Protocol for assisted sperm fusion insemination of human oocytes using zona pellucida-bound sperm

Oocyte degeneration is a critical and persistent limitation of intracytoplasmic sperm injection (ICSI). Here, we present assisted sperm fusion insemination (ASFI), a protocol for fertilizing human oocytes using zona pellucida-bound sperm (ZP-sperm) to minimize oocyte degeneration while preserving cellular integrity. We describe steps for co-culturing immature or degenerated oocytes with sperm, harvesting ZP-sperm, and facilitating their attachment to and fusion with oocytes. This protocol uses standard ICSI equipment and provides a reproducible approach for minimizing the mechanical stress during fertilization. For complete details on the use and execution of this protocol, please refer to Hatakeyama et al. 1