Comparison of techniques for selection of motile spermatozoa*†

Vol. 43, No.2, February 1985 Printed in U.8A.

FERTILITY AND STERILITY Copyright © 1985 The American Fertility Society

Comparison of techniques for selection of motile spermatozoa*t

Trish Berger, Ph.D.:j:§11 Richard P. Marrs, M.D.:j: Dean L. Moyer, M.D.~ University of Southern California, Los Angeles, and University of California, Davis, California

Procedures to separate motile sperm with high rates of recovery may have clinical application in in vitro fertilization and intrauterine insemination in increasing the probability of fertilization by a normal sperm and subsequent normal embryonic development. A two-step continuous Percoll gradient was an effective means of separating motile sperm which also had enhanced ability to penetrate zona-free hamster ova. However, the requirement for a high-speed centrifuge and rotor makes the procedure impractical in many cases. A one-step discontinuous Percoll gradient was also effective in separating a population of motile sperm. Comparison of the discontinuous Percoll gradient with other techniques for separation of motile sperm indicated the discontinuous Percoll gradient had advantages in terms of recovery, enhancement of motility, and increased ability to penetrate zona-free hamster ova. The velocity of selected sperm was not significantly different among techniques. The onestep discontinuous Percoll gradient appears to have value both for increasing homogeneity of human sperm populations used for basic research and in clinical practice for male subfertility. Fertil Steril43:268, 1985

Human cervical mucus has been reported to differentially select motile sperm and act as a barrier to nonmotile sperm. I, 2 However, in recent years, intrauterine insemination and in vitro fertilization have developed as treatments for both female factor and male factor infertility.3 In Received June 11, 1984; revised and accepted October 22, 1984. *Supported in part by National Institutes of Health grants 5R23HD16306 and 7R23HD18892 (to T. B.). tPresented in part at the Thirty-First Annual Meeting of the Pacific Coast Fertility Society, October 12 to 16, 1983, Rancho Mirage, California. :j:Department of Obstetrics and Gynecology, University of Southern California. §Department of Animal Science, University of California, Davis. IIReprint requests: Trish Berger, Ph.D., Department of Animal Science, University of California, Davis, California 95616. ~Department of Pathology, University of Southern California. 268

Berger et al. Selection of motile spermatozoa

both treatments, cervical selection of motile sperm is bypassed. Several laboratory techniques have been suggested as procedures for selecting motile sperm, including albumin gradients,4-6 Percoll gradients,7.8 swim-up procedures,9 and glass wool filtration. IO A high yield of motile sperm as well as enhancement of motility are necessary for a method to be practical-particularly with oligospermic ejaculates. Although the in vitro fertilization rate of mature human ova is quite high (80% to 90%)11 with essentially unselected sperm, the implantation rate is low. The decreased implantation rate may in part be due to fertilization by defective sperm. Possibilities of improving implantation rates following in vitro fertilization and of improving the fertilizing potential of sperm used for intrauterine insemination by improving sperm quality exist. These possibilities, as well as the need to increase the homogeneity of samples used for basic Fertility and Sterility

research, led to this study comparing techniques for selection of motile sperm. Recovery of motile sperm, enhancement of motility, and sperm quality assessed by the hamster penetration test were evaluated.

EXPERIMENTAL DESIGN

In the first experiment, the effect of the modified two-step continuous Percoll gradient separation was compared with the control washing procedure. Ejaculates from five presumed normal donors were used to compare enhancement of motility and penetration of zona-free hamster ova. In the second experiment, recovery and enhancement of motility in ejaculates from 21 donors were examined after a discontinuous one-step Percoll gradient. In the third experiment, four washing techniques were compared with regard to enhancement of motility, recovery of motile sperm, and penetrating ability using ejaculates from eight fertile and subfertile men. Subsamples were fixed and processed for electron microscopy following separation and washing in four of these ejaculates. Percentage of motility was estimated subjectively with phase-contrast optics at x 200 magnification. Data were subjected to analysis of variance using a mixed model with treatment as fixed and ejaculate as random independent variables. Percentage data were analyzed before and after transformation to the arc sin square root. Results of the analysis of variance of the untransformed data are presented. Differences in significance with the transformed data are noted in the text. SPERM PROCESSING

In the first and third experiments, sperm washing began within 1 hour of ejaculation. In the second experiment, washing began within 2.5 hours of ejaculation. The percent recovery was calculated as x 100.

(concentrationinitial x volumeinitial x motilitYinitial)

All separation techniques were performed at ambient temperature. Modified Ham's F-I0 medium (GIBCO, Grand Island, NY) was prepared and supplemented as previously described 12 and supVol. 43, No.2, .February 1985

CONTROL WASHING

An aliquot of semen was washed twice by centrifugation at 160 x g for 10 minutes with two volumes of medium containing 10% serum. The final pellet was then resuspended to 1 x 107 motile spermlml.

MATERIALS AND METHODS

(concentrationfinal x volUmefinal x motilitYfinal)

plemented with heat-inactivated frozen human fetal cord serum. All media were sterilized by filtration with a 0.2-,....m pore size.

PERCOLL GRADIENTS

Preliminary studies followed the procedures of Gorus and Pipeleers. 7 Ham's F-I0 (10 x) was prepared by dissolving a package of powdered medium sufficient for 11 in only 100 ml of double-distilled water. This was modified by supplementing with 18.75 mg penicillin (1790 U/mg), 12.50 mg streptomycin sulfate (740 mg/g) , 1680.4 mg Na bicarbonate, 507.6 mg K bicarbonate, and 183.9 mg Ca lactate 5 H 2 0 and adjusting the final volume to 109 ml for an osmolarity of 2800 mOsm. Nine parts of Percoll were added to one part of modified Ham's F-I0 (10 x).to make isotonic PercolI. The isotonic Percoll was further diluted with 1 x Ham's F-I0 for Percoll of the desired density (d). Semen was layered above 3 ml of 1.05 d PercolI and centrifuged for 20 minutes at 200 x g. A continuous Percoll gradient was formed with 4 ml of 1.10 d Percoll by centrifugation for 20 minutes at 20,000 x g. The pellet from the first step was resuspended in 0.5 ml and layered on top of the continuous gradient. After centrifugation for 20 minutes at 300 x g, the tube was pierced, and the first 20 drops were collected. This fraction was diluted to 1 x 107 motile spermlml without further washing. The requirement for a specialized centrifuge and rotor and the increased difficulty in maintaining sterility led to an adaptation of the discontinuous Percoll gradient system. 8 In this modification, isotonic Percoll was diluted with 1 x Ham's F-I0 for 90%, 80%, 70%, 55%, and 40% isotonic Percoll. Discontinuous gradients were made in 16 x 125-mm tissue culture tubes (Falcon 3033, Falcon Plastics, Oxnard, CA) by carefully pipetting 1.5 ml of 100%, 90%, 80%, 70%, and 55% Percoll such that interfaces were visible between the layers. Two milliliters of 40% Percoll was added in the same manner. One milliliter of semen was layered on top, and the gradient was ce!ltrifuged for 20 minutes at 300 x g. The seBerger et al. Selection of motile spermatozoa

269

men, 40%, 55%, and 70% layers were pipetted off and discarded. The 80%, 90%, and 100% layers were combined, further diluted with two volumes of Ham's F-10, divided into two tubes, and centrifuged at 160 x g for 10 minutes. The pellets were each suspended in 1 ml of medium containing 10% serum, recentrifuged, and resuspended to 1 x 107 motile sperm/ml.

DETERMINATION OF VELOCITY

A 7-1.Ll aliquot of sperm was placed on a warmed (37°C) microscope slide and covered with a warmed coverslip. Using phase-contrast illumination, 2-second exposures were taken of random fields. Negatives were projected on white paper, sperm tracks were traced, and velocity was calculated. Twenty-three to 156 tracks were measured for each treatment in each of eight ejaculates.

ALBUMIN GRADIENTS

Both bovine and human serum albumin gradients have been previously used with similar results4 -6 ; therefore, the more economical bovine serum albumin was used in this study. Two-step albumin gradients were prepared in 12 x 75-mm tissue culture tubes (Falcon 2054, Falcon Plastics) using 17% albumin and 7.5% albumin in Ham's F-10 medium. An aliquot of semen was washed once and resuspended to the original volume, and 0.5 ml of washed sperm was layered on top of each gradient. After 30 minutes, the washed sperm layer was removed by pipette. After an additional 60 minutes, the 7.5% albumin layer was also aspirated, leaving the highly motile sperm in the 17% albumin. These sperm were washed once by dilution with two volumes of medium and resuspended to 1 x 10 7 motile sperm/ ml in medium + 10% serum. SWIM-UP PROCEDURE

Semen (0.5 ml) was layered beneath 2 ml of medium in 12 x 75-mm culture tubes. After 60 minutes, the upper interface was gently aspirated with a Pasteur pipette. The aspirate was washed twice in serum-supplemented medium and resuspended to 1 x 10 7 motile sperm/ml. After an additional 60 minutes, the upper interface was again aspirated. HAMSTER TEST

The hamster penetration test was performed as previously described. 12 Sperm were preincubated for 3 hours at 37°C in 5% CO 2 in air prior to insemination of the zona-free ova. Gametes were then coincubated for 3 hours. Fixed and stained ova were subsequently examined for the enlarged sperm heads and associated tails characteristic of penetrated sperm. Both the percentage of ova penetrated and the average number of sperm which penetrated each ovum were evaluated. 270

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Selection of motile spermatozoa

DETERMINATION OF WHITE BLOOD CELLS (WBCs) IN SEMEN

A sperm smear was made of ejaculated sperm and air-dried. Slides were stained according to the procedure of Couture et al. 13 One thousand sperm and the associated WBCs were counted for each ejaculate. EVALUATION OF ELECTRON MICROGRAPHS

Micrographs were examined without knowledge of experimental treatment. Cell images with at least one dimension of 2.4 JJ.m which was not a longitudinal section of tail were tabulated according to the following criteria: (1) normal heads; (2) cytoplasm in the tail (probably cytoplasmic droplets) when either a cross-section of the midpiece was surrounded by cytoplasm or the posterior portion of the head had excess cytoplasm; (3) excess cytoplasm in the head when cytoplasm was present in the anterior portion of the head or in the subaerosomal space, or granularity indicative of cytoplasm was present in vesicles surrounded by nuclear material; (4) disintegrating cytoplasm with and without sperm for cytoplasmic vesicles with diffuse plasma membranes and contents; (5) multiple heads for a cytoplasmic vesicle containing a minimum of two separate heads in one section; and (6) cytoplasmic vesicles when normal cytoplasm appeared to be surrounded by an intact plasma membrane with no evidence of sperm contents. Seventy-nine to 245 sperm cells and the associated nonsperm cells were scored for each treatment in each of four ejaculates in the third experiment.

RESULTS As Table 1 shows, continuous Percoll gradients separated a sperm fraction with enhanced motility (79% versus 60% at insemination; P < 0.05) Fertility and Sterility

Table 1. Penetrating Ability and Motility of Sperm After Separation on a Continuous Percoll Gradient" % P~gres-

Sperm selection

sive motility

% Ova penetrated

60 79

40 70

Control Continuous PercolI gradient SEM b P

3 < 0.05

5 < 0.05

Sperm penetrated per ovum 0.49 1.37 0.38 < 0.01

aEach value represents the mean of five ejaculates. bStandard error of the mean.

and increased penetrating ability assessed by both percentage of ova penetrated (70% versus 40%; P < 0.05) and average number of sperm penetrating each ovum (1.37 versus 0.49; P < 0.01). The average initial motility of these five ejaculates was 60%, consistent with normal semen quality. In four ejaculates, dilution of the washed sperm with 50% isotonic Percoll to 1 X 107 motile sperm/ml had no effect on penetration (51 % versus 58%; standard error of the mean = 8; P> 0.25). In 20 of 21 ejaculates in the second experiment, the average recovery of motile sperm from a discontinuous Percoll gradient was 49%, with the range from 7% to 90%. Preseparation motility ranged from 10% to 70%, while the postseparation motility was 86% (40% to 95%), with an average increase in percentage of motility of 47. In one ejaculate with an initial motility of 5%, there was no enhancement of motility. In the third experiment, there was no difference in recovery of motile sperm between the control procedure (68%) and the Percoll separation (59%; Table 2). However, recovery of motile sperm was lower after separation on the albumin gradient (38%; P < 0.01 versus the control procedure and P < 0.05 versus the Percoll procedure) and after the swim-up technique (18%; P < 0.001,

compared with control and Percoll procedures). Approximately 68% of the motile sperm collected by the swim-up procedure were recovered in the first hour, and 32% in the second hour. The percentage of progressive motility after the Percoll and albumin procedures (88% and 81%, respectively) was higher than that observed with the control procedure (44%; P < 0.001) or in the fraction recovered from the swim-up technique (62%; P < 0.01). The average velocity appeared higher in the selected sperm fractions obtained from the albumin and Percoll gradients (0.27 mm -1 and 0.26 mm -1), compared with that observed for the control and swim-up fractions (0.24 mm- 1 and 0.22 mm -1); but the effect was not significant (0.05 < P < 0.10). The sperm fractions. recovered from the Percoll procedure had increased penetrating ability (P < 0.05), compared with that'in other procedures (36% of the ova penetrated versus 18%, 17%, and 14% with the control, albumin, and swim-up procedures, respectively). The effect on penetrating ability was also reflected in the increased number of sperm penetrated per ovum (0.48 in the Percoll-selected fraction versus 0.20, 0.24, and 0.17 following the control, albumin, and swim-up procedures, respectively). In general, the cell distribution following separation was similar between the Percoll and albumin gradients. There was a trend toward a higher percentage of normal heads, compared with the control (32% and 31 % ofthe cells versus 20%; P < 0.10), an increase in percentage of sperm with cytoplasm in the tail (40% versus 23%; P < 0.05), and a decrease in the number of sperm with excess cytoplasm in the head to 22% and 20% from 27% in the control (P < 0.05). There was also a slight trend toward a decrease in the number of disintegrating cytoplasmic vesicles (P < 0.25) and a decrease in the number of intact cytoplasmic vesicles (P < 0.10 for the untransformed data; P

Table 2. Effect of Separation Technique on Recovery, Motility, and Penetrating Abilitya Separation technique Control Discontinuous Percoll gradient Albumin gradient Swim-up technique SEM b

pc

% Progressive

motility

% Ova penetrated

Sperm penetrated per ovum

% Recovery of

motile sperm

Sperm attached per ovum

44 88

18 36

0.20 0.48

68 59

4.85 3.19

81 62 4 0.001

17 14 4 0.01

0.24 0.17 0.07 0.05

38 18 6 0.00'1

2.80 2.63 0.62 0.10

aEach value represents the mean of eight ejaculates. bStandard error of the mean. cp values are from analysis of variance of a significant effect of the separation technique. Vol. 43, No.2, February 1985

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< 0.01, transformed data). The cell distribution following the swim-up procedure was generally similar to that observed in the control unseparated fraction. However, there appeared to be a decrease in the proportion of sperm with excess cytoplasm in the head. In the eight ejaculates, there were 0 to 6 WBCs/1000 sperm.

DISCUSSION

The percent motility of the sperm fraction harvested from the swim-up technique in this study is lower than that observed by others or previously achieved in this laboratory when minimal sperm numbers were recovered. However, in attempts to improve the recovery rate, some nonmotile sperm were also aspirated, with a subsequent decline in motility. The swim-up procedure still had the lowest recovery rate, even with this overzealous collection. The continued presence of cytoplasmic vesicles after the swim-up procedure might be due to a tendency for these vesicles to float. The recovery rate from discontinuous Percoll gradients was similar to that reported for a preparative procedure. 14 However, selection of motile sperm did not occur to the same extent. The enhancement of motility is similar to that reported by Forster et al.,8 but recovery rates were not reported. Both the albumin and Percoll techniques enhanced motility, and the cell distributions observed with electron microscopy were also similar. However, in general, sperm separated from the albumin gradient did not have enhanced penetrating ability, although in one donor there was enhanced penetrating ability (32% greater than in the control procedure and 11% less than in the Percoll procedure). The difference in the penetrating ability of the fractions separated by the two techniques may indicate that different or slightly different sUbpopulations of motile sperm are selected by the two procedures. Since only 0.1% of the sperm in the culture dish have penetrated the ova, this difference could be difficult to detect. An alternative explanation is that Percoll has two effects: one is separating sperm on the basis of density, and the other is an effect on the sperm cell during the short-term exposure. Since dilution of sperm with isotonic Percoll did not increase penetrating ability, it would appear that both effects are necessary. 272

Berger et al. Selection of motile spermatozoa

The nonbeneficial effect of separation on an albumin gradient agrees with the previous report by Weeda and Cohen 15 that sperm separated by an albumin gradient did not have enhanced penetrating ability. Although the Percoll samples consistently had increased motility with respect to the standard washing procedure, increased penetrating ability was observed in some, but not all, samples. In this study, the increase in penetrating ability was not related to the incidence ofWBCs present, because all samples had < 1 WBC/100 sperm. These results suggest that the use of a Percoll gradient for separation of motile sperm may increase the fertilizing potential of some ejaculates. Due to the adequate recovery of motile sperm and the rapidity of the procedure, sperm separation on a Perc oIl gradient may be a useful procedure for intrauterine insemination and in vitro fertilization as well as for isolation of subpopulations of sperm for basic research.

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