ACTL9 (actin like 9) is a testis-specific protein essential for male fertility, primarily functioning in spermatogenesis and fertilization. Mechanistically, ACTL9 plays a critical role in organizing the perinuclear theca (PT), a cytoskeletal structure in the sperm head that properly positions phospholipase C zeta (PLCζ) 1. This correct localization of PLCζ is essential for triggering normal calcium oscillations in oocytes during fertilization 1. ACTL9 mutations cause spermatogenic failure 53, characterized by abnormal sperm morphology including coiled flagella, thickened midpieces, and irregular mitochondrial sheath arrangements, resulting in reduced sperm motility 2. At the ultrastructural level, ACTL9 mutations disrupt the '9+2' microtubular organization in flagella 2. Clinically, homozygous ACTL9 variants cause total fertilization failure (TFF) even after intracytoplasmic sperm injection (ICSI), occurring in approximately 3.70% of male patients with post-ICSI fertilization failure 3. However, assisted oocyte activation (AOA) using ionomycin or calcium ionophore successfully overcomes TFF in ACTL9 mutation carriers, enabling successful pregnancy and live births 13. These findings establish ACTL9 as a critical genetic marker for diagnosing unexplained fertilization failure and identifying candidates for AOA therapy.