L26/O-312 Artificial oocyte activation with ionomycin in TESA-ICSI cycles for non-obstructive azoospermia: fertilization, embryo competence and reproductive outcomes

Abstract Study question In TESA-ICSI cycles for non-obstructive azoospermia, does ionomycin-assisted artificial oocyte activation influence fertilization, embryo development and downstream reproductive competence? Summary answer Ionomycin improved fertilization without enhancing blastocyst-yield; embryos reaching blastocyst stage demonstrated preserved reproductive competence and no early safety-signal, supporting a selective rather than enhancing role. What is known already Artificial oocyte activation (AOA) using calcium ionophores is primarily indicated for rescue after fertilization failure, and its broader application remains controversial. In non-obstructive azoospermia, testicular sperm often exhibit impaired oocyte-activating capacity related to deficient PLCζ expression. While AOA improves fertilization, its impact on embryo developmental competence, downstream reproductive outcomes and safety particularly in biologically defined NOA populations remains incompletely characterized. Study design, size, duration This non-randomized observational cohort study was conducted between 2019 and 2024 with ethical approval and waiver of informed consent. TESA-ICSI cycles using ionomycin-assisted AOA were compared with contemporaneous cycles without AOA (1282 vs 603 oocytes, respectively). Women aged <37 years undergoing self-gamete, non-PGT TESA-ICSI cycles for non-obstructive azoospermia with successful sperm retrieval (TESA or micro-TESE) were included; cycles with uterine pathology were excluded. Participants/materials, setting, methods Ionomycin calcium salt was applied immediately post-ICSI for 10 minutes per manufacturer protocol, followed by washing and standard culture. All frozen-embryo-transfer-cycles involved transfer of one/two good-quality-blastocysts. Outcomes included fertilization, blastocyst-development, and frozen-embryo-transfer outcomes (clinical pregnancy, implantation, miscarriage and live birth). Neonatal-outcomes were reviewed for major congenital-anomalies and adverse perinatal events. Exploratory multivariable-logistic regression was performed for clinical pregnancy adjusting for female age, blastocyst day at transfer and number of embryos transferred. Main results and the role of chance Fertilization rates were higher following ionomycin-assisted activation compared with controls (88.1% vs 85.7%), consistent with its established role in overcoming impaired oocyte activation associated with testicular sperm in NOA. Baseline oocyte maturity rates were comparable, and no oocyte degeneration attributable to ionomycin exposure was observed. Despite improved fertilization, ionomycin use was not associated with enhanced embryo development. Total blastocyst formation was lower in the ionomycin group (52.9% vs 59%), driven predominantly by fewer Day-5 blastocysts, while Day-6 blastocyst development was comparable, consistent with previous reports that AOA does not enhance blastulation. Following frozen embryo transfer, reproductive outcomes were broadly comparable. Clinical pregnancy rates were higher in the ionomycin group (81% vs 52.3%; OR 3.94, 95% CI 1.72–9.04), while live birth rates were not inferior (43.3% vs 36%; OR 1.37, 95% CI 0.64–2.94). Miscarriage rates were numerically lower (OR 0.22, 95% CI 0.05–0.95; underpowered). Implantation rates were higher (58% vs 39%), without formal embryo-level testing due to clustering. In exploratory multivariable analysis, the association between ionomycin use and clinical pregnancy remained directionally consistent. Neonatal assessment identified no major congenital anomalies, imprinting disorders or adverse perinatal events. Limitations, reasons for caution The observational, non-randomized design limits causal inference. Residual confounding and clustering by couple cannot be excluded, and safety conclusions are limited to early neonatal outcomes. Wider implications of the findings In NOA-TESA cycles, ionomycin improves fertilization without enhancing embryo development. The absence of adverse reproductive or early neonatal signals suggests preserved embryo competence rather than biological enhancement, supporting cautious, biologically targeted use and underscoring the need for prospective controlled studies before broader adoption. Trial registration number No