Abstract
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).
Highlights
Human sperm cryopreservation has been widely used for human reproduction
To explore whether freezing sperm at −80°C freezer is feasible for human ejaculate and testicular spermatozoa, we evaluated the effects of freezing sperm at −80°C freezer and vapor rapid freezing techniques on sperm quality, and simplified the procedure and equipment for freezing human ejaculate and testicular spermatozoa
In the −80°C freezer, the sample was placed in liquid nitrogen vapor for 5 min, the cooling rate was approximately -12°C/ min down from room temperature (RT) to −40°C, and the drop from RT to −80°C took approximately 1990 s (Figure 2, Supplementary Table 1)
Summary
Human sperm cryopreservation has been widely used for human reproduction. Autologous sperm freezing is the general method applied in vitro fertilization (IVF) laboratories in a variety of circumstances ranging from fertility preservation for cancer patients to the clinical management of male infertility (Trottmann et al, 2007).The first attempt to freeze semen dates can be traced back to 1776 when Abbot et al (Royere, et al, 1996) reported that snow could store sperm by cooling. A variety of freezing methods have been discovered with the development of cryopreservation technology: slow freezing, liquid nitrogen vapor rapid freezing, and vitrification (Tao et al, 2020). The standard cryopreservation method normally involves freezing human sperm in liquid nitrogen vapor to −80°C and stored in liquid nitrogen. Vapor rapid freezing cannot control the rate of temperature drop caused by the volatilization of liquid nitrogen (Di Santo et al, 2012). Another limitation of liquid nitrogen vapor rapid freezing is that it cannot cool many semen samples simultaneously
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