O-094 Utilization of ultrastructural analysis and genomics of spermatozoa to better characterize subtle forms of male factor infertility

Study question Can sequencing the sperm genome provide insight into the various forms of male factor infertility caused by sperm organelle ultrastructural defects? Summary answer A comprehensive genomic assessment of spermatozoal DNA is able to identify genetic causes of ultrastructural defects visualized by transmission electron microscopy (TEM). What is known already To evaluate a man’s reproductive potential, a conventional semen analysis through the assessment of concentration, motility, and morphology can indicate the proficiency of male gametes. Among those, conventional morphology assay can only provide indirect information on the different components of the sperm cell. The assessment of nanoscopic details such as chromatin, centriolar, mitochondrial, and axonemal components can only be observed by TEM. Indeed, TEM has been used to identify defects in the acrosome, chromatin compaction, and axonemal/periaxonemal structures. Furthermore, exome sequencing of spermatozoal DNA may identify novel causes and candidate genes for these ultrastructural defects. Study design, size, duration In the past 2 years, 20 men with history of fertilization failure or severe astheno-/terato-zoospermia were selected for TEM analysis of their spermatozoa, while 3 fertile men served as controls. Sperm head characteristics, intactness of fibrous sheath, and axonemal/periaxonemal structure were examined by diagnostic TEM. For consenting patients, NGS assessment was concurrently performed to identify mutations responsible for the structural abnormalities identified by TEM. Participants/materials, setting, methods TEM was performed on the ejaculates of 20 infertile patients and 3 fertile controls. Post-centrifugation cell pellets were resuspended, fixed, and dehydrated to be infiltrated and embedded onto the resin. Fixed specimens were sliced by ultramicrotome to 100-nm sections, then viewed by JEOL-1400 electron microscope at 300,000X magnification. At least 100 spermatozoa were evaluated by TEM. For consenting patients, DNA was extracted and amplified from at least 500 spermatozoa for concurrent NGS analysis. Main results and the role of chance Four types of sperm ultrastructural defects were observed, including globozoospermia, dysplasia of fibrous sheath (DFS), proximal centriole defect, and primary ciliary dyskinesia (PCD). One combined case of globozoospermia and DFS was identified. In globozoospermic patients (n = 13), 97-100% of the spermatozoa displayed characteristic spherical heads with absence of acrosomes, dispersed chromatin, and perinuclear theca deformities. Centrosomal and axonemal structures were conserved. NGS identified gene deletions (DPY19L, PICK1, SPATA16) directly related to the globozoospermic phenotype. In patients with DFS (n = 4), complete absence of flagellum was observed in 90-100% of spermatozoa. These defective gametes also displayed mitochondria disorganization, microtubular deformities, and cytoplasmic residues containing coiled flagellum with deformed capitulum within the plasma membrane. Contrary to the globozoospermia, acrosomes and nuclei appeared normal, indicating incomplete late spermiogenesis. Indeed, NGS confirmed gene deletions involved in flagellar development/function (AKAP4, SPAG16, CATSPER1). For the patient with proximal centriole defect (n = 1), sperm nucleus, fibrous sheath, and flagellar structure were conserved. However, 90% of proximal centrioles assessed exhibited microtubular disorganization, confirmed by ODF2 mutation per NGS. In the PCD patient (n = 1), chaotic flagellar microtubule arrangement and absence of outer dynein arms were prevalent in 90% of axonemal cross-sections examined, which was explained by a DNAH5 gene deletion. Limitations, reasons for caution While TEM can overcome the limitations of conventional semen analysis by providing direct visualization of the inner organelle arrangement of spermatozoa to accurately diagnose rare sperm pathologies, it is not routinely applied in clinics due to its high cost and technical specifications. Therefore, confirmatory NGS can provide additional diagnostic value. Wider implications of the findings Ultrastructural analysis with a concurrent genomic assessment characterized phenotypes and genotypes of rare sperm pathologies in infertile men. The utilization of TEM, corroborated by genomic assay, is therefore crucial for clinical and translational reproductive medicine to better characterize male factor infertility. Trial registration number N/A