A novel homozygous chromosomal deletion encompassing the ACTL9 gene is associated with cIVF polyspermy and male infertility

Actin-like 9 (ACTL9) is a testis-specific actin-like protein located in the perinuclear theca of the sperm head and neck that plays a role in acrosome formation and sperm head shaping. Pathogenic mutations in ACTL9 have been reported to be associated with male infertility and fertilization failure after conventional in vitro fertilization (cIVF) and intracytoplasmic sperm injection (ICSI). In this study, we identified a homozygous chromosomal microdeletion within the ACTL9 gene in an infertile male characterized by polyspermy in cIVF and nearly total fertilization failure in ICSI. Transmission electron microscopy revealed that spermatozoa from the affected individual exhibited a ruffled acrosome, and the inner acrosomal membrane was detached from the nuclear envelope. Immunofluorescence staining revealed the absence of phospholipase C zeta 1 (PLCZ1) protein in mutant spermatozoa, which explains the characteristic multipronuclei observed in cIVF. ICSI and artificial oocyte activation treatment successfully rescued the cIVF polyspermic phenotype and resulted in a live birth. These findings expand the spectrum of ACTL9 mutations and could facilitate the diagnosis of sperm-borne polyspermy.