Cryopreservation is an expanding strategy to allow not only fertility preservation for individuals who need such procedures because of gonadotoxic treatments, active duty in dangerous occupations or social reasons and gamete donation for couples where conception is denied, but also for animal breeding and preservation of endangered animal species. Despite the improvement in semen cryopreservation techniques and the worldwide expansion of semen banks, damage to spermatozoa and the consequent impairment of its functions still remain unsolved problems, conditioning the choice of the technique in assisted reproduction procedures. Although many studies have attempted to find solutions to limit sperm damage following cryopreservation and identify possible markers of damage susceptibility, active research in this field is still required in order to optimize the process. Here, we review the available evidence regarding structural, molecular and functional damage occurring in cryopreserved human spermatozoa and the possible strategies to prevent it and optimize the procedures. Finally, we review the results on assisted reproduction technique (ARTs) outcomes following the use of cryopreserved spermatozoa.

Study question Could Reactive Oxygen Species (ROS) detected in human spermatozoa represent a predictive marker of fertilization ability? Summary answer ROS detected by CellROX® Orange probe is related to a better sperm quality and function, indicating the sensitivity of the probe to identify physiological ROS. What is known already Oxidative stress (OS), defined as an unbalance between ROS production and antioxidant defenses, is one of the causes of male infertility. A small amount of ROS is necessary for the physiological sperm function, however high ROS levels could impair fertility potential inducing damages at membrane, protein and DNA levels. Previous studies performed by using different methods and probes for OS evaluation in semen or in spermatozoa highlighted the negative role of ROS on sperm functions. However, such studies were not conclusive because of the small number of included subjects and of high variability in the cohorts. Study design, size, duration Observational study conducted on 73 male partners of infertile couples attended consecutively to the Andrology Laboratory of Careggi University Hospital of Florence from September to December 2021. Analyses were performed on both washed and Swim-up selected spermatozoa. Participants/materials, setting, methods After routine semen analysis, washed and Swim-up selected spermatozoa were incubated with CellRox®Orange (a fluorescent probe able to reveal OS in viable cells), at the concentration of 1 µM for 30 minutes at 37°C, 5%CO2, and revealed by flow cytometry. Sperm DNA fragmentation (by TUNEL/PI method), sperm kinematic parameters and hyperactivated motility (by C.A.S.A. system) were also assessed. In some samples a double staining with CellRox®Orange and Annexin V (which stains phosphatidylserine externalization) was performed. Main results and the role of chance We found that the percentage of spermatozoa positive to CellRox® Orange is positively correlated with routine seminal parameters. Although this result appears in contrast with most studies in literature reporting negative correlations between OS and semen parameters, it can be explained by the fact that the probe is specific for viable spermatozoa characterized by a better motility and morphology. Considering the importance of distinguishing between positive and negative ROS in spermatozoa, we further investigated the significance of ROS detected by this probe. To understand whether the probe marks spermatozoa with a better quality, CellRox® Orange positivity was evaluated in Swim-up selected spermatozoa finding significantly higher levels of CellRox® Orange positivity respect to unselected samples. Furthermore, the fact that most of the CellRox® Orange positive spermatozoa were negative for Annexin V (which reveals cells with early signs of apoptosis) is a further confirmation of the good quality of CellRox® Orange positive spermatozoa. Another evidence is represented by the finding that we observed a negative correlation between OS detected by CellRox® Orange and sperm DNA fragmentation, although several studies have shown a positive relationship between these two parameters (when OS is measured by different probes). Limitations, reasons for caution Up to now, the study involved a limited number of subjects. Further experiments should be performed to increase the number of subjects in order to confirm the results. Wider implications of the findings Results of this study together with those previously published evidence that different OS is detected depending on which probe is used. CellRox® Orange seems to be sensible for physiological ROS detection, therefore, it could be employed in future studies aimed to evaluate the association between OS and assisted reproduction outcomes. Trial registration number not applicable

BackgroundOxidative stress is defined as the unbalance between reactive oxygen species (ROS) production and antioxidant defences. Whereas low levels of ROS are necessary for physiological sperm functions, high levels impair fertility damaging membranes, proteins and DNA. In this study, we used two probes, CellROX® Orange and Dihydroethidium (DHE), which reveal different intracellular ROS species, to evaluate the association between the percentage of oxidized viable spermatozoa and sperm functions.MethodsThe percentage of oxidized spermatozoa was evaluated by flow cytometry with the two probes concomitantly with standard semen parameters and sperm DNA fragmentation (sDF, by TUNEL/PI). Phosphatidylserine membrane exposure, caspase 3,7 activity, sperm kinematic parameters and hyperactivated motility were evaluated by Annexin V, FLICA™ and CASA system respectively.ResultsOxidized viable spermatozoa, evaluated with both probes, were positively associated with sperm basal parameters and negatively with sDF. Also, we found that a consistent percentage of CellROX® positive viable spermatozoa were selected from whole semen during swim up procedure. Double staining of CellROX® Orange with Annexin V and FLICA™ demonstrated that viable oxidized spermatozoa do not show apoptotic features.ConclusionOverall, our results suggest that CellROX® Orange and DHE allows identification of the viable oxidized sperm fraction related to better performances.

Background: Sperm cryopreservation is recommended to preserve male fertility for cancer patients or other medical conditions at risk of sperm decline. Whether motility and viability recovery rates vary depending on the medical conditions requiring cryopreservation is poorly known. We report here on the 24-year experience of our semen bank. Methods: Motility and viability recovery rates were evaluated in 1973 collections from patients with various medical conditions and 67 collections from donors, and the results were related to basal semen quality. Results: Motility and viability recovery were highly related to basal semen quality and varied between cancer and non-cancer conditions, independently of the duration of cryopreservation and patient age. In samples with a sperm number below 2 × 106/mL, recovery rates approximated to zero. The highest recovery rates were found in donor collections. Cut-off values for the recovery of at least 1% motile spermatozoa were established based on initial semen quality. Conclusions: Our results indicate that the occurrence of any pathological or medical condition resulted in lower recovery rates with respect to donors, indicating that intrinsic sperm characteristics drive susceptibility to cryodamage. Established cut-off values for motility recovery can be useful for patient counseling as well as for ART laboratories to decide the type of procedure.