Due to assisted reproductive techniques like intracytoplasmic sperm injection (ICSI), many infertile couples are helped to conceive their own genetically related child. Unfortunately, fertilization failure (FF) still occurs in 1 to 3% of all ICSI cycles due to oocyte activation deficiencies. Infertility has an enormous social, psychological and financial impact on the couples’ lives. Therefore, it is necessary to provide ways to identify the causal factor (sperm and/or oocyte) and suggest whether assisted oocyte activation (AOA) treatment would be beneficial, so that in the end the couple can achieve a genetically related child. Up until now, twenty-five PLCZ1, seven ACTL7A and three ACTL9 variants have been reported to cause FF or embryo developmental arrest. This research broadens the spectrum of known variants in these three male genes. With diagnostic tests such as MOAT, MOCA and HOCA, a distinction can be made whether or not sperm cells are causing the FF phenotype. Furthermore, the detected variants were subjected to functional tests such as immunostaining or transmission electron microscopy, to assess their causality. Additionally, this study provides new insights into the efficiency of AOA on the newly and previously identified variants.