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
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Abstract |
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Key words: cryopreservation/oligozoospermia/sperm/ultra-rapid freezing
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Introduction |
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Two techniques for sperm cryopreservation have been described. A slower freezing method consists of gradually cooling the sperm over a period of 24 h in two or three steps either manually (Thachil and Jewett, 1981
) or using a programmable freezer (Serafini and Marrs, 1986
). 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, 1984
). 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, 1981
; Mahadevan and Trounson, 1984
; Serafini and Marrs, 1986
). Additional research has suggested that holding the specimen at 5°C for 10 min and seeding the specimen may improve cryosurvival (Critser et al., 1987
). Alternatively a more rapid method consists of manually placing the sperm in liquid nitrogen vapour for 530 min prior to submersion into liquid nitrogen (Sherman, 1963
; Kobayashi et al., 1991
; Rofeim et al., 2001
). Although some research has shown the slower freezing method to be superior (Mahadevan and Trounson, 1984
), others have published data favouring more rapid cooling rates (Sherman, 1963
). 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., 1999
). 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., 1984a; Weidel and Prins, 1987
; Henry et al., 1993
; Royere et al., 1996
). Additionally, the quality of thawed sperm can be improved by concentrating the ejaculates prior to freezing (Perez-Sanchez et al., 1994
). 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., 2001) 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., 1997
; Hsieh et al., 2000
). 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, 1995
; Gil-Salom et al., 2000
). 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.
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Materials and methods |
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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). Testyolk 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 (1954), 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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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., 1984b) 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.
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Results |
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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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Discussion |
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In subsequent studies, Sherman showed a significant improvement in sperm survival when glycerol-treated samples were placed into liquid nitrogen vapour for 510 min prior to placing them at 196°C (Sherman 1963). 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, 1963; Mahadevan and Trounson, 1984
) while others have reported the techniques to be comparable (Fernandez-Cano et al., 1964
). 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, 1984
). 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., 1995
). 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., 1993). 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., 1964
). 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 (1985) 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, 2000
).
Currently several techniques exist for cryopreserving limited numbers of sperm. Kupker et al. (2000) 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, 1995
; Gil-Salom et al., 2000
). An additional method for freezing small numbers of isolated sperm include using mouse or human zona pellucida as storage containers (Cohen et al., 1997
; Hsieh et al., 2000
). Although labour intensive and expensive, recovery rates of motile sperm for ICSI are 82% (Hsieh et al., 2000
). 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 freezethaw cycle (Rofeim et al., 2001). 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. (1992) failed to show a correlation between sperm motility or viability with acrosomal damage. Experiments are currently underway focusing on functional tests of ultra-rapid frozenthawed 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., 1964
; Sawetawan et al., 1993
). In a similar population of patients with poor quality sperm, Ragni et al. (1990
) 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.
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Submitted on April 19, 2002; resubmitted on September 19, 2002; accepted on December 17, 2002.