Evaluation of Nycodenz* and Percoll† density gradients for the selection of motile human spermatozoa‡

Vol. 49, No.2, February 1988 Printed in U.S.A.

FERTILITY AND STERILITY Copyright c 1988 The American Fertility Society

Evaluation of Nycodenz* and Percollt density gradients for the selection of motile human spermatozoa*

Sharon T. Gellert-Mortimer, B.Sc.§ll Gary N. Clarke, M.Sc.V H. W. Gordon Baker, M.D. PhD.**

Ross V. Hyne, PhD.tt W. Ian H. Johnston, F.R.A.C.O.G.§

Royal Women's Hospital, Carlton, Victoria, and Epworth Hospital, Richmond, Victoria, Australia

Two high-density media, Percoll (Pharmacia, Uppsala, Sweden) and Nycodenz (Nyegaard & Co., Oslo, Norway), were assessed for efficacy of selection of motile spermatozoa from oligozoospermic and asthenozoospermic semen samples. The results indicated initially that the media were equivalent for motile sperm selection. However, only the spermatozoa selected from discontinuous Nycodenz gradients retained their motility for at least 21 hours (60% motility compared with 5% motility). A significantly higher number of motile spermatozoa were harvested from discontinuous Nycodenz gradients than from matched control samples using the swim-up procedure (12.0 ± 3.0 standard error of the mean [SEM] X 106 /ml versus 1.9 ± 0.7 SEM X 106 /ml; 27 experiments, P < 0.001). Discontinuous Nycodenz gradients may be useful for selection of motile spermatozoa from poor-quality semen for in vitro fertilization. Fertil Steril 49:335, 1988

In vitro fertilization and embryo transfer (IVFET) is being considered more often as a treatment for infertility due to a "male factor" (e.g., oligozoospermia, <20 X 106 spermatozoa/ml, or asthenozoospermia, <40% total motility), because of the relatively low numbers of spermatozoa required (50,000 to 100,000 motile spermatozoa/oocyte). However, success rates are often poor. For example, in the IVF-ET program at the Royal Women's Hospital in 1985, 60% of the failed fertilization

Received July 27, 1987; revised and accepted October 7, 1987. * Nyegaard & Co., Oslo, Norway. t Pharmacia, Uppsala, Sweden. :j: Supported by The Royal Women's Hospital Research Fund. §Reproductive Biology Unit, Royal Women's Hospital. II Reprint requests and present address: Sharon T. GellertMortimer, B.Sc., Endocrine/Infertility Clinic, Department of Obstetrics and Gynaecology, University of Calgary Health Sciences Centre, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada. ~Department of Andrology, Royal Women's Hospital. **Department of Obstetrics and Gynaecology, Royal Women's Hospital. tt Infertility Medical Centre, Epworth Hospital. Vol. 49, No.2, February 1988

cycles were attributable to male factors and low fertilization rates of only 19% were obtained when the motility of the spermatozoa for insemination was <20%. Therefore, improved methods of sperm preparation are needed for the treatment of male infertility by IVF-ET. Sperm washing is necessary prior to oocyte insemination in vitro, first, to remove the seminal plasma, which contains factors that inhibit fertilization, and second, to select the motile spermatozoa. These procedures occur at the cervix in vivo. 1 Our sperm washing procedure involves the centrifugation of semen diluted with culture medium. The seminal plasma remains in the supernatant, which is discarded, and the motile spermatozoa swim up into fresh medium, which is carefully layered over the pellet. The yield is approximately 18% of the motile spermatozoa originally present in the semen. 2 While this is an acceptable procedure when a normal semen sample (>20 X 106 spermatozoa/ ml and >40% motility) is used, insufficient numbers of motile spermatozoa for IVF may be obtained when using abnormal semen. Alternative methods to swim-up techniques have been suggested for the isolation of motile sperma-

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tozoa from poor semen samples. The most successful of these involve the passage of spermatozoa through concentration gradients of high-density media, such as albumin 3 •4 or Percoll (Pharmacia, Uppsala, Sweden). 5-10 Sperm selection on Percoll gradients has been reported to yield up to 60% of the motile, morphologically normal spermatozoa from a normal semen sample. 2 However, the recovery of normal, motile spermatozoa from oligozoospermic and asthenozoospermic samples was not found to be as great nor as consistent as for normal samples. 11 We have investigated the potential of an alternative density gradient medium, Nycodenz (Nyegaard & Co., Oslo, Norway), for sperm selection. It is an iodinated organic molecule, N,N'-bis-2,3-dihydroxypropyl- 5- [N- (2,3- dihydroxypropyl)acetamido ]-2,4,6-triiodoisophthalamide, dissolved in a Tris buffer. In this form, it is a biologically inert and nontoxic medium used for gradient fractionation of biologic samples. This compound, in a similar buffer, also has been marketed as an x-ray contrast medium, iohexol or Omni-paque. Studies of iohexol have revealed a low incidence of adverse reactions during angiography, 12-14 and no adverse effects in experimental animals on reproductive function in males or females or on embryo development.15 The aims of this study were to compare the efficacy of Percoll and Nycodenz gradients, and to optimize the latter and to compare it with swim-up procedures in the selection of motile spermatozoa from oligoasthenozoospermic semen samples. MATERIALS AND METHODS Preparation of Gradients

Stock isotonic Percoll was prepared by mixing 9.2 parts of Percoll (Pharmacia South Seas, Melbourne, Victoria, Australia) with 0.8 parts of lOX concentrated Ham's F-10 medium (Flow Laboratories, Melbourne, Victoria, Australia). Stock isotonic Nycodenz was used as supplied, in a sterile, isotonic Tris buffer and supplemented with 1 mg/ml human serum albumin Cohn fraction V (Calbiochem-Behring, La Jolla, CA). To achieve the required densities, the stock solutions of both Percoll and Nycodenz were diluted in supplemented Ham's F-10 medium (SHF-10), containing 1 mM calcium lactate, 20 mM NaHC0 3 , 5 mM KHC0 3 , 0.5 mM MgS0 4 (AnalaR, Ajax Chemicals, Sydney, NSW, Australia), 50 mg/1 pen336

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icillin G, 50 mg/l streptomycin, and 1 mg/ml human serum albumin (Calbiochem-Behring, La Jolla, CA). The continuous Percoll gradients were generated by centrifugation of 9-ml volumes of 60% stock Percoll at 30,000 X g for 15 minutes at 20°C, according to the method of Bolton and Braude. 9 Discontinuous gradients were generated in 12-ml tissue culture tubes (Falcon 2001, Falcon Plastics, Oxnard, CA) by layering 1.6-ml aliquots of 35, 50, 65, 80, 90, and 100% isotonic stock Percoll, following the method of Hyne et al. 11 Continuous Nycodenz gradients were formed by the diffusion 16 of discontinuous gradients consisting of 1.6-ml volumes of 35, 50, 65, 75, and 85% stock Nycodenz in 12-ml tissue culture tubes. The tubes were capped and laid horizontally for 60 minutes at room ternperature, then returned to the vertical position. Discontinuous Nycodenz gradients were prepared in the same way as the discontinuous Percoll gradients. The discontinuous gradient that gave the optimal motile sperm selection consisted of 1.6 ml 35%, 1.6 ml 50%, 3.2 ml 65%, and 1.6 ml 100% Nycodenz. Comparison of Percoll and Nycodenz Gradients for Sperm Selection

The semen samples used for the media comparison were from patients attending the Royal Women's Hospital Andrology Laboratory for semen analysis. All of the samples selected for the study were asthenozoospermic, with <40% motile spermatozoa. Other parameters, such as sperm concentration and morphologic features, were not considered in detail, although all samples had >20 X 106 spermatozoa/ml and average to high numbers of abnormal forms (range, 50% to 96%). Semen was collected by masturbation and allowed to liquefy for 30 minutes at 37°C before an initial assessment of sperm concentration and motility was made. The liquefied semen was diluted in an equal volume of SHF -10. With 1 ml of diluted semen serving as a control, the remainder was divided into equal portions and centrifuged through continuous or discontinuous gradients of Percoll or Nycodenz at 350 X g for 12 minutes at room temperature. Fractions collected from the gradients after centrifugation then were diluted in 2 volumes of SHF-10, centrifuged again (350 X g for 12 minutes), and the pellets resuspended in 1 ml fresh SHF-10 containing 10 mg/ml human serum albumin. The control

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samples were diluted in the same volumes of medium and centrifuged at the same time as the test samples. The sperm suspensions then were stored at 37°C in a humidified 5% C02 atmosphere. Aliquots were taken after 0, 3, and 21 hours of incubation for subjective motility assessment. Objective Sperm Motility and Velocity Assessments

After selection of discontinuous Nycodenz gradients, six samples were assessed for motility and velocity. The controls were washed spermatozoa from the same semen samples. Objective assessment of these parameters was made by a modification of the technique of Overstreet et al. 17 using a Makler sperm counting chamber 18 (Sefi Medical Instruments, Haifa, Israel), a warm stage set at 37°C, and Polaroid 400 ASA, 35-mm instant slide film (Polaroid HC.135.12, Polaroid Corporation, Cambridge, MA) to obtain a positive image. The film was projected onto an Apple graphics tablet and more than 100 sperm tracks were traced with a magnetic tracing pen connected to an Apple I /e computer (Apple Computer Inc., Cupertino, CA). The tracks were analyzed using a program that gave the percentage of spermatozoa with progressive motility (linear velocity> 10 p,m/sec), a velocity histogram, and the average velocity of the progressive spermatozoa. 19 Sperm morphology also was assessed on these six cases. For this, the sperm concentration was adjusted to 5 to 10 X 106 spermatozoa/ml and 50-p,l aliquots of each suspension were allowed to air dry onto glass slides. The slides were placed in methanol for 30 minutes, then stained in hematoxylin/ acetic acid (Medos, Australia) for 10 minutes. Excess stain was removed by washing with water. Assessment of the unmounted slides was made under oil using 1000X magnification by a trained technician unaware of the experimental group.

semen (n = 27), the swim-up procedure was carried out at the same time as a control. If there was insufficient semen for both procedures, retrospective comparisons were made with past swim-up results, if they were available (n = 12). At the time of the study, a few of the samples had better motility than expected from previous results. The swim-up procedure used was as follows: 1 ml liquefied semen was diluted with 3 ml SHF-10 and centrifuged at 350 X g for 10 minutes. The supernatant was removed and 1 ml fresh medium was layered over the pellet. Motile spermatozoa were allowed to swim up into the fresh medium during an incubation period of 60 minutes at 37°C in a humidified 5% C0 2 atmosphere. RESULTS Comparison of Percoll and Nycodenz Gradients

Sperm selection was equivalent on both continuous and discontinuous Percoll gradients, with over 60% of spermatozoa being motile in the 100% stock Percoll fractions (Fig. 1). A comparable harvest of motile spermatozoa also was obtained from the 65% Nycodenz fraction of discontinuous Nycodenz gradients (Fig. 2). No fraction with enhanced sperm motility was harvested from the continuous Nycodenz gradients (Fig. 3).

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Selection of Motile Spermatozoa from Oligoasthenozoospermic Patients using Nycodenz Gradients

10

Semen samples were collected from a total of 42 patients (47 samples) known to be asthenozoospermic (mean sperm motility< 40%) or oligoasthenozoospermic (mean sperm motility < 40% and mean sperm concentration< 20 X 106 /ml), and processed on discontinuous Nycodenz gradients, as described previously. When there was > 1 ml of Vol. 49, No. 2, February 1988

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Figure 1 Motility of spermatozoa (mean ± SEM) assessed at 0 (solid line), 3 (broken line), and 21 (dotted line) hours after selection on Percoll gradients. The histograms represent the motility of spermatozoa in the control samples following 0 (heavy hatched column), 3 (hatched column), and 21 (open column) hours' incubation.

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and the results compared with those of control samples. The spermatozoa selected on the Nycodenz gradients had significantly higher motility than their controls (P < 0.05 by Wilcoxon signed ranks test, see Table 1). There was a small reduction in the number of abnormal forms of spermatozoa following Nycodenz selection, which just reached statistical significance (P = 0.041 by Wilcoxon signed ranks test, see Table 1). The morphologic types excluded were the immotile forms, such as ring-tails.

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Selection of Motile Spermatozoa from Oligoasthenozoospermic Patients Using Nycodenz Gradients 0

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65

75

100

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Figure 2 Motility of spermatozoa (mean ± SEM) assessed at 0 (solid line), 3 (broken line), and 21 (dotted line) hours after selection on discontinuous Nycodenz gradients. The histograms represent the motility of spermatozoa in the control samples following 0 (heavy hatched column), 3 (hatched column), and 21 (open column) hours' incubation.

After overnight incubation (21 hours) at 37°C, the Nycodenz-selected sperm samples had better motility than the parallel controls (60.9 ± 2.9% versus 28.5 ± 2.8%, P < 0.001 by paired t-test, all values mean± standard error of the mean [SEM]). There was no significant difference between the initial motility of the samples recovered from the 65% Nycodenz fraction and their motility after 21 hours' incubation (61.9 ± 2.2% versus 60.9 ± 2.9%, P > 0.7 by paired t-test). However, the motility of the Percoll-selected samples decreased significantly between 3 and 21 hours of incubation (61.7 ± 3.7% versus 12.7 ± 2.8% for Fraction 5 and 64.6 ± 3.6% versus 5.0 ± 1.3% for Fraction 6, both P < 0.001 by paired t-tests), and were significantly lower than the control values after 21 hours (22.4 ± 3.6%; Fraction 5 P < 0.02 and Fraction 6 P < 0.001, by paired t-tests). Following these results, further studies were made of sperm selection on discontinuous Nycodenz gradients. Improvement in Motility, Velocity, and Morphology with Discontinuous Nycodenz Gradients

Spermatozoa harvested from the 65% layer of discontinuous Nycodenz gradients were assessed objectively for progressive motility and velocity 338

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Motile spermatozoa were selected from oligoasthenozoospermic samples using discontinuous Nycodenz gradients. The motility was assessed before selection (initial) and after recovery from the 65% fraction of Nycodenz gradients (final). Comparison of these results revealed that the final motility was greater than the initial motility in 46/47 cases (Fig. 4). Of the 47 samples tested, the final motility showed a 20% improvement in 39 (83%) and a >50% improvement in 25 (53%). Linear regression revealed a direct positive relationship between the initial and final sperm motility (final = [0.851 X initial] + 28.24, r = 0.68, P < 0.001). Two patients had semen samples collected on several different occasions (points marked a and b, 60

6 Fraction No.

Figure 3 Motility of spermatozoa (mean ± SEM) assessed at 0 (solid line), 3 (broken line), and 21 (dotted line) hours after selection on continuous Nycodenz gradients. The histograms represent the motility of spermatozoa in the control samples following 0 (heavy hatched column), 3 (hatched column), and 21 (open column) hours' incubation.

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Table 1

Objective Assessment of Nycodenz-Selected Spermatozoa in Six Patients %Motility

Velocity

% Abnormals

(~tmfsec)

Patient

Control

Test

Control

Test

Control

Test

A B

6 9 18 18 11 6

13 31 32 19 21 9

13 25 25 45 31 18

25 39 38 44 34 23

66 84 50 95 92

58 76 44 94 88

c

D E F

Fig. 4). The proximity of results for each patient's samples suggested that the selection procedure could be reproducible over time, although this would require further studies. The mean yield of motile spermatozoa from the Nycodenz gradients for all samples was 39 ± 3% of the motile spermatozoa applied to the gradients, with a range from 4% to 92%. Following selection of the subgroup of ten samples with marked oligozoospermia (4 to 9 X 106 spermatozoa/ml) on these gradients, sperm concentrations of 1 to 5 X 106 /ml were recovered, with a range of 34% to 85% motile. The mean yield of motile spermatozoa for these samples was 42 ± 6% (range, 11% to 73%). Comparison of Discontinuous Nycodenz Gradients with the Swim-up Procedure

matozoa harvested following each procedure were compared (Fig. 5). A line was drawn between the axes to represent the outcome if equivalent numbers of motile spermatozoa were recovered. However, a greater number of motile spermatozoa were collected in 26 of the samples by Nycodenz selection (12.0 ± 3.0 X 106 /ml) than by swim-up (1.9 ± 0.7 X 106 /ml). This represented a significant difference (P < 0.001 by Wilcoxon signed ranks test) and indicated that, at least for poor-quality semen, a sufficient number of motile spermatozoa for IVF would be retrieved by density gradient centrifugation on Nycodenz, but not by the swim-up procedure. Retrospective controls for the remaining cases revealed a similar improvement over the expected outcome, based upon previous swim-up results (data not shown).

Swim-up controls were performed for 27 patients' samples. The total numbers of motile sperDISCUSSION

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Figure 4 Comparison of sperm motility before and after selection on discontinuous Nycodenz gradients for samples with initial sperm concentrations of>20 X 106 /ml (solid circles), 10 to 20 X 106 /ml (open circles), and <10 X 106 /ml (open squares). Points a and b represent samples collected from two patients, respectively, on different occasions. The line is the line of equivalence between the initial and final motility values (slope= 1.0). Vol. 49, No.2, February 1988

A low number of motile spermatozoa in the final sperm preparation has been shown to be correlated with poor IVF performance. 2° Consequently, if we could improve the final motility and number of motile spermatozoa available for insemination from male factor patients, we may be able to obtain better results with IVF. Various procedures, including swim-up, washing, and density gradients, have been used recently in attempts to improve sperm motility for IVF or intrauterine insemination treatments. Because of poor results when swim-up or simple washing methods were applied to male factor semen, we have been evaluating the use of density gradient procedures. Percoll has been used previously for density gradient centrifugation of spermatozoa and several reports note good yields and final motilities. 2•6- 10 However, only one report has dealt with initially poor semen specimens. 11 In this study, we chose to compare the efficacy of motile sperm selection by Percoll with that by Nycodenz, a density gradient

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Figure 5 Comparison of the number of motile spermatozoa harvested by Nycodenz selection with the number harvested by swim-up. The line is the line of equivalence between the two preparative methods (slope= 1.0).

medium whose constituent molecule has been approved for internal human use as an x-ray contrast medium. The results obtained in this study indicated that the two density gradient media were initially equivalent for sperm selection from poor semen samples. Overnight incubation then was carried out in order to determine whether the selection procedures affected sperm longevity, as motility must be maintained for 3 to 21 hours for successful IVF. 21 •22 The significantly larger time-dependent decrease in motility of spermatozoa selected on Percoll gradients suggested that this procedure may cause more membrane damage than Nycodenz selection. These findings would agree with the work of Arcidiacono et al., who reported membrane swelling and enzyme leakage following selection of motile human spermatozoa on continuous Percoll gradients.7 The results of this investigation also indicated a direct relationship between the initial sperm motility and the final motility following selection on discontinuous Nycodenz gradients (Fig. 4). Extrapolation of the regression line suggests that a final motility of 88% would be obtained if the initial motility was 70%, as is common for normal semen samples. It is therefore apparent that Nycodenz warrants further investigation as a density gradient medium for the selection of motile human spermatozoa from poor semen samples. In conclusion, using Nycodenz gradients for sperm selection from samples with low numbers of 340

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motile spermatozoa is successful and results in the collection of sufficient numbers of motile spermatozoa for IVF. A greater number of motile spermatozoa were harvested from Nycodenz gradients than from swim-up procedures. We have commenced a controlled trial of discontinuous Nycodenz gradients for sperm selection from oligoasthenozoospermic samples in our IVF program. So far, this has resulted in the establishment of three pregnancies, one of which was a successful twin pregnancy. The findings from this trial will be presented in detail in a subsequent publication. Investigations are proceeding in an attempt to further refine the Nycodenz selection procedure and to evaluate its use on semen samples from patients with spermatozoal autoantibodies, and on cryopreserved semen.

Acknowledgments. We thank Mr. Cvetko Smaila, Mr. Peter Elliott, and Mr. Elias Sokratis for assistance with semen analyses.

REFERENCES 1. Mortimer D: The male factor in infertility. Part II: Sperm function testing. In Current Problems in Obstetrics, Gynaecology and Fertility, Vol VIII, No.8. Chicago, Year Book Medical Publishers, 1985, 75 pp 2. Berger T, Marrs RP, Moyer DL: Comparison of techniques for selection of motile spermatozoa. Fertil Steril 43:268, 1984 3. Ericsson RJ, Langevin CN, Nishino M: Isolation of fractions rich in human Y sperm. Nature 246:421, 1973 4. Urry RL, Middleton RG, McNamara L, Vikari CA: The effect of single-density bovine serum albumin columns on sperm concentration, motility and morphology. Fertil Steril 40:666, 1983 5. Gorus FK, Pipeleers DG: A rapid method for the fractionation of human spermatozoa according to their progressive motility. Fertil Steril 35:662, 1981 6. Forster MS, Smith WD, Lee WI, Berger RE, Karp LE, Stenchever MA: Selection of human spermatozoa according to their relative motility and their interaction with zona-free hamster eggs. Fertil Steril 40:655, 1983 7. Arcidiacono A, WaltH, Campana A, Balerna M: The use of Percoll gradients for the preparation of subpopulations of human spermatozoa. Int J Androl 6:433, 1983 8. Lessley BA, Garner DL: Isolation of motile spermatozoa by density gradient centrifugation in Percoll. Gamete Res 7:49, 1983 9. Bolton VN, Braude PR: Preparation of human spermatozoa for in vitro fertilization by isopycnic centrifugation on selfgenerating density gradients. Arch Androl13:167, 1984 10. Dravland JE, Mortimer D: A simple discontinuous Percoll gradient for washing human spermatozoa. IRCS Med Sci 13:16, 1985 11. Hyne RV, Stojanoff A, Clarke GN, Lopata A, Johnston

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12. 13.

14. 15. 16.

WIH: Pregnancy from in vitro fertilization of human eggs after separation of motile spermatozoa by density gradient centrifugation. Fertil Steril 45:93, 1986 Almen T: Experimental investigations with iohexol and their clinical relevance. Acta Radiol Suppl 366:9, 1983 Assem ESK, Bray K, Dawson P: The release of histamines from human basophils by radiological contrast agents. Br J Radiol56:647, 1983 Dawson P: Some aspects of contrast medium chemotoxicity. Acta Radiologica Suppl 366:174, 1983 Nyegaard & Co., Norway: Unpublished data Rickwood D: Properties of iodinated density gradient media. In Iodinated Density Gradient Media: A Practical Approach, Edited by D Rickwood. Oxford, IRL Press, 1983, p8

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17. Overstreet JW, Katz DF, Hanson FW, Fonseca JR: A simple inexpensive method for objective assessment of human sperm characteristics. Fertil Steril31:162, 1979 18. Makler A: The improved ten-micrometer chamber for rapid sperm count and motility evaluation. Fertil Steril 33:337, 1980 19. Clarke GN: Unpublished data 20. Yates C, de Kretser DM: Male factor infertility and IVF. J In Vitro Fert Embryo Transfer. 4:141, 1987 21. Lopata A, McMaster R, McBain JC, Johnston WIH: Invitro fertilization of preovulatory human eggs. J Reprod Fertil 52:339, 1978 22. Perreault SD, Rogers BJ: Capacitation pattern of human spermatozoa. Fertil Steril 38:258, 1982

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