Nanosecond pulsed electric field precisely manipulates cytoplasmic Ca ²⁺ oscillations during oocyte activation

Oocyte activation deficiency (OAD) is a primary cause of fertilization failure following intracytoplasmic sperm injection (ICSI), a problem that can potentially be overcome through artificial oocyte activation (AOA). However, concerns persist regarding the safety and efficacy of AOA techniques in clinical practice. In this study, nanosecond pulsed electric field (nsPEF) is proposed as a safe and controllable method for oocyte activation in vitro by precisely manipulating Ca ²⁺ signaling in the ooplasm. Mouse oocytes collected from oviducts were exposed to nsPEF stimulation at varying intensities to induce distinct Ca ²⁺ signaling patterns. Subsequently, these oocytes underwent parthenogenetic activation and were cultured to assess developmental potential up to the blastocyst stage. The sperm-initiated physiological Ca ²⁺ oscillations are successfully mimicked by one series of Ca ²⁺ signals induced by nsPEF at low or medium intensities, and improve activation efficiency and developmental potential compared to calcium ionophore A23187. Low-intensity nsPEF pulses mediated repetitive extracellular Ca ²⁺ influx in a non-invasive, electro-permeable manner. Medium-intensity nsPEF stimulation triggered periodic Ca ²⁺ release from the endoplasmic reticulum (ER) via the PIP ₂ -IP ₃ -IP ₃ R pathway, producing physiological-like Ca ²⁺ oscillations. The non-invasive nsPEF method ensures safe oocyte activation by preserving cellular integrity and minimizing stress responses. The efficacy of nsPEF in precisely manipulating Ca ²⁺ signaling patterns was further validated in human unfertilized oocytes. Our findings present a novel AOA approach with enhanced safety and efficacy, especially for patients experiencing repeated fertilization failures. It is anticipated that nsPEF is emerging as a promising technology for addressing male infertility issues, and may offer a potential alternative fertility treatment, bypassing sperm for initiation of oocyte activation.