**3.1 Background on vitrification of spermatozoa**

Vitrification is an alternative method of freezing based on the rapid coolling of water to a glassy state through extreme elevation of viscosity without intracellular ice crystallization (Fahy, 1986; Katkov et al., 2006). The relationship between the size of different cells, particularly, different spermatozoa species, and the ability of cells to be vitrified are discussed in details in the paper by Katkov (Katkov et al., 2006).

The earliest experiments on vitrification from the 1930s was not successful because critical rates of cooling were unachievable at that time. With the use of LN2 and the discovery of cryoprotectants, however, it became possible to vitrify many types of cells. The five basic ways to achieve vitrification have been described in details by Katkov et al.: equilibrium freezing-out of the bulk of water with the use of CPAs and storage at ultra low temperature; lyophilization using slow freezing to moderately low (-40 °C) followed by secondary drying at +30°C (mostly used in food and pharmaceutical industries); ice-free vitrification at high rates and high concentration of CPAs; ice-free vitrification at very fast rates without permeable agents ("CPAs-free vitrification"); high temperature' vitrification by air/vacuum drying at temperature above 0°C (Katkov et al., 2006).

However, until only recently, vitrification of spermatozoa was unsuccessful, possibly due to high concentrations of permeable CPAs (30-50% compared to 5-7% with slow freezing) and low tolerance of spermatozoa to permeable agents. Even brief exposure to a high concentration of CPAs can lead to toxic and osmotic shock and would be lethal for spermatozoa. One possible strategy to lower the concentration of CPAs could be to increase the speed of cooling and warming temperatures as higher rates of cooling and warming, require lower concentrations of CPAs; these conditions can help eliminate intracellular ice crystallization, and facilitate the formation of a glassy state (Katkov et al., 2006). Another option is to add non-permeable CPAs--such as carbohydrates--to permeable CPAs to minimize osmotic shock by decreasing osmotic pressure and stabilizing the nuclear membrane. Since the intracellular matrix of human spermatozoa contains large amounts of proteins and sugars, they can be successfully frozen in the absence of permeable CPAs using protein- and sugar-rich non-permeable agents (Koshimoto et al., 2000).

Cryopreservation of Human Spermatozoa

Lulat, 2000).

**Rapid motility (% of motile)**

**VAP** 

**VSL** 

**VCL** 

**ALH** 

**LIN (ratio of VSL/VCL)** 

vitrification vs. slow freezing.

vitrification and slow freezing.

**Parameters Prior to** 

**freezing** 

**Post vitrif.** 

by Vitrification *vs.* Slow Freezing: Canadian Experience 85

damage between semen samples cryopreserved by standard vapour freezing verses vitrification protocols (Moskovtsev et al., 2011). Semen samples from 11 patients presenting for infertility were washed by density gradient centrifugation and evaluated by Computer-Aided Sperm Analysis (CASA). Subsequently kinematic parameters were assessed as previously described: sperm motility, average path velocity (VAP) curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), amplitude of lateral head displacement (ALH) (Moskovtsev et al., 2009). However, kinematic parameters are averages of values obtained from analyzing the entire motile fraction of cells in a sample and include absolute (actual) parameters (VAP, VCL, VSL, ALH) and relative (derived) such as LIN. When cryopreserved samples are evaluated after thawing, the CASA-paradox can take place, when despite of deterioration of semen samples after cryopreservation "pseudoenhancement" of kinematics characteristics is observed (Katkov & Lulat, 2000). Modified Kinematic Parameters (MKP) were calculated as previously described: Kinematic Parameters (KP) x Motility/ 100% To account for this phenomenon, modifications of actual CASA-parameters are recommended and are incorporated into our data (Table 1). (Katkov&

> **Post vitrif. MKP**

**Motility (%)** 68.0 ± 10.79 25.4 ± 13.6 14.6 ± 10.2 < 0.05

**(microns/sec)** 60.73 ± 8.93 43.09 ± 14.24 10.9 ± 3.6 38. 00 ± 8.57 5.5 ± 1.2 < 0.05

**(microns/sec)** 47.73 ± 9.39 35.45 ± 13.98 9.0 ± 3.5 31. 00 ± 8.27 4.5 ±1.2 < 0.05

**(microns/sec)** 96.18 ± 3.68 81.18 ± 21.31 20.6 ± 5.4 68.73 ± 13.01 10.0 ± 1.9 < 0.05

**(microns)** 4.42 ± 0.79 4.3182 ± 0.80 1.1 ± 0.2 3.67 ± 0.85 0.5 ± 0.1 < 0.05

**TUNEL (%)** 7.5 ± 5.5 9.6 ± 4.4 9.5 ± 5.1 NS

**Note:** \* Statistically significant P values, when compared between MKP of samples frozen by

Table 1. Comparison of CASA and TUNEL resuls between semen samples frozen by

49.91 ± 0.55 42.45 ± 7.43 10.8 ± 1.9 45.91 ± 5.59 6.7 ± 0.8 < 0.05

50.64 ± 4.52 19.45 ± 12.98 4.9 ± 3.3 10.45 ± 8.49 1.5 ± 1.2 < 0.05\*

**Post slow freezing** 

**Post slow MKP** 

**P (vitrif. vs. slow freezing)** 

Successful vitrification of human spermatozoa was first reported by the Isaschenkos' group (Nawroth et al, 2002; E. Isachenko et al, 2003). The high viscosity of the intracellular milieu due to large amounts of proteins, nucleotides and sugars and low water in human spermatozoa content determines the ability of human sperm to be vitrified at relatively low cooling rates (Katkov et al., 2006). It was noted that human spermatozoon is one of the smallest germ cells among mammals, has almost no residual histones and has very compacted DNA (Holt, 2000), which indirectly confirms this hypothesis (see also Chapter by Katkov et al in this Book).

As we mentioned above, the major breakthrough in successful vitrification of *human* spermatozoa without the use of permeable CPAs was reported only recently by the Isachenko group (Nawroth et al., 2002), who actually re-invented the work of the "pioneers" in the 1930-40s mentioned above. The combination of extremely high rates of cooling/warming and utilization of vitrification media containing proteins and polysaccharides made it feasible to avoid de-vitrification during warming without use of toxic CPAs. The same group compared viability, survival rate and sperm DNA damage between slow freezing and vitrification and found that DNA integrity was independent from the mode of cooling and the presence of cryoprotectants in thawed spermatozoa (V. Isachenko et al., 2004). The acrosome reaction, capacitiation and mitochondrial activity of spermatozoa were compared vis-a-vis slow freezing and vitrification (E. Isachenko et al., 2008). The group reported that changes in the mitochondrial membrane potentials relate to the type of vitrification media with the best achieved results when both sugar and albumin were added to the media. To achieve high cooling rates the vitrification specimen volume needs to be kept to a minimum. Specially designed freezing carriers such as cryoloops and electron microscope copper grids have been suggested for vitrification of human spermatozoa (E. Isachenko et al., 2003; Nawroth et al., 2002). However, placing drops of semen directly into LN2 raises the issue of the potential risk of microbial or viral cross contamination during freezing and storage (Katkov, 2002). The development of aseptic techniques of vitrification allowing to freeze 5-10 µl of sperm suspension in open-pulled straws (OPS) or 1-2 µl of sample cut standard straw (CSS) placed inside of insemination straw further advanced the methodology of human sperm vitrification (V. Isachenko et al., 2005). The ultra-high freezing rates utilized for vitrification, via direct plunging of specimens into LN2, leads to solidification of a solution by an intense increase in viscosity during cooling which avoids water crystallization and damaging ice formation (Katkov et al., 2006).

Most importantly, vitrified spermatozoa were successfully utilized in ICSI treatment with clinical pregnancy resulting in delivery of healthy twins (E. Isachenko et al., 2011). While only a small volume 0.2 to 40 µl of sample suspension was frozen in the past, recently larger amounts of spermatozoa (100 µl) were successfully vitrified using newly developed straw packaging system (SPS) made from cut in half 0.25 ml plastic straw (E. Isachenko et al., 2011). A first live birth was reported following intrauterine insemination of semen vitrified without permeable cryoprotectants from patient with oligoasthenozoospermia making this freezing technique even more attractive in clinical practice (Sanchez al., 2011).

#### **3.2 Vitrification of human spermatozoa: Canadian experience**

Encouraged by the findings of the German group, we have also looked at possibilities to utilize vitrification in our laboratory. We have compared sperm motility, kinetics and DNA

Successful vitrification of human spermatozoa was first reported by the Isaschenkos' group (Nawroth et al, 2002; E. Isachenko et al, 2003). The high viscosity of the intracellular milieu due to large amounts of proteins, nucleotides and sugars and low water in human spermatozoa content determines the ability of human sperm to be vitrified at relatively low cooling rates (Katkov et al., 2006). It was noted that human spermatozoon is one of the smallest germ cells among mammals, has almost no residual histones and has very compacted DNA (Holt, 2000), which indirectly confirms this hypothesis (see also Chapter

As we mentioned above, the major breakthrough in successful vitrification of *human* spermatozoa without the use of permeable CPAs was reported only recently by the Isachenko group (Nawroth et al., 2002), who actually re-invented the work of the "pioneers" in the 1930-40s mentioned above. The combination of extremely high rates of cooling/warming and utilization of vitrification media containing proteins and polysaccharides made it feasible to avoid de-vitrification during warming without use of toxic CPAs. The same group compared viability, survival rate and sperm DNA damage between slow freezing and vitrification and found that DNA integrity was independent from the mode of cooling and the presence of cryoprotectants in thawed spermatozoa (V. Isachenko et al., 2004). The acrosome reaction, capacitiation and mitochondrial activity of spermatozoa were compared vis-a-vis slow freezing and vitrification (E. Isachenko et al., 2008). The group reported that changes in the mitochondrial membrane potentials relate to the type of vitrification media with the best achieved results when both sugar and albumin were added to the media. To achieve high cooling rates the vitrification specimen volume needs to be kept to a minimum. Specially designed freezing carriers such as cryoloops and electron microscope copper grids have been suggested for vitrification of human spermatozoa (E. Isachenko et al., 2003; Nawroth et al., 2002). However, placing drops of semen directly into LN2 raises the issue of the potential risk of microbial or viral cross contamination during freezing and storage (Katkov, 2002). The development of aseptic techniques of vitrification allowing to freeze 5-10 µl of sperm suspension in open-pulled straws (OPS) or 1-2 µl of sample cut standard straw (CSS) placed inside of insemination straw further advanced the methodology of human sperm vitrification (V. Isachenko et al., 2005). The ultra-high freezing rates utilized for vitrification, via direct plunging of specimens into LN2, leads to solidification of a solution by an intense increase in viscosity during cooling which avoids water crystallization and damaging ice formation (Katkov et

Most importantly, vitrified spermatozoa were successfully utilized in ICSI treatment with clinical pregnancy resulting in delivery of healthy twins (E. Isachenko et al., 2011). While only a small volume 0.2 to 40 µl of sample suspension was frozen in the past, recently larger amounts of spermatozoa (100 µl) were successfully vitrified using newly developed straw packaging system (SPS) made from cut in half 0.25 ml plastic straw (E. Isachenko et al., 2011). A first live birth was reported following intrauterine insemination of semen vitrified without permeable cryoprotectants from patient with oligoasthenozoospermia making this

Encouraged by the findings of the German group, we have also looked at possibilities to utilize vitrification in our laboratory. We have compared sperm motility, kinetics and DNA

freezing technique even more attractive in clinical practice (Sanchez al., 2011).

**3.2 Vitrification of human spermatozoa: Canadian experience** 

by Katkov et al in this Book).

al., 2006).

damage between semen samples cryopreserved by standard vapour freezing verses vitrification protocols (Moskovtsev et al., 2011). Semen samples from 11 patients presenting for infertility were washed by density gradient centrifugation and evaluated by Computer-Aided Sperm Analysis (CASA). Subsequently kinematic parameters were assessed as previously described: sperm motility, average path velocity (VAP) curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), amplitude of lateral head displacement (ALH) (Moskovtsev et al., 2009). However, kinematic parameters are averages of values obtained from analyzing the entire motile fraction of cells in a sample and include absolute (actual) parameters (VAP, VCL, VSL, ALH) and relative (derived) such as LIN. When cryopreserved samples are evaluated after thawing, the CASA-paradox can take place, when despite of deterioration of semen samples after cryopreservation "pseudoenhancement" of kinematics characteristics is observed (Katkov & Lulat, 2000). Modified Kinematic Parameters (MKP) were calculated as previously described: Kinematic Parameters (KP) x Motility/ 100% To account for this phenomenon, modifications of actual CASA-parameters are recommended and are incorporated into our data (Table 1). (Katkov& Lulat, 2000).


**Note:** \* Statistically significant P values, when compared between MKP of samples frozen by vitrification vs. slow freezing.

Table 1. Comparison of CASA and TUNEL resuls between semen samples frozen by vitrification and slow freezing.

Cryopreservation of Human Spermatozoa

(Moskovtsev et al., 2010) (Figure 2).

by Vitrification *vs.* Slow Freezing: Canadian Experience 87

(Thomson et al., 2009). A slide-based technique for the assessment of sperm DNA was performed as previously described (TUNEL: TdT-mediated dUTP nick end labelling)

**Note:** Brown (TUNEL-positive): damaged DNA; gray-green (TUNEL-negative): undamaged cells.

sperm DNA damage between slow freezing and vitrification (9.6 ± 4.4 vs. 9.5 ± 5.1).

We found statistically significant increase in sperm DNA damage after both methods of sperm freezing (P < 0.05). However, the increase in DNA damage was minimal and to a degree probably irrelevant to clinical concerns. No significant differences were observed in

We can now confirm previous reports that human spermatozoa can be successfully vitrified without the use of potentially toxic cryoprotectants. The vitrification protocol showed significantly better results in preserving motility rates of spermatozoa when compared to slow vapour freezing. No significant differences were observed in post thaw sperm DNA damage in comparison to the standard slow freezing method. While our results are based on the freezing of a small volume of specimens, we are evaluating vitrification of larger volumes of spermatozoa with a proprietary mixture developed in our laboratory in CBS. We have achieved comparable results with both small volume (5 µl) and relatively large volume

Human semen cryobanking can be divided into two broad categories: autologous banking for personal fertility preservation and donor sperm banking. Semen banking is useful in many situations and can be considered a safeguard against unforeseen future circumstances. These may include: prior to chemotherapy or radiation therapy; pre- vasectomy; before certain types of pelvic or testicular surgery; in cases of degenerative illnesses such as diabetes or multiple sclerosis, spinal cord disease or injury; high risk occupations or sports;

Fig. 2. Sperm DNA damage assessment by TUNEL assay.

of 200 µl semen samples (unpublished data).

**4.1 Referring patients to a sperm bank** 

**4. Sperm banking** 

Our results indicate that sperm motility was significantly reduced for both types of frozenthawed samples (P <0.03) (Table 1). Mean motility of vitrified samples was 25.4% ± 13.6 (a decrease of 36.4% compared to samples prior to freezing), which was almost two-fold higher compared to motility of samples frozen by standard slow vapour protocol (14.6% ± 10.2, decrease of 47.2% compared to samples prior to freezing), (P <0.05). Sperm kinematics such as VCL, VSL, and LIN were not significantly different between the two types of cryopreservation protocols without taking into account CASA- paradox. However, when MKP were calculated, it was revealed that indeed vitrified samples had superior recovery of sperm kinematic parameters in comparison to slow freezing.

Samples for slow vapour freezing were diluted 3:1 with commercial cryoprotectant medium and frozen by standard protocol in CBS. Aliquots of samples for vitrification were diluted 1:1 with a G-IVF medium (Vitrolife, Göteborg, Sweden) supplemented with 0.25M sucrose and 1% of LSPS (Life Global Protein Supplement, IVF Online, Guelph, ON, Canada). We have used 0.5 ml OPS and loaded 5 µl of vitrified sample in each straw by capillary; OPS were inserted into 0.5 CBS straws and sealed (Figure 1).

Fig. 1. Comparison of 0.5 ml CBS straw and 0.5 cc OPS straw and schematic of OPS inserted and sealed inside a CBS.

Samples were immediately plunged into LN2 and stored there for several days. For thawing procedure, OPS were rapidly removed from CBS straws, and plunged into 2 ml of the same medium used for vitrification at 37ºC for 10 seconds.

We have evaluated the effect of cryopreservation on sperm DNA damage as the subject remains controversial. |While several reports indicate no negative effect of freezing on sperm DNA integrity (Duty et al., 2002; V. Isachenko et al., 2004). others have reported significant negative effect of sperm cryopreservation and DNA damage and chromatin stability (Hammadeh et al., 1999; Said et al., 2010). Significant increase in percentage of DNA fragmentation was associated with an increase in oxidative stress during cryopreservation

Our results indicate that sperm motility was significantly reduced for both types of frozenthawed samples (P <0.03) (Table 1). Mean motility of vitrified samples was 25.4% ± 13.6 (a decrease of 36.4% compared to samples prior to freezing), which was almost two-fold higher compared to motility of samples frozen by standard slow vapour protocol (14.6% ± 10.2, decrease of 47.2% compared to samples prior to freezing), (P <0.05). Sperm kinematics such as VCL, VSL, and LIN were not significantly different between the two types of cryopreservation protocols without taking into account CASA- paradox. However, when MKP were calculated, it was revealed that indeed vitrified samples had superior recovery of

Samples for slow vapour freezing were diluted 3:1 with commercial cryoprotectant medium and frozen by standard protocol in CBS. Aliquots of samples for vitrification were diluted 1:1 with a G-IVF medium (Vitrolife, Göteborg, Sweden) supplemented with 0.25M sucrose and 1% of LSPS (Life Global Protein Supplement, IVF Online, Guelph, ON, Canada). We have used 0.5 ml OPS and loaded 5 µl of vitrified sample in each straw by capillary; OPS

Fig. 1. Comparison of 0.5 ml CBS straw and 0.5 cc OPS straw and schematic of OPS inserted

Samples were immediately plunged into LN2 and stored there for several days. For thawing procedure, OPS were rapidly removed from CBS straws, and plunged into 2 ml of the same

We have evaluated the effect of cryopreservation on sperm DNA damage as the subject remains controversial. |While several reports indicate no negative effect of freezing on sperm DNA integrity (Duty et al., 2002; V. Isachenko et al., 2004). others have reported significant negative effect of sperm cryopreservation and DNA damage and chromatin stability (Hammadeh et al., 1999; Said et al., 2010). Significant increase in percentage of DNA fragmentation was associated with an increase in oxidative stress during cryopreservation

sperm kinematic parameters in comparison to slow freezing.

were inserted into 0.5 CBS straws and sealed (Figure 1).

medium used for vitrification at 37ºC for 10 seconds.

and sealed inside a CBS.

(Thomson et al., 2009). A slide-based technique for the assessment of sperm DNA was performed as previously described (TUNEL: TdT-mediated dUTP nick end labelling) (Moskovtsev et al., 2010) (Figure 2).

**Note:** Brown (TUNEL-positive): damaged DNA; gray-green (TUNEL-negative): undamaged cells.

Fig. 2. Sperm DNA damage assessment by TUNEL assay.

We found statistically significant increase in sperm DNA damage after both methods of sperm freezing (P < 0.05). However, the increase in DNA damage was minimal and to a degree probably irrelevant to clinical concerns. No significant differences were observed in sperm DNA damage between slow freezing and vitrification (9.6 ± 4.4 vs. 9.5 ± 5.1).

We can now confirm previous reports that human spermatozoa can be successfully vitrified without the use of potentially toxic cryoprotectants. The vitrification protocol showed significantly better results in preserving motility rates of spermatozoa when compared to slow vapour freezing. No significant differences were observed in post thaw sperm DNA damage in comparison to the standard slow freezing method. While our results are based on the freezing of a small volume of specimens, we are evaluating vitrification of larger volumes of spermatozoa with a proprietary mixture developed in our laboratory in CBS. We have achieved comparable results with both small volume (5 µl) and relatively large volume of 200 µl semen samples (unpublished data).
