Assessing the predictive value of hyaluronan binding ability for the freezability potential of human sperm

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1 Assessing the predictive value of hyaluronan binding ability for the freezability potential of human sperm Leah Yogev, Ph.D., Sandra E. Kleiman, Ph.D., Ron Hauser, M.D., Amnon Botchan, M.D., Ofer Lehavi, M.D., Gedalia Paz, Ph.D., and Haim Yavetz, M.D. The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel Objective: To evaluate the predictive value of a sperm maturation test using the hyaluronan-binding assay (HBA) for freezability potential; and to determine the effect of freezing thawing on HBA results. Design: Prospective study. Setting: Andrology laboratory at a teaching hospital. Patient(s): Candidates for sperm bank donation (n ¼ 113) and active sperm bank donors (n ¼ 16). Intervention(s): Semen analyses including HBA and sperm freezing thawing. Main Outcome Measure(s): Percentage of sperm HBA results and other sperm parameters in relation to freezing thawing results. Result(s): The predictive value of HBA for high freezability value (R40% postthaw motility) was significant. However, 1- and 4-hour percentage of motility had a higher predictive value for good freezability. A better prognostic value than that of HBA resutlts was also found for sperm concentration and percentage of normal morphology. Freezing thawing had no significant influence on HBA results. Conclusion(s): To the best of our knowledge this is the first demonstration that sperm maturation, determined by the HBA test, has a low value for predicting freezing thawing sperm survival. (Fertil Steril Ò 2010;93: Ó2010 by American Society for Reproductive Medicine.) Key Words: Freezability, sperm bank donor, sperm hyaluronan-binding assay Cryopreservation of human semen is a basic service of the fertility clinic; it facilitates the use of cryopreserved semen from oncology patients, patients who have undergone testicular or epididymal sperm extraction or aspiration, and sperm bank donors. Unfortunately, cryodamage is a general phenomenon (1). During the last 2 decades only negligible progress has been made in improving the freezing methods and minimizing the associated decrease in sperm quality (2). To date, freezability success of sperm samples is unpredictable. Accordingly, each sperm donor candidate should be evaluated before acceptance as a donor. Prerequisite for sperm bank donation is normal sperm quality (3) and minimal deterioration of sperm quality after the process of freezing and thawing. Satisfactory postthaw motility was reported to be R40% (4). Recently, the sperm hyaluronan-binding assay (HBA) was introduced as a commercial diagnostic kit for assessing sperm maturity and function. This test is based on hyaluronic acid (a linear repeating polymer of disaccharides) that selectively binds to mature sperm. It has been reported that normal sperm maturation can also be identified by extensive heat shock protein (HspA2) expression (5). Other characteristic markers were absence of cytoplasmic retention and enzymes, plasma membrane remodeling, and the formation of zona pellucida binding sites, with intact acrosome and good morphology (6 9). Sperm HBA results were found to highly correlate with sperm motility and morphology but proved to be less significant than sperm morphology in relation to the fertilization rate in IVF (10). It has been suggested that HBA may be a useful additional tool in the evaluation of male infertility. The purpose of the present study was to correlate HBA results and several other parameters of fresh specimens before freezing with sperm freezability potential. The effect of freezing thawing itself on HBA results was also determined. Received July 16, 2008; revised August 22, 2008; accepted September 16, 2008; published online November 19, L.Y. has nothing to disclose. S.E.K. has nothing to disclose. R.H. has nothing to disclose. A.B. has nothing to disclose. O.L. has nothing to disclose. G.P. has nothing to disclose. H.Y. has nothing to disclose. This study was carried out under the auspices of the Alan and Ada Selwyn Chair in Clinical Infertility Research and Molecular Medicine (Melbourne, Australia), awarded to G.P. Reprint requests: Leah Yogev, Ph.D., The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel (FAX: ; layogev@zahav.net.il). MATERIALS AND METHODS Between April 2006 and May 2007 semen was obtained from candidates for sperm bank donation and active sperm bank donors. Recruitment into the study was randomly performed (i.e., all candidates who provided informed consent for participation were included). The study comprised young (20 30 years) unmarried men, most of them students, all of Caucasian origin. All participants were instructed to observe 2 to 3 days of sexual abstinence before each donation. 154 Fertility and Sterility â Vol. 93, No. 1, January /10/$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 All sperm bank donors fulfilled the criteria for sperm donors as previously described (4, 11) (i.e., concentration > / ml and sperm motility percentage at first hour >50%, most in progressive motility). Percentage of normal sperm morphology was >12%, and postthaw motility R40%. The study was approved by the local institutional review board committee in accordance with the Helsinki Declaration of Sperm specimens of all candidates were examined for semen quality according to the World Health Organization (WHO) guidelines (3) before and immediately after the freezing and thawing procedure. Detailed semen analysis has already been described (12). Briefly, each ejaculate was allowed to liquefy for at least 20 minutes at 34 C. The volume was determined using a 5-mL graduated disposable pipette. A sample was taken to evaluate sperm concentration (by Makler chamber; Sefi Medical Instruments, Haifa, Israel) and percentage of motile spermatozoa. Percentage of morphologically normal sperm was assessed in accordance with the recommendations of the WHO guidelines, by strict criteria on Papanicolaou-stained smears (3). Degree of motility, graded from 1 (poor, WHO grade C) to 4 (excellent, WHO grade A), and percentage of motility in thawed samples of each specimen were also evaluated. Our andrology laboratory operates under the guidance of the United Kingdom National External Quality Assessment Scheme, Manchester, United Kingdom. Sperm HBA Procedure Each slide (Biocoat, Fort Washington, PA) contained two HBA tests, with a molecular layer of hyaluronan bound to it. A 7-mL drop of freshly mixed semen was loaded onto one of the chambers, covered with a Cell-Vu gridded cover slip (Biocoat), incubated at room temperature for 15 minutes, and counted for the percentage of bound motile sperm. The result of each sample was the mean value achieved by two laboratory technicians. The mean values were used after the evaluation of the correlation between the scores of the two technicians (r ¼ 0.94, Pearson correlation coefficients) and the difference between the two (not significant, paired t-test). Sample Parameters and HBA of Fresh and Frozen Thawed Specimens The effect of the cryopreservation process on HBA values was evaluated in semen specimen of candidates for sperm bank donation and of active donors. Each specimen was analyzed for sperm motility and HBA result before freezing and after thawing. Obviously, for the HBA evaluation, only motile sperm cells were examined. Washing Procedure This process was performed by dilution of 1 ml semen with human tubal fluid (HTF) medium (1:2 vol/vol) (Irvine Scientific, Santa Ana, CA) and centrifugation at 300 g for 10 minutes 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 HTF medium. Freezing and Thawing Procedures The specimen was diluted by the addition of droplets in equal volume of the freezing medium test yolk buffer (Irvine Scientific). The mixture was equilibrated for 15 minutes at room temperature, sealed in 0.5-mL straws (IMV, Paris, France), cooled gradually in a semiprogrammable freezer (Nicool LM-10; Air Liquid, Paris, France), and then transferred directly to liquid nitrogen for 30 minutes (12). For thawing, the straws were left at 34 C for 5 minutes and assessed immediately for sperm parameters. Statistical Evaluation Logistic regression models followed by receiver operator characteristic (ROC) curve analyses were used to evaluate the discriminative power and determine the best cutoff point for each of the semen parameters for diagnosis of high-levels of cryofreezability. Means were compared with t-test, paired t-test, and Mann-Whitney test, as appropriate. The association between continuous parameters was evaluated by Pearson correlation coefficients. Multivariate regression models were constructed to study the independent association of different factors with postthaw motility (linear regression) and with the probability of high postthaw motility (R40%, linear regression). Commercial software (SAS for Windows; SAS Institute, Cary, NC) was used for all statistical analyses. RESULTS During an approximately 1-year period, a total of 113 men were tested as candidates for sperm bank donation. Of these, only 45 were found to have specimens of good, acceptable, quality according to the postthaw motility of their sperm. For the first part of our study, to evaluate the prognostic value of HBA results for cryofreezability, a total of 90 men were recruited. For evaluation of the freezing effect on HBA values, an additional 23 men participated in the second part of the study. Prognostic Value of HBA Result and Additional Sperm Parameters Acceptable cryofreezability was identified by a postthaw motility of R40%. The predictive value of HBA for high freezability was significant (P¼.024), with a cutoff point of 80.5% and area under the ROC curve (AUC) (Table 1, Fig. 1). However, 1- and 4-hour percentage of motility had the highest predictive value for good freezability, with a cutoff point of 59% and 49% and ROC values of and 0.889, respectively. A good prognostic value was also found for the parameters concentration ( /ml cutoff point; AUC) and percentage of normal morphology (12% cutoff point, AUC). Multivariate regression models were applied to examine the association between postthaw motility and the different sperm parameters. Logistic regression modeling for the probability of high postthaw motility (R40%) revealed two significant factors, 1-hour motility and percentage of normal morphology (c ¼ 0.95). The HBA score was not significant. 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3 TABLE 1 Receiver operator characteristic curve analysis: AUC and cutoff with best sensitivity and specificity for different fresh-sperm parameters. Parameter AUC Cutoff Sensitivity (%) Specificity (%) HBA (%) h motility (%) h motility grade (1 4) 4-h motility (%) h motility grade (1 4) Normal morphology (%) Concentration (10 6 /ml) Volume (ml) The linear regression model for postthaw motility (as a continuous parameter) included the following significant parameters: 1-hour motility, concentration, and percentage of normal morphology (R 2 ¼ 0.68). The HBA entered the model with borderline significance (P¼.063) and increased the proportion of explained variance by 1.4%. Comparing the difference between values of specimens with high (as determined by R40% thawed specimen motility) and low freezability (<40% motility) revealed that HBA had borderline significance (P¼.052; Table 2). A comparison of these two groups for additional sperm parameters revealed a highly significant difference (P<.0001) for 1- and 4-hour percentage of motility, 4-hour degree of motility, concentration, and morphology and a slight but still significant difference for the 1-hour degree of motility (P¼.047; Table 2). When comparing the two groups, high and low freezability, after changing the cutoff value from 40% to 35% and 45%, the HBA significance level increased (P¼.019 and 0.01, respectively). Another parameter with a changed significance was the 1-hour degree of motility (P¼.018 and.097, respectively); all other parameters remained with the same high significance values (P<.0001). Freezing Effect on HBA and Motility Results Considering the possible effect of freezing thawing on HBA results (second part of our study), it was found that HBA values in the candidates (n ¼ 23) and sperm bank donors (n ¼ 16) did not differ significantly (by Mann-Whitney test) before (82.4% 1.72% and 83.0% 1.86%, respectively) and after cryopreservation (84.8% 1.56% and 86.7% 1.06%, respectively). Obviously, a significant decrease was found in the percentage of motility of frozen thawed sperm cells of both candidate and donor groups (30.7% 2.65% and 45.4% 2.20%, respectively) compared with fresh samples (54.9% 1.80% and 63.1% 1.41%, respectively) as the result of the freezing thawing procedure (P<.0001). The candidate and donor groups differed significantly from each other regarding percentage of motility of fresh and thawed specimens (P¼.03 and P¼.003, respectively) but not for HBA and other specimen parameters (data not shown). DISCUSSION Prediction of cryofreezability using semen analysis and sperm maturation evaluation, as determined by the HBA, was studied. It was found that 1- and 4-hour percentage of motility had the best predictive value for sperm cryofreezability, whereas sperm maturation as identified by the HBA had borderline significance for evaluating sample survival during cryopreservation. The significance of high values of initial sperm motility 1 hour after delivery (R55%) with most sperm cells in forward motility was previously reported as a good prognostic indicator for successful sperm freezing (4). In that study, low decay in percentage of motility after specimen delivery and maintenance of degree of forward motility, as measured at 4 hours after delivery, were also found to have a good predictive value. Mitochondria play a major role in diverse cellular functions, such as energy production, that influence sperm motility. It was suggested that damage to the mitochondria plays a major role in cryoinjury (13). Accordingly, a number of reports have suggested that sperm mitochondria are the cellular structures most sensitive to cooling and rewarming in boars (14). Because glycolysis in the principal piece of the flagellum induces motility in addition to the mitochondria, it can be assumed that frozen thawed spermatozoa with cryoinjured mitochondria may still participate in the fertilization process. Concerning the effect of cryopreservation on the HBA values, previously it was shown that prefreezing sperm manipulation and the freezing thawing process did not impair the capability of the rescued sperm to bind to the zona pellucida (15). In the hemizona assay, as in the HBA test, only 156 Yogev et al. Sperm hyaluronan-binding assay and freezability Vol. 93, No. 1, January 2010

4 FIGURE 1 Receiver operating characteristic curves for HBA (%), normal morphology (NF, %), concentration (10 6 /ml), and percentage of motility 1 and 4 hours after ejaculation (Mot 1h and Mot 4h, respectively). motile spermatozoa are involved. Thus it is reasonable that the same conclusion regarding the absent effect of freezing is relevant for both tests. In the first part of the present studies, two groups were defined using postthaw motility cutoffs of 35%, 40%, and 45%. These groups differed from each other in most prefreezing parameters, except for specimen volume and 1-hour grade of motility with the 45% of cutoff. However, concerning the HBA results for sperm maturity, only borderline or relatively low significance could be shown. Interestingly, sperm concentration in the high-freezability group was almost twofold greater than that of the low-freezability group. Indeed, the association between sperm concentration and postthaw percentage of motility was evident in a linear regression model for postthaw motility as a continuous parameter. The concentration factor was found to contribute significantly, in addition to the 1-hour motility and percentage of normal morphology. Fertility and Sterility â 157

5 TABLE 2 Semen parameters of fresh specimens with high and low freezability. Parameter High freezability (n [ 31) Low freezability (n [ 59) P value HBA (%) h motility (%) < h motility grade (1 4) h motility (%) < h motility grade (1 4) <.0001 Normal morphology (%) <.0001 Concentration (10 6 /ml) <.0001 Volume (ml) Note: Values are mean SE. Postthaw motility of 40% served as the freezability cutoff. Cryodamage is still a general phenomenon, and more research is warranted to improve the methods and consequently diminish its impact on sperm quality. Whatever technique is applied, it is generally accepted that some cryodamage occurs. These cryoinjuries include damage at different cellular levels (1). It could be expected that maturation of sperm cells will be the main contributor for withstanding this process. However, to the best of our knowledge, in the present study we were able to show for the first time that there are other parameters, probably associated with the mitochondria organelle, that play a far more dominant role in the nature of sperm freezability than its maturation status. Acknowledgement: The authors thank Mrs. B. Gore, M.Sc., F. Feldhoon, M. Ilatov, B.Sc., and L. Ariely, M.Sc., Institute for the Study of Fertility, Tel Aviv Medical Center, for their excellent and skillful technical assistance; and E. Shabtai, The Statistics Service, Tel Aviv Medical Center, for statistical assistance. The slides for the hyaluronic acid test were a generous gift from Biocoat, Horsham, PA. REFERENCES 1. Desrosiers P, Legare C, Leclerc P, Sullivan R. Membranous and structural damage that occur during cryopreservation of human sperm may be time-related events. Fertil Steril 2006;85: Paras L, Freisinger J, Esterbauer B, Schmeller N, Szlauer R, Jungwirth A. Cryopreservation technique: comparison of test yolk buffer versus SpermCryo and vapor versus computerized freezing. Andrologia 2008;40: World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge, UK: Cambridge University Press, Yavetz H, Yogev L, Homonnai ZT, Paz G. Prerequisites for successful human sperm cryobanking: sperm quality and prefreezing holding time. Fertil Steril 1991;55: Ergur AR, Dokras A, Giraldo JR, Habana A, Kovanci E, Huszar G. Sperm maturity and treatment choice of in vitro fertilization (IVF) or intracytoplasmic sperm injection: diminished sperm HspA2 chaperone levels predict IVF failure. Fertil Steril 2002;77: Ranganathan S, Ganguly AK, Datta K. Evidence for presence of hyaluronan binding protein in spermatozoa and its possible involvement in sperm function. Mol Reprod Develop 1994;38: Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratio. Biol Reprod 1997;56: Huszar G, Celik-Ozenic C, Cayli S, Zayaczki Z, Hansch E, Vigue L. Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status. Fertil Steril 2003;79(Suppl 3): Huszar G, Ozkavukcu S, Jakab A, Celik-Ozencil C, Sati GL, Cayli S. Hyaluronic acid binding ability of human sperm reflects cellular maturity and fertility potential: selection of sperm for intracytoplasmic sperm injection. Curr Opin Obstet Gynecol 2006;18: Ye H, Huang G, Gao Y, Liu DY. Relationship between human sperm-hyaluronan binding assay and fertilization rate in conventional in vitro fertilization. Hum Reprod 2006;21: Botchan A, Hauser R, Gamzu R, Yogev L, Paz G, Yavetz H. Results of 6139 artificial insemination cycles with donor spermatozoa. Hum Reprod 2001;16: Yogev L, Kleiman S, Shabtai E, Botchan A, Gamzu R, Paz G, et al. Seasonal variations in pre- and post- thaw donor sperm quality. Hum Reprod 2004;19: Ortega-Ferrusola C, Sotillo-Galan Y, Varela-Fernandez E, Gallardo- Bola~nos JM, Muriel A, Gonzalez-Fernandez L, et al. Detection of apoptosis-like changes during the cryopreservation process in equine sperm. J Androl 2008;29: Pe~na FJ, Johannisson A, Wallgrem M, Rodriguez Martinez H. Antioxidant supplementation in vitro improves boar sperm motility, and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Anim Reprod Sci 2003;78: Yogev L, Gamzu R, Paz G, Kleiman SE, Botchan A, Hauser R, et al. Prefreezing sperm preparation does not impair thawed spermatozoa binding to the zona pellucida. Hum Reprod 1999;14: Yogev et al. Sperm hyaluronan-binding assay and freezability Vol. 93, No. 1, January 2010