SPERMFREE: integrated analytical workflow for cell-free DNA (cfDNA)-based assessment of testicular function in severe male infertility

Severe male infertility affects approximately 1 % of men; however, its diagnosis and therapeutic decision-making often remain uncertain. The need for a reliable tool to classify severe male infertility and guide timely therapeutic choices can significantly shorten the time from a man’s first clinic visit to a family-planning solution while reducing costs associated with consultations and procedures (exceeding €2 000 per patient). Therefore, the SPERMFREE project proposes a non-invasive method for evaluating testicular function. In short, cell-free DNA (cfDNA) isolated from ejaculate is sequenced and bioinformatically analyzed across several molecular layers - fragmentomic, pseudotranscriptomic, and microbiome. The resulting parameters from each layer are integrated into a rule-based computational model that enables the assessment of testicular functionality, patient classification, and subsequent optimization of treatment decisions. During the project, existing samples from different infertility subcategories and controls (n = 40) will be used to identify the most discriminative parameters for classification and to develop the model. Additional new patient samples will be collected to test and refine the workflow under laboratory conditions. Within 8 months, the goal is to reach TRL 4, to develop and validate the SPERMFREE analytical workflow, prepare an open-access scientific publication, and submit a patent application.