Table 2.

Studies examining the correlation between DNA damage and increased levels of ROS.

Method Used for DNA Damage Detection	Method and Cryoprotectant Used Cryopreservation	Patients Included in the Study	Results	References
Flow cytometry	Programmable slow freezing with glycerol as a cryoprotectant.	18 normozoospermic patients (>20 × 106/mL and motility ≥50%)	Levels of ROS were increased after cryopreservation.	Wang et al., (1997) [60]
Flow cytometry	Programmable slow freezing with glycerol as a cryoprotectant.	60 patients (34 with abnormal semen results and 26 with normal semen results)	The process of cryopreservation resulted in an increase in DNA fragmentation. The dominant pathway to DNA fragmentation during cryopreservation is the ROS pathway.	Thomson et al., (2009) [61]
Flow cytometry	Freezing on liquid nitrogen vapor with glycerol as a cryoprotectant.	30 normozoospermic patients (>20 × 106/mL and motility ≥50%)	The levels of ROS detected via flow cytometry increased significantly compared with the fresh control group.	Li et al., (2010) [62]
Flow cytometry	Freezing on liquid nitrogen vapor with glycerol as a cryoprotectant.	15 normozoospermic patients (>20 × 106/mL and motility ≥50%)	They found no relationship between DNA fragmentation and ROS levels; they suggest cryopreservation-induced DNA damage happens through other pathways.	Zribi et al., (2010) [49]
Flow cytometry	freezing on liquid nitrogen vapor and vitrification with glycerol as a cryoprotectant	49 patients of infertile couples undergoing routine semen analysis	Both cryopreservation methods induced higher levels of ROS production. Results with the vitrification method were poorer than results achieved via vapor freezing.	Arciero et al., (2021) [63]