BACKGROUND: Single sperm cryopreservation (SSC) is a method that preserves the limited number of spermatozoa in testicular sperm. But testicular spermatozoa are characterized with low movement, which is not ideal for sperm selection before SSC. OBJECTIVE: This study was designed to investigate in vitro incubation (IVC) as a sperm selection technique before SSC on biological factors of testicular spermatozoa. MATERIALS AND METHODS: Testicular tissue was obtained from 15 azoospermia men. One part of the testicular samples was used as a Control group, which was assessed fresh. One portion was cryopreserved by a vitrification (Vit) method and the two other portions were in vitro cultured for 24 h, with (IVC-Vit) or without (IVC) vitrification. Sperm motility, viability, morphology, DNA fragmentation and mitochondrial membrane potential were evaluated. RESULTS: Sperm motility and viability were better maintained in the IVC-Vit group compared to the Vit group (P=0.04 and P= 0.003, respectively). Sperm morphology, the fresh, Vit, IVC, and IVC-Vit groups all showed similar results (P >
0.05). Mitochondrial activity was significantly lower in the Vit group compared to the Control fresh group (P = 0.0001). The IVC group had a significantly higher DFI as compared to the Control (P <
0.0001). Compared to the IVC group, the IVC-Vit sperm had a significant increase in DFI (P= 0.0009). There was a statistically significant difference between post warm DFI of the Vit group and IVC-Vit group (P <
0.0001). CONCLUSION: IVC as a sperm selection method increased motility and viability of testicular spermatozoa before single sperm vitrification. As DNA fragmentation increased by this technique, this method is not ideal for selecting viable sperm. https://doi.org/10.54680/fr25110110512.