Seasonal variations in pre- and post-thaw donor sperm quality

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1 Human Reproduction Vol.19, No.4 pp. 880±885, 2004 Advance Access publication February 27, 2004 DOI: /humrep/deh165 Seasonal variations in pre- and post-thaw donor sperm quality L.Yogev 1,2, S.Kleiman 2, E.Shabtai 3, A.Botchan 2, R.Gamzu 2, G.Paz 2, H.Yavetz 2 and R.Hauser 2 2 The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, af liated to Sackler Faculty of Medicine, Tel Aviv University, Israel 1 To whom correspondence should be addressed at: The Institute for the Study of Fertility, Lis Maternity Hospital, 3 Statistic department Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel. layogev@zahav.net.il BACKGROUND: The present study was conducted to evaluate seasonal variability in the quality of pre- and postthaw semen parameters among sperm bank donors. METHODS: The rst two consecutive ejaculates during the months March (spring, 92 males), June (summer, 97 males), September (autumn, 81 males) and December (winter, 97 males) were analysed. A comparison was made between sperm parameters from the same sperm donor at different seasons. Only males who donated semen samples during at least two seasons were enrolled in the study group (n = 103). Sperm specimens were cryopreserved in aliquots with xed range of 8± /ml of progressive motile sperm concentration after thawing. RESULTS: Differences between months were found in sperm concentration (P = 0.030) and normal morphology (P = 0.038); highest values were found in March and December, and the lowest in September. Mean specimen volume and percent of motile sperm cells did not vary throughout the seasons. The freezability of the donors' sperm dropped dramatically from March to September, as determined by the number of straws ( xed aliquots of 0.5 ml) and total thawed progressive motile sperm that were cryopreserved for each male (P = and P = 0.002, respectively). CONCLUSIONS: Cryopreservation of donor sperm is more effective during winter and spring than during the rest of the year. Key words: cryopreservation/donor sperm/season/sperm bank Introduction Seasonal variations in human reproduction have been studied intensively (Rojansky et al., 1992). Most reports demonstrate an increase in sperm count and several other parameters during winter and spring (Tjoa et al., 1982; Saint Pol et al., 1989; Levine et al., 1992; Gyllenborg et al., 1999; Centola and Eberly, 1999; Andolz et al., 2001; Chen et al., 2003), whereas other studies have been inconclusive (Mortimer et al., 1983; Ombelet et al., 1996). During summer, a similar reduction in semen quality among indoor and outdoor workers was reported, excluding the possibility that summer heat is the only detrimental factor for seasonal variability (Levine et al., 1992). With regard to the female partner, seasonal variations were shown in the natural and IVF set-up. During the four seasons, a variance was observed in ovum quality, fertilization rate, embryo quality and endometrial receptivity, as well as conception rate following arti cial inseminations using sperm donation (Paraskevaides et al., 1988; Rojansky et al., 2000). Arti cial insemination using donor sperm is still the treatment of choice for certain severe male factor infertilities (e.g. necrozoospermia), repeated IVF/ICSI failures, genetic indications and for single women. Although the quarantining of frozen semen has a negligible impact on sperm quality, the freezing and thawing process is usually associated with diminished motility, viability and some functional ability of the spermatozoa (Sharma et al., 1997). Overall, a correlation between these parameters and pregnancy rate has been demonstrated (Mahadevan and Trounson, 1984; Johnston et al., 1994). It has been established previously that the quality of thawed donor sperm can vary (Kolon et al., 1992); however, its seasonality dependence has, as yet, not been documented. The aim of this study was to determine seasonal variability in the pre- and post-thawed sperm parameters of sperm bank donors. For this purpose, a comparison was made between parameters of sperm donated by the same donor during different seasons. Materials and methods Data for this study were derived from analyses of 734 semen samples of sperm bank donors collected during the months March (spring, 92 males), June (summer, 97 males), September (autumn, 81 males) and December (winter, 97 males). Month selection was made a priori 880 Human Reproduction vol. 19 no. 4 ã European Society of Human Reproduction and Embryology 2004; all rights reserved

2 Donor sperm banking at different seasons Table I. Median, 5th and 95th percentiles for all sperm parameters in the different months March (n = 92) June (n = 97) September (n = 81) December (n = 97) Median 5th 95th Median 5th 95th Median 5th 95th Median 5th 95th Volume (ml) Concentration (10 6 /ml) Total counts (10 6 ) Motility (%) Normal forms (%) No. of frozen straws Prog. motility conc. (10 6 /ml) Total prog. froz. count (10 6 ) Thawed sperm motility (%) Prog. = progressive; conc. = concentration; froz. = frozen. before analysis of the data, according to the calendar seasons in Israel. The rst two consecutive ejaculates during each of the aforementioned months were analysed during the period 1995±2003. Mean values in the four seasons were calculated. To compare sperm parameters in the same sperm donor at different seasons, only males who donated sperm during at least two seasons were enrolled in the study group (n = 103 men). The order of entering the study was random for season. A total of 59 men donated samples during all four seasons. The participants comprised young (21±27-year-old) unmarried students, all of Caucasian origin, and almost all of whom were born in Israel. A total of 69% of the participants had proven fertility judging from pregnancies achieved by using their sperm donations. The specimens of the remaining donors had not yet been used. All participants ful lled the strict criteria for sperm donors as previously described (Yavetz et al., 1991; Botchan et al., 2001), including hemizona assay for sperm binding, and the mucus penetration test. All participants were instructed to observe 2±3 days of abstinence before each donation. Sperm quality was assessed according to World Health Organization guidelines (WHO, 1999) prior to and immediately after each specimen freezing procedure (by thawing a sample) to assure it conformed to the expected quality. At the time of giving their informed consent to use the samples they donated, the donors also provided their permission to use their specimens for future studies to be conducted. As this was a retrospective study of the anonymous donor specimen database, Institute Review Board submission was not required. Each ejaculate was allowed to liquefy for at least 30 min at 37 C. The volume was determined by drawing up the entire sample into a 5 ml graduated disposable pipette. A sample was taken to evaluate sperm concentration (by Makler chamber) and percentage of motile spermatozoa. Percentage of morphologically normal sperm was assessed according to strict criteria (Menkveld and Kruger, 1995; WHO, 1999) on Papanicolaou stained smears. Number of frozen straws ( xed 0.5 ml aliquots), progressive [type A according to WHO (1999)] motility concentration and percentage of motility in thawed sample of each cryopreserved ejaculate were also evaluated. The laboratory has been under the external quality assessment scheme for Andrology by United Kingdom National External Quality Assessment Schemes, Manchester, UK (UKNEQAS) since Freezing procedure: optimizing the progressive motile sperm cell concentration The objective of our freezing procedure was to obtain 8± /ml progressive motile sperm concentration in each thawed straw. We found this quantity adequate for cervical and intrauterine inseminations. To achieve this goal, a total count was calculated for each ejaculate by multiplying the volume and concentration. In the previous freezing procedure, the number that was found appropriate to achieve one thawed straw with 8± /ml progressive motility concentration was used to divide the total count result. The quotient was indicative of the number of straws to be prepared. The specimen was washed with human tubal uid medium (Irvine Scienti c, Santa Ana, CA, USA) supplemented with 1% human serum albumin (Kamapharm Human Albumin; Kamada, Kibbutz Beit Kama, Israel), and an aliquot of the medium was added to the pellet in a volume suf cient for the previously calculated number of straws, plus a specimen to be thawed immediately after the freezing process. For the rst freezing of a sample from a new donor, a total of /ml was used. The results of the tested freezing±thawing process facilitated calibration of the following freezing procedures. When the concentration of the raw specimen was high enough to achieve the minimum of /ml thawed progressive motility concentration without any need for further concentration (as determined when previously tested, usually with more than /ml of concentration), the freezing process was performed without pre-freezing sperm preparation. The quantity of sperm cells required to achieve one straw with the desired progressive motility concentration was monitored after each freezing, and was altered according to the uctuations of each sperm donor. Samples with less than /ml were discarded from the bank, although they are included in the Results section of the present study. Freezing process In both procedures (pre-treated and raw samples), the specimen was carefully diluted by the addition of equal volume of the freezing medium test yolk buffer (Irvine Scienti c). After dilution, the mixture was equilibrated for 15±20 min at room temperature, then sealed in 0.5 ml straws (I.M.V., Paris, France) and cooled in a semiprogrammable freezer (Nicool LM-10; Air Liquid, Paris, France). The straws were cooled from room temperature to ±6 C, 1.7 C/min, and then to ±100 C, 5 C/min. The straws were then transferred directly to liquid nitrogen (±196 C) for storage (Yogev et al., 1999). Thawing procedure The specimen designated to be evaluated immediately after the freezing process was allowed to thaw at 37 C for 5 min and assessed for percentage of motility and number of progressive motile sperm concentration. Statistical analysis Results were given as median and 5th and 95th percentiles, by `box and whisker plot' that graphically presents measurements of central tendency and variability, and by mean 6 SE. Analysis for normal 881

3 L.Yogev et al. Figure 1. Seasonal differences in nine sperm parameters, before and after freezing, plotted with `box and whiskers', for the months March, June, September and December. Sperm parameters before freezing are volume, concentration, total count, motility percentage and percentage of morphologically normal forms. Parameters after freezing process are number of frozen straws, progressive motility concentration in thawed straws, total count of progressive motile frozen sperm and percentage of thawed sperm motility. The box encloses the middle half of the data (supplying rst and third quartile), and is bisected by the median value. The vertical lines at the top and bottom of the box indicate the range of `typical' data values. Extreme values are displayed as `dots' for possible outliers (values that are outside the box boundaries by >1.5 times the size of the box) and `stars' for probable outliers (values that are outside the box boundaries by >3 times the size of the box). distribution was performed, and log transformation was used whenever necessary. To compare sperm parameters in the same sperm donor at different seasons, sperm count data were analysed applying the mixed model for analysis of variance (ANOVA) with repeated measures. This model was chosen to account for missing data in some of the donors. The covariance structure used was the compound symmetry, as determined by the Akaike criterion. Several contrasts between seasons were tested, and Sidak's method was applied to control the overall signi cance level at The Wilcoxon test was used to compare the difference between September and December for pre-freezing total count and post-freezing total progressive motility count, and between summer and winter for the 59 men who donated specimens in all four seasons. All analyses were performed using the SAS system for Windows (Cary, NC, USA), release Results Sperm parameters such as volume, concentration, and percentage of motility and normal morphology were evaluated in the same sperm donors in March, June, September and December (Table I; Figures 1 and 2). The analysis for normal distribution revealed skewed distribution for three parameters: volume, number of straws and total progressive motility count 882 of frozen sperm. Consequently, these parameters were log transformed before analysing for seasonal differences. Differences between months were found in sperm concentration (P = 0.035; highest level in December and lowest in September), and percentage of normal morphology (P = 0.038; highest in March and lowest in September, Table II). Mean specimen volume and percentage of motile spermatozoa did not vary throughout the seasons. The average number of straws ( xed 0.5 ml aliquots) cryopreserved for each donor varied between the seasons (P = 0.033; higher in March±December than in June±September (P = 0.003; Table II). The freezability of the donors' sperm, as judged by the cryopreserved total progressive motility count (i.e. number of frozen straws multiplied by thawed progressive motility concentration), increased dramatically from September to December. This increase (25%) was much higher than the increase in pre-freezing total count (15%; P = 0.012, by Wilcoxon test). Interestingly, the percentage of motility of the thawed specimens remained constant throughout all seasons. Analysis for the 59 men who donated samples during all four seasons revealed a signi cant difference between summer (June and September) and winter (December and March)

4 Donor sperm banking at different seasons Figure 2. Seasonal differences in nine sperm parameters, before (A±E) and after (F±I) freezing. Each of the nine illustrations shows the upper 30% of its scale for the months March, June, September and December. Sperm parameters are detailed in Figure 1. Values are presented as mean 6 SEM. The degree of signi cance of variations between months is indicated in Table II. Table II. In uence of seasonal variations on sperm parameters Parameter Between seasons Sept. ± Dec. b Sept. ± March b June + Sept. ± Dec. + March b Concentration Total count Normal form No. of frozen straws a Thawed progressive motility concentration Total count of progressive motile frozen sperm (10 6 ) a The table exhibits the parameters with signi cant differences (P) among seasons. Level of signi cance between all seasons, September and December, September and March, and between June and September compared with December and March are shown. P values are according to mixed model for ANOVA with repeated measurements. a This parameter was log transformed before using ANOVA with repeated measurements. b Differences are signi cant when P < (after adjustment for three contrasts between seasons according to Sidak's method for ANOVA with repeated measurements). seasons for the following six parameters: volume, total count, normal form, number of frozen samples and total progressive frozen sperm count (Table III). Obviously, the signi cance was lower than the entire group of 103 men (Table II), even though the results exhibited the same tendency, because of the smaller group size. Discussion A comparison was performed between sperm parameters in the same sperm donors during different seasons. The participants in the present study comprised a homogenous group of young men (21±27 years old) with a constant period of abstinence (2± 3 days) and xed duration from ejaculation to freezing (~1 h). 883

5 L.Yogev et al. Table III. The difference between summer (June and September) and winter (December and March) regarding nine sperm parameters of the 59 men who donated samples during all four seasons Parameter P (June and September ± December and March) Volume (ml) Concentration (10 6 /ml) Total count (10 6 ) Motility (%) Normal form (%) Number of frozen straws Progressive motility concentration (10 6 /ml) Total count of progressive motile frozen sperm (10 6 ) Thawed sperm motility (%) P values according to Wilcoxon test. By keeping these confounders constant, their contribution to seasonal differentiation could be ignored. The results of this study revealed seasonal dependence of sperm donation on sperm concentration, normal morphology and freezability, as expressed by the total progressive motility count of cryopreserved samples. This study con rmed previous reports of such seasonal variations by Tjoa et al. (1982), Saint Pol et al. (1989), Henkel et al. (2001) and others, who observed signi cantly higher sperm concentration in winter and spring compared with autumn. Although the aforementioned studies were performed in different latitudes with variable temperature and humidity, the same tendency was observed (Rojansky et al., 1992). Seasonal changes in freezability of sperm bank donors are shown, to the best of our knowledge, for the rst time. A high value of sperm nuclear maturity was reported in winter, in addition to the aforementioned parameters (Henkel et al., 2001). Nevertheless, the mechanism of in uence, and the exact impact on freezability and on reproduction performance in general, have yet to be evaluated. Although seasonal uctuation was found for sperm quality as well as for fecundity, there was no overlapping of the two (Mahadevan et al., 1982; Paraskevaides et al., 1988). Nonetheless, it was reported that in the IVF program, ovulation, fertilization, embryo quality and pregnancy rates were higher in winter and spring compared with summer and autumn (Stolwijk et al., 1994). It was shown previously that the pre-freezing sperm manipulation and freezing±thawing process did not impair the capability of rescued sperm to bind to the zona pellucida (Gamzu et al., 1992; Yogev et al., 1999). Sperm manipulation prior to the freezing process makes it possible to optimize the progressive motile sperm cell concentration, an important parameter for successful fertilization (Tomlinson et al., 1999). This facilitates the storage of samples with good quality, even when the features of the raw semen are suboptimal. By maintaining the quality of the sperm in each straw within a xed range, the comparison between the number of frozen straws at different seasons proved most informative. Thus it was found that more straws could be stored from each sperm 884 donor during winter and spring compared with summer or autumn. Interestingly, the increase in the total frozen sperm count with progressive motility in December compared with September was higher than the increase of total pre-freezing counts. Bearing in mind that the motility percentage of thawed samples did not uctuate between seasons, it could be speculated that the seasonal changes in the percentage of cells with normal morphology [as reported in the present study, and also by Levine et al. (1992), Andolz et al. (2001) and others] and in chromatin condensation (Henkel et al., 2001) could contribute to the seasonal change in freezability, as was expressed by the quality of motility (local, sluggish or progressive), as well as some other contributing factors. In conclusion, cryopreservation of donor sperm is more effective during winter and spring than the remainder of the year. In our previous study, use of frozen±thawed sperm for intrauterine insemination resulted in a pregnancy rate of 12.1% (Botchan et al., 2001). Analysing pregnancy rate according to the season in which the sperm samples were frozen, and the season in which the cryopreserved samples had been used for inseminations, will further contribute to understanding the in uence of seasonality. Acknowledgements We wish to thank Mrs B.Gore (MSc), F.Feldhoon, M.Ilatov (BSc) and L.Ariely (MSc), of the Institute for the Study of Fertility Sperm Bank, for their excellent and skillful technical assistance. We appreciate the expert graphic illustration of S.Siso. References Andolz P, Bielsa MA and Andolz A (2001) Circadian variation in human semen parameters. Int J Androl 24,266±271. Botchan A, Hauser R, Gamzu R, Yogev L, Paz G and Yavetz H (2001) Results of 6139 arti cial insemination cycles with donor spermatozoa. Hum Reprod 16,2298±2304. 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