all cases. Yet current diagnostic tools remain limited. Now, researchers from the Human Reproduction group at the University of the Basque Country (search) (EHU) have developed a robust new methodology that enables, for the first time, comprehensive metabolomic profiling directly from sperm cells—an advance that could transform how male infertility (search) is diagnosed and treated.
"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. The standard semen analysis measures general parameters such as sample volume, sperm concentration, motility, and morphology under a microscope, but provides no molecular insight into underlying metabolic dysfunction.
Overcoming Methodological Hurdles in Sperm Analysis
Most prior research in this field has relied on seminal plasma rather than sperm cells themselves. The EHU team recognized that metabolites—the products and intermediates of metabolic reactions including lipids, amino acids, proteins, nucleic acids, and carbohydrates—could offer far richer diagnostic information when analyzed directly from sperm. "For example, by analysing metabolites, you can find out whether a metabolic pathway is altered in a disease," Calzado noted.
However, extracting metabolites from sperm posed significant technical challenges. "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.
An additional obstacle involved patients who ejaculate very few sperm, making metabolite detection even more difficult. The researchers systematically determined the minimum sample size required for reliable results. 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," Calzado added.
A Landmark Catalog: 955 Metabolites and 473 Lipids
Using the optimized sonication protocol combined with mass spectrometry, the EHU team performed a comprehensive analysis of lipids and metabolites from a minimal quantity of cells. For the first time, they identified 955 metabolites and 473 lipids that could be present in sperm, creating the most extensive catalog of sperm metabolites reported to date.
"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," explained Calzado. She elaborated that in certain disorders, specific lipids or proteins may be absent, or present in abnormally high or low quantities—patterns that remain to be systematically investigated.
Toward Clinical Applications
The EHU's Human Reproduction group has established a robust platform for discovering biomarkers with potential clinical applications in male infertility (search). "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," Calzado concluded.
The research forms part of Irune Calzado-Lopez's PhD thesis in the EHU Physiology Department, supervised by Nerea Subirán-Ciudad and Manu Araolaza-Lasa of the EHU Faculty of Medicine and Nursing. Samples were obtained from Galdakao Hospital (search) through the Biobizkaia Institute for Health Research (search).
Calzado also highlighted a broader context: historically, most infertility research has focused 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," she said. With approximately 30% of cases related to female factors, another 30% having male origin, and the remainder involving combined or unexplained causes, the researchers argue that male-factor infertility has been underexplored. "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."