- Email: [email protected]
Effect of cryopreservation on quality and fertilization capacity of human sperm
European Journal of Obstetrics & Gynecology and Reproductive Biology 116 (2004) 63–66
Effect of cryopreservation on quality and fertilization capacity of human sperm N. Marcus-Braun*, G. Braun, G. Potashnik, I. Har-vardi IVF Unit, Department of Obstetric and Gynecology, Faculty of Health Sciences, Soroka University Medical Center, Ben-Gurion University of the Negev, P.O. Box 151, Beer-Sheva 84101, Israel Received 10 January 2003; received in revised form 27 October 2003; accepted 14 January 2004
Abstract Objective: To analyze retrospectively the effect of cryopreservation on donor’s sperm. Study design: Data were collected on 178 cryopreserved-thawed sperm specimens from 44 donors and 624 oocytes from 62 women, which underwent in vitro fertilization-embryo transfer (IVF-ET) treatments with donor’s sperm. Data on fresh sperm, 175 sperm specimens from 76 couples which underwent IVF-ET treatments, were used as a control group. Semen analysis was done by cell concentration, percent of motility, and quality of motility according to the World Health Organization (WHO) recommendation. Sperm quality parameters which had the strongest impact on fertilization capacity were determined using the statistical response surface model and conjoint analysis. Results: Passing sperm through Percoll column decreased sperm concentration, with no improvement in sperm motility but with a slight increase in quality of motility. Quality of motility of donor’s sperm had the strongest impact on fertilization capacity. Conclusion: Current freezing–thawing protocols of sperm cause a decrease in sperm parameters without affecting fertilization capacity. Furthermore, quality of motility of frozen–thawed sperm seems to be a significant measure of sperm fertilization capacity. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Sperm; Donor; Fertility rate; Cryopreservation; Quality; Conjoint analysis
1. Introduction The use of donor sperm is an integral part of in vitro fertilization-embryo transfer (IVF-ET) in couples with male infertility and in single women. Freezing donor’s sperm, in order to prevent sexually transmitted diseases, enhances the importance of defining the quality and the fertilization capacity of sperm, following processes of freezing and thawing (1). The fertilization potential of cryopreservedthawed sperm has been studied for more than 50 years [2–5]. Until recently there has been no sufficient information available in the literature and no conclusive evidence regarding sperm fertilization capacity following freezing and thawing as compared with fresh sperm. In some clinical studies, frozen–thawed sperm had a lower fertilization capacity when compared with fresh sperm [6,7]. However, in other studies no significant difference between frozen– thawed and fresh sperm was observed [8,9]. Sperm morphology was analyzed in a few studies. In most of the cases, sperm morphology was found to be unaffected by the * Corresponding author. Tel.: þ972-8-6518954; fax: þ972-8-6519492. E-mail address: [email protected] (N. Marcus-Braun).
freezing–thawing processes [10–12]. It is known that passage of sperm cell through Percoll column in preparation for IVF-ET improves sperm motility and fertilization capacity of the sperm [13]. However, the linkage between sperm quality following freezing–thawing procedures and its fertilization capacity has not yet been well established [1,14]. The present study was designed to investigate the effect of the freezing–thawing process on sperm quality parameters. An attempt was also made to identify the most sensitive parameter which can predict sperm fertilization capacity.
2. Materials and methods This study was performed at the IVF unit of the Soroka Medical Center, Beer-Sheva, Israel. The study was retrospective by nature, based on data which was collected between the years 1990 and 1998. 2.1. Study population Sperm quality parameters and sperm data were collected from 178 sperm specimens obtained from 44 donors. All
0301-2115/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2004.01.036
64
N. Marcus-Braun et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 116 (2004) 63–66
sperm data collected referred to frozen–thawed sperm specimens which were used for IVF-ET treatments. In order to determine the fertilization capacity of the corresponding frozen–thawed sperm, data of 624 oocytes were collected from 62 women. The women were between 25 and 35 years old with indication for IVF-ET using donor sperms. Control group data of 677 oocytes were collected from 76 women between 22 and 35 years old. The women in the control group underwent IVF-ET treatments with fresh husband sperms, 175 specimens. The sperm characteristics were similar to the donors group sperm. 2.2. Analysis methods All semen samples were analyzed according to the World Health Organization (WHO) recommendation [15]. Sperm morphology was observed before freezing and was found to be in the normal range according to the WHO criteria. Sperm quality was examined by the following parameters: sperm concentration, percent of motility and quality of motility. Quality of motility was based on an increasing scale of 1–5, where grade 5 referred to maximal linear motility. These parameters were evaluated before freezing, immediately after thawing and after passage through Percoll column. In the control group, the parameters were examined before and after passage through Percoll column. In order to determine fertilization capacity of the sperm, the following parameters were examined for each cycle: number of oocytes and number of fertilized oocytes. Mean fertilization value was determined through the number of oocytes that had two pronuclei 16–18 h after insemination. Mean fertilization value obtained in the frozen–thawed group was compared with the mean value obtained in the fresh sperm group. 2.3. Statistical analysis The conjoint analysis and response surface model [16,17] was used in order to determine which of the sperm parameters had the strongest impact on fertilization capacity. The model allows assessing the relative importance of few parameters on a dependent variable. In an analogy, we used the model to determine the relative importance of the sperm parameters on fertilization capacity. Comparison of fertilization rate, the woman’s age and number of oocytes between the donors and the control groups was done by ANOVA. Paired t-test was used to compare sperm parameters between three states: before freezing, after thawing and after Percoll column. Cross tabulation test was used to find the distribution of diagnosis between both groups.
3. Results Data of 178 sperm specimens from 44 donors were examined. Each specimen was analyzed for concentration, percent of motility and quality of motility. Those parameters
were examined before freezing, after thawing and after passage through Percoll column. The comparison of sperm parameters at different stages of treatment is presented in Table 1. No change in sperm concentration was observed before freezing and after thawing (ð106 51Þ 106 ml1 versus ð99 45Þ 106 ml1). This was followed by a significant decrease by 77.8% after Percoll treatment (from ð99 45Þ 106 ml1 to ð22 16Þ 106 ml1; P < 0:001). Percent of motility decreased significantly by 29.5%, immediately following thawing (from 58 13 to 44 12; P < 0:001). It was then almost entirely recovered to its pre-freezing value after passage through Percoll column. Quality of motility was unaffected by the freezing–thawing procedure and was further improved by 11.1% after Percoll column. Data of 175 sperm specimens from 76 husbands were examined in the control group. Each specimen was analyzed for concentration, percent of motility and quality of motility before and after passage through Percoll column. All specimens were in the normal range according to the WHO criteria. Sperm parameters before and after Percoll in the control group are presented in Table 2. Sperm concentration decreased significantly by 54.9% after passage through Percoll column (ð82 48Þ 106 ml1 versus ð37 25Þ 106 ml1; P < 0:001). This decrease is comparable to the 77.8% decrease observed in the donors group (Table 1). The percent of motility in the control group improved by 18.5% after Percoll treatment (Table 2) compared with 29.5% improvement in the donors group (Table 1). The quality of motility improved after Percoll in the control group by 20% compared with 11% improvement in the donors group. No significant difference existed between the mean values of women’s age in the donors group as compared with the control group (31:4 2:3 versus 29:8 3, respectively). No significant clinical difference was expected as long as the women were less then 35 years old [18]. Mean number of oocytes was not significantly different, as compared between the two groups (10 5 versus 8 4). The diagnosis for IVFET treatments in the donors group was distributed as follows: 47% unexplained, 19% mechanical problems, 34% male factor. In the control group, the diagnosis was distributed as follows: 33% unexplained, 65% mechanical problems and other 2%. Mean fertilization value, determined through the number of oocytes that had 2 pronuclei 16–18 h after insemination Table 1 Comparison of mean averages of sperm parameters, 178 samples of donors, before freezing, after thawing and after Percoll
Concentration (106 ml1) Percent of motility Quality of motility (1–5) *
P ¼ NS (non significant). P < 0:001.
**
Before freezing
After thawing
After Percoll
106 51 58 13 3.7 0.5
99 45* 44 12** 3.6 0.6*
22 16** 57 19* 4 0.5**
N. Marcus-Braun et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 116 (2004) 63–66 Table 2 Comparison of mean averages of sperm parameters, 175 samples in the control group, before and after Percoll
6
1
Concentration (10 ml ) Percent of motility Quality of motility (1–5)
Before Percoll
After Percoll
82 48 54 14 3.5 0.6
37 25 64 21 4.2 0.6
65
Table 3 Comparison of mean fertilization percentage between the donors and control groups Percent of fertilization Donors (N ¼ 178) Control group (N ¼ 175)
63.3 25 61.8 21
P ¼ NS.
P < 0:001.
was compared between the two groups as presented in Table 3. The mean percents of fertilization were identical in both groups. The impact of sperm parameters on fertilization capacity in both groups was examined by using the surface response model, adopted from the marketing research field [16,17]. The model allows assessing the relative importance of few parameters on a dependent variable. The parameter values taken were those corresponding to sperm after passing through Percoll column. In each group, for each sperm parameter separately, the partial derivative @S/@x (relative slope) of fertilization rate was calculated. The higher the value of the slope, the stronger impact the sperm parameter has on fertilization capacity. The @S/@x for concentration was 0.01 in the donors group versus 0.01 in the control group. The @S/@x for percent of motility was 0.05 in the donors group versus 0.14 in the control group. The @S/@x for quality of motility was 4.19 in the donors group versus 1.00 in the control group. The quality of motility had the steepest ascent towards fertilization capacity, thus having the strongest impact on fertilization capacity (Fig. 1).
4. Discussion Reduction of sperm quality was pronounced in all three parameters that were examined. Sperm quality was expected Relative slope
to decrease by the freezing–thawing process, due to previously observed changes in biochemical and physical parameters [19–21]. Reduction of sperm parameters due to the freezing–thawing process was also pronounced in the surface response analysis. While quality of motility after freezing–thawing had the strongest impact on fertilization capacity, sperm concentration and percent of motility had relatively very little impact. In the control group, the quality of motility had the strongest impact as well, although less dramatic than in the frozen–thawed sperm. The difference in the effect of quality of motility between the control and frozen–thawed groups, as pronounced in the surface response analysis, may be linked to the finding that quality of motility was the least damaged parameter in the freezing– thawing process (Table 1). Only quality of motility of the frozen–thawed sperm was improved after Percoll column, while the concentration and percent of motility decreased and did not improve. It is thus not surprising that the effect of quality of motility on fertilization capacity of the frozen– thawed sperm was relatively larger than in the control group. Recently, the gene responsible for sperm motility has been studied [22,23]. It has been demonstrated that knocking this gene down resulted in infertile sperm, in accord with our clinical indications. Therefore, a connection between sperm motility and its fertilization capacity is strongly supported and may lead to a stronger attention to sperm motility in IVF treatments. In previous studies, the connection between sperm quality and its fertilization capacity was not well established. Some
4.5
DONORS
4
CONTROL
3.5 3 2.5 2 1.5 1 0.5 0 -0.5
Cocentration
Percent of motility
Quality of motility
Fig. 1. The effect of sperm parameters on fertilization capacity.
66
N. Marcus-Braun et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 116 (2004) 63–66
clinical evidence has been accumulated, indicating that fertilization capacity was not affected by cryopreservation [8,9]. Controversially, other studies pointed out that cryopreservation did affect fertilization capacity [6,7]. Our analysis has shown (Table 3) that the percents of fertilization were identical in the frozen–thawed group as compared with the control group. Therefore, despite the freezing–thawinginduced decrease in sperm quality, which was pronounced in all parameters, fertilization capacity remained unaffected. Averages of sperm concentration and percent of motility before freezing in the donors group were higher than in the control group. This was expected since donor sperm was carefully selected to be of high quality. However, in the control group, the sperm specimens were not expected to be of such a high quality. The data used in the present study was of sperm from husbands with no male infertility. Nevertheless, all sperm samples were in the normal range according to the WHO criteria. Mean number of oocytes was slightly higher in women that went through IVF-ET treatments with donor’s sperm. We conclude that cryopreservation of human sperm, using standard protocols, decrease sperm parameters as assessed by sperm concentration, percent of motility and quality of motility. However, fertilization capacity of frozen–thawed sperm is identical to fertilization capacity of fresh sperm. Our findings suggest that the quality of motility is a positive predictor for fertilization capacity in frozen–thawed sperm. References [1] Barratt CLR, Clements S, Kessopoulou E. Semen characteristics and fertility tests required for storage of spermatozoa. Hum Reprod 1998;13:1–7. [2] Polge C, Smith AU, Parker AS. Revival of spermatozoa after vitrification and dehydration at low temperature. Nature 1949;164: 666–7. [3] Bunge RG, Sherman JK. Fertilization capacity of frozen human spermatozoa. Nature 1953;172:767. [4] Ragni G, Lombroso Finzi GC, Caccamo A, Dalla Serra A, Crosignani PG. Enhanced sperm quality with TEST-yolk buffer: preliminary results in intraperitoneal insemination for male infertility. In: Proceedings of the Abstracts of the 7th Annual Meeting of the European Society of Human Reproduction and Embryology. Paris; Hum Reprod 1991;6:421. [5] Royere D, Barthelemy C, Hamamah S, Lansac J. Cryopreservation of spermatozoa: a 1996 review. Hum Reprod Update 1996;2:553–9. [6] Smith KD, Rodriguez-Rigau LJ, Steinberger E. The influence of ovulatory dysfunction and timing of insemination on the success of artificial insemination donor (AID) with fresh or cryopreserved semen. Fertil Steril 1981;36:496–502.
[7] Subak LL, Adamson GD, Boltz NL. Therapeutic donor insemination: a prospective randomized trial of fresh versus frozen sperm. Am J Obstet Gynecol 1992;70:313–6. [8] Ashkenazi J, Dicker D, Feldberg D, Goldman JA. Fresh versus frozen thawed semen for initial and late insemination in IVF-ET cycles. Eur J Obstet Gynecol Reprod Biol 1991;42:115–7. [9] Lansac J, Thepot F, Mayaux MJ, Czyglick F, Wack T, Selva J, et al. Pregnancy outcome after artificial insemination or IVF-ET with frozen semen donor: a collaborative study of the French CECOS Federation on 21597 pregnancies. Eur J Obstet Gynecol Reprod Biol 1997;74:223–8. [10] Stanic P, Tandara M, Sonicki Z, Simunic V, Radakovic B, Suchanek E. Comparison of protective media and freezing techniques for cryopreservation of human semen. Eur J Obstet Gynecol Reprod Biol 2000;91:65–70. [11] Ranganathan P, Mahran AM, Hallak J, Agarwal A. Sperm cryopreservation for men with nonmalignant, systemic diseases: a descriptive study. J Androl 2002;23:71–5. [12] Hammadeh ME, Greiner S, Rosenbaum P, Schmidt W. Comparison between human sperm preservation medium and TEST-yolk buffer on protecting chromatin and morphology integrity of human spermatozoa in fertile and subfertile men after freeze-thawing procedure. J Androl 2001;22:1012–8. [13] Kelly MP, Corson SL, Gocial B, Batzer FR, Gutmann JN. Discontinuous Percoll gradient preparation for donor insemination: determinants for success. Hum Reprod 1997;12:2682–6. [14] Jeyendran RS, Gunawardana VK, Barisic D, Wentz AC. TEST-yolk media and sperm quality. Hum Reprod 1995;1:73–9. [15] Van den Eede B. Investigation and treatment of infertile cauples ESHRE guidelines. Hum Reprod Update 1995;10:1246– 71. [16] Rust RT, Oliver RL. On the measurement of perceived service quality. In: DeSarbo WS, Huff L, Rolandelli MM, editors. Service quality: new direction in theory and practice. London: Sage; 1994. p. 201–22. [17] Danaher PJ. Using conjoint analysis to determine the relative importance of service attributes measured in customer satisfaction surveys. J Retailing 1997;73:235–60. [18] Moon Kang A, Tsung-Chieh J. Effect of age on intrauterine insemination with frozen donor sperm. Obstet Gynecol 1996;88: 93–8. [19] Ericsson SA, Garner DL, Redelman D, Ahmad K. Assessment of the viability and fertilizing potential of cryopreserved bovine spermatozoa using dual fluorescent staining and two flow cytometric systems. Gamete Res 1989;22:355–68. [20] MacLaughlin EA, Ford WCL, Hull MGR. Adenosine triphosphate and motility characteristics of fresh and cryopreserved human spermatozoa. Int J Androl 1994;17:71–6. [21] Ragni G, Vegrtti W. In: Fertility and Sterility Proceedings of the 15th world Congress of FDS (France). The Parthenon Publishing Group; 1995. p. 317–20. [22] Ren D, Navarro B, Perez G, Jackson AC, Hsu S, Shi Q. A sperm ion channel required for sperm motility and male fertility. Nature 2001;413:603–9. [23] Quill TA, Ren D, Clapham DE, Garbers DL, Quill TA. A voltagegated ion chanel expressed specifically in spermatozoa. Proc Natl Acad Sci USA 2001;98:12527–31.
Effect of cryopreservation on quality and fertilization capacity of human sperm N. Marcus-Braun*, G. Braun, G. Potashnik, I. Har-vardi IVF Unit, Department of Obstetric and Gynecology, Faculty of Health Sciences, Soroka University Medical Center, Ben-Gurion University of the Negev, P.O. Box 151, Beer-Sheva 84101, Israel Received 10 January 2003; received in revised form 27 October 2003; accepted 14 January 2004
Abstract Objective: To analyze retrospectively the effect of cryopreservation on donor’s sperm. Study design: Data were collected on 178 cryopreserved-thawed sperm specimens from 44 donors and 624 oocytes from 62 women, which underwent in vitro fertilization-embryo transfer (IVF-ET) treatments with donor’s sperm. Data on fresh sperm, 175 sperm specimens from 76 couples which underwent IVF-ET treatments, were used as a control group. Semen analysis was done by cell concentration, percent of motility, and quality of motility according to the World Health Organization (WHO) recommendation. Sperm quality parameters which had the strongest impact on fertilization capacity were determined using the statistical response surface model and conjoint analysis. Results: Passing sperm through Percoll column decreased sperm concentration, with no improvement in sperm motility but with a slight increase in quality of motility. Quality of motility of donor’s sperm had the strongest impact on fertilization capacity. Conclusion: Current freezing–thawing protocols of sperm cause a decrease in sperm parameters without affecting fertilization capacity. Furthermore, quality of motility of frozen–thawed sperm seems to be a significant measure of sperm fertilization capacity. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Sperm; Donor; Fertility rate; Cryopreservation; Quality; Conjoint analysis
1. Introduction The use of donor sperm is an integral part of in vitro fertilization-embryo transfer (IVF-ET) in couples with male infertility and in single women. Freezing donor’s sperm, in order to prevent sexually transmitted diseases, enhances the importance of defining the quality and the fertilization capacity of sperm, following processes of freezing and thawing (1). The fertilization potential of cryopreservedthawed sperm has been studied for more than 50 years [2–5]. Until recently there has been no sufficient information available in the literature and no conclusive evidence regarding sperm fertilization capacity following freezing and thawing as compared with fresh sperm. In some clinical studies, frozen–thawed sperm had a lower fertilization capacity when compared with fresh sperm [6,7]. However, in other studies no significant difference between frozen– thawed and fresh sperm was observed [8,9]. Sperm morphology was analyzed in a few studies. In most of the cases, sperm morphology was found to be unaffected by the * Corresponding author. Tel.: þ972-8-6518954; fax: þ972-8-6519492. E-mail address: [email protected] (N. Marcus-Braun).
freezing–thawing processes [10–12]. It is known that passage of sperm cell through Percoll column in preparation for IVF-ET improves sperm motility and fertilization capacity of the sperm [13]. However, the linkage between sperm quality following freezing–thawing procedures and its fertilization capacity has not yet been well established [1,14]. The present study was designed to investigate the effect of the freezing–thawing process on sperm quality parameters. An attempt was also made to identify the most sensitive parameter which can predict sperm fertilization capacity.
2. Materials and methods This study was performed at the IVF unit of the Soroka Medical Center, Beer-Sheva, Israel. The study was retrospective by nature, based on data which was collected between the years 1990 and 1998. 2.1. Study population Sperm quality parameters and sperm data were collected from 178 sperm specimens obtained from 44 donors. All
0301-2115/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2004.01.036
64
N. Marcus-Braun et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 116 (2004) 63–66
sperm data collected referred to frozen–thawed sperm specimens which were used for IVF-ET treatments. In order to determine the fertilization capacity of the corresponding frozen–thawed sperm, data of 624 oocytes were collected from 62 women. The women were between 25 and 35 years old with indication for IVF-ET using donor sperms. Control group data of 677 oocytes were collected from 76 women between 22 and 35 years old. The women in the control group underwent IVF-ET treatments with fresh husband sperms, 175 specimens. The sperm characteristics were similar to the donors group sperm. 2.2. Analysis methods All semen samples were analyzed according to the World Health Organization (WHO) recommendation [15]. Sperm morphology was observed before freezing and was found to be in the normal range according to the WHO criteria. Sperm quality was examined by the following parameters: sperm concentration, percent of motility and quality of motility. Quality of motility was based on an increasing scale of 1–5, where grade 5 referred to maximal linear motility. These parameters were evaluated before freezing, immediately after thawing and after passage through Percoll column. In the control group, the parameters were examined before and after passage through Percoll column. In order to determine fertilization capacity of the sperm, the following parameters were examined for each cycle: number of oocytes and number of fertilized oocytes. Mean fertilization value was determined through the number of oocytes that had two pronuclei 16–18 h after insemination. Mean fertilization value obtained in the frozen–thawed group was compared with the mean value obtained in the fresh sperm group. 2.3. Statistical analysis The conjoint analysis and response surface model [16,17] was used in order to determine which of the sperm parameters had the strongest impact on fertilization capacity. The model allows assessing the relative importance of few parameters on a dependent variable. In an analogy, we used the model to determine the relative importance of the sperm parameters on fertilization capacity. Comparison of fertilization rate, the woman’s age and number of oocytes between the donors and the control groups was done by ANOVA. Paired t-test was used to compare sperm parameters between three states: before freezing, after thawing and after Percoll column. Cross tabulation test was used to find the distribution of diagnosis between both groups.
3. Results Data of 178 sperm specimens from 44 donors were examined. Each specimen was analyzed for concentration, percent of motility and quality of motility. Those parameters
were examined before freezing, after thawing and after passage through Percoll column. The comparison of sperm parameters at different stages of treatment is presented in Table 1. No change in sperm concentration was observed before freezing and after thawing (ð106 51Þ 106 ml1 versus ð99 45Þ 106 ml1). This was followed by a significant decrease by 77.8% after Percoll treatment (from ð99 45Þ 106 ml1 to ð22 16Þ 106 ml1; P < 0:001). Percent of motility decreased significantly by 29.5%, immediately following thawing (from 58 13 to 44 12; P < 0:001). It was then almost entirely recovered to its pre-freezing value after passage through Percoll column. Quality of motility was unaffected by the freezing–thawing procedure and was further improved by 11.1% after Percoll column. Data of 175 sperm specimens from 76 husbands were examined in the control group. Each specimen was analyzed for concentration, percent of motility and quality of motility before and after passage through Percoll column. All specimens were in the normal range according to the WHO criteria. Sperm parameters before and after Percoll in the control group are presented in Table 2. Sperm concentration decreased significantly by 54.9% after passage through Percoll column (ð82 48Þ 106 ml1 versus ð37 25Þ 106 ml1; P < 0:001). This decrease is comparable to the 77.8% decrease observed in the donors group (Table 1). The percent of motility in the control group improved by 18.5% after Percoll treatment (Table 2) compared with 29.5% improvement in the donors group (Table 1). The quality of motility improved after Percoll in the control group by 20% compared with 11% improvement in the donors group. No significant difference existed between the mean values of women’s age in the donors group as compared with the control group (31:4 2:3 versus 29:8 3, respectively). No significant clinical difference was expected as long as the women were less then 35 years old [18]. Mean number of oocytes was not significantly different, as compared between the two groups (10 5 versus 8 4). The diagnosis for IVFET treatments in the donors group was distributed as follows: 47% unexplained, 19% mechanical problems, 34% male factor. In the control group, the diagnosis was distributed as follows: 33% unexplained, 65% mechanical problems and other 2%. Mean fertilization value, determined through the number of oocytes that had 2 pronuclei 16–18 h after insemination Table 1 Comparison of mean averages of sperm parameters, 178 samples of donors, before freezing, after thawing and after Percoll
Concentration (106 ml1) Percent of motility Quality of motility (1–5) *
P ¼ NS (non significant). P < 0:001.
**
Before freezing
After thawing
After Percoll
106 51 58 13 3.7 0.5
99 45* 44 12** 3.6 0.6*
22 16** 57 19* 4 0.5**
N. Marcus-Braun et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 116 (2004) 63–66 Table 2 Comparison of mean averages of sperm parameters, 175 samples in the control group, before and after Percoll
6
1
Concentration (10 ml ) Percent of motility Quality of motility (1–5)
Before Percoll
After Percoll
82 48 54 14 3.5 0.6
37 25 64 21 4.2 0.6
65
Table 3 Comparison of mean fertilization percentage between the donors and control groups Percent of fertilization Donors (N ¼ 178) Control group (N ¼ 175)
63.3 25 61.8 21
P ¼ NS.
P < 0:001.
was compared between the two groups as presented in Table 3. The mean percents of fertilization were identical in both groups. The impact of sperm parameters on fertilization capacity in both groups was examined by using the surface response model, adopted from the marketing research field [16,17]. The model allows assessing the relative importance of few parameters on a dependent variable. The parameter values taken were those corresponding to sperm after passing through Percoll column. In each group, for each sperm parameter separately, the partial derivative @S/@x (relative slope) of fertilization rate was calculated. The higher the value of the slope, the stronger impact the sperm parameter has on fertilization capacity. The @S/@x for concentration was 0.01 in the donors group versus 0.01 in the control group. The @S/@x for percent of motility was 0.05 in the donors group versus 0.14 in the control group. The @S/@x for quality of motility was 4.19 in the donors group versus 1.00 in the control group. The quality of motility had the steepest ascent towards fertilization capacity, thus having the strongest impact on fertilization capacity (Fig. 1).
4. Discussion Reduction of sperm quality was pronounced in all three parameters that were examined. Sperm quality was expected Relative slope
to decrease by the freezing–thawing process, due to previously observed changes in biochemical and physical parameters [19–21]. Reduction of sperm parameters due to the freezing–thawing process was also pronounced in the surface response analysis. While quality of motility after freezing–thawing had the strongest impact on fertilization capacity, sperm concentration and percent of motility had relatively very little impact. In the control group, the quality of motility had the strongest impact as well, although less dramatic than in the frozen–thawed sperm. The difference in the effect of quality of motility between the control and frozen–thawed groups, as pronounced in the surface response analysis, may be linked to the finding that quality of motility was the least damaged parameter in the freezing– thawing process (Table 1). Only quality of motility of the frozen–thawed sperm was improved after Percoll column, while the concentration and percent of motility decreased and did not improve. It is thus not surprising that the effect of quality of motility on fertilization capacity of the frozen– thawed sperm was relatively larger than in the control group. Recently, the gene responsible for sperm motility has been studied [22,23]. It has been demonstrated that knocking this gene down resulted in infertile sperm, in accord with our clinical indications. Therefore, a connection between sperm motility and its fertilization capacity is strongly supported and may lead to a stronger attention to sperm motility in IVF treatments. In previous studies, the connection between sperm quality and its fertilization capacity was not well established. Some
4.5
DONORS
4
CONTROL
3.5 3 2.5 2 1.5 1 0.5 0 -0.5
Cocentration
Percent of motility
Quality of motility
Fig. 1. The effect of sperm parameters on fertilization capacity.
66
N. Marcus-Braun et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 116 (2004) 63–66
clinical evidence has been accumulated, indicating that fertilization capacity was not affected by cryopreservation [8,9]. Controversially, other studies pointed out that cryopreservation did affect fertilization capacity [6,7]. Our analysis has shown (Table 3) that the percents of fertilization were identical in the frozen–thawed group as compared with the control group. Therefore, despite the freezing–thawinginduced decrease in sperm quality, which was pronounced in all parameters, fertilization capacity remained unaffected. Averages of sperm concentration and percent of motility before freezing in the donors group were higher than in the control group. This was expected since donor sperm was carefully selected to be of high quality. However, in the control group, the sperm specimens were not expected to be of such a high quality. The data used in the present study was of sperm from husbands with no male infertility. Nevertheless, all sperm samples were in the normal range according to the WHO criteria. Mean number of oocytes was slightly higher in women that went through IVF-ET treatments with donor’s sperm. We conclude that cryopreservation of human sperm, using standard protocols, decrease sperm parameters as assessed by sperm concentration, percent of motility and quality of motility. However, fertilization capacity of frozen–thawed sperm is identical to fertilization capacity of fresh sperm. Our findings suggest that the quality of motility is a positive predictor for fertilization capacity in frozen–thawed sperm. References [1] Barratt CLR, Clements S, Kessopoulou E. Semen characteristics and fertility tests required for storage of spermatozoa. Hum Reprod 1998;13:1–7. [2] Polge C, Smith AU, Parker AS. Revival of spermatozoa after vitrification and dehydration at low temperature. Nature 1949;164: 666–7. [3] Bunge RG, Sherman JK. Fertilization capacity of frozen human spermatozoa. Nature 1953;172:767. [4] Ragni G, Lombroso Finzi GC, Caccamo A, Dalla Serra A, Crosignani PG. Enhanced sperm quality with TEST-yolk buffer: preliminary results in intraperitoneal insemination for male infertility. In: Proceedings of the Abstracts of the 7th Annual Meeting of the European Society of Human Reproduction and Embryology. Paris; Hum Reprod 1991;6:421. [5] Royere D, Barthelemy C, Hamamah S, Lansac J. Cryopreservation of spermatozoa: a 1996 review. Hum Reprod Update 1996;2:553–9. [6] Smith KD, Rodriguez-Rigau LJ, Steinberger E. The influence of ovulatory dysfunction and timing of insemination on the success of artificial insemination donor (AID) with fresh or cryopreserved semen. Fertil Steril 1981;36:496–502.
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