Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique

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1 Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique Giuseppe Ricci, M.D., a Sandra Perticarari, B.Sc., b Rita Boscolo, B.Sc., a Marcella Montico, B.Sc., c Secondo Guaschino, M.D., a and Gianni Presani, B.Sc. b a Assisted Reproduction Unit, Department of Obstetrics and Gynecology, b Clinical Analysis Unit, Department of Laboratory Medicine, and c Epidemiology Unit, Institute of Child Health IRCCS Burlo Garofolo, and University of Trieste, Trieste, Italy Objective: To compare the effects of density-gradient centrifugation and swim-up on sperm apoptosis by using a multiparameter flow cytometric method. Design: Autocontrolled split-sample study. Setting: Tertiary infertility center. Patient(s): Sixty-two male partners of couples undergoing infertility investigations. Intervention(s): Each sample was analyzed both before and after semen preparation by optical microscopy and by flow cytometry. Main Outcome Measure(s): Percentage of viable, apoptotic, and necrotic sperm and recovery rate of total motile, progressive motile, and viable sperm before and after the two sperm preparation methods. Result(s): Compared with the original semen, the mean percentages of apoptotic and necrotic sperm were significantly lower after both sperm preparation methods. The mean percentage of viable sperm was significantly higher after swim-up compared with gradient centrifugation. The recovery rates of total motile, progressive motile, and viable sperm were significantly higher using gradient centrifugation compared with swim-up. The viable sperm percentage and the progressive sperm motility were significant predictors for negative difference between the two methods in terms of viable sperm percentage after preparation. Conclusion(s): Both sperm preparation methods allow obtaining a sperm population with a low percentage of apoptotic sperm. Therefore, the risk of using apoptotic sperm for clinical treatment seems to be rather low. The choice of method will depend on whether IVF/ICSI or intrauterine insemination is to be performed. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Apoptosis, density-gradient centrifugation, flow cytometry, fluorospheres, semen analysis, sperm, swim-up, Syto 16 Although several studies have been published on the effectiveness of different semen preparation methods, there is insufficient evidence to recommend any specific sperm preparation technique (1). Comparative studies on sperm preparation methods have essentially investigated outcomes such as recovery rates and conventional semen parameters (1, 2). Over the last years, the effects of sperm preparation procedures on sperm quality have been evaluated using new laboratory methods. Earlier studies have suggested that sperm preparation techniques involving centrifugal pelleting of unselected sperm or density-gradient centrifugation of sperm may result in generation of reactive oxygen species (ROS) and sperm DNA damage (3 5). Furthermore, the percentage of normal chromatin-condensed sperm was found to be significantly lower after swim-up compared with the ejaculate (6). In subsequent reports, however, it has been shown that swimup sperm separation may improve some of the sperm chromatin structure assay related parameters (7) and does not induce Received September 2, 2007; revised and accepted November 19, Reprint requests: Giuseppe Ricci, M.D., Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Institute of Child Health IRCCS Burlo Garofolo and University of Trieste, Via dell Istria, 65/1, 34137, Trieste, Italy (FAX: þ39 (0) ; ricci@burlo.trieste.it). sperm DNA damage (8). Furthermore, it has been reported that motile sperm prepared by density-gradient centrifugation had higher mitochondrial membrane potential and lower DNA fragmentation, generated lower ROS and were more viable than those of the whole semen (9, 10). Other studies have compared the effects of swim-up and density-gradient centrifugation on sperm integrity. Sakkas et al. (11) reported that in using density-gradient centrifugation techniques there was a significant decrease in the percentage of sperm with DNA damage, whereas in using the swim-up method the recovered sperm showed no significant improvement. In contrast, Zini et al. (12) found that the percentage of sperm with denaturated DNA was reduced significantly in swimup treated but not in density-gradient centrifugation treated sperm compared with the whole semen. Thus, there is no consensus about which method is superior for isolating functionally normal sperm. In recent years, growing attention has been paid to apoptosis markers as indicators of sperm integrity (13 24). Some studies have investigated apoptosis in prepared sperm by swim-up (25, 26) and by density-gradient centrifugation (27, 28), but comparisons between different methods have not been carried out. Recently, we proposed a new 632 Fertility and Sterility â Vol. 91, No. 2, February /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 multiparameter flow cytometric method for semen analysis that includes sperm viability and apoptosis evaluation (29). The aim of the present study was to compare the effects of density-gradient centrifugation and swim-up on sperm quality by using this new method. MATERIALS AND METHODS Semen Analysis Semen samples were obtained from 62 men (mean age years) undergoing semen analysis. All subjects were caucasian and partners of women who failed to conceive after 24 months of unprotected intercourse. The study was approved by the Institutional Review Board. Routine semen analysis was performed according to World Health Organization (WHO) guidelines (30). Sperm morphology was assessed using WHO criteria at a cut-off point of 30% normal sperm. The sperm were stained using Testsimplets (Waldeck & Co., M unster, Germany). From each ejaculate two aliquots were taken for density-gradient centrifugation and swim-up preparation and another aliquot for multiparameter flow cytometric analysis. Swim-Up An aliquot of 0.5 ml whole semen was washed with 1 ml medium (Quinn s Advantage Medium w/hepes [Sage Bio- Pharma, Bedminster, NJ] supplemented with 0.5% human serum albumin [Sage Assisted Reproduction Products, CooperSurgical, Trumbull, CT]) in a 5 ml Falcon conical tube and then centrifuged at 300g for 10 min. The supernatant was discarded and the pellet resuspended in 0.5 ml medium. Then 0.5 ml medium was gently layered on the sperm suspension and the tube was inclined at an angle of 45 degrees and incubated at 37 C for at least 45 min. The tube was gently set upright and the upper interface was then gently aspirated with a Pasteur pipette. An aliquot was examined for sperm concentration and motility, and another aliquot was used for the multiparameter flow cytometric analysis. Density-Gradient Centrifugation Because Percoll is no longer available, silane-coated silica particles have been used for gradient separation (PureSperm 40/80; Nidacon International, Goteborg, Sweden). In fact, Centola et al. (31) demonstrated that PureSperm was as effective as Percoll for the recovery of good progressively motile sperm. A two-layer gradient was prepared by using readyto-use solutions of 80% and 40% PureSperm. Media were warmed to 37 C. Using a sterile pipette, 0.5 ml liquefied semen sample was placed on top of the upper layer into a conical 5 ml centrifuge tube. The tube was centrifuged at 300g for 20 min. The supernatant was then removed and the pellet was suspended in a volume of 1 ml medium and again centrifuged at 500g for 10 min. The pellet was resuspended in a volume of 0.5 ml medium. An aliquot was examined for sperm concentration and motility, and another aliquot was used for the multiparameter flow cytometric analysis. Flow Cytometric Analysis A multiparameter flow cytometric analysis to assess simultaneously sperm concentration, viability, apoptosis, and necrosis was carried out as we have previously described (29) with minor modifications. Briefly, 100 ml whole semen or prepared sperm were stained for 20 min in the dark at room temperature using 2 ml of a 10-mmol/L solution of Syto 16 greenfluorescent nucleic acid stain from Molecular Probes (Eugene, OR; final concentration 200 nmol/l) and 10 ml 7- amino-actinomycin-d (7-AAD Via-Probe; BD Pharmingen, San Diego, CA). The sperm in whole semen were counted and diluted in medium to reach approximately the same concentration of prepared sperm ( /ml). A Flow- Count fluorospheres vial (Beckmann-Coulter, Fullerton, CA; lot ) at a concentration of 1,016 beads/ml was gently mixed for s, and 100 ml of fluorospheres were accurately pipetted before analysis. After the incubation period, 1 ml cold phosphate-buffered saline was added to each tube and the samples analyzed by flow cytometry. Flow cytometric analysis was performed using a four-color FACSCalibur (Becton Dickinson, San Jose, CA) equipped with a 488 nm argon laser with 530 nm (FL1), 585 nm (FL2), and 670 nm (FL3) band-pass fluorescence filters and a 635 nm red diode laser with a 661 nm band-pass filter (FL4). Statistical Analysis Comparisons between whole semen and prepared sperm from the same ejaculate, as well as between the two sperm preparation methods, were performed using the Wilcoxon matched pairs test. Relationship between semen analysis parameters and flow cytometry results were analyzed using the Spearman rank or the Pearson correlation test where appropriate. For each sperm preparation method, univariate and multivariate analyses were used to explore relationships between progressive motile and viable sperm recovery rates and neat semen variables. To assess differences between the two sperm preparation methods, two new variables were created by subtracting the values of swim-up recovery rate from the values of density-gradient recovery rate and the values of viable sperm percentage after swim-up from the values of viable sperm percentage after density-gradient centrifugation. These differences were then dichotomized as negative differences (better result for swim-up method) versus positive difference (better result for density gradient method). Odds ratios (OR) and 95% confidence intervals (CIs) were calculated, with OR >1.0 corresponding to positive associations and OR <1.0 corresponding to negative associations. P<.05 was considered to be statistically significant. RESULTS An example of flow cytometric analysis before and after sperm preparation is shown in Figure 1. There was an extremely significant correlation between flow cytometry and optical microscopy results in sperm counting for both neat semen (r ¼.970, P<.0001) and capacitated fractions (r ¼.927, P<.0001, after density-gradient centrifugation; r ¼.941, Fertility and Sterility â 633

3 FIGURE 1 Cytofluorimetric analysis of dot-plot with log of green fluorescence (Syto16) versus log of red (7-AAD) fluorescence (A, B, C) displaying viable (R2), apoptotic (R3) and dead (R4) sperm cells. Plots D, E, F represent FSC versus SSC parameters on the basis of which a gate R5 sperm was set up to eliminate cell debris from analysis. Region R6 represents the bead population for absolute count. Cytograms A and D represent the analysis of semen sample before preparation, B and E after density-gradient centrifugation, C and F after swim-up technique. 634 Ricci et al. Semen preparation methods and apoptosis Vol. 91, No. 2, February 2009

4 TABLE 1 Conventional and flow cytometry sperm parameters before and after density-gradient centrifugation and swim-up. Whole semen Density-gradient centrifugation Swim-up P value Concentration (10 6 ) <.0001 Total motility (%) <.0001 Progressive motility (%) <.0001 Progressive motile <.0001 sperm (10 6 ) Viable sperm (%) <.0001 Apoptotic sperm (%) Necrotic sperm (%) <.0001 Note: Values are mean SEM. P<.0001, after swim-up). The concentration of progressive motile sperm was significantly higher after density-gradient centrifugation compared with swim-up preparation (Table 1). Compared with the original semen, the percentages of apoptotic and necrotic sperm were significantly lower after both sperm preparation methods. The percentage of viable sperm was significantly higher after swim-up compared with density-gradient centrifugation preparation (Table 1). The recovery rate of total motile, progressive motile, and viable sperm were significantly higher using density-gradient centrifugation compared with swim-up preparation (Table 2). There was no significant correlation between the percentage of viable, apoptotic, and necrotic sperm and progressive motility in whole semen (Table 3). After semen processing, a significant correlation between the percentage of viable sperm and progressive motility was found. Furthermore, there was a negative correlation between the percentage of apoptotic and necrotic sperm and progressive motility in both swim-up and density-gradient centrifugation prepared samples (Table 3). In whole semen, a significant correlation between the concentration of progressive motile sperm and the concentration of viable sperm was found (r ¼.812, P<.0001). In prepared sperm, this correlation was extremely significant (r ¼.946, P<.0001, after density-gradient centrifugation; r ¼.977, P<.0001, after swim-up). A linear regression analysis was applied to determine which parameters (age, sperm concentration, total sperm, progressive motility, normal sperm morphology, and viable, apoptotic, and necrotic sperm percentages) were independent in the prediction of progressive motile sperm recovery rate. There was no correlation between age and progressive motile sperm recovery rate. Statistically significant positive correlations were found between progressive motile sperm recovery rate after swim-up and basal sperm concentration (b ¼ 0.05, P¼.021), sperm morphology (b ¼ 0.36, P¼.008), and viable sperm percentage (b ¼ 0.19, P¼.034), whereas the correlation between progressive motile recovery rate and apoptotic sperm percentage was negative (b ¼ 0.27, P¼.027). When basal sperm concentration, progressively motile sperm percentage, sperm morphology, and viable sperm percentage were tested as independent variables in a linear multivariate regression analysis, none of these parameters was found to be an independent predictor of progressive motile sperm recovery rate. Multivariate regression was not performed for progressive motile recovery rate after the density-gradient preparation method, because none of the variables analyzed showed a significant correlation in univariate analysis. To compare the two sperm preparation methods, regression analysis was performed using as outcomes the differences between the two methods in terms of viable sperm percentage and progressive motile and viable sperm recovery rates after semen preparation. Simple logistic regression analysis showed that increasing total and progressive sperm motility and viable sperm percentage produced significant reduction in positive difference between the two methods in terms of TABLE 2 Recovery rate (%) after density-gradient centrifugation and swim-up. Total motile sperm Progressive motile sperm Viable sperm Densitygradient centrifugation Note: Values are mean SEM. Swimup P value < < <.0001 Fertility and Sterility â 635

5 TABLE 3 Correlation between conventional sperm parameters and flow cytometry parameters in whole semen and in density-gradient centrifugation and swim-up fractions. Viable Apoptotic Necrotic r P r P r P Whole semen Concentration Progressive motility Density-gradient centrifugation Concentration Progressive motility < <.001 Swim-up Concentration < < <.001 Progressive motility <.001 viable sperm percentage after preparation (OR 0.93 [95% CI ], P¼.008, for total motility; OR 0.91 [95% CI ], P¼.011, for progressive motility; and OR 0.93 [95% CI ], P¼.002, for viable sperm percentage). Finally, multiple logistic regression, in which several variables were considered simultaneously, was applied. Again, progressive sperm motility and viable sperm percentage remained significant predictors for negative difference between density-gradient and swim-up in terms of viable sperm percentage (OR 0.90 [95% CI ], P¼.040, for progressive motility; OR 0.93 [95% CI ], P¼.012, for viable sperm percentage). For progressive motile and viable sperm recovery rate differences, logistic regression was not applicable, because differences between density-gradient centrifugation and swim-up methods were positive in almost all cases. DISCUSSION Guidelines for the standardization of the human semen examination have been proposed by the WHO and are periodically reviewed (30). However, the results of conventional semen analysis can be very subjective, and a high intra- and interobserver variability has been described in many reports (32, 33). Furthermore, the conventional semen analysis does not adequately represent sperm functional status. Current pregnancy and live birth success rates of assisted reproduction techniques (ART) are not completely satisfactory. The use of apoptotic sperm during ART may be one of the causes for these suboptimal results (34). The negative association between sperm apoptosis and fertilization rate has been documented in clinical and experimental studies (28, 34). Sperm apoptosis seems to have a negative impact on sperm oocyte penetration (34). Therefore, the selection of nonapoptotic sperm should be one of the prerequisites for achieving optimal conception rates after ART. Recently, we introduced a novel multiparameter flow cytometry method that offers the possibility of a simultaneous assessment of several semen parameters, including functional parameters (29). In the present study, we have extended multiparameter flow cytometry analysis to capacitated sperm. A strong correlation between conventional optical microscopy and flow cytometry results was found also in semen samples after both swim-up and density-gradient preparation, confirming that flow cytometry is a useful and reliable method for sperm counting. Using flow cytometric analysis we found that the percentage of apoptotic sperm was significantly lower after both the swim-up and the gradient density preparation compared with whole semen. The low percentage of apoptotic sperm found both in swim-up and density-gradient fractions suggests that both methods allow removing most apoptotic sperm. It can be hypothesized that the steps of both methods, such as incubation or centrifugation, either do not induce apoptosis or induce a negligible percentage of apoptosis. Therefore, the risk of selecting apoptotic sperm during clinical ART seems to be low. An extremely significant correlation between viable sperm (nonapoptotic and nonnecrotic sperm) concentration and motile progressive sperm concentration was observed in processed samples, whereas this correlation was lower in whole semen. These findings suggest that, probably, most apoptotic sperm in prepared semen have no progressive motility. This might be a physiologic mechanism to prevent oocyte fertilization by damaged sperm. Moreover, prepared sperm should always be used for intracytoplasmic sperm injection (ICSI), because in raw semen early apoptotic sperm might have progressive motility. A recent review showed that density-gradient technique seems to result in a higher sperm concentration and a higher progressive motile sperm recovery rate than swim-up technique (1). In contrast, the swim-up technique seems to result in a better motility than the density-gradient technique. The results of the present study obtained using optical microscopy were consistent with the literature data (1). Flow cytometry data showed that the mean percentage of apoptotic 636 Ricci et al. Semen preparation methods and apoptosis Vol. 91, No. 2, February 2009

6 and necrotic sperm in swim-up processed samples was significantly lower than those in gradient-density processed samples. In contrast, density-gradient semen processing resulted in a significant higher mean recovery rate of viable sperm compared with swim-up preparation. We concluded that an ideal method probably does not exist and that, theoretically, either method should be used on the basis of basal semen parameters. We attempted to identify semen parameters that were independent predictors of better semen processing outcome. However, multivariate linear regression analysis showed that, when controlled for the other variables, none of basal semen parameters were independent predictors of progressive motile recovery rate. In the present study, densitygradient centrifugation method in almost all cases resulted in higher progressive motile and viable sperm recovery rate. However, we found that in a significant number of cases swim-up provided better results in terms of viable sperm percentage, and basal viable sperm percentage was an independent predictor for the difference between the two methods. A recent meta-analysis concluded that there is not enough evidence to recommend any specific preparation technique for intrauterine insemination (IUI) (1). Therefore, the method selection for ART can only be based on theoretic arguments. For IUI, where it has been demonstrated that the processed total motile sperm count independently predicts success (35), according to the present results density-gradient method in most cases should be preferred. For IVF techniques, it has been demonstrated that sperm count after processing is not correlated with fertilization rate (36), whereas a negative association between sperm apoptosis and fertilization rate has been documented in clinical and experimental studies (28, 34). Therefore, according to the present results, swim-up method in most cases should be preferred. In conclusion, this is the first study that compares the effects of gradient-density centrifugation and swim-up techniques on sperm apoptosis using flow cytometry. Flow cytometry analysis results suggest that gradient-density centrifugation provides a better semen sample from the quantity point of the view, although swim-up is preferable from the quality point of the view. However, both sperm preparation methods allow obtaining a sperm population with a low percentage of apoptotic sperm. Therefore, the risk of using apoptotic sperm for clinical treatment seems to be rather low. Preliminary multiparameter flow cytometry analysis might be helpful in selecting the most effective semen preparation method for individual couples undergoing ART. However, this technology is not presently available at most assisted reproduction laboratories. 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