**1. Introduction**

Vitrification, as a fast cryopreservation method, has been used to cryopreserve human oocytes and embryos for many years and has become the routine and major method for cryopreservation of human oocytes and embryos as high survival rates can be obtained after warming [1, 2] However, for human sperm freezing, the current methods still mainly rely on slow freezing [3, 4]. Various methods for vitrification or fast freezing of human sperm have been tested, especially for cryopreservation of small number of sperm [5–12]. However, so far, no standard and practical vitrification/fast freezing method has been developed. This may be due to the facts that the number of sperm is quite large in most semen samples and current slow-programmed sperm freezing method can provide acceptable post recovery rate and survival rate. Furthermore, vitrification did not offer significantly superior results than slow freezing for sperm cryopreservation in the previous study [13].

Clinical uses of rare sperm or small number of sperm in human in vitro fertilization (IVF) are very common, especially when sperm are collected from testicle biopsy. Due to very small number of sperm in these samples, many clinics chose to freeze the sperm during the initial semen analysis or testicle biopsy. However, sperm numbers and/or sperm motility may decrease after the traditional slow freezing. Occasionally, there are not enough sperm survival to inseminate all oocytes from a cycle. Therefore, methods for cryopreservation of small number of human sperm with high recovery and survival rates need to be investigated.

Currently, there are two methods for cryopreservation of small number of human sperm. One is to freeze all tissue or samples in cryogenic storage vials (1–2 ml) or straws (0.25–0.5 ml) by slow freezing [3]. Another is to find motile sperm and then the sperm were placed in some small devices to perform fast freezing or vitrification [5–8]. However, most protocols do not offer satisfactory recovery and motility rates after thawing. Because sperm are very small, it is difficult to process sperm for freezing. Devices and methods for oocyte and embryos vitrification are not suitable to freeze sperm. Therefore, it is necessary to develop devices and methods that are easy to use, and can also provide high recovery rate and good motility, to freeze small number of human sperm. Recently, a SpermVD, a sperm vitrification device, has been reported to freeze small number of human sperm from men with non-obstructive azoospermia [13]. Although it has been reported that high sperm recovery rates can be obtained with this device, sperm motility rates after thawing varied from 0 to 100% among samples from different patients [14]. It is still unknown whether the big differences between samples were resulted from freezing method, different samples, or technical difficulties. Therefore, in the present study, to avoid the differences among different samples, we used normal sperm samples to examine some factors that affect the post thawing sperm motility after fast freezing.
