**4. Discussion**

Vitrification has been used to cryopreserve human oocytes and embryos in human infertility clinics for many years. For oocyte and embryo cryopreservation, vitrification requires high concentration (30%) of cryoprotectants in the vitrification solutions [1, 2]. Cryoprotectants are macromolecules added to the freezing medium to protect cells from the detrimental effects of intracellular ice crystal formation during the process of freezing and thawing. When embryo vitrification solution is cooled to −196°C in liquid nitrogen, substances in the solution is transformed into a glass, not ice crystal. However, in the present study, we used slow sperm freezing solution with low concentration of cryoprotectant (7.5% glycerol) and sperm still survived after freezing. It is still unknown whether ice crystal is formed inside sperm or not.

Although the procedure used in the present study is similar to vitrification, cooling time from room temperature to −196°C is longer (~2 minutes) than vitrification, thus the cooling rate is lower than vitrification. In fact, direct immersion of sperm to liquid nitrogen (that is similar as vitrification) did not support sperm

#### *A Method for Small Number of Human Sperm Cryopreservation DOI: http://dx.doi.org/10.5772/intechopen.98674*

survival, which is completely different from vitrification of oocytes and embryos. Our data indicate that this fast sperm freezing method with regular concentration of cryoprotectant in the freezing solution can bring about ~90% recovered rate of sperm motility after thawing. This rate is higher than those reported in the previous studies with other methods [4–12]. The results in our study were very stable after freezing of a total of 16 semen samples, further indicating that this method is practicable in human IVF clinics.

A few previous publications indicated that vitrification or fast sperm freezing with different concentrations of sucrose as cryoprotectant also supported sperm motility after thawing, but the overall sperm motility rates were less than 40% [4–12]. Reduced sperm motility after freezing/thawing is mainly caused by the injury of sperm membrane [12–15]. Sperm DNA can also be further damaged by different cryopreservation methods or cryopreservation medium [12, 16, 17]. We did not examine sperm DNA fragmentation before vitrification and after warming in the present study, it remains necessary to further examine whether high survival rate is correlated with low DNA fragmentation.

Some devices for embryo vitrification have been used for vitrification/fast freezing of sperm but they are not the ideal devices for sperm freezing [5, 6]. Cryogenic storage vials may be suitable for normal sperm freezing because large volume is required for these kinds of freezing, but not suitable for small number of sperm freezing [10, 12–15]. Recently, a report indicated that direct pellet vitrification of human sperm with 0.25 M sucrose and thawing at 42°C increased sperm motility to ~70% and the authors considered that their high post thawing survival rate was resulting from a higher thawing temperature [10]. However, this method may not be suitable for freezing of small number of sperm.

Based on our study, we found that the following technical aspects are important to obtain high rates of sperm motility after fast freezing:

First, the traditional slow sperm freezing solution containing glycerol and sucrose works well for fast sperm freezing. It is not necessary to use higher concentrations of cryoprotectants, like those used for oocyte and embryo vitrification. However, cryoprotectants are still necessary for fast sperm freezing because very low motile sperm was obtained in the solution without cryoprotectants. This result indicates that the current commercial sperm freezing solution is appropriate for fast sperm freezing.

Second, time for equilibration of sperm in the freezing solution and volume of solution did not affect the post thawing sperm motility. These advantages allow this fast sperm freezing procedure to be easily used in human infertility clinics. For example, if there are very few sperm, freezing solution drop can be as small as 1 μl, and if there are more sperm, the volume of micro drop can be increased to 5 μl. However, it may not be necessary to freeze more than 20 sperm (if there is small number of sperm in a sample) in one device as high recovery and survival rates can be obtained with this method. Also, the time range (1–5 min) for equilibration of sperm in the sperm freezing solution allows laboratory technicians to process the freezing without rushing. We did not examine whether longer equilibration time has similar post thawing sperm motility or not, but it appears that up to 5 minutes is sufficient for technicians to load and freeze sperm.

Third, direct immersion of sperm to liquid nitrogen does not support post thawing sperm motility. This result indicates that fast cooling rate, like that for embryo vitrification, does not work for sperm freezing. By contrast, slow cooling rate is better than rapid cooling rate. When the devices are sealed in the cryogenic storage vial and then immersed to liquid nitrogen, cooling rate is reduced 60 times (120 sec vs. 2 sec) as compared with direct immersion of device to liquid nitrogen. These results indicate that vitrification does not work for human sperm freezing with the

current sperm freezing solutions. When the device was placed in the vial and then immersed to liquid nitrogen, the cooling rate was much slower than vitrification.

High survival rates (>90%) can be obtained when human oocytes and embryos were vitrified [1, 2]. Here we provide evidence that ~90% of recovered sperm motility can also be obtained with this fast sperm freezing method. In the present study, we examined sperm motility, but did not examine sperm vitality because ~90% of sperm were motile after thawing. Usually, sperm survival rates after freezing and thawing are higher than motility, indicating that sperm membrane has been injured in some sperm and these sperm cannot move but they are still live [4–12]. However, in the present study, high post thawing sperm motility indicates that sperm membrane was not injured during freezing and thawing, thus the current method may have less injury to sperm and/or sperm membrane than the previous methods [4–12].

The advantage of the devices used in the present study is that these devices can be directly placed in a culture dish, so sperm loading before freezing and sperm picking up after thawing can be done directly under a phase contrast microscope. Therefore, it is not necessary to transfer sperm between devices or wash sperm by centrifugation.

Furthermore, this method is a closed system. The vials must be closed tightly before immersion to liquid nitrogen to avoid liquid nitrogen entering the vials. Therefore, this method not only guarantees high rate of sperm motility after freezing, but also ensures samples not to have cross contamination during freezing and storage.

## **5. Conclusion**

In conclusion, our data indicate that high post thawing sperm motility can be obtained after fast sperm freezing with the current commercial sperm freezing solution and commercially available devices, such as SpermVD and Microdevice. Our results also indicate that this method is safe as the devices are sealed in the storage vials during freezing and storage, which can avoid cross contamination. Furthermore, this method is simple and easy to learn.

#### **Acknowledgements**

This research was supported by a grant from Nanning Scientific Research and Technology Development project, (20173017-8) and Guangxi Medical and Health Department (20190862).
