ASTL (astacin-like metalloendopeptidase) encodes ovastacin, a zinc metalloprotease with primary function in fertilization and polyspermy prevention 1. Upon sperm-egg fusion, ovastacin is released during cortical granule exocytosis and cleaves ZP2, a zona pellucida glycoprotein, preventing additional sperm binding and causing zona pellucida hardening 1. This cleavage is essential for early embryonic development and normal fertilization 2. The enzyme's activity is tightly regulated: unfertilized oocytes release small amounts of inactive ovastacin, bound by its inhibitor Fetuin-B (FETUB) 1, and premature ASTL activity without FETUB inhibition causes infertility 3. Bi-allelic ASTL variants cause female infertility through impaired polyspermy blocking. Frameshift mutations significantly reduce ASTL protein quantity, while missense variants impair enzymatic activity in cleaving ZP2 4. Pathogenic variants disrupt the catalytic domain and critical interaction sites with ZP2 and FETUB 1. Clinical manifestations include abnormal fertilization, oocyte maturation defects, and polyspermy in vitro 5. Affected individuals exhibit subfertility due to reduced embryonic developmental potential 4. Notably, intracytoplasmic sperm injection with assisted oocyte activation successfully bypasses polyspermy defects and enables live birth 56. ASTL mutations should be included in female infertility diagnostic panels 2.