1 Andrology Research & Clinical Laboratories, Department of Urology, The Cleveland Clinic Foundation, Cleveland OH 44195, USA and 2 Andrology & Human Reproduction Clinic (ANDROFERT), Rua Tiradentes, 289/61, Campinas, São Paulo, Brazil 13023190
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Abstract |
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Key words: acrosome/cryopreservation/human spermatozoa/sperm processing/swim-up
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Introduction |
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Under normal physiological conditions, ejaculated spermatozoa must be washed free from seminal plasma in order to undergo capacitation, which is necessary before they can undergo acrosome reaction (AR) and penetrate the zona pellucida (Zaneveld et al., 1991; Henkel et al., 1993
; Wang et al., 1993
). Seminal plasma is removed as the spermatozoa traverse the cervical mucus and capacitation occurs as they are transported across the cervix, uterus and Fallopian tubes.
Prolonged exposure to seminal plasma results in marked decline in both motility and viability. The presence of seminal plasma in an IVF system inhibits the AR, which is a critical component of the fertilizing ability of human spermatozoa (Liu and Baker, 1988; Polanski and Lamb, 1988
; Henkel et al., 1993
; Calvo et al., 1994
; Parinaud et al., 1995
). In addition, ejaculated spermatozoa are damaged by reactive oxygen species emanating from seminal leukocytes and damaged spermatozoa (Aitken and Clarkson, 1988
). It is essential, therefore, that in assisted reproduction procedures such as intrauterine insemination (IUI) and IVF, ejaculated spermatozoa are separated from the seminal environment as soon as possible.
The swim-up procedure accomplishes this, and enables the selection of populations of highly motile spermatozoa for assisted reproduction procedures. It has been used by many laboratories as the main sperm washing technique (Berger et al., 1985; Brandeis and Manuel, 1993
). It seems reasonable to speculate, therefore, that removing the seminal plasma and selecting highly motile spermatozoa by the swim-up technique, before freezing, may reduce the loss of viability, motility and acrosome integrity during cryopreservation.
The purpose of this study was to determine if using swim-up to select a sperm population with better motion characteristics before freezing can minimize the deleterious effects of cryopreservation, as examined by sperm motion and functional measures.
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Materials and methods |
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A stock solution of FITC-PNA was prepared by dissolving 2 mg of FITC-PNA in 1 ml PBS, and it was stored in 600 µl aliquots at 20°C. Hoechst-33258 was prepared by dissolving 1 mg of H33258 in 1 ml PBS, and it was stored in 10 µl aliquots at 20°C.
Semen collection, sperm processing and cryopreservation procedures
Sperm specimens from 15 healthy volunteers were obtained by masturbation into sterile specimen cups. All subjects were asked to observe 23 days of sexual abstinence before donating. Semen samples had normal volume, count and motility according to the World Health Organization criteria (WHO, 1999). After the initial semen analysis, each semen specimen was divided into two equal aliquots. The first aliquot received no treatment (control). The second aliquot was processed by the swim-up method from a washed sperm preparation (indirect method) to give a highly motile sperm population (Russell and Rogers, 1987). It should be noted that the centrifugation speed and duration were reduced as far as possible to minimize the potential damage caused by this process. Briefly, the liquefied semen sample was diluted with an equal volume of BWW. After centrifugation at 300 g for 10 min, the supernatant was removed and the pellet resuspended in 2 ml of BWW. A second centrifugation was followed by resuspension to a final volume of 600 µl of BWW supplemented with 0.3% bovine serum albumin (BSA) factor V (Irvine Scientific). This fraction was divided in three equal aliquots of 200 µl. Each aliquot was underlayered beneath 800 µl of BWW supplemented with 0.3% BSA in 15 ml centrifuge tubes. The tubes were loosely capped and placed in a 37°C incubator, under 5% CO2 in air, at a 45° angle for 1 h. During this period, motile spermatozoa migrated from the underlayered sperm suspension to the upper layer. The top 600700 µl of the supernatant containing the actively motile spermatozoa was removed with extreme care. Control and swim-up specimens were analysed by computer-assisted semen analysis using a computer-assisted semen analyser (CASA, Motion Analysis, Cell-Trak, model VP110, Santa Rosa, CA, USA). Sperm motion characteristics such as percent motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and amplitude of lateral head displacement (ALH) were measured. A small aliquot (0.1 ml) was removed from control and swim-up specimens for assessment of acrosome status.
Both aliquots were then cryopreserved by the liquid nitrogen vapour method. TEST-yolk buffer with glycerol was used as a freezing agent for cryopreservation (Esteves et al., 1998). In brief, a 5 ml vial of the freezing agent was thawed by incubation at 37°C. An aliquot of the medium equal to 25% of the original specimen volume was then added to the specimen. The specimen was gently mixed for 5 min using the Hema-Tek aliquot mixer (Miles, Elkhart, IN, USA). This process was repeated until the ratio of freezing medium to ejaculate was 1:1. Cryovials were placed at 20°C for 8 min and submerged in liquid nitrogen vapour (N2) at 79°C for 2 h. Vials were then immersed in liquid N2 at 196°C for long-term storage. After at least 48 h in liquid nitrogen, the vials were incubated at 37°C for 20 min, washed twice in BWW to remove the cryomedium, and reconstituted in 0.5 ml BWW medium with 0.3% BSA (Agarwal et al., 1995
). A small aliquot (0.1 ml) was removed from each specimen for measurement of percentage motility, motion characteristics and frequency of spontaneous AR assessment after thawing.
Calibration set-up of the sperm motion analyser
The CASA calibration set-up was as follows: 2-well, 20 µm, duration of data capture (frames): 15 (control) and 30 (swim-up); minimum motile speed (µm/s): 600 (control) and 800 (swim-up); distance scale factor (µm/pixel): 0.9457; centroid cell size minimum (pixels): 2; centroid cell size maximum (pixels): 8; number of cells to find per well: 200; minimum number of fields per sample: 3. A high degree of correlation was seen between the CASA and both manual sperm counts (r2 = 1, slope = 1) and motility (r2 = 0.97, slope = 0.97), thereby establishing the accuracy of our CASA measurements. The reproducibility of the analyser results was determined by using a calibration videotape recording. The baseline values for the raw specimens were: sperm count was 38.342.5x106/ml (30 frame/s) and motility: 61.675.0% (30 frame/s); the washed specimens had baseline values of 33.134.7x106/ml (60 frame/s) for sperm count and 78.191.5% (60 frame/s) for motility. For these computations, results >2 SD were rejected.
Sperm capacitation and ionophore challenge
After thawing, a highly motile sperm population from each specimen was isolated by the swim-up method as described previously. The spermatozoa were then capacitated for 3 h by incubation in a BWW medium with 3% BSA at 37°C, under 5% CO2 in air. Each capacitated sample was divided into two aliquots: one was challenged with 2.5 µmol/l calcium ionophore A23187 solution, and the other was treated with diluted dimethylsulphoxide (DMSO) alone to serve as a control. Both aliquots were then incubated at 37°C for 1 h under the same conditions.
Assessment of viability, acrosome status and ionophore-induced AR
Spermatozoa with intact acrosomes were counted before freezing and after thawing. Calcium ionophore-induced AR was measured only after cryopreservation.
Acrosome status was assessed with FITC-PNA, in conjunction with a nuclear stain, bis-benzimide as a viability test (Cross et al., 1986; Mortimer et al., 1990
; Aitken et al., 1993
). Viability and acrosome status were assessed simultaneously by mixing 100 µl of sperm specimen with 100 µl of 2 µg/ml Hoechst-33258 solution and incubating for 10 min in the dark. Spermatozoa were then washed in PBS solution by centrifugation at 300 g for 5 min to remove excess stain, and the pellet was resuspended in 100 µl of BWW. A 20 µl aliquot of this solution was smeared on a microscope slide and allowed to dry. Air-dried slides were immersed in ice-cold methanol for 30 s to permeabilize the sperm membranes and allowed to air dry. The fixed smears were immersed in a 40 µg/ml FITC-PNA solution, incubated at room temperature for 20 min in a foil Coplin jar (Alleigance Healthcare Corp., McGaw Park, IL, USA), and washed gently in PBS to remove the excess label. Scoring was completed within 48 h of staining.
A Leitz Orthoplan fluorescence microscope (Leitz, Wetzlar, Germany) equipped with a Ploemopak epi-illumination module and a mercury ultraviolet source was used to examine the slides at x1000 magnification in the presence of an anti-quenching agent (Cargille immersion oil, type DF, Fisher Scientific, Pittsburgh, PA, USA) to minimize the loss of fluorescence. Filter cube I.2 was used for FITC-PNA, which fluoresces apple-green, and cube A.2 for Hoechst-33258, which fluoresces a bright medium blue. Hoechst-33258 stains the nuclei of damaged cells (dead spermatozoa), which show a bright blue-white fluorescence, and is excluded from viable cells (live spermatozoa), which show a pale blue fluorescence. The same spermatozoa were examined for FITC-PNA labelling and for Hoechst-33258 staining by interchanging the two filters.
Categorization of staining patterns
Acrosome staining on FITC-PNA labelling was classified as follows. In an intact acrosome, the acrosomal region of the sperm head exhibited a uniform apple-green fluorescence. In a reacted acrosome, only the equatorial segment of the acrosome was stained.
Viability staining on Hoechst-33258 was classified as follows. In viable spermatozoa, the sperm head showed a pale-blue fluorescence, and in dead spermatozoa, the sperm head showed a bright blue-white fluorescence. A total of 200 spermatozoa per sample was scored. The frequency of spermatozoa with intact and reacted acrosomes was based on viable cells only.
Reproducibility of the acrosome scores
To evaluate the inter-observer reproducibility of the acrosome scores, in a blind study one slide from each donor (n = 15) was evaluated by two observers. To determine the intra-observer reproducibility of the acrosome scores, the same slides were re-evaluated by one observer.
Statistical analysis
The Student's paired t-test was used to analyse statistical differences in percentage motility, motion characteristics, viability and frequency of acrosome-intact spermatozoa in control and swim-up groups before and after cryopreservation, since the data were normally distributed as assessed by the KolgomorovSmirnov test for homogeneity and normality. Pearson's correlation coefficient was used to assess inter-observer and intra-observer reproducibility for acrosome scores. An alpha level of 0.05 or less was considered statistically significant and all tests were two-tailed. Statistical analysis was performed using the SAS statistical software package (Cary, NC, USA).
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Results |
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Discussion |
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Intact ejaculated spermatozoa may be damaged by oxygen radicals emanating from seminal leukocytes and damaged spermatozoa (Aitken and Clarkson, 1988), so that the stress caused by the cryopreservation procedure itself may add to damage inflicted already by free oxygen radicals. It is not known if the processes of freezing and thawing damage all spermatozoa equally, or if there is selective damage to a more susceptible population such as those suffering lipid peroxidation. If damage is selective, removing intact spermatozoa immediately from potentially damaging leukocytes and damaged spermatozoa may improve sperm survival after cryopreservation.
The swim-up procedure eliminates the seminal plasma and immotile and dead spermatozoa along with exfoliated epithelial cells, cellular debris, leukocytes and amorphous material. Spermatozoa selected by the swim-up method in an experimental model perform better in the sperm penetration assay (SPA) than untreated specimens (Russell and Rogers, 1987).
In our study, all ejaculates had normal pre-freeze sperm characteristics. Cryopreservation shifted these characteristics in both control and swim-up specimens into the subnormal or sub-optimal range, as showed by decreased sperm motility and viability. However, specimens processed by swim-up from a washed sperm preparation before freezing had better post-thaw motion characteristics (VSL, VCL, VAP, and ALH). Spermatozoa separated by the SpermPrep method (Zavos et al., 1991) had higher percentages of motile cells and the subjective motility ratings after the freezethaw process were better than the same characteristics in spermatozoa from the same ejaculates frozen in semen, even though cells still suffered damage. It was found (Kobayashi et al., 1991
) that post-thaw motility was higher in samples concentrated by Percoll centrifugation before freezing. Another study (Pérez-Sánchez et al., 1994
) demonstrated that spermatozoa selected by the swim-up technique, post-thaw, contained more morphologically normal forms, fewer totally degenerated spermatozoa with damaged head and tail membranes, and more progressively motile spermatozoa with greater velocities and amplitudes of lateral head displacement than did untreated frozenthawed semen. However, the proportion of damaged viable cells was identical in both groups. Specimens prepared by Percoll separation techniques before freezing retained motility longer than did controls, although comparison with pre-freeze values revealed that selected and unselected spermatozoa lost viability from the freeze-thaw process to the same extent (Sharma and Agarwal, 1996
).
In our study, although swim-up separation before freezing resulted in a lower total and motile sperm count compared to the control specimens, the results of motile sperm count after thawing were comparable between the control and swim-up specimens. The optimum concentration of total motile spermatozoa that can result in successful fertilization and pregnancy in IUI varies from 1 to 5x106. However, pregnancies have been achieved with as few as 0.1x106 motile spermatozoa (Berg et al., 1997). Greater sperm concentration does not result in a corresponding increase in fecundity rates (Ombelet et al., 1995
).
Our results, as well as those obtained previously (Pérez-Sánchez et al., 1994; Sharma and Agarwal, 1996
), show that any improvements in sperm motion characteristics after selection by a swim-up or Percoll procedure do not seem to be a consequence of the selected spermatozoa being more resistant to the freezethaw process. First, the selected spermatozoa had higher absolute changes in some sperm characteristics after cryopreservation because they had higher initial values for motility, rather than better resistance to cryo-injury. Second, both sperm preparations (control and selected) had significant loss of viability after thawing, and the proportion of viable cells damaged was identical in both groups. The post-thaw swim-up group was not included in our study, since there are reports of its use in the literature (Graczykowski and Siegel, 1991
). These authors reported low recovery rates after swim-up from cryopreserved specimens, as well as decreased motion characteristics. In a previous study, we also compared the effectiveness of swim-up to recover motile spermatozoa in fresh and cryopreserved specimens. Our results indicated that swim-up provides similar yields of spermatozoa from cryopreserved specimens (recovery rate: 13%; range: 9.729.4) compared to fresh ones (22%; range: 1143; not significant) in normozoospermic individuals, although sperm motion characteristics and acrosome status were significantly lower in the former (Esteves et al., 1999
). In addition, cryopreserved spermatozoa are known to be more fragile than fresh spermatozoa. Swim-up washing after thawing takes >1.5 h (thawing takes 20 min, removal of cryomedia takes ~20 min, and swim-up takes another 60 min) compared with <30 min in our protocol.
On the other hand, we found that specimens processed by swim-up from a washed sperm preparation before freezing had higher percentages of intact acrosomes after thawing. This effect cannot be explained by higher initial values for acrosome integrity, because pre-freeze acrosome integrity was similar in both sperm preparations. Along the same lines, the percentage of cryopreserved spermatozoa with intact acrosomes that were able to undergo AR in response to calcium ionophore was significantly higher in swim-up specimens, and these findings are relative changes rather than absolute changes. To our knowledge, this is the first study to report a beneficial effect of pre-freeze swim-up processing on post-thaw acrosome status. The reason for this is unclear, but we speculate that the highly motile sperm population selected by swim-up may not be affected by oxygen radicals emanating from damaged leukocytes and from other spermatozoa susceptible to or damaged by lipid peroxidation. To support this hypothesis, a recent study from our laboratory has demonstrated that ejaculates frozen with antioxidant-supplemented cryoprotectant had improved ability to respond to calcium ionophore post-thaw (Esteves et al., 1998). In a study using transmission electron microscopy, it was demonstrated (Barthelemy et al., 1990
) that the deleterious effects of cryopreservation on sperm acrosomes were less evident if spermatozoa had been frozen without seminal plasma. These investigators postulated that the buffering effects of semen vary among individuals, and washing spermatozoa eliminates the important individual variable of seminal plasma.
Only spermatozoa with normal intact acrosomes can undergo AR, which is essential for the spermatozoa to penetrate the zona pellucida and to fuse with the oolemma. Also, the AR must occur at the correct time for the spermatozoa to be able to penetrate the zona pellucida (ZP). The correct timing of AR is ensured by physiological AR inducers in the cumulus oophorus and in the ZP (Tesarik et al., 1992). A normal sperm sample should contain a high proportion of spermatozoa with intact acrosomes capable of undergoing AR after incubation in capacitating conditions. Therefore, the difference between the percentage of intact acrosomes after ionophore treatment and the baseline value is important (Takahashi et al., 1992
). There is now sufficient evidence that specialized sperm function tests, such as the AR, are better predictors of the fertility potential than traditional semen characteristics (Takahashi et al., 1992
; Henkel et al., 1993
; Calvo et al., 1994
; Yovich et al., 1994
). Using an AR to ionophore challenge (ARIC) cut-off at 10%, the sensitivity of the test in predicting subfertility was 54% with a positive predictive value of 64% and specificity of 85% (Cummins et al., 1991
). Patients with ARIC scores above 10% have consistently better fertilization rates than those with lower scores (Henkel et al., 1993
; Calvo et al., 1994
). In our study, ARIC scores were >10% in 86.7% of the cryopreserved swim-up specimens, and 60% of controls. When a specimen was compared to its own control, swim-up processing improved the ARIC scores in 83% (5/6) of the subjects whose unprocessed specimen scores were <10%. It should be noted, however, that in a previous study (Esteves et al., 1998
), the variation in the AR in fresh and cryopreserved specimens from the same individuals was analysed in two different germinative cycles (median interval 5 months; range 58 months). It was concluded that acrosome results differ significantly in the same individual at different time intervals.
In conclusion, the swim-up method using a washed sperm preparation before freezing offers the possibility of selecting spermatozoa with better post-thaw motility, acrosome integrity, and ability to undergo AR. These effects may be relevant to cancer patients as well as to donor spermatozoa for use in artificial donor insemination. Although our data were obtained from individuals with normal sperm characteristics, the results are not likely to be substantially different in semen cryopreserved for other reasons, such as before cancer treatments; cancer patients' specimens freeze as effectively as those from normal men (Agarwal et al., 1995). Cancer patients generally have poor semen quality before cryopreservation. However, it has also been shown that 1260% of men with cancer, depending on the type of disease, present with normal or slightly abnormal sperm parameters at the time of sperm banking (Agarwal et al., 1995
; Hallak et al., 1999
). Because the yield of spermatozoa obtained by the swim-up procedure is low, oligozoospermic specimens may not be suitable, and swim-up procedures may have to be performed in multiple tubes to maximize the number of motile spermatozoa from a given sample. In addition, the possibility of providing multiple ejaculates can be considered, particularly in cancer patients, who usually have poor pre-freeze semen characteristics.
Whether swim-up processing before freezing also improves fertilization and pregnancy outcomes with assisted reproductive procedures such as IUI or conventional IVF warrants further investigation.
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Acknowledgments |
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Notes |
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3 To whom correspondence should be addressed at: Andrology Research & Clinical Laboratories, Department of Urology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, A19.1, Cleveland, Ohio 44195, USA. E-mail: agarwaa{at}ccf.org
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References |
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Submitted on February 21, 2000; accepted on June 19, 2000.