**2. Materials and methods**

#### **2.1 Ethical statement**

All patients undergoing sperm cryopreservation signed informed consents and the research was approved by ethics committee at the Reproductive Hospital of Guangxi Zhuang Autonomous Region.

#### **2.2 Samples and devices**

Semen samples were obtained from patients who were requested to freeze their semen for later IVF. A small portion of samples was used for fast freezing after swim-up for the study and the remaining was frozen by slow programmed freezing method. Samples were used after swim-up to obtain high percentage of motile sperm so that motility assessment is more accurate with swim-up samples than the original samples in which there were more immotile sperm and debris.

Two devices, as shown in **Figure 1**, SpermVD (MFC Global, Israel) and Microdevice (Xinchang Medical, China) were used in the experiments. For device holding, 2.0 ml externally threaded cryogenic storage vials (Fisher Scientific, USA) were used.

Sperm were washed and processed for swim-up with sperm washing medium (Fujifilm-Irvine Scientific, CA, USA) and then were frozen after being mixed with different ratios of Quinn's advantage sperm freezing medium (Origio, CT, USA). In the present study, to avoid the result variations between good samples and poor samples, we used normal sperm samples for all experiments. For loading the sperm to devices, we used 140 μl flexible pipette to add different volumes of samples to the devices and then processed the freezing.

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

#### **Figure 1.**

*Fast sperm freezing devices and set up for freezing. A: SpermVD, B: Microdevice, C: 2 ml externally threaded cryogenic storage vial. D: SpermVD in the vial, and E: Microdevice in the vial. Red arrows indicate the location of sperm drops in the devices.*

#### **2.3 Fast sperm freezing**

Two methods were used for fast sperm freezing. Method I: after sperm were loaded to the devices and equilibrated in the sperm freezing solution for different times, the devices were directly immersed to liquid nitrogen and then each device was inserted to the cryogenic storage vial for storage. Method II: after sperm were loaded to the device, the device was inserted to the vial, the vial was tightly closed with the cap and then immersed to liquid nitrogen.

#### **2.4 Sperm thawing and motility assessment**

For sperm thawing, cryogenic vials were removed from liquid nitrogen, and the devices were taken out of the vials with forceps, placed on a 60 mm culture dish. After the frozen drop was thawed or melted within ~1 min at room temperature, the device was directly placed in a 60 mm culture dish with warm oil for sperm motility assessment. Because the volume is very small, it is not necessary to transfer sperm to other sperm count chamber to do motility assessment. Motility was assessed under a phase contrast microscope at ×200 magnifications.

#### **2.5 Experiment design**

In experiment 1, post thawing sperm motility was compared between Method I and Method II as well as between two devices, SpermVD and Microdevice. Each 1 μl of micro drop of equilibrated sperm for 5 min in 1:1 of sperm washing solution and sperm freezing medium was frozen in the SpermVD and Microdevice with either Method I or Method II. Three replications were done for this experiment. Temperature changes for two methods during sperm cooling were measured with a digital thermometer.

In experiment 2, post thawing sperm motility was compared among different volumes of micro drops. Each 1 μl, 2 μl and 5 μl of micro drops of equilibrated sperm (5 min in 1:1 of sperm washing solution and sperm freezing medium) was frozen in the SpermVD with the Method II. Five replications were done for this experiment.

In experiment 3, post thawing sperm motility was compared after different equilibration times. Each 1 μl of micro drop of sperm equilibrated for 1, 2 and 5 min

#### *Infertility and Assisted Reproduction*

in 1:1 of sperm washing solution and sperm freezing medium was frozen in the SpermVD with the Method II. Three replications were done for this experiment.

In experiment 4, post thawing sperm motility was compared among different ratios of sperm freezing solutions. Each 1 μl of micro drop of equilibrated sperm for 5 min with different proportions of sperm freezing solution (0:1, 1:1, 2:1 and 5:1 of sperm freezing medium and sperm washing solution) was frozen in SpermVD with the Method II. Five replications were done for this experiment.

#### **2.6 Statistical analysis**

Each experiment was repeated three to five times, and each time with a different semen sample being used. For motility assessment, 100 sperm in each sample were counted. Mean ± SD of percentages of motile sperm before freezing and after thawing were obtained with replications. The recovered rates of sperm motility were calculated after dividing the post thawing sperm motility by the original sperm motility. All data were analyzed by ANOVA. If there were differences among groups, the differences between groups were further compared with chi-square test. If the P value was less 0.05, the difference was considered statistically significant.
