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Human autologous sperm freezing involves ejaculated sperm, and testicular or epididymal puncture sperm freezing, and autologous sperm freezing is widely used in assisted reproductive technology. In previous studies, researchers have tried to cryopreserve sperm from mammals (rats, dogs, etc.) using a −80°C freezer and have achieved success. It is common to use liquid nitrogen vapor rapid freezing to cryopreserve human autologous sperm. However, the operation of this cooling method is complicated, and the temperature drop is unstable. In this study, we compared the quality of human ejaculation and testicular sperm after liquid nitrogen vapor rapid freezing and −80°C freezing for the first time. By analyzing sperm quality parameters of 93 ejaculated sperm and 10 testicular sperm after liquid nitrogen vapor rapid freezing and −80°C freezing, we found reactive oxygen species (ROS) of sperm of the −80°C freezer was significantly lower than liquid nitrogen vapor rapid freezing. Regression analysis showed that progressive motility, ROS, and DNA fragmentation index (DFI) in post-thaw spermatozoa were correlated with sperm progressive motility, ROS, and DFI before freezing. For the freezing method, the −80°C freezer was positively correlated with the sperm progressive motility. Among the factors of freezing time, long-term freezing was negatively correlated with sperm progressive motility and ROS. Although freezing directly at −80°C freezer had a slower temperature drop than liquid nitrogen vapor rapid freezing over the same period, the curves of the temperature drop were similar, and slight differences in the freezing point were observed. Furthermore, there were no statistically significant differences between the two methods for freezing testicular sperm. The method of direct −80°C freezing could be considered a simplified alternative to vapor freezing for short-term human sperm storage. It could be used for cryopreservation of autologous sperm (especially testicular sperm) by in vitro fertilization centers.Clinical Trial Registration: (website), identifier (ChiCTR2100050190).

Human autologous sperm freezing involves ejaculated sperm, and testicular or epididymal puncture sperm freezing, and autologous sperm freezing is widely used in assisted reproductive technology. In previous studies, researchers have tried to cryopreserve sperm from mammals (rats, dogs, etc.) using a −80°C freezer and have achieved success. It is common to use liquid nitrogen vapor rapid freezing to cryopreserve human autologous sperm. However, the operation of this cooling method is complicated, and the temperature drop is unstable. In this study, we compared the quality of human ejaculation and testicular sperm after liquid nitrogen vapor rapid freezing and −80°C freezing for the first time. By analyzing sperm quality parameters of 93 ejaculated sperm and 10 testicular sperm after liquid nitrogen vapor rapid freezing and −80°C freezing, we found reactive oxygen species (ROS) of sperm of the −80°C freezer was significantly lower than liquid nitrogen vapor rapid freezing. Regression analysis showed that progressive motility, ROS, and DNA fragmentation index (DFI) in post-thaw spermatozoa were correlated with sperm progressive motility, ROS, and DFI before freezing. For the freezing method, the −80°C freezer was positively correlated with the sperm progressive motility. Among the factors of freezing time, long-term freezing was negatively correlated with sperm progressive motility and ROS. Although freezing directly at −80°C freezer had a slower temperature drop than liquid nitrogen vapor rapid freezing over the same period, the curves of the temperature drop were similar, and slight differences in the freezing point were observed. Furthermore, there were no statistically significant differences between the two methods for freezing testicular sperm. The method of direct −80°C freezing could be considered a simplified alternative to vapor freezing for short-term human sperm storage. It could be used for cryopreservation of autologous sperm (especially testicular sperm) by in vitro fertilization centers. Clinical Trial Registration: (website), identifier (ChiCTR2100050190). [ABSTRACT FROM AUTHOR]

Background: We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a −80°C freezer, maintaining their ability to fertilize mouse eggs. Objectives: The main objective here was to evaluate the effects of five years at −80°C compared to liquid nitrogen storage (LN2, control condition) on mouse sperm viability, physiological parameters, and fertilization capacity. Materials and methods: Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR‐14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3‐AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability. Results: Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at −80°C for 5 years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN2. Discussion: Long‐term mouse sperm storage at −80°C (five years) could be considered an ideal alternative to the most common LN2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking. Conclusion: It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a −80°C freezer with no significant differences compared to the storage in LN2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity. [ABSTRACT FROM AUTHOR]

In this study, we summarize our long-term follow-up data of 24 patients who underwent autologous peripheral blood stem cell transplantation (PBSCT) using the dump-freezing method in a -80 degrees C freezer. Collected peripheral blood mononuclear cells were mixed with a cryoprotectant solution consisting of autologous plasma and 20% dimethyl sulfoxide, then placed in a -80 degrees C freezer. The recovery rate of mononuclear cells (MNCs), colony-forming unit-granulocyte/macrophage (CFU-GM) colonies, and CD34+ cells were calculated. Engraftment time (with neutrophil count > 0.5 x 10(9)/L, platelet count > 50 x 10(9)/L) and normal hemopoiesis (neutrophil count > 2 x 10(9)/L, platelet count > 100 x 10(9)/L) were evaluated. Median duration of cryopreservation was 76 days. The mean recovery rates of MNCs, CFU-GM colonies, and CD34+ cells were 93.4%, 78.4%, and 95.3%, respectively. The median engraftment times of neutrophils and platelets were 8 and 27 days, respectively. The median normal hemopoiesis times of neutrophil and platelet were 31 and 45 days, respectively. Nine patients are alive and in complete remission (CR). Seven patients in first CR sustained normal hemopoiesis with a median duration of 35 months. Two patients, who achieved second CR after salvage chemotherapy due to a leukemia relapse after PBSCT, maintained engraftment status for 24 and 28 months, and 1 reached normal hemopoiesis. These results demonstrate that PBSCT using the dump-freezing method in a -80 degrees C freezer leads to acceptable long-term engraftment stability. [ABSTRACT FROM AUTHOR]