The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Israel
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Abstract |
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Key words: cryopreservation/hemizona assay/spermatozoa preparation
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Introduction |
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Frequently, particularly in men with a malignant disease, the quality of the semen sample for cryopreservation is poor in terms of concentration, motility and morphology (Botchan et al., 1997a, 1997b
). Under such circumstances, the prospect for an acceptable quality of the frozenthawed specimen is low. In view of the possibility that sperm separation after thawing may be impaired (Graczykowski and Siegel, 1991
), it was suggested that the sample be treated prior to the freezing procedure, in order to select and enrich the specimen with higher-quality sperm cells (Barthelemy et al., 1990
). However, the contribution of the treatment before freezing is not obvious because sperm manipulation may damage the cells and reduce their cryo-freezability and fertility potential.
This study was, therefore, conducted to assess the fertility potential of frozenthawed sperm cells which had been cryopreserved after separation on Percoll gradients, or washed out of seminal plasma in comparison with that of the traditional method. This was evaluated using conventional sperm parameters combined with the zona pellucida binding assay, which was found to be an important predictor of fertilizing potential (Liu and Baker, 1992; Gamzu et al., 1994
).
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Materials and methods |
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Percoll separation procedure
The column was prepared by layering 1-ml aliquots of 40% upper and 80% lower isotonic Percoll (Pharmacia, Uppsala, Sweden). Up to 1 ml of the ejaculate was then layered on the Percoll gradient and centrifuged for 20 min at 300 g. The sperm pellet was collected and re-suspended in 1 ml of human tubal fluid medium (HTF; Irvine Scientific, Santa Ana, CA, USA) supplemented with 1% human serum albumin (Kamapharm Human Albumin; Kamada, Kibbutz Beit Kama, Israel). After centrifugation, the pellet was resuspended in 0.3 ml of HTF medium.
Washing procedure
This was performed by diluting 1 ml semen with HTF medium (1:2 v/v) and centrifuging at 300 g for 10 min to separate the spermatozoa from seminal plasma. The pellet was then suspended in 1 ml HTF medium, centrifuged once more, and resuspended in 0.3 ml of HTF medium.
Freezing procedure
The specimen was carefully diluted by the addition of droplets in equal volume to the freezing medium test yolk buffer (Irvine Scientific, Santa Ana, CA, USA). After dilution, the mixture was equilibrated for 15 min at room temperature, then sealed in 0.5 ml straws (I.M.V., Paris, France) and cooled in a semi-programmable freezer (Nicool, LM-10; Air Liquid, Paris, France). The straws were cooled from room temperature to 6°C, 1.7°C/min, then to 100°C, 5°C/min. The straws were then transferred directly to liquid nitrogen (196°C) for storage (Yavetz et al., 1991).
Thawing procedure
The straws were allowed to thaw at 37°C for 5 min and assessed immediately for sperm parameters and binding capacity by the hemizona assay (HZA).
Hemizona assay: Thawed samples were washed twice with HTF medium and the pellet was overlaid with 0.5 ml medium and incubated at 37°C for 1 h to facilitate swim-up separation. After separation, the samples were diluted with the medium to achieve a motile sperm concentration of 500 000 cells/ml. The control sample for all the HZA performed in this study consisted of a cryopreserved pool of 12 ejaculates from different fertile donors, selected by virtue of good freezability. The pooled sample (31 ml), with 124x106 spermatozoa/ml and 51% motile spermatozoa, was diluted 1:1 with freezing medium and transferred into 0.5-ml straws for freezing. The straws contained 55x106 spermatozoa/ml and 40% motile sperm cells after thawing. Before the HZA was performed, a straw was thawed and the sperm cells were washed and prepared by swim-up separation as described previously.
Unfertilized oocytes from failed cycles were stored in a salt solution, as described elsewhere (Yanagimachi et al., 1979). On the day of the assay, the oocytes were removed from the salt solution and rinsed three times in HTF medium. Leitz micromanipulators (Leica, Wetzl, Germany) were used for cutting the oocytes as described previously (Burkman et al., 1988
; Gamzu et al., 1992
). The number of sperm cells that could not be removed from the matching hemizonae was counted to enable subtraction when the hemizona index (HZI) was calculated. The matching hemizonae were separately co-incubated in 50-µl droplets containing spermatozoa derived from the tested sample, and from the control sample with a motile sperm concentration of 0.5x106/ml. The hemizona and spermatozoa were co-incubated for 4 h at 37°C. After the co-incubation period, the hemizonae were pipetted vigorously to dislodge all loosely attached sperm cells. The same two technicians counted the number of tightly bound sperm cells. Two oocytes were used for each assay. The results of the HZA were expressed by the HZI, which was calculated by dividing the number of those tested by the number of control sperm cells attached to the hemizonae. The final HZI, expressed as a percentage, was the average of the two hemizona indices. The intra- and inter-assay coefficients of variation were 8% and 14% respectively.
Statistical evaluation
The results are given as mean ± SE. A significant difference was defined using Student's t-test and paired t-test, as appropriate.
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Results |
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Discussion |
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Another study failed to establish any significant difference when the dilution with the cryoprotective medium was carried out with the seminal plasma, or after its removal. Likewise, removal of seminal plasma, which requires centrifugation, had no striking effect on the status of the acrosome. On the contrary, less acrosomal injury was observed when the spermatozoa were cryopreserved after removal of the seminal plasma (Barthelemy et al., 1990).
As far as the separation technique before freezing was concerned, Zavos et al. (1991) demonstrated that spermatozoa selected by SpermPrep columns showed a slower decline of motility after the freezingthawing process, compared with that of unselected spermatozoa. The same phenomenon was observed by Kobayashi et al. (1991) who used a Percoll gradient. Percoll-selected spermatozoa before freezing retained greater longevity than raw samples (Sharma and Agarwal, 1996). Notwithstanding, spermatozoa selected by swim-up were as susceptible to the stress caused by freezing and thawing as unselected spermatozoa in the original semen sample (Perez-Sanchez et al., 1994).
It has been suggested that sperm processing alone can damage spermatozoa because of reactive oxygen species (ROS) production (Aitken and Clarkson, 1988). In this regard, Percoll-gradient centrifugation selected spermatozoa with a better functional competence than swim-up from a washed pellet, due to enhanced protection against peroxidative damage caused by the centrifugation process (Griveau and Lannou, 1994
). Moreover, sperm washing by repeated centrifugation and resuspension increased the level of ROS by 2050-fold, when compared with that of the original sample, but centrifugation without removing the seminal plasma reduced the levels of ROS, suggesting a protective role of seminal plasma (Iwasaki and Gagnon, 1992
). These phenomena may be the basis for the high recovery of progressive motile spermatozoa in the Percoll-treated samples when compared with that of the washed samples. However, it should be borne in mind that, in poor specimens, the selective feature of the Percoll gradient decreases the yield of the number of progressive motile spermatozoa (Ng et al., 1992
). Despite these data, the present study demonstrated that, with regard to zona pellucida binding, no difference was found between the two sperm preparation techniques, both of which did not change the zona pellucida binding ability when compared with that of unprocessed specimens.
Although it is acceptable that freezing and thawing are associated with a decrease in sperm motility and viability, several functional tests, such as the bovine mucus penetration, zona-free hamster egg penetration, hypo-osmotic swelling test and triple-stain technique for acrosome reaction, demonstrated that frozen spermatozoa maintained their fertilizability (Yoshida et al., 1990). In this regard, we reported previously that the freezingthawing process does not impair the capability of rescued spermatozoa to bind to the zona pellucida (Gamzu et al. 1992
). Our present finding that fertilizing capacity, as indicated by zona pellucida binding capability, is the same in spermatozoa manipulated before freezing and those that were not manipulated is substantial for sperm banking of poor specimens. In such cases, optimizing the concentration of progressive motile sperm cells before the freezing process is recommended to ameliorate the fertilizing capacity of the frozen spermatozoa.
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Notes |
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References |
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Submitted on March 26, 1998; accepted on September 24, 1998.