P-069 A male factor panel of NGS can identify the genetic cause of more than half patients with seminal alterations

Abstract Study question Is Next Generation Sequencing (NGS) an effective diagnostic tool for sperm abnormalities? Summary answer NGS male factor gene panel can be a potent diagnostic tool for discovering pathogenic variants linked to sperm abnormalities. What is known already Male infertility, responsible for half of infertility cases, often shows as absence or decreased sperm count (azoospermia, criptozoospermia or oligozoospermia), poor motility (asthenozoospermia) or a higher proportion of morphologically abnormal sperm (teratozoospermia). While the spermiogram remains the gold standard for evaluating male fertility, the potential to identify the molecular basis of sperm abnormalities may make NGS a valuable approach for such patients. The aim of our study was to assess NGS panel's effectiveness in diagnosing patients with abnormal sperm count, motility and morphology. Study design, size, duration A prospective study was conducted from May 2021 to May 2022 in 82 patients including 20 patients with oligozoospermia, 19 with asthenozoospermia and 23 with teratozoospermia. No other sperm alterations were taken into account in each group. The control group consisted of 20 normozoospermic healthy donors selected on the basis of normal sperm parameters according to the WHO criteria (2010). Patients carrying Y-chromosome microdeletions or abnormal karyotype were excluded. Participants/materials, setting, methods Genomic DNA extraction from blood-EDTA of the patients was performed using the commercial MagMax DNA MultiSample Ultra kit and the King-Fisher automated extractor (ThermoFisher®). Next Generation Sequencing (NGS) was done using a panel with 426 genes involved in the spermatogenesis process. Panel sequencing for identification of genetic variants was performed using Nextera Enrichment technology (Illumina®). FASTAQ data were processed using BWA and GATK algorithms. VCF files were analysed using Variant Interpreter software and in silico predictors. Main results and the role of chance Data analysis showed that thirty-seven of the sixty-two patients were carriers of pathogenic mutations in at least one of the genes included in the panel (37/62,59.6%). In the oligozoospermic group, eleven patients (11/20,55%) were carriers of pathogenic mutations in:CFTR, CEP290, WDR66, ESR1, DNAI2, POLG, PIWIL3, GNRHR, MSH5 and CTNS. In the asthenozoospermic group, ten out of nineteen patients (10/19,52.6%) were carriers of pathogenic mutations in:HS6ST1, PMS2, CYP19A1, DNAI2, POLG, LRRC6, G6PD, CCDC39 and PIWIL3. In the teratozoospermic group, six of the twenty-three patients (6/23,26%) were carriers of pathogenic mutations in:CFTR, PIWIL3, CYP21A2, SRD5A. No pathogenic mutations were detected in the control group. The analysis showed comparable results among the various spermiogram alterations with no significant difference (p > 0.05). Each variant was found in a single patient per group, except POLG and CCDC39, which were found in two cases in the oligozoospermic and asthenozoospermic group, respectively, also CFTR and PIWIL3 in two teratozoospermic patients. Besides, it is remarkable that some of the pathogenic variants were present in genes in more than one group:CFTR (3/62), DNAI2 (2/62), POLG (3/62) and PIWIL3 (4/62). All identified variants were linked with processes of spermatogenesis such as dynein assembly and DNA integrity. Limitations, reasons for caution The main limitation of this study is the limited number of patients included. Functional studies with a larger cohort of males with seminal disorders is warranted to confidently correlate the genetic variants identified in this analysis with spermatogenic failure. Wider implications of the findings The gene list included in our panel represents a step-forward in the screening of males with altered sperm parameters. Our results may add in the knowledge of male factor infertility to provide etiologic factors towards a personalized treatment and adequate genetic counselling. Trial registration number Not applicable