Ultra-rapid freezing of very low numbers of sperm using cryoloops

Timothy G. Schuster1, Laura M. Keller2, Rodney L. Dunn1, Dana A. Ohl1 and Gary D. Smith1,2,3,4

Departments of 1 Urology, 2 Obstetrics and Gynecology and 3 Physiology, University of Michigan, Ann Arbor, MI 48109, USA

4 To whom correspondence should be addressed. e-mail: gdsmith{at}umich.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: With the availability of ICSI, men with severe oligozoospermia (<5x106/ml) are able to reproduce. Current methods for cryopreservation of severe oligozoospermic samples are labour intensive and costly. The objective of this study was to evaluate whether freezing small numbers of motile sperm (~100) was feasible using cryoloops. METHODS: Initial tests assessed the effect of various dilutions of cryoprotectants on pre-freezing sperm motility. Several solutions were further evaluated for their ability to cryoprotect sperm during ultra-rapid freezing. Sperm were placed on cryoloops and held in liquid nitrogen vapour for 5 min prior to freezing (ultra-rapid freezing) or directly submerged into liquid nitrogen. Using the optimal cryoprotectant and technique from these experiments, ultra-rapid and standard slow-rate freezing protocols were compared. RESULTS: Optimal sperm survival was seen when sperm in cryoloops were placed in liquid nitrogen vapour in test yolk buffer with 12% v/v glycerol versus other cryoprotectants. Using this cryoprotectant, post-thaw sperm motility is comparable between ultra-rapid and slow-rate freezing methods. CONCLUSION: Ultra-rapid freezing of very low numbers of sperm is feasible using cryoloops suspended in liquid nitrogen vapour for 5 min.

Key words: cryopreservation/oligozoospermia/sperm/ultra-rapid freezing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cryopreservation of human sperm with the possibility of post-thaw pregnancy initiation has been available for >40 years (Bunge and Sherman, 1953Go). Since that time a significant amount of research has been devoted to understanding better the events that occur during cryopreservation (Mazur, 1984Go; Royere et al., 1996Go). As the temperature of a semen sample is lowered, extracellular ice crystals form causing solutes in the extracellular fluid to become more concentrated. The resulting osmotic gradient causes dehydration of the cell as water effluxes. Damage can occur to the cell if the osmotic gradient is too steep, resulting in water efflux that is too rapid. Additionally, excessive dehydration causes increased intracellular solute concentration that can be toxic to cells. Alternatively, if not enough intracellular water is removed prior to the formation of intracellular ice, potential for damage to organelles and the cytoskeleton exists (Mazur et al., 1972Go). Penetrating cryoprotectants act by maintaining intracellular and extracellular solute concentrations as the specimen is frozen. With the addition of these compounds to the extracellular fluid there is an initial water efflux followed by water influx as the cryoprotectant equilibrates. Common intra cellular cryoprotectants utilized include glycerol, dimethyl sulphoxide, propylene glycol, and ethylene glycol (Gilmore et al., 1997Go). Conversely, extracellular cryoprotectants are large molecules (typically sugars) that act as osmotic agents to dehydrate the cells and decrease the amount of intracellular water available for ice formation. Examples of such cryoprotectants include glucose, xylose, sucrose, trehalose and hydroxyethyl starch (McGann, 1978Go; Critser et al., 1988Go).

Two techniques for sperm cryopreservation have been described. A slower freezing method consists of gradually cooling the sperm over a period of ~2–4 h in two or three steps either manually (Thachil and Jewett, 1981Go) or using a programmable freezer (Serafini and Marrs, 1986Go). Initial cooling rates of the specimen from room temperature to 5°C have been shown to be optimal at ~0.5 to 1°C per min (Mahadevan and Trounson, 1984Go). The sample is then frozen at a rate of 1 to 10°C per min from 5°C to –80°C after which the specimen is plunged into liquid nitrogen (Thachil and Jewett, 1981Go; Mahadevan and Trounson, 1984Go; Serafini and Marrs, 1986Go). Additional research has suggested that holding the specimen at –5°C for 10 min and seeding the specimen may improve cryosurvival (Critser et al., 1987Go). Alternatively a more rapid method consists of manually placing the sperm in liquid nitrogen vapour for 5–30 min prior to submersion into liquid nitrogen (Sherman, 1963Go; Kobayashi et al., 1991Go; Rofeim et al., 2001Go). Although some research has shown the slower freezing method to be superior (Mahadevan and Trounson, 1984Go), others have published data favouring more rapid cooling rates (Sherman, 1963Go). Despite these findings, the more rapid cooling method has not gained universal acceptance. This may be due to the unavailability of suitable ultra-rapid cryopreservation containers. Recently, the use of a cryoloop containerless technique to vitrify mouse and human blastocysts was described (Lane et al., 1999Go). This new technique offers the potential for use in cryopreserving sperm.

Although cryopreservation of sperm is feasible, the recovery rate of motile sperm after thawing can vary widely. Factors influencing sperm recovery include rate of freezing and thawing as well as choice and concentration of cryoprotectants (Jeyendran et al., 1984aGo; Weidel and Prins, 1987Go; Henry et al., 1993Go; Royere et al., 1996Go). Additionally, the quality of thawed sperm can be improved by concentrating the ejaculates prior to freezing (Perez-Sanchez et al., 1994Go). For patients with oligozoospermia, having motile sperm concentrated in thawed specimens would be valuable for use during ICSI.

With the advent of ICSI, theoretically only one sperm is required per harvested oocyte, allowing men with severe oligozoospermia (<5x106/ml; Ohashi et al., 2001Go) the opportunity to reproduce. However, the practicality of isolating, freezing and finding the sperm after thawing is problematic. Recently the ability to cryopreserve individual sperm in isolated zonae pellucidae was described (Cohen et al., 1997Go; Hsieh et al., 2000Go). Drawbacks of the procedure include it being labour intensive, the potential need for non-human biological samples, and the substantial cost. Alternatively, small numbers of motile sperm can be stored in microdroplets, although the recovery rate can be variable (Craft and Tsirigotis, 1995Go; Gil-Salom et al., 2000Go). These techniques are the only currently available options to cryopreserve and recover sperm from men with severe oligozoospermia. Using a cryoloop enclosed in a vial could offer an alternative option.

Because of increased demand for fertility intervention in cases of male factor infertility due to severe oligozoospermia, and the limitations associated with cryopreserving sperm from this population, we established three objectives and tested them with four sequential experiments. First, we sought to determine the effects of several cryoprotectant solutions on human sperm motility and viability. Second, several of the selected solutions were assessed for their ability to cryoprotect oligozoospermic samples of sperm on cryoloops held in liquid nitrogen vapour for 5 min prior to freezing (ultra-rapid freezing) or directly submerged into liquid nitrogen. Finally, using the optimal cryoprotectant and freezing technique from initial experiments, thawed sperm motility after ultra-rapid freezing with cryoloops was compared with standard slow freezing.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Semen samples
Semen samples were obtained from men undergoing evaluation for infertility after a minimum of 3 days of abstinence. Samples were allowed to liquefy for 30 min at room temperature prior to analysis. Standard semen analyses were performed manually by a single individual and consisted of assessment of semen volume, pH, viscosity, liquefaction, sperm count, sperm motility, sperm agglutination, strict sperm morphology, and cell contamination. Semen samples with normal parameters (volume 2–6 ml, pH 7.2–8.0, sperm count >20x106/ml, sperm motility >50%, strict sperm morphology >14%, and insignificant sperm agglutination and cell contamination) were selected for experimental use. Approval for utilizing semen samples for all experiments was obtained from the University of Michigan Institutional Review Board (IRB).

Experiment 1
Motility assessment in various concentrations of cryoprotectants
Initial experiments evaluated sperm survival in several concentrations of cryoprotectants currently used in our laboratory for oocyte preservation. For the initial two experiments, semen samples were prepared by density gradient separation using Isolate (Irvine Scientific, USA). The supernatant was removed after centrifugation at 300 g for 20 min and the pellet resuspended in 500 µl of HEPES-buffered human tubal fluid with 0.2% bovine serum albumin. Initial sperm motility was manually assessed by a single individual in duplicate for each sample by evaluating 100 sperm. Cryoprotectant A (CryoA) contained 40% ethylene glycol, 0.5 mol/l trehalose, 5% dextran serum substitute (DSS) and 10% test yolk buffer (TYB). DSS contained human serum albumin (50 mg/ml) and dextran (20 mg/ml) in a saline solution (Irvine Scientific). Test–yolk buffer contained 20% egg yolk, 1000 IU/ml penicillin-G and 1000 µg/ml streptomycin sulphate (Irvine Scientific). Cryoprotectant B (CryoB) contained 25% ethylene glycol, 0.5 mol/l trehalose, 5% DSS, 10% TYB and 25% glycerol.

Ten samples were diluted 1:1 in CryoA and CryoB. Additionally, serial dilutions of each cryoprotectant were made with TYB 1:1, 3:1 and 4:1 to assess whether altering the concentrations of cryoprotectant would improve initial sperm motility. Sperm samples were mixed 1:1 in each dilution and motility was assessed immediately.

Experiment 2
Time-dependent changes in sperm motility
All solutions evaluated in Experiment 1 with sperm motility >20% at time 0 were further tested using five additional semen samples after timed intervals of 1, 2 and 4 min solution exposure. The two solutions with the highest percentage of sperm motility after 4 min exposure were selected for further testing and called Cryoprotectant 1 and 2.

Experiment 3
Cryoprotectants
Four separate solutions were tested for their ability to cryoprotect sperm during freezing using 14 additional semen samples. The first two solutions were the selected cryoprotectants from the initial experiments. These solutions were CryoA in a 1:1 dilution with TYB (Cryoprotectant 1) and CryoA in a 3:1 dilution with TYB (Cryoprotectant 2). Additionally, freezing medium containing TYB with 12% v/v glycerol (Cryoprotectant 3; Irvine Scientific, Santa Ana, CA, USA) diluted 1:1 with semen was tested. Finally, similar to prior work by Sherman (1954Go), the innate ability of homologous seminal plasma to cryoprotect unprocessed semen samples without the addition of exogenous cryoprotectants was evaluated (Cryoprotectant 4). After mixing with cryoprotectant, each specimen was allowed to equilibrate for 4 min prior to freezing.

Seminal plasma was prepared by centrifugation of an aliquot of each processed semen sample at 3500 g for 3 min to remove the sperm. The supernatant was then aliquoted into a new Eppendorf tube and centrifugation was repeated. The final supernatant suspension was examined under light microscopy at x40 to verify the absence of sperm.

Freezing techniques
Nylon cryoloops (Hampton Research, USA) were used for ultra-rapid freezing (Figure 1 and Figure 2). A clean cryoloop was inoculated in ~5 µl of sperm suspension and then placed into a cryovial. The vial was then either (i) directly submerged into liquid nitrogen or (ii) placed in non-circulating liquid nitrogen vapour for 5 min prior to submersion (ultra-rapid freezing). During suspension in vapour, the bottoms of the cryovials were 3 cm above the surface of the liquid nitrogen. Vials were left submerged in the liquid nitrogen for a minimum of 10 min prior to thawing.



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Figure 1. Cryoloop and cryovial used for ultra-rapid freezing (A). Cryoloops can be magnetically attached to a metal wand for easier manipulation (B). Loops were inoculated with ~100–200 sperm and then placed into cryovials for freezing.

 


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Figure 2. Empty cryoloop prior to sperm inoculation (A) (x100). Inoculated cryoloops at x100 (B), x200 (C), x400 (D1) and x640 (E1). To enhance visualization of individual spermatazoa, embossed photographs of x400 and x640 are shown in D2 and E2 respectively.

 
Thawing and post-thaw sperm assessment
Because the cryoloops only hold a film of fluid, the loops must be resuspended in liquid droplets to evaluate sperm motility. After freezing, cryoloops were removed from liquid nitrogen and immediately resuspended in 2 µl of TYB (Cryoprotectant 1–4) or homologous seminal plasma (Cryoprotectant 4) at room temperature.

Sperm motility was evaluated by a single individual by counting 100 sperm using light microscopy at x40 magnification. To assess viability, 5 µl of 5% eosin Y and 5 µl of 10% nigrosin were mixed with the thawed sperm and then smeared over a glass slide. Using light microscopy with x40 magnification, 100 sperm were counted per slide. Unstained sperm were considered viable. Viability stains were not performed on samples frozen in solution 3 because of reagent incompatibility.

Experiment 4
Comparison of ultra-rapid freezing and slow rate freezing
Using Cryoprotectant 3 (determined by initial studies to be optimal), ten semen samples were divided and frozen and thawed using the ultra-rapid method (determined to be optimal in Experiment 3) and with a standard slow rate freezing protocol. The method for slow rate freezing consisted of diluting 100 µl of the semen sample 1:1 with three aliquots of Cryoprotectant 3 over 10 min. Samples were then placed into cryovials and equilibrated in an ice bath at 5°C for 45 min. The vials were then placed into liquid nitrogen vapour for 20 min before being plunged into liquid nitrogen. Slow rate frozen samples were thawed at room temperature. All semen samples were left in liquid nitrogen for a minimum of 10 min and sperm motility was assessed by a single individual in triplicate for each sample.

Cryoprotectant 3 was incompatible with the viability stain. To assess sperm viability for this cryoprotectant, a hypo-osmotic swelling (HOS) test (Jeyendran et al., 1984bGo) was performed. HOS medium contained 0.02 mol/l sodium citrate and 0.07 mol/l fructose in water. Ten additional semen samples were prepared by density gradient separation as described in Experiment 1. A direct swim-up was performed from the pellet and the final concentration of sperm diluted to 20x106 motile sperm/ml. Aliquots were frozen and thawed using the ultra-rapid method and with the slow rate freezing protocol described above. Sperm motility was assessed in both the fresh and thawed samples by a single individual. For the fresh and slow rate frozen aliquots, 300 µl of the samples were mixed with 1 ml of HOS media. Ultra-rapid frozen aliquots were thawed in 1 µl TYB and then mixed with 10 µl HOS medium. After the addition of HOS medium, all samples were incubated at 37°C for 30 min. A minimum of 25 sperm per sample were then assessed for the presence of the characteristic coiled tails of viable sperm exposed to HOS treatment.

Statistical analysis
Comparisons of sperm motility and viability between tested samples were performed using a paired t-test and signed-rank test. Because no outlying variables were present in the data, similar results were obtained with both tests. Only the paired t-test results are reported. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experiment 1
For initial motility assessments, 10 semen samples which had been processed with density gradient separation were evaluated. Initial sperm motility was 87 ± 1.5% (mean ± SEM). Sperm motility in pure CryoA and CryoB were each 1 and 0% respectively. Sperm motility in 1:1 dilution (CryoA:TYB) was 77 ± 2.4%; 3:1 dilution was 51 ± 4.3%; and 4:1 dilution was 39 ± 5.7%. Sperm motility in 1:1 dilution (CryoB:TYB) was 41 ± 6.3; 3:1 dilution was 11 ± 3.5%; and 4:1 dilution was 2 ± 1.7% (Figure 3). Sperm motility in each solution was significantly lower when compared with sperm motility in control samples (P < 0.05). Comparisons of sperm motility between various solutions were also significantly different (P < 0.05). The least detrimental effects on sperm motility when compared with control were seen with solutions CryoA 1:1 dilution, CryoA 3:1 dilution, CryoA 4:1 dilution and CryoB 1:1 dilution.



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Figure 3. Mean sperm motility after placement into various dilutions of CryoA [freezing solution (FS) 1] and CryoB (FS 2) (n = 10). Error bars represent SEM. TYB = test–yolk buffer.

 
Experiment 2
Four cryoprotectants from Experiment 1 (CryoA 1:1 dilution, CryoA 3:1 dilution, CryoA 4:1 dilution and CryoB 1:1 dilution) were selected to evaluate changes in motility with time. Five fresh semen samples which had been processed with density gradient separation were evaluated (Table I). Sperm in solution CryoA 1:1 dilution appeared to show a slight decrease in motility with time from 71.2 ± 6.0% (mean ± SEM) to 66.4 ± 8.1%, although this was not significantly different. Conversely, sperm showed a gradual non-significant increase in motility with time in CryoA 3:1 dilution (42.2 ± 6.8 to 48.6 ± 4.4%) and CryoA 4:1 dilution (26.4 ± 5.0 to 34.8 ± 6.6%). In CryoB 1:1 dilution, motility significantly increased over time of exposure (20.4 ± 3.7 to 45.8 ± 5.1%; P < 0.05). At each time point, sperm motility assessed in each solution was statistically lower than sperm motility in the control sample (P < 0.05).


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Table I. Time-dependent sperm motility in various cryoprotectants (Cryo) after density gradient separation (n = 5)
 
Experiment 3
Freezing using cryoloops was performed on 14 semen samples using Cryoprotectants 1–4 (Cryoprotectant 1 or CryoA in a 1:1 dilution with TYB; Cryoprotectant 2 or CryoA in a 3:1 dilution with TYB; Cryoprotectant 3 or freezing medium containing TYB with 12% v/v glycerol; and Cryoprotectant 4 or unprocessed semen samples). Prior to freezing, mean sperm motility and viability were 54 ± 3.4 and 66 ± 3.8% respectively. Regardless of solution, survival of sperm submerged directly into liquid nitrogen was 0%.

Of samples initially suspended in liquid nitrogen vapour (ultra-rapid freezing), post-thaw sperm motility was 5 ± 1.9% (Cryoprotectant 1); 0% (Cryoprotectant 2); 40 ± 3.2% (Cryoprotectant 3); 16 ± 3.6 and 21 ± 4.7% (Cryoprotectant 4 resuspended in seminal plasma or TYB, respectively; Figure 4). Viabilities of thawed sperm were 4 ± 2.0% (Cryoprotectant 1); 0% (Cryoprotectant 2); not assessed because of reagent incompatibility (Cryoprotectant 3); and 17 ± 3.9 and 21 ± 4.7% (Cryoprotectant 4 resuspended in seminal plasma and TYB respectively). Sperm motility and viability were significantly lower after ultra-rapid freezing when compared with sperm motility and viability in the initial sample (P < 0.05). In addition, post-thaw sperm motility in Cryoprotectant 3 was significantly better when compared with the other cryoprotectants tested (P < 0.05).



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Figure 4. Mean sperm motility and viability before and after ultra-rapid freezing on cryoloops (n = 14). No survival was seen when using Cryoprotectant 2. Viability stains could not be performed with Cryoprotectant 3 because of reagent incompatibility. Error bars represent SEM. Columns with different letters of the same case are significantly different (P < 0.05). Upper case letters are used for motility comparisons, lower case letters are used for viability comparisons.

 
Experiment 4
Comparisons of thawed sperm motility from the same semen sample frozen in Cryoprotectant 3 with the ultra-rapid freezing technique and standard slow rate freezing are shown in Figure 5. Sperm motility of initial samples was 55 ± 2.2% (mean ± SEM). Post-thaw sperm motility was 45 ± 3.1 and 45 ± 3.4% for slow rate and ultra-rapid freezing methods respectively. A significant difference was noted between initial sperm motility and thawed sperm motility for each freezing method tested (P < 0.05); however, there was no significant difference in thawed sperm motility between methods.



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Figure 5. Comparison of thawed sperm motility before and after two different freezing protocols using Cryoprotectant 3. After assessing sperm motility in a fresh semen sample, the sample was divided and frozen using ultra-rapid and slow-rate freezing (n = 10). Sperm motility was assessed after thawing and comparisons were made between the two freezing techniques as well as with the fresh sample. Columns with different letters are significantly different (P < 0.05). Values are means ± SEM.

 
No significant difference was found between sperm motility and hypo-osmotic swelling in the fresh or post-thaw slow rate and ultra-rapid freezing methods.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Vitrification is a process whereby an object is supercooled rapidly enough to cause the solution to become solid, or glass-like, without ice crystal formation (Fahy et al., 1984Go). Vitrification can be achieved by direct submersion into liquid nitrogen; however, several problems currently limit the practical use of vitrification for biological specimens. After vitrification, if rewarming occurs too slowly, ice crystals will form with thawing and cause cellular damage (Mazur, 1984Go). Additionally, to vitrify an object requires high concentrations of cryoprotectants which are often cytotoxic. Of the commonly used intracellular cryoprotectants, ethylene glycol has been demonstrated to permeate sperm at the fastest rate (Gilmore et al., 1997Go). Additionally, compared with other intracellular cryoprotectants, ethylene glycol lowers activation energy and increases hydraulic conductivity allowing sperm to remain closer to equilibrium with changes in temperature (Gilmore et al., 2000Go). With this information, CryoA and CryoB were designed containing ethylene glycol. However, given the potential cytotoxic effects of the high concentrations of ethylene glycol, we additionally tested several dilutions of each cryoprotectant with the intention of selecting the least toxic solutions and using them as cryoprotectants during direct submersion of sperm into liquid nitrogen. We did not perform the initial two experiments with Cryoprotectant 3 because of prior publications showing a lack of sperm survival with direct plunging into liquid nitrogen using comparable solutions (Sherman, 1963Go). Because we did not directly measure temperatures within the cryovials, it is impossible to state definitively whether cooling was fast enough for vitrification to occur when specimens were plunged into liquid nitrogen; however, since no survival was observed with this technique, we did not pursue precise temperature measurements during freezing and thawing. Sherman (1954Go) described attempts to cryopreserve human sperm with direct submersion into cooled isopentane and liquid nitrogen; however, similar to our experience, very few sperm survived this technique. Studies suggest that the observed poor survival of the sperm with direct submersion into liquid nitrogen is due to the high concentration of intracellular water which freezes causing damage to intracellular organelles and the cytoskeleton (Mazur et al., 1972Go).

In subsequent studies, Sherman showed a significant improvement in sperm survival when glycerol-treated samples were placed into liquid nitrogen vapour for 5–10 min prior to placing them at –196°C (Sherman 1963Go). With this additional knowledge, we included a similar protocol as part of Experiment 3. Similar to prior work, we found that a significant percentage of sperm did survive submersion after only 5 min in liquid nitrogen vapour.

Several studies have compared rapid freezing with slower protocols and found contradictory results (Sherman, 1963Go; Mahadevan and Trounson, 1984Go) while others have reported the techniques to be comparable (Fernandez-Cano et al., 1964Go). Similar to the results of this latter study, we demonstrated equivalent results for both slower and ultra-rapid freezing methods when comparing the same specimen using both techniques. Possible explanations for the discrepancy with other studies may include use of different concentrations of cryoprotectant, freezing methods (biological freezers versus liquid nitrogen vapour) and thawing methods (room temperature versus 37°C) (Mahadevan and Trounson, 1984Go). An additional factor may be the specimen container used for freezing. When a cryovial is placed into a cold environment, the liquid on the periphery of the container will be exposed to colder temperatures first. As a result, a temperature gradient exists from the perimeter to the middle of the vial causing water on the periphery of the container to freeze faster than that in the centre (Gao et al., 1995Go). As this occurs, the solute towards the centre of the vial becomes more concentrated, which will affect osmotic gradients across cell membranes and the ability of sperm to survive cryopreservation. The cryoloop used in the above experiments eliminates the temperature gradient since the sperm are suspended in a thin film. As a result, sperm on the cryoloop have a similar exposure to the change in temperature that occurs in the cryovial which may account for their ability to survive more rapid freezing.

Studies have also demonstrated an interaction between cooling and rewarming rates, whereby specimens cooled slowly survive better when warmed slowly and specimens frozen rapidly have improved survival with rapid thawing (Henry et al., 1993Go). Although specimens frozen on cryoloops and by the slow freeze method were each thawed at room temperature, the rate of thawing was different. Samples frozen on cryoloops were removed from the liquid nitrogen and placed directly into media at room temperature. This allowed the frozen film of media on the loop containing the sperm to thaw immediately and be assessed. Conversely, slow rate frozen samples contained 200 µl in a cryovial and were thawed at room temperature which takes several minutes for the samples to thaw completely before they can be assessed. Consequently, although samples frozen by each method were thawed at room temperature, the rate of thawing was clearly faster in the ultra-rapidly frozen samples. The combination of these freeze/thaw rates may account for our comparable results for slow and ultra-rapid sperm cyropreservation (Fernandez-Cano et al., 1964Go). It is possible that the rewarming rate for specimens directly plunged into liquid nitrogen was not rapid enough to prevent ice crystals from forming with subsequent cellular damage.

A few observations from our experiments require additional comment. Although solution CryoB 1:1 dilution was not selected for further evaluation as a cryoprotectant, the significant increase in sperm motility over time seen with exposure is interesting. An explanation could be that the solution created an initial non-toxic osmotic shock that allowed the sperm to recover motility with time, probably from an increase in intracellular calcium. It is possible that 4 min did not allow enough time for equilibrium to occur and that extending the exposure time would have further improved sperm motility. Additionally, it is unclear from our experiments why Cryoprotectant 3 was not compatible with the sperm viability stain that was used. Glycerol was the only chemical present in this solution not found in the other cryoprotectants tested. Despite this, Mahadevan (1985Go) showed that a viability stain could be performed on sperm frozen in 15% glycerol with 0.5% eosin yellow in 0.15 mol/l phophate buffer. The cause of eosin/nigrosin precipitation in our experiments remains to be determined. Finally, the cryovials used contain ventilation ports in their lids which allowed liquid nitrogen to ‘leak’ into vials. This raises concerns over potential viral cross contamination. In addition, consideration must be given to the potential for cryovial explosion when using leak-proof vials. The port holes may not be essential. Experiments are underway to assess whether similar results can be obtained using cryoloops in leak-proof cryovials. Alternatively, one can eliminate the chance for cryovial explosion and obviate cross-contamination by storing samples in liquid nitrogen vapour (Tomlinson and Sakkas, 2000Go).

Currently several techniques exist for cryopreserving limited numbers of sperm. Kupker et al. (2000Go) described freezing testicular tissue with the subsequent isolation of motile sperm after thawing. Although reasonable for patients undergoing testicular biopsy, this technique does not allow for freezing residual sperm from epididymal aspirations or isolated sperm from ejaculated specimens. An alternative technique to freeze testicular sperm in small droplets has also been reported with recovery rates of motile sperm up to 90% (Craft and Tsirigotis, 1995Go; Gil-Salom et al., 2000Go). An additional method for freezing small numbers of isolated sperm include using mouse or human zona pellucida as storage containers (Cohen et al., 1997Go; Hsieh et al., 2000Go). Although labour intensive and expensive, recovery rates of motile sperm for ICSI are 82% (Hsieh et al., 2000Go). Compared with the above methods, ultra-rapid freezing using cryoloops offers the advantage of being less time-consuming and easy to perform while maintaining a similar recovery rate of motile sperm of 82% as demonstrated in Experiment 4.

The ability to cryopreserve oligozoospermic samples has many potential areas for application including for patients with severe oligozoospermia or asthenozoospermia. Sperm motility has been shown to continually decrease with each freeze–thaw cycle (Rofeim et al., 2001Go). With the use of cryoloops, multiple vials containing enough sperm for ICSI can be individually thawed without the need to refreeze unused portions. Additionally, sperm obtained from testicular biopsies not used for ICSI on the day of isolation could be easily stored for future use, thus eliminating the need for the patient to undergo a repeat biopsy. This technology could also allow the surgeon to rapidly cryopreserve the few sperm obtained from the epididymal fluid at the time of vasoepididymostomy. Finally, when freezing semen samples with normal counts, the residual few microlitres in the preparation vials could be quickly stored on cryoloops as a back-up for ICSI if situations where future intrauterine insemination or IVF methods fail, the number of cryopreserved samples is limited, and the patient was unable to produce more sperm for assisted reproduction.

Although using cryoloops to perform ultra-rapid freezing of oligozoospermic samples is feasible, many questions remain to be answered prior to widespread implementation. Similar to many studies evaluating cryopreservation of sperm, we used sperm motility and viability stains to evaluate outcomes of the freezing technique. However, McLaughlin et al. (1992Go) failed to show a correlation between sperm motility or viability with acrosomal damage. Experiments are currently underway focusing on functional tests of ultra-rapid frozen–thawed sperm, as well as morphological studies using electron microscopy. Finally, it should be noted that although we evaluated feasibility of freezing samples containing small amounts of sperm, the samples utilized were aliquots from normal semen samples. It has been shown that semen from patients with oligozoospermia and/or asthenozoospermia have a tendency for reduced cryosurvival using standard freezing techniques when compared with normal controls (Fernandez-Cano et al., 1964Go; Sawetawan et al., 1993Go). In a similar population of patients with poor quality sperm, Ragni et al. (1990Go) showed improved cryosurvival when samples were frozen using a computerized slow-rate technique versus rapid liquid nitrogen vapour freezing. Clearly, oligozoospermic semen samples will need to be evaluated for their ability to be cryopreserved with cryoloops. These studies are currently underway. However, presented data have demonstrated the feasibility and success of ultra-rapid cryopreservation of very low numbers of sperm using cryoloops, which paves the way for future experimentation in this line of andrological studies.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bunge, R.G. and Sherman, J.K. (1953) Fertilizing capacity of frozen human spermatozoa. Nature, 172, 767–768.

Cohen, J., Garrisi, G.J., Congedo-Ferrara, T.A., Kieck, K.A., Schimmel, T.W. and Scott, R.T. (1997) Cryopreservation of single human spermatozoa. Hum. Reprod., 12, 994–1001.[CrossRef][ISI][Medline]

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Critser, J.K., Huse-Benda, A.R., Aaker, D.V., Arneson, B.W. and Ball, G.D. (1987) Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction. Fertil. Steril., 47, 656–663.[ISI][Medline]

Critser, J.K., Huse-Benda, A.R., Aaker, D.V., Arneson, B.W. and Ball, G.D. (1988) Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility. Fertil. Steril., 50, 314–320.[ISI][Medline]

Fahy, G.M., MacFarlane, D.R., Angell, C.A. and Meryman, H.T. (1984) Vitrification as an approach to cryopreservation. Cryobiology, 21, 407–426.[ISI][Medline]

Fernandez-Cano, L., Menkin, M.F., Garcia, C.R. and Rock, J. (1964) Refrigerant preservation of human spermatozoa. Fertil. Steril., 15, 390–406.[ISI][Medline]

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Submitted on April 19, 2002; resubmitted on September 19, 2002; accepted on December 17, 2002.