Pronuclear morphology seems to be an important predictive value of zygote development and integrity. In this study we want to evaluate the effect of insemination technique, male factor and oocyte cryopreservation on pronuclear morphology of zygotes derived from sibling oocytes in our Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova, Reggio Emilia, Italy. Subjects (n = 190) were submitted to IVF cycles with non-frozen and frozen sibling oocytes. Morphological evaluations were assessed using zygote pronuclear morphology (pronuclei, nucleoli and axis) in four groups: Group 1: 144 zygotes from 85 conventional IVF cycles with non-frozen oocytes; Group 2: 164 zygotes from 85 intracytoplasmic sperm injection (ICSI) cycles with Group 1 patients' sibling frozen oocytes; Group 3: 221 zygotes from 123 ICSI cycles with non-frozen oocytes; Group 4: 197 zygotes from 123 ICSI cycles with Group 3 patients' sibling frozen oocytes. No differences between Group 1 and Group 2 were seen. Group 3 was statistically different from Group 4 in relation to the nucleolar morphology. Oocyte cryopreservation procedure modified the nucleolar morphology of zygotes only in the presence of poor semen quality.