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OBJECTIVE To investigate the extent to which assisted oocyte activation (AOA) improves clinical outcomes in patients diagnosed with oocyte activation deficiencies (OADs). DESIGN Retrospective cohort study comparing AOA cycles and previous intracytoplasmic sperm injection (ICSI) cycles in couples experiencing low or total failed fertilization after ICSI. Importantly, the sperm-related oocyte-activating capacity was examined in all patients before AOA with the use of the mouse oocyte activation test (MOAT). SETTING Infertility center at a university hospital. PATIENT(S) A total of 122 couples with a history of low or total failed fertilization after ICSI. INTERVENTION(S) ICSI, MOAT, AOA, and embryo transfer. MAIN OUTCOME MEASURE(S) Fertilization, pregnancy, and live birth rates. RESULT(S) MOAT revealed 19 patients with a sperm-related OAD (MOAT group 1), 56 patients with a diminished sperm-related oocyte-activating capacity (MOAT group 2), and 47 patients with a suspected oocyte-related OAD (MOAT group 3). AOA (191 cycles) significantly improved fertilization, pregnancy, and live birth rates in all MOAT groups compared with previous ICSI attempts (243 cycles). Fertilization rates after AOA were significantly different among MOAT groups 1 (70.1%), 2 (63.0%), and 3 (57.3%). Between MOAT group 1 and 3, significant differences in pregnancy (49.0% vs. 29.4%) and live birth (41.2% vs. 22.1%) rates were observed. In total, 225 embryo transfers resulted in 60 healthy live births following AOA. CONCLUSION(S) Patients undergoing diagnostic testing before AOA show a significant improvement in clinical outcomes compared with previous cycles. Our findings highlight that AOA should be reserved for patients with clear OADs.