2 The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, affiliated to Sackler Faculty of Medicine, Tel Aviv University, Israel 3 Statistic department Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel. e-mail: layogev{at}zahav.net.il
1 To whom correspondence should be addressed at: The Institute for the Study of Fertility, Lis Maternity Hospital,
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Abstract |
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Key words: cryopreservation/donor sperm/season/sperm bank
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Introduction |
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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 artificial inseminations using sperm donation (Paraskevaides et al., 1988; Rojansky et al., 2000
).
Artificial 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.
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Materials and methods |
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The participants comprised young (2127-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 fulfilled 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 23 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 (fixed 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 1997.
Freezing procedure: optimizing the progressive motile sperm cell concentration
The objective of our freezing procedure was to obtain 812 x 106/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 812 x 106/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 fluid medium (Irvine Scientific, 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 sufficient for the previously calculated number of straws, plus a specimen to be thawed immediately after the freezing process. For the first freezing of a sample from a new donor, a total of 60 x 106/ml was used. The results of the tested freezingthawing process facilitated calibration of the following freezing procedures.
When the concentration of the raw specimen was high enough to achieve the minimum of 8 x 106/ml thawed progressive motility concentration without any need for further concentration (as determined when previously tested, usually with more than 100 x 106/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 fluctuations of each sperm donor. Samples with less than 7 x 106/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 Scientific). After dilution, the mixture was equilibrated for 1520 min at room temperature, then sealed in 0.5 ml straws (I.M.V., Paris, France) and cooled in a semi-programmable 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 ± SE. Analysis for normal 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 Sidaks method was applied to control the overall significance level at 0.05. 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 8.02.
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Results |
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Analysis for the 59 men who donated samples during all four seasons revealed a significant difference between summer (June and September) and winter (December and March) 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 significance 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.
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Discussion |
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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 confirmed previous reports of such seasonal variations by Tjoa et al. (1982), Saint Pol et al. (1989)
, Henkel et al. (2001)
and others, who observed significantly 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 first 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 influence, and the exact impact on freezability and on reproduction performance in general, have yet to be evaluated.
Although seasonal fluctuation 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 freezingthawing 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 fixed 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 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 fluctuate 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 frozenthawed 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 influence of seasonality.
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Acknowledgements |
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References |
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Submitted on November 27, 2003; accepted on January 8, 2004.