Department of Obstetrics and Gynecology, University of Göteborg, SU/Sahlgrenska, SE 413 45 Göteborg, Sweden
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Abstract |
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Key words: IVF/Percoll/PureSperm®/sperm morphology/swim-up
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Introduction |
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The aim of this study was to evaluate PureSperm® as an alternative to Percoll for gradient separation of spermatozoa for use in assisted reproduction. Since different numbers of layers can be used in gradient centrifugation, and the routine in our laboratory has been to use a four-layer Percoll, this was compared to a four-layer and also to the more standard use of a two-layer PureSperm® gradient. Progressive motility, normal morphology before and after preparation and yield from the initial semen samples were evaluated. In the second part of the study fertilization and pregnancy rates in our in-vitro fertilization (IVF) programme after using PureSperm® gradient separation versus swim-up procedure during a 6 month period were compared.
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Material and methods |
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Gradient centrifugation with Percoll and PureSperm ®respectively.
A total of 22 semen samples were included in the study. Ejaculates were collected after 35 days sexual abstinence and were left to liquefy for 2030 min. Semen analysis was performed according to World Health Organization guidelines (WHO, 1992) except for the morphology analysis which will be described separately. After this basic evaluation the samples were divided into three equal parts and put onto the different gradients; four-layer Percoll and four- and two-layer PureSperm® respectively.
Percoll gradient centrifugation technique
Percoll, 100% (Pharmacia, Uppsala, Sweden) was supplemented with Earle's balanced salt (Sigma-Aldrich, Stockholm, Sweden), sodium bicarbonate, NaHCO3 (Sigma S-4019), penicillin (Sigma) and HSA (human serum albumin 200 mg/ml, Pharmacia & Upjohn). Osmolarity was adjusted to 290295 mOsmol/kg. This stock solution (100% Percoll) was diluted with IVF medium IVFTM-50 (Scandinavian IVF Science AB, Göteborg, Sweden) to 90, 80, 70 and 45% Percoll solutions. The density gradients were performed layering 1 ml of each concentration into a 15 ml, conical Falcon tube. Gradients were pre-incubated overnight to obtain 37°C, and after liquefaction 0.51.0 ml of the ejaculate was layered on the top of each gradient and centrifuged for 30 min at 300 g.
After centrifugation the upper layer (seminal plasma plus 45% Percoll) was aspirated and the 90% layer was collected from the bottom of the tube using a clean pipette and was transferred to a new, clean tube and washed twice in IVF medium (300 g, 10 min). The pellet was resuspended in exact volumes of IVF medium before concentration and motility were evaluated. Recovery rates of motile, progressive (grade A and B) spermatozoa from the initial semen samples were calculated by dividing the retrieved number of motile spermatozoa by the initial number of available motile spermatozoa.
PureSperm® gradients
A two-layer gradient was prepared by using ready-to-use solutions 90% and 45% PureSperm®. The four-layer gradient was made by diluting 90% PureSperm® with IVF medium to 80% and 70% respectively. The preparation and evaluation technique was the same as for Percoll preparation.
Morphology slide preparation and morphology evaluation
Smears for morphology evaluation according to strict criteria (Kruger et al., 1986, 1988
) were prepared before and after Percoll and PureSperm® centrifugation respectively. Slides were washed in 70% ethanol before use. For each smear 520 µl of the sample were used. The slides were air-dried before fixation and staining was performed with a modified Papanicolaou technique as previously described (Menkveld et al., 1991
).
Sperm morphology evaluation was always performed by the same trained technician. A total of 200 spermatozoa were evaluated on each slide under oil immersion at a magnification x1000. Head, neck and tail defects or combinations of these were recorded separately. Teratozoospermia index (TZI) (WHO, 1992), i.e. the average number of defects per abnormal spermatozoon, which is equivalent to the multiple anomalies index (MAI) (Jouannet et al., 1988), was also calculated.
Fertilization and pregnancy rates after using PureSperm® or swim-up procedure
This part of the study included 88 conventional IVF cycles where swim-up was performed and 63 IVF cycles where PureSperm® was used for the sperm preparation. Handling of semen samples and preparation techniques was the same as described above. A four-layer PureSperm® gradient was used for this part of the study. Due to practical reasons in the laboratory, a randomization was performed by alternating between swim-up procedure and gradient separation every other day.
The women were down-regulated with gonadotrophin releasing hormone (GnRH) agonist buserelin (Suprefact® or Suprecur®; Hoechst, Frankfurt am Main, Germany) for 35 weeks, starting on cycle day 21 or 1 and then stimulated with daily s.c. injections of human menopausal gonadotrophin (HMG; Pergonal®; Serono Laboratories, Geneva, Switzerland), or with daily s.c. injections of purified or recombinant follicle stimulating hormone (FSH; Fertinorm® or Gonal-F®; Serono), followed by human chorionic gonadotrophin (HCG; Profasi® 10 000 IU i.m.; Serono). Oocyte retrieval was performed 3638 h later via transvaginal ultrasound-guided retrieval. The oocytes were inseminated with a sperm concentration of 200 000/ml 45 h after oocyte aspiration. Embryos were cultured in IVFTM-50 medium 23 days until embryo transfer with one to three embryos (mean 2.0) was carried out.
Fertilization and pregnancy rates were calculated for each group. The percentage of good quality embryos was also calculated for the two groups (number of embryos good enough for embryo transfer or freezing/number of fertilized embryos).
Swim-up technique
Swim-up was performed by adding 0.51.0 ml of the ejaculate to the bottom of a Falcon tube (15 ml) containing 1.5 ml of IVF medium. The tube was placed in a 45° angle and incubated at 37°C in 5% CO2 for 45 min. After incubation 1.0 ml of the supernatant was collected, IVF medium added and the supernatant was washed once at 300 g for 10 min. Evaluation was performed in the same way as after gradient centrifugation.
Statistics
In the comparisons of Percoll and PureSperm® gradients the MannWhitney U test with the Bonferroni correction was used. Student's t-test was used when comparing IVF results between the different sperm preparation methods. 2 test was used for categorical data. A P value < 0.05 was considered statistically significant.
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Results |
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Discussion |
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Since it was not possible to compare Percoll and PureSperm® in a clinical situation, i.e. using the prepared semen samples for fertilization, the outcome after using spermatozoa obtained by gradient separation with PureSperm® was instead compared to the outcome after using the swim-up technique for the semen preparation. No differences were found between the two groups in fertilization rate, pregnancy rate, term pregnancy rate or percentage of good quality embryos (Table III). A higher pregnancy rate has been reported in an IVF programme (Vanderzwalmen et al., 1991
) after using gradient separation with Percoll than after swim-up, which could not be confirmed in the present study using PureSperm®. However, the fact that Vanderzwalmen washed the semen twice by centrifugation before swim-up may have caused damage to the spermatozoa previously discussed (Aitken and Clarkson, 1988
; Mortimer, 1991
; Alvarez et al., 1993
), resulting in a lower fertilization rate after swim-up than after gradient centrifugation of samples with moderate sperm parameters.
As has been shown earlier (Englert et al., 1992; Ng et al., 1992
), we also found that the swim-up procedure yielded a higher percentage of motile spermatozoa than density gradient separation with PureSperm®, P = 0.01 (Table II
). Ng et al. (1992) also showed that the swim-up method resulted in the recovery of spermatozoa with significant higher straight linear velocity (VSL) and average path velocity (VAP). It was reported recently that the fertilization rate was significantly increased when the fastest moving spermatozoa were used for intracytoplasmic sperm injection (ICSI) (Van den Bergh et al., 1998
). These findings may support the use of the swim-up technique apart from the fact that no `additional substances' are introduced when swim-up is performed. Other findings (Mortimer, 1991
; Alvarez et al., 1993
), suggesting that centrifugation of semen samples may cause damage to the membranes of the spermatozoa, also support the use of the direct swim-up technique where usually only one centrifugation is performed.
In conclusion, the results from this study show that PureSperm® can be used to replace Percoll when density gradient separation is performed, although a higher percentage of morphologically abnormal spermatozoa are obtained than after using Percoll. The results also indicate that it may be of importance to use at least a three layer gradient when PureSperm® gradient separation is used, since this resulted in a slightly better motility and morphology than the two-step gradient. It was also found that using PureSperm®-treated spermatozoa resulted in similar fertilization and pregnancy rates as using spermatozoa obtained after the swim-up procedure. However, the direct swim-up technique has the advantage of selecting a higher percentage of motile spermatozoa without using any chemicals other than the IVF culture medium, and therefore we still recommend the swim-up technique to be the method of choice when separation of spermatozoa for IVF and ICSI is performed, particularly for cases likely to yield sufficient sperm numbers after preparation.
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Acknowledgments |
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Notes |
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References |
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Alvarez, J.G., Lasso, J.L., Blasco, L. et al. (1993) Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime. Hum. Reprod., 8, 10871092.[Abstract]
Centola, G.M., Herko, R., Andolina, E. and Weisensel, S. (1998) Comparison of sperm separation methods: effect on recovery, motility, motion parameters, and hyperactivation. Fertil. Steril., 70, 11731175.[ISI][Medline]
Chen, M-J. and Bongso, A. (1999) Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception. Hum. Reprod., 14, 759764.
Claassens, O.E., Menkveld, R. and Harrison, K.L. (1998) Evaluation of three substitutes for Percoll in sperm isolation by density gradient centrifugation. Hum. Reprod., 13, 31393143.[Abstract]
Englert, Y., Van den Bergh, M., Rodesch, C. et al. (1992) Comparative auto-controlled study between swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization. Hum. Reprod., 7, 399402.[Abstract]
Ericsson, R.J. (1977) Isolation and storage of progressively motile human sperm. Andrologia, 9, 111114.[ISI][Medline]
Fishel, S., Jackson, P., Webster, J. et al. (1988) Endotoxins in culture medium for human in vitro fertilization. Fertil. Steril., 49, 108111.[ISI][Medline]
Gorus, F.K. and Pipeleers, D.G. (1981) A rapid method for the fractionation of human spermatozoa according to their progressive motility. Fertil. Steril., 35, 662665.[ISI][Medline]
Jouannet, P., Ducot, B., Feneux, D. and Spira, A. (1988) Male factors and the likelihood of pregnancy in fertile couples. I. Study of sperm characteristics. Int. J. Androl., 11, 379394.[ISI][Medline]
Koper, A., Evans, P.R., Witherow, R.O.N. et al. (1979) A technique for selecting and concentrating the motile sperm from semen in oligozoospermia. Br. J. Urol., 51, 587590.[ISI][Medline]
Kruger, T.F., Menkveld, R., Stander, F.S.H. et al. (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil. Steril., 46, 11181123.[ISI][Medline]
Kruger, T.F., Acosta, A.A., Simmons, K.F. et al. (1988) Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil. Steril., 49, 112117.[ISI][Medline]
Lopata, A., Patullo, M.J., Chang, A. and James, B. (1976) A method for collecting motile spermatozoa from human semen. Fertil. Steril., 27, 677684.[ISI][Medline]
McClure, R.D., Nunes, L. and Tom, R. (1989) Semen manipulation: improved sperm recovery and function with a two-layer Percoll gradient. Fertil. Steril., 51, 874877.[ISI][Medline]
Menkveld, R., Swanson, R.J., Kotze, T.J.vW. et al. (1990) Comparison of a discontinuous Percoll gradient method versus a swim-up method: effects on sperm morphology and other semen parameters. Andrologia, 22, 152158.[ISI][Medline]
Menkveld, R., Oettle, E.E., Kruger, T.F. et al. (1991) In Brown, C-L. (ed.), Atlas of Human Sperm Morphology. Williams & Wilkins, Baltimore, p. 117.
Menkveld, R., Claassens, O.E., Harrison, K.L. et al. (1998) Evaluation of three substitutes for Percoll in sperm isolation by density gradient centrifugation. Hum. Reprod., 13 (Abstract book 1), 140.
Mortimer, D. (1991) Sperm preparation techniques and iatrogenic failures of in-vitro fertilization. Hum. Reprod., 6, 173176.[ISI][Medline]
Ng, F.L.H., Liu, D.Y. and Baker, H.W.G. (1992) Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples. Hum. Reprod., 7, 261266.[Abstract]
Paulson, J.D., Polakoski, K. and Leto, S. (1979) Further characterization of glasswool column filtration of human serum. Fertil. Steril., 32, 125126.[ISI][Medline]
Perry, G., Glezerman, M. and Insler, V. (1977) Selective filtration of abnormal spermatozoa by the cervical mucus in vitro. In Insler, V. (ed.), The Uterine Cervix in Reproduction. G. Thieme, Stuttgart, pp. 118128.
Pertoft, H., Rubin, K., Kjellen, L. et al. (1977) The viability of cells grown or centrifuged in a new density gradient medium, PercollTM. Exp. Cell Res., 110, 449457.[ISI][Medline]
Pertoft, H., Laurent, T.C. and Laas, T. (1978) Density gradients prepared from collodial silica particles coated by polyvinylpyrrolidone (Percoll). Anal. Biochem., 88, 271277.[ISI][Medline]
Svalander, P.C., Lundin, K. and Holmes, P.V. (1995) Endotoxin levels in Percoll® density-gradient media used to prepare sperm for human IVF treatment. Hum. Reprod., 10 (Abstract book 2), 130.
Van den Bergh, M., Emiliani, S., Biramane, J. et al. (1998) A first prospective study of the individual straight line velocity of the spermatozoon and its influences on the fertilization rate after intracytoplasmic sperm injection. Hum. Reprod., 13, 31033107.[Abstract]
Vanderzwalmen, P., Bertin-Segal, G., Geerts, L. et al. (1991) Sperm morphology and IVF pregnancy rate: comparison between Percoll gradient centrifugation and swim-up procedures. Hum. Reprod., 6, 581588.[Abstract]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge.
Submitted on October 8, 1999; accepted on January 6, 2000.