CONCURRENT ULTRASTRUCTURAL ANALYSIS AND GENOMIC ASSESSMENT OF SPERMATOZOA TO ACCURATELY DIAGNOSE RARE AND SEVERE FORMS OF MALE INFERTILITY

Abstract

In men with ART failure, sperm DNA sequencing with ultrastructural analysis can identify specific sperm defects and guide ART treatment. In the past 2 years, 22 consenting men who failed IVF cycles due to severe astheno-/teratozoospermia were recruited; specimens from 3 fertile men were used as controls. To assess ultrastructure by transmission electron microscopy (TEM), pelleted spermatozoa were fixed, embedded, sectioned to 100-nm slides by ultramicrotome, and viewed with an electron microscope (JEOL-1400) at 300,000X magnification. Sperm head morphology, fibrous sheath intactness, and axonemal/periaxonemal structure were examined on at least 100 spermatozoa to diagnose specific sperm organelle defects. DNA was extracted and amplified from at least 500 spermatozoa for next-generation sequencing (NGS) to identify mutations contributing to the ultrastructural abnormalities. Four types of sperm ultrastructural defects were identified: globozoospermia, dysplasia of fibrous sheath (DFS), proximal centriole defect, and primary ciliary dyskinesia (PCD). One case of combined globozoospermia and DFS and one case of combined DFS and proximal centriole defect were identified. In 13 globozoospermic cases, 97–100% of the spermatozoa displayed complete spherical heads with the absence of acrosomes, perinuclear theca deformities, and dispersed chromatin. Microtubular arrangement of centrosome and flagellum was conserved. NGS identified deletions (DPY19L, PICK1, SPATA16) directly responsible for the globozoospermic phenotype. Based on the findings, assisted oocyte activation was performed for 7 couples with an oocyte-activating agent, boosting fertilization from 5.7% to 40.2% (p<0.00001), resulting in 5 pregnancies and 4 deliveries. In patients with DFS (n=6), flagellar deformities were observed in 90–100% of spermatozoa with mitochondria disorganization, disarranged microtubules, and cytoplasmic remnant enveloping coiled flagella, indicating incomplete late spermiogenesis. Normal acrosomes and nuclei were seen. NGS identified deletions (AKAP4, SPAG16, CATSPER1) involved in flagellar development and function. For patients with proximal centriole defects (n=2), the sperm nuclei and fibrous sheath appeared normal. Approximately 90% of proximal centrioles exhibited microtubular disorganization, confirmed by an ODF2 mutation identified by NGS. For a patient with PCD, chaotic flagellar microtubular arrangement and the absence of outer dynein arms prevailed in 90% of the axonemal cross-sections examined, confirmed by a DNAH5 gene deletion. Nonetheless, a few motile spermatozoa were identified and used for ICSI, yielding 80% fertilization and the delivery of a healthy twin. Concurrent ultrastructural and genomic analysis can pinpoint sperm organelle disruption and lead to successful ART cycles.