Development of a technique for monitoring the contamination of human semen samples with leukocytes

FERTILITY AND STERILITY

Vol. 57, No.6, June 1992

Copyright c 1992 The American Fertility Society

Printed on acid-free paper in U.S.A.

Development of a technique for monitoring the contamination of human semen samples with leukocytes

Csilla Krausz, M.D.* Katrine West, B.Sc.t Donna Buckinghamt R. John Aitken, Ph.D.t:\: University of Edinburgh, Edinburgh, Scotland, and Universit6 di Firenze, Florence, Italy

Objective: To determine whether receptors for the N-formyl chemotactic peptide, FMLP, exist on the surface of human spermatozoa and regulate reactive oxygen species generation by these cells. Design: Chemiluminescent analysis of reactive oxygen species generation by suspensions of human spermatozoa before and after removal of leukocytes using a magnetic cell separation technique. Setting: Academic Research Institute. Patients: Unselected male volunteers. Results: Human sperm suspensions responded to FMLP and phorbol ester (PMA) with a burst of reactive oxygen species production. Autoradiographic analyses employing 3H FMLP and chemiluminescence studies involving the selective removal of leucocytes with anti -CD 45-coated magnetic beads demonstrated that the FMLP responses were because of leukocyte contamination. In contrast, reactive oxygen species production in response to PMA appeared to reflect the oxidant-generating capacity of both leukocytes and spermatozoa. Conclusion: The only cells present in the human ejaculate possessing detectable receptors for FMLP and capable of generating reactive oxygen species in response to this reagent come from the leukocyte population. Luminol-dependent, FMLP-induced, chemiluminescence provides a rational basis for monitoring the presence of leukocytes in suspensions of human spermatozoa. Fertil SteriI1992;57:1317-25 Key Words: Leucocytospermia, reactive oxygen species, FMLP

A highly specialized, biochemical attribute of the human spermatozoon is the capacity of this cell type to generate reactive oxygen species, including superoxide anion and hydrogen peroxide (1, 2). This activity is clinically significant because oxidative stress, created by the excessive generation of reactive oxygen species, is believed to playa significant role in the etiology of defective sperm function (1-6). The mechanism by which spermatozoa produce reReceived October 22, 1991; revised and accepted February 24, 1992. * Dipartimento di Fisiopatologia Clinica, Universit8. di Firenze. t Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, University of Edinburgh. Reprints requests: R. John Aitken, Ph.D., MRC Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, Scotland.

*

Vol. 57, No.6, June 1992

active oxygen species is believed to involve the mediation of a nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase in the sperm plasma membrane that catalyzes the transfer of electrons from cytoplasmic NADPH to molecular oxygen, achieving a one-electron reduction to superoxide anion, which then dismutates to hydrogen peroxide under the influence of superoxide dismutase. This biochemical pathway for superoxide production by human spermatozoa is very similar, in principle' to the mechanism by which activated polymorphonuclear leukocytes (PML) generate reactive oxygen species. The biochemical parallels between human spermatozoa and leukocytes are further reflected in the claim that both cell types contain specific receptor sites for synthetic N-formylmethionyl Krausz et al.

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peptides, such as N-formyl-methionyleucylphenylalanine (FMLP), that resemble the chemotactic factors produced by bacteria (7-9). The presence of such receptors on polymorphonuclear neutrophils (PMN) is in keeping with the biological role of these cells in the phagocytosis and destruction of pathogenic organisms. Accordingly, activation of the FMLP receptors on human leukocytes induces a powerful chemotactic response and the generation of reactive oxygen species (10). Because a chemotactic response is also elicited from human spermatozoa by the presence of FMLP (7), the possibility that N-formyl peptides might trigger the generation of reactive oxygen species by the sperm NADPH oxidase was investigated. If such a relationship existed, it would afford a mechanism by which bacteria, which are known (11) to have a direct immobilizing action on human spermatozoa, might exert a direct cytotoxic effect on these cells by the localized creation of oxidative stress. MATERIALS AND METHODS Sperm Preparation

The study population consisted of a cohort of 60 un selected donors who were normozoospermic according to the criteria laid down by the World Health Organization (12). The semen samples were produced by masturbation and collected into a sterile container for immediate transportation to the laboratory. After allowing at least 30 minutes for liquefaction to occur, the spermatozoa were fractionated on a discontinuous two-step Percoll gradient comprising a 3-mL volume of isotonic, (100%) PercolI overlaid by an additional 3-mL volume of 50% Percoll in a 10-mL conical-based sterile centrifuge tube. Isotonic (100%) Percoll was created by the addition of 300 mg of bovine serum albumin, 3 mg of sodium pyruvate, and 0.37 mL of a sodium lactate syrup to 10 mL of lOX concentrated medium 199 (Flow Laboratories, Irvine, Scotland) and mixing the resultant solution with 90 mL of Percoll (Pharmacia, Uppsala, Sweden): 50% Percoll was obtained by dilution with the medium developed by Biggers, Whitten, and Whittingham (BWW) (13), as described by Aitken and Clarkson (3). One to 3 mL of semen was layered on the top of each gradient and centrifuged at 500 X g for 20 minutes. Thereafter, the seminal plasma was discarded and the cells collected from the 50%/100% Percoll interface (50% fraction) and the base ofthe isotonic Percolliayer (100% fraction), respectively. The cells from each fraction were resuspended in 7 mL of 1318

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FMLP and human sperm

BWW, centrifuged at 500 X g for 5 minutes, and finally resuspended in BWW at a sperm concentration of 1 X 107 cells/mL. Leukocyte Removal

To remove the leukocytes from the sperm suspensions, a magnetic cell separation technique was used. For this purpose, magnetic Dynabeads (M450), coated with sheep anti-mouse immunoglobulin G (Dynal AS., Oslo, Norway) were labeled with an antibody against the common leukocyte antigen, CD45 (Scottish Antibodies Production Unit, Carluke, Scotland). Twenty-five microliters of Dyna beads were added to 500 ,uL sperm samples and incubated on a rotating wheel at room temperature for 2 hours. The leukocytes, bound to the Dyna beads, were finally separated from the spermatozoa using a magnetic field. The number of samples on which the magnetic cell separation technique could be performed was dependent on the number of spermatozoa recovered from the Percoll gradients for each individual specimen. Before magnetic bead separation, 33 and 41 samples were recovered for analysis from the 50% and 100% Percoll fractions, respectively, whereas after magnetic bead separation the corresponding figures were 39 and 60. Reactive Oxygen Species

The measurement of reactive oxygen species was normally carried out on a Berthold LB 9505 Luminometer (Berhold, Wildbad, Germany), at a chamber temperature of 37°C over a total time period of 30 minutes. The probe used was a 5-amino2,3 dehydro-1,4 phthalazinedone (luminol; Sigma Chemical Company, St Louis, MO) (1, 2). A 400-,uL aliquot of the cell suspension (at a sperm concentration of 1 X 107/mL) was incubated with 25 ,uM luminol, supplemented with 12.4 U horseradish peroxidase (Type VI, 310 U/mg; Sigma Chemical Company) for 10 minutes to sensitize the assay for the generation of extracellular hydrogen peroxide (14, 15). The basal signal obtained in this way was considered to be representative of the steady state situation. After allowing 10 minutes to capture this basal luminol-dependent signal, cells were stimulated with FMLP and monitored for an additional 20 minutes to determine the magnitude of the peak chemiluminescent response and allow the system to return to baseline. The cell suspensions were then stimulated with 100 nM 12-myristate, 13-acFertility and Sterility

etate, phorbol ester (PMA) and monitored for 10 minutes to assess the residual capacity of the cell population for reactive oxygen species generation, PMA representing the optimal stimulus for reactive oxygen species production by both spermatozoa and leukocytes. The responses to FMLP and PMA were recorded as the peak chemiluminescent signal after subtraction of the basal, steady state signal using the subtract function on the LB9505 luminometer. Where indicated in the figure legends, measurements of chemiluminescence were also made with a Berthold 9500T luminometer, in which case the chemiluminescent counts represented the integrated signal over a 10second period. Leukocyte Immunocytochemistry

The cells were pelleted by centrifugation at 500 X g for 5 minutes and resuspended in Dulbecco's

phosphate-buffered saline (PBS; Flow Laboratories) at half the original volume. Five microliters of each sperm suspension were then air dried in duplicate on the separate wells of a 12-spot Henley slide (C. A. Henley Ltd., Loughton, United Kingdom) and stored at -70°C until used. The staining procedure described by Aitken and West (14) was employed in these studies using an antibody against the common leukocyte antigen, CD45 (Scottish Antibodies Production Unit). The outcome of this immunocytochemical procedure was expressed relative to the concentration of spermatozoa, i.e., leukocytes X 10 4/10 7 spermatozoa. However, because the final sperm concentration employed in these studies was uniformly maintained at 1 X 107/mL, this method of expressing the results equated with the number of leukocytes X 104/mL. The resolution of the immunocytochemical technique at the sampling frequency employed in this study was 0.01 X 10 4 leukocytes/mL. Nucleated round cells that did not stain with the monoclonal antibody and exhibited the appropriate morphology (12) were taken to be precursor germ cells. Autoradiography

The presence of specific receptor sites for FMLP on human spermatozoa was examined using 60 nM 3H FMLP (1.48 to 3.22 TBq/mmol; New England Nuclear, Du Pont, Wilmington, DE) and sperm samples from both the 50% and isotonic (100%) regions ofthe Percoll gradients at a concentration of 1 X 107/mL. After a I-hour incubation in the Vol. 57, No.6, June 1992

presence of 3H FMLP, the cells were centrifuged at 500 X g for 5 minutes, washed, and finally resuspended in 4% glutaraldehyde; control incubations contained 40 JIM cold FMLP. The fixed cells were finally air dried onto cleaned microscope slides, washed twice with PBS, dehydrated through a series of ethyl alcohols (70%, 90%, 100%), and coated with L4 emulsion (Bford, Knutsford, United Kingdom). The emulsioncoated slides were then sealed with light-tight boxes and maintained at 4°C for 55 days. The slides developed by emersion in D19 developer (Kodak, Baxter Scientific Products, Irvine, CA) for 5 minutes, washed with distilled water, dried, stained with hematoxylin (10 seconds) and eosin yellow (0.5% in PBS for 3 to 4 minutes), and mounted with coverslips using the mounting medium, DPX (British Drug Houses, Poole, United Kingdom). RESULTS Unfractionated Cell Populations Response to FMLP

The unfractionated cell populations recovered from both the 50% and isotonic (100%) regions of discontinuous Percoll gradients responded to the presence of FMLP with a sudden burst of reactive oxygen species generation that was virtually instantaneous. Dose-dependent studies indicated that the amplitude of the chemiluminescent response increased with FMLP concentrations into the JIM range and, as a result, all subsequent experiments were conducted using a peptide concentration of 50 JIM. It was characteristic of the FMLP responses that they peaked rapidly and then gradually decayed taking 10 minutes to return to baseline. The mean response was 2 log orders of magnitude (P < 0.001) greater for the low density cells recovered from the 50%/100% Percoll interface (3.95 ± 1.42 X 106 counts per minute [CPM)) than those pelleting to the base of the isotonic (100%) Percolllayer (0.048 ± 0.015 X 106 cpm). Response to PMA

Once the cells had responded to FMLP, they were relatively refractory to further stimulation with this reagent, although they remained fully responsive to a reagent exhibiting a different Krausz et al.

FMLP and human sperm

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mechanism of action in the form of PMA, which bypasses surface receptors and directly stimulates protein kinase C. Thus, subsequent administration of PM A to the FMLP-treated samples consistently generated chemiluminescent responses in both the 50% and isotonic (100%) Percoll fractions. As with FMLP, the PMA-induced rise in chemiluminescence occurred without a detectable lag period but differed from the former in that the response was more sustained. Moreover, the peak response to PMA was lOX greater (P < 0.001) than that induced by FMLP (25.36 ± 7.60 X 106 and 0.48 ± 0.19 X 106 for the 50% and isotonic [100%] Percoll fractions, respectively). This was not because of

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o 2 4 6 8 Log (1 + x ) FMLP Chemiluminescence (counts) Figure 2 Chemiluminescent responses in the un fractionated, isotonic (100%) Percoll samples. (a), FMLP and (b), PMA responses in relation to leukocyte concentration; (c), relationship between PMA and FMLP responses. Elipse surrounds a cohort of 11 samples that responded to PMA but neither contained leukocytes, (panel b), nor exhibited a response to FMLP (panel c). Circle surrounds a cohort of 15 samples that did not contain leukocytes and failed to respond to FMLP (panel a) and in 4 cases, also failed to respond to PMA (panels b and c).

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1320

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FMLP and human sperm

a priming effect of the FMLP because a similar difference in the magnitude of the chemiluminescent responses was observed when the PMA and FMLP were added to separate aliquots of the same suspension (data not shown). Despite this difference in the magnitude of the responses to FMLP and PMA, the chemiluminescent signals induced by these reagents were highly (P < 0.001) correlated, giving r values of 0.826 and 0.761 for the 50% and isotonic (100%) Percoll fractions, respectively (Figs. 1 and 2). Within the unFertility and Sterility

fractionated, isotonic (100%) Percoll samples, 15 specimens did not exhibit a chemiluminescent response to FMLP, and 4 of these also failed to respond to PMA (Fig. 2). Leukocytes and Germ Cells

The 50% Percoll fractions were not only characterized by high levels of reactive oxygen species generation but also a relative abundance of leukocytes and germ cells, compared with the cell populations isolated on isotonic (100%) Percoll. Hence, although the purified sperm suspensions pelleting to the base of the isotonic (100%) Percoll fraction possessed a minimal concentration of leukocytes (0.026 ± 0.005 X 104 /10 7 spermatozoa) and precursor germ cells (0.274 ± 0.078 X 10 4 /10 7 spermatozoa), the sperm suspensions recovered from the 50%:100% Percoll interface contained significantly (P < 0.001) greater numbers of both cell types (0.380 ± 0.85 and 0.953 ± 0.117 X 104 /10 7 spermatozoa, respectively). Leukocytes and Chemiluminescence

The presence of leukocytes in the 50% Percoll fractions was significantly (P < 0.001) correlated with the generation of reactive oxygen species in response to both FMLP (r = 0.740) and PMA (r = 0.664) (Fig. 1). Similarly, within the isotonic (100%) Percoll fraction (Fig. 2) significant (P < 0.001) correlations were noted between the low concentration of leukocytes in these samples and the chemiluminescent response to PMA (r = 0.526) and FMLP (r = 0.677). Of the 41 unfractionated, isotonic (100%) Percoll fractions on which measurements of FMLP responses were undertaken, 15 exhibited no chemiluminescent response to FMLP whatsoever, and all 15 were free of leukocyte contamination (Fig. 2). Four of these refractory specimens also failed to exhibit a chemiluminescent response to PMA (Fig. 2), whereas the reminder exhibited PMA responses of variable intensity. Because leukocytes were not present in this remaining subgroup of samples, these PMA-induced signals must have originated from the spermatozoa. These results suggested that the chemiluminescent responses to FMLP were entirely dependent on the presence of leukocytes. When significant numbers of leukocytes were present, as in the 50% Percoll fractions, chemiluminescent responses to FMLP were observed that were highly correlated with leukocyte number (Fig. 1). When leukocytes were absent, as in the case of approximately one Vol. 57, No.6, June 1992

third of the samples prepared on isotonic (100%) Percoll, FMLP responses were not obtained (Fig. 2). In contrast, the chemiluminescent responses to PMA, although correlated with leukocyte number (Figs. 1 and 2), were not entirely dependent on the presence of these cells (Fig. 2). Fractionated Cell Populations

Magnetic Bead Separation

To further explore the relationship between leukocyte contamination and the chemiluminescent responses given by human sperm suspensions, the former were selectively removed using a magnetic cell separation technique and the consequences, in terms of reactive oxygen species generation, examined. Treatment of both the 50% and isotonic (100%) Percoll fractions with anti-CD45-coated magnetic beads significantly (P < 0.001) and selectively reduced the concentration ofleukocytes (0.027 ± 0.007 and 0.002 ± 0.001 X 104 /10 7 for the 50% and isotonic, 100%, fractions, respectively) without having any impact on the number of precursor germ cells present in these fractions. This reduction in leukocyte numbers was associated with a highly significant decline (P < 0.001) in the chemiluminescent responses recorded in both the 50% and isotonic (100%) Percoll fractions in response to both FMLP (0.042 ± 0.017 X 106 and 0.0003 ± 0.0002 X 106 cpm for the 50% and isotonic [100%] Percoll fractions, respectively) and PMA (0.314 ± 0.154 X 106 and 0.0146 ± 0.0053 cpm for the 50% and isotonic [100%] Percoll fractions, respectively). In the case of 50% Percoll fractions, sufficient numbers of leukocytes remained after treatment with the anti-CD45-coated magnetic beads for significant (P < 0.01) linear correlations to be observed between the residual concentration of these cells in the sperm suspensions and the responses to FMLP (r = 0.655; Fig. 3) and PMA (r = 0.431; Fig. 3). In these 50% fractions, the responses to the agonists were also correlated with each other (r = 0.68; Fig. 3), presumably because of the common influence of leukocyte contamination on both types of response. Of the 39, 50% Percoll fractions subjected to the magnetic cell separation technique, 16 were rendered leukocyte-free by this treatment, and although none of these samples exhibited a chemiluminescent response to FMLP (Fig. 3),12 responded positively to stimulation with PMA (Fig. 3). Treatment of the isotonic (100%) Percoll fractions with anti-CD45-coated beads removed all traces of detectable leukocytes from 56 of the 60 samples on Krausz et al.

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The above results indicated that the stimulation of reactive oxygen species production by FMLP was invariably tied to the presence of leukocytes; the more leukocytes in the sperm suspension, the greater the FMLP response. These results could be explained in one of two ways: either the FMLP responses were a direct result of reactive oxygen species production by leukocytes or this peptide induced leukocytes to produce cytokines that, in turn, ini-

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which this technique was performed. In all 56 samples there was no detectable FMLP response (Fig. 4) although in 26 specimens a chemiluminescent response to PMA could still be elicited (Fig. 4). Two samples containing leukocytes gave positive responses to both FMLP and PMA. Two further samples in which low concentrations ofleukocytes (0.014 and 0.02 X 104/107 spermatozoa) were detected failed to exhibit a detectable response to either PMA or FMLP (Fig. 4). 1322

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Figure 4 Chemiluminescent responses after treatment of the isotonic (100%) Percoll fractions with anti-CD45 coated beads. (a), FMLP and (b), PMA responses in relation to leukocyte contamination; (e), relationship between PMA and FMLP responses. Elipse surrounds a cohort of 26 samples that responded to PMA but neither contained leukocytes (panel b) nor responded to FMLP (panel c). Circle surrounds a cohort of 56 samples that did not contain leukocytes and failed to exhibit a response to FMLP (panel a); 30 of these samples also failed to respond to PMA (panel b). Fertility and Sterility

tiated reactive oxygen species production by the spermatozoa. To examine the second possibility, an experiment was conducted involving the coincubation of purified suspensions of human spermatozoa with peripheral leukocytes that had, or had not been, activated by a 5-minute pre-exposure to 50 ~M FMLP. In this study, peripheral PMN, at a concentration of 1 X 106/mL, were pre incubated with FMLP or diluent control medium for 5 minutes before being washed and resuspended in BWW. The control and FMLP -treated leukocytes were then added to purified suspensions of human spermatozoa such that the final concentration of leukocytes and spermatozoa was 1 X 106 and 1 X 107/mL, respectively: control incubations consisted of 1 X 107 spermatozoa/mL in isolation. During the ensuing 2-hour incubation period, chemiluminescent measurements confirmed that the suspensions containing FMLPactivated leukocytes were at least 10 times more active in terms of reactive oxygen species production than the control incubations containing spermatozoa alone or spermatozoa cocultured with nonactivated leukocytes. Once the 2-hour incubation period was over, the spermatozoa were recovered from these coincubations by centrifugation through isotonic (100%) Percoll and re-examined for their capacity to generate reactive oxygen species. The experiment was repeated six times, and on each occasion the spermatozoa recovered from the cocultures of spermatozoa and FMLP-activated leukocytes were no more active in generating reactive oxygen species than either the untreated controls or the spermatozoa coincubated with nonactivated leukocytes. This result suggested that the association between the presence of leukocytes and FMLP-induced chemiluminescence was because of the direct stimulation of reactive oxygen species production by the PMN and did not involve the indirect stimulation of reactive oxygen species production by the spermatozoa. This conclusion was further supported by the auto radiographic studies that failed to detect FMLP receptors on human spermatozoa while confirming the presence of a high concentration of binding sites on the surface of infiltrating leukocytes (Fig. 5). DISCUSSION

The results obtained in this study have clearly demonstrated that human spermatozoa do not possess receptors for the formyl chemotactic peptide, FMLP. This contradicts the results obtained in a Vol. 57, No.6, June 1992

Figure 5 Autoradiography of two human sperm suspensions with 3H-FMLP illustrating lack of ligand binding to the spermatozoa (open arrow) in contrast to the heavily labeled leukocytes (black arrow).

previous study (7) that described a single class of high-affinity, low-capacity binding sites on human spermatozoa, on the basis of kinetic studies using radiolabeled ligand. In the present study, both the auto radiographic analyses and the cell separation experiments indicated that the only cell type in the human ejaculate that exhibits a detectable capacity to bind formyl peptides and respond to such reagents with a burst of reactive oxygen species production are the leukocytes that contaminate every human semen sample (16). In light ofthese data, the most plausible explanation for the apparent detection of FMLP receptors on human spermatozoa (7) is that the sperm suspensions used in such studies were contaminated with leukocytes. The use of anti-CD45 magnetic beads to remove the leukocyte population appeared to be an extremely effective means of purifying human sperm suspensions, particularly when the latter had previously been isolated by centrifugation through a column of isotonic (100%) Percoll. After a protocol involving recovery of the spermatozoa from isotonic (100%) Percoll and magnetic bead separation, 56 of the 60 samples analyzed were free of leukocyte contamination. Of the 4 remaining samples, 2 that still contained a low concentration of active leukocytes could readily be identified by virtue of their responKrausz et al.

FMLP and human sperm

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siveness to both FMLP and PMA. For such samples, another round of magnetic cell separation would seem appropriate. The 2 remaining samples contained extremely low concentrations of leukocytes «0.02 X 104/mL) and yet did not exhibit a chemiluminescent response to FMLP or PMA. There are two possible explanations for such findings: (1) that the leukocytes identified by the panleukocyte monoclonal antibody were not polymorphs but another species of leukocyte, such as a lymphocyte that does not generate reactive oxygen species and (2) that the leukocytes were polymorphs, but they were no longer viable and hence were incapable of giving a response to any form of agonist, including PMA. Regardless of which explanation is correct, the presence of small numbers of lymphocytes or nonviable leukocytes in occasional sperm samples from which FMLP responses are not obtained does not significantly compromise the validity of the FMLP provocation test as a means of monitoring the contamination of sperm samples with leukocytes capable of creating oxidative stress. Thus, in the present study, a total of 87 independent leukocyte-free samples were generated from the two Percoll fractions, and none of these samples exhibited a positive FMLP response. Conversely, 86 samples were analyzed in which leukocytes were positively identified by immunocytochemistry, and 83 gave a positive FMLP response. Notwithstanding the possibility discussed above, that the leukocytepositive/FMLP-negative samples may have contained lymphocytes or nonviable polymorphs, the FMLP provocation test would appear to be at least 98% (170/173) accurate in detecting the presence or absence of low concentrations of leukocytes. In light of these findings, a preparative protocol involving: (1) isolation of partially purified sperm suspensions by centrifugation through isotonic (100%) Percoll; (2) leukocyte removal using antiCD45-coated magnetic beads; and (3) a chemiluminescent FMLP provocation test to assess the purity ofthe sample would seem a rational and effective way to prepare leukocyte-free sperm samples. This protocol should be of value whether the objective is therapeutic, in terms of preparing purified suspensions of human spermatozoa from patients exhibiting leukocytospermia or scientific, in terms of preparing pure sperm suspensions for investigating the mechanisms regulating reactive oxygen species production. Although it is certainly true that leukocytes constitute a major source of reactive oxygen species in the unfractionated ejaculate (17), the use ofthis pu1324

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FMLP and human sperm

rification strategy in the present study has emphasized the potential that human spermatozoa possess for reactive oxygen species generation. Hence, 12 of the 16 samples isolated from the 50%:100% Percoll interface and 26 of the 56 samples recovered in the isotonic (100%) Percoll fraction were shown to generate reactive oxygen species in response to PMA in the absence of leukocyte contamination, as indicated by both immunocytochemical analysis and the absence of detectable FMLP responses. The extreme variability in PMA-induced reactive oxygen species production by purified suspensions of human spermatozoa is intriguing and is reflective of a phenomenon that in its most extreme form constitutes a biochemically definable pathology in which a loss of sperm function is associated with peroxidative damage induced by excessive reactive oxygen species generation (1). Although the original studies in this area (1, 4, 6) may have been influenced by the fact that leukocytes were not removed from the sperm suspensions used for analysis, recent studies in which this criterion was met (2) have confirmed the importance of reactive oxygen species derived from the spermatozoa in pathological conditions such as oligozoospermia. In diagnostic terms, the use of magnetic cell separation techniques, in conjunction with the FMLP-provocation test to monitor the purity of the samples, should provide a convenient means of preparing purified sperm suspensions with which to monitor the source and etiology of reactive oxygen species produced by the ejaculates of subfertile patients.

REFERENCES 1. Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species. J Reprod Fertil 1987;81:459-69. 2. Aitken RJ, Buckingham D, West K, Wu FC, Zikopoulos K, Richardson DW. On the contribution ofleucocytes and spermatozoa to the high levels of reactive oxygen species recorded in the ejaculates of oligozoospermic patients. J Reprod Fertil. In press. 3. Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J AndroI1989;9:367-76. 4. Jones R, Mann T, Sherins RJ. Peroxidative breakdown of phopholipids in human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil Steril1979;31:531-7. 5. Aitken RJ, Irvine DS, Wu FC. Prospective analysis of spermoocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol 1991;164:542-51. 6. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide

Fertility and Sterility

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8.

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and superoxide in human spermatozoa. J AndroI1987;8:338-48. Gnessi L, Fabbri A, Silvestroni L, Moretti C, Fraioli F, Pert CB, et al. Evidence for the presence of specific receptors for N-formylchemotactic peptides on human spermatozoa. J Clin Endocrinol Metab 1986;63:841-6. Williams LT, Snyderman R, Pike MC, Leftowitz RJ. Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes. Proc Nat! Acad Sci USA 1977;74: 1204-8. Bass DA, Dechatelet LR, McCall CEo Independent stimulation of motility and the oxidative metabolic burst of human polymorphonuclear leukocytes. J Immunol 1978;121:172-6. Wymann MP, von Tschamer V, Deranleau DA, Baggiolini M. The onset of the respiratory burst in neutrophils. J BioI Chem 1987;26:12048--53. Teague NS, Boyarsky S, Glenn JF. Interference of human spermatozoa motility by Escherichia coli. Fertil Steril1971;22: 281-5. World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus in-

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