t Diagnostic Andrology Service, Bourn Hall London. Csilla Krausz, M.D.*t Carla Mills, Ph.D.t Shaun Rogers, B.Sc. t

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1 FERTILITY AND STERILITY Copyright e 1994 The American Fertility Society Printed on acid-free paper in U. S. A. Stimulation of oxidant generation by human sperm suspensions using phorbol esters and formyl peptides: relationships with motility and fertilization in vitro Csilla Krausz, M.D.*t Carla Mills, Ph.D.t Shaun Rogers, B.Sc. t S.L. Tan, M.D.:j: R. John Aitken, Ph.D. II Universita di Firenze, Florence, Italy; Bourn Hall London; The London Women's Clinic, London; and Medical Research Council (MRC) Reproductive Biology Unit, Edinburgh, United Kingdom Objectives: To investigate the influence of reactive oxygen species generated by human spermatozoa and contaminating leukocytes on sperm movement and fertilization in vitro. Design: A chemiluminescence technique, using luminol and peroxidase, was used to monitor the generation of reactive oxygen species by human sperm suspensions and the results were correlated with sperm movement and the fertilization of human ova in vitro. Setting: Diagnostic Andrology Laboratory and IVF Clinic. Patients: Infertile couples undergoing IVF therapy. Results: An N-formyl-methionyl-leucyl-phenylalanine () provocation test was used to demonstrate that the presence of leukocytes in 28.5% of the sperm preparations was associated with elevated levels of spontaneous reactive oxygen species production, impaired movement, and a reduced capacity for fertilization in vitro. In the absence of leukocytes, exposure to phorbol ester stimulated a burst of reactive oxygen species generation by human spermatozoa, the magnitude of which was correlated highly with a loss of sperm motility but not with fertilization rates observed in the concurrent IVF cycle. Conclusion: Leukocyte contamination of human sperm preparations can be detected readily by -induced, luminol-dependent chemiluminescence and the results have an important bearing on the fertilizing capacity of the spermatozoa in vitro. Fertil Steril 1994;62: Key Words: Leukocytes, reactive oxygen species, motility, IVF Received December 14, 1993; revised and accepted April 21, * Dipartimento di Fisiopatologia Clinica, Universita di Firenze. t Diagnostic Andrology Service, Bourn Hall London. t The London Women's Clinic. MRC Reproductive Biology Unit, Centre for Reproductive Biology, University of Edinburgh. II Reprint requests: John Aitken, Ph.D., MRC Reproductive Biology Unit, 37 Chalmers Street, Edinburgh EH3 9EW, United Kingdom (FAX: ). The ability of reactive oxygen species to initiate the peroxidation of unsaturated fatty acids in the sperm plasma membrane is thought to play a key role in the etiology of male infertility ( 1-7). Experimental studies using a xanthine oxidase-based, free radical-generating system have shown that the oxygen metabolite that is the most damaging to human spermatozoa is hydrogen peroxide, the presence of which leads to a loss of motility and the capacity for sperm-oocyte fusion (8-10). Within human sperm suspensions, the reactive oxygen species that might induce such peroxidative damage could emanate from the spermatozoa themselves or any phagocytic leukocytes contaminating the sperm preparations (2, ) In clinical terms, it is important to differentiate between these two sources of reactive oxygen species because this information has a bearing on the strategies that might be used to reduce the oxidative stress experi- Krausz et al. Reactive oxygen species and IVF 599

2 enced by the spermatozoa during the course of IVF therapy. To address this issue, a simple, rapid method for screening human sperm preparations and differentiating between reactive oxygen species emanating from contaminating leucocytes or spermatozoa is required. An important reagent in this regard is formyl leucyl phenylalanine (), a peptide that has been shown to bind specifically to receptor sites on polymorphonuclear leukocytes and stimulate the generation of reactive oxygen species by these cells and not spermatozoa (14). As a result of such specificity, can be used as a means of selectively triggering the generation of reactive oxygen by leukocytes to monitor the potential of these cells for damaging the fertilizing capacity of human spermatozoa (14). Alternatively, the capacity of the sperm population to generate reactive oxygen species can be monitored in leukocyte-free sperm suspensions by adding the 12-myristate, 13-acetate phorbol ester, the most powerful stimulus for oxidant generation by human spermatozoa yet identified (14). In this study we have used these strategies for assessing the potential influence of reactive oxygen species generated from these two cellular sources on human sperm function and correlated the results with the outcome of IVF therapy. Study Population MATERIALS AND METHODS The study population comprised the male partners of 27 unselected couples and a single donor, who produced semen specimens for IVF therapy at the London Women's Clinic. Twelve couples were affected by pure female factor infertility (nine cases of tubal damage, two cases of tubal damage with associated endometriosis, and one case of tubal damage in a patient who also was exhibiting the polycystic ovarian syndrome), six couples presented with male factor infertility, and five couples exhibited both tubal damage and a male factor. In four cases the infertility was of unknown etiology. The semen profiles for this cohort of patients were volume, 2.87 ± 0.28 ml; motility, 41.0% ± 4.03%; sperm concentration 69.2 ± 8.7 X 10 6 /ml; total count, ± 23.4 X 10 6 /ejaculate; and morphology, 54.8% ± 2.2% normal. Sperm Preparation The semen samples were produced by masturbation. After allowing at least 30 minutes for liquefac- tion to occur, the spermatozoa were fractionated on a discontinuous three-step Percoll gradient comprising: 0.3 ml 85% Percoll, 0.5 ml 65% Percoll, and 0.5 ml 50% Percoll, respectively, in a 10-mL conical-based, sterile centrifuge tube. Isotonic (100%) Percoll was created by the addition of 3 mg sodium pyruvate and 0.37 ml of a sodium lactate syrup to 10 ml of lox concentrated Earle's medium (Flow Laboratories, Irvine, United Kingdom) and mixing the resultant solution with 90 ml of Percoll (Pharmacia, Uppsala, Sweden). To this solution was added 9 ml of a 5% human serum albumin (HSA) preparation (Albuminar; Armour Pharmaceutical Co., Eastbourne, United Kingdom) and 2 ml of 1 M HEPES salts (Flow Laboratories), creating a basic stock of 89% Percoll. This stock then was diluted with Earle's medium (Flow Laboratories) supplemented with 0.5 mg/ml HSA to give the final concentrations used in the gradient. One half to 1 ml of semen was layered on the top of three to four gradients and centrifuged at 400 X g for 15 minutes. Thereafter, the seminal plasma and the 50% and 65% Percoll fractions were discarded and the cells were collected from the base of the 85% Percolllayer. The cells were resuspended in 1 ml of supplemented Earle's medium, centrifuged at 200 X g for 10 minutes, and finally resuspended in supplemented Earle's medium at a sperm concentration of 4 X 10 6 cells/ml. An aliquot of the preparation was used to inseminate the oocytes collected under transvaginal ultrasound guidance using the long protocol for luteinizing hormone-releasing hormone administration described by Tan et al. (15), and the remaining material was used for the assessment of motility and chemiluminescence. For this cohort of subjects, an average of ± 1.06 ova were recovered during the oocyte recovery procedure. Reactive Oxygen Species The measurement of reactive oxygen species was carried out on a Berthold Luminometer (Berthold, Wildbad, Germany) at a chamber temperature of 37 C over a total time period of 20 minutes. The probe used was a 5-amino-2,3 dehydro-1,4 phthalazinedone (luminol; Sigma Chemical Company, St. Louis, MO). A 400-L aliquot of the cell suspension used for the insemination of the oocytes was incubated with 25 M luminol supplemented with 12.4 U horseradish peroxidase (Type VI, 310 U/mg; Sigma Chemical Company) for 6 minutes to sensitize the assay 600 Krausz et al. Reactive oxygen species and IVF Fertility and Sterility

3 for the generation of extracellular hydrogen peroxide (16). After allowing 6 minutes to capture this basal luminal-dependent signal, cells were stimulated with and monitored for an additional 7 minutes to determine the magnitude of the peak chemiluminescent response and to allow the system to return to baseline. The cell suspensions then were stimulated with 100 nm 12-myristate, 13-acetate phorbol ester and monitored for 7 minutes to assess the residual capacity of the cell population for reactive oxygen species generation (14). Theresponses to and phorbol ester were recorded as the integration of the chemiluminescence counts over a 5-minute observation period. Motility Analysis The motility analysis was carried out on a computer-aided sperm analysis system (version 8; Hamilton-Thorne, Beverly, MA). The movement characteristics were recorded at 37 C for an untreated aliquot of each sample, 1 hour after the spermatozoa had been prepared for IVF. In addition, the sperm populations that had been subjected to chemiluminescence analysis were assessed, 40 minutes after exposure of the spermatozoa to 100 nm phorbol ester, on the specimens that did not give aresponse to. Because the spermatozoa analyzed under these circumstances had been exposed to luminol and peroxidase before the addition of phorbol ester, the influence ofthese reagents on the movement characteristics of human spermatozoa also were assessed carefully on nine independent samples and were found to be without significant effect. The measurements were conducted in 200- J,Lm flat capillary tubes (Camlab, Cambridge, United Kingdom) and at least 100 motile cells were assessed for each determination. These determinations were carried out in duplicate and the results were averaged. The movement characteristics analyzed included percentage motile, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and amplitude of lateral sperm head displacement (ALH). "Per cent rapid" represented cells exhibiting a VAP > 25 JLm/s whereas "per cent progressive motility" equated with a straightness of >75%. The sort criteria used for identifying hyperactivated motility were curvilinear velocity > 100 J.Lm/s, linearity (VSL/VCL) < 65%, and ALH > 7.5 J.Lm. Statistics The data were transformed where necessary to normalize their distribution and then were sub- jected to linear regression analysis; nonparametric statistics (Mann-Whitney U, Wilcoxon signed ranks) were used to assess the significance of differences between groups using the Statview 2 program (Abacus Concepts, Berkeley, CA) on an Apple Macintosh Centris 650 computer. RESULTS Sperm populations that had been prepared for IVF therapy were first treated with to stimulate any leukocytes contaminating the cell suspensions to generate reactive oxygen species (14). Addition of was found to stimulate an increase in reactive oxygen species generation in 8 of 28 samples analyzed (28.5%). For this subgroup of leukocyte-contaminated samples, the generation of reactive oxygen species was elevated significantly over the remainder of the study population not only with respect to the -induced chemiluminescent response (P < 0.001) but also with respect to the basal chemiluminescent signal (P < 0.05; Fig. 1) and the responses to phorbol ester (P < 0.001). The chemiluminescent signal observed in the presence of also was correlated highly with the basal, spontaneous levels of reactive oxygen species generation by these sperm preparations (r = ; P < 0.001). Moreover, the magnitude of the induced increase in reactive oxygen species generation (integrated signal - integrated basal signal) also was correlated significantly with the spontaneous, basal level of activity (r = 0.513, p < 0.01). Within this subgroup of leukocyte-contaminated samples, the movement characteristics of the spermatozoa were impaired significantly relative to the uncontaminated, -negative specimens, with respect to several aspects of sperm movement, including percentage motility (P < 0.05), progressive motility (P < 0.05), V AP (P < 0.01), and VSL (P < 0.01), as illustrated in Figure 1. Moreover, this cohort of leukocyte-contaminated samples also were characterized by significantly (P < 0.05) lower fertilization rates in the simultaneous IVF cycle (Fig. 1) and a significant negative correlation was observed between the intensity of the log-transformed chemiluminescent signal after treatment and the percentage of human ova fertilized (r = ; P < 0.001). These results indicated that the generation of reactive oxygen species by contaminating leukocytes was associated with impaired sperm function. To determine whether the poten- Krausz et al. Reactive oxygen species and IVF 601

4 ?l ::; Ci.lso > 50 a) b) + + c) d).l!! I!! c:.2 ' Cl) u. + + Cl) e) f) u c: 6.5 Cl) - :5 5.5.&:; u 5i..!D. ' = ) but not with any of the other criteria examined. To determine whether the reactive oxygen species actually generated by the spermatozoa in the presence of phorbol ester-influenced sperm function, the movement characteristics of the sperm populations after phorbol ester treatment were examined in relation to the magnitude of the phorbol ester-induced chemiluminescent responses. This analysis revealed that the addition of phorbol ester to leukocyte-free samples induced a highly significant increase in the generation of reactive oxygen species and a corresponding decline in the quality of sperm movement (Fig. 2). Thus, the addition of phorbol ester was associated with significant declines in motility (P < 0.01), progressive motility (P < 0.001), VAP (P < 0.001), VCL (P < 0.001), VSL (P < 0.001), ALH (P < 0.01), and hyperactivation (P < 0.01) relative to the untreated controls. Moreover, there was a striking correlation between the levels of reactive oxygen species generated by the a) b) Figure 1 The samples characterized by -positive chemiluminescent signals were associated with significant decreases in a number of movement characteristics relative the -negative specimens. Such differences were observed with every movement characteristic examined, including (A) percentage motility, (B) percentage progressive motility, and (C) VSL. (D), The fertilization rates observed in the positive samples were also significantly suppressed whereas both (E) the basal, spontaneous level of reactive oxygen species generation and (F) the 12-myristate, 13-acetate phorbol esterinduced activity were significantly elevated. * P < 0.05; ** P < 0.02; ***P < c) d) tial of the spermatozoa to generate reactive oxygen species was associated similarly with sperm dysfunction, the responses of the remaining 20 leukocyte-free samples to phorbol ester stimulation were analyzed. Within this subset of samples, 14 (70%) gave a chemiluminescent response to the phorbol ester. In contrast to the signals, these phorbol ester-induced responses were not correlated with the basal, spontaneous level of reactive oxygen species generation. As a consequence, the responses to phorbol ester were correlated only weakly with the movement characteristics recorded in untreated aliquots of the same samples, exhibiting significant negative correlations with both straightness (r = ) and beat cross frequency (r 80 e) f) Figure 2 Stimulation of human spermatozoa with the 12- myristate, 13-acetate phorbol ester () in the presence of peroxidase leads to the suppression of sperm movement as evidenced by statistically significant reductions in (A) percentage motility, (B) percentage progressive motility, (C) VAP, (D) VCL, (E) VSL, and (F) ALH. **P < 0.01; ***P < Krausz et al. Reactive oxygen species and IVF Fertility and Sterility

5 a) y = x , r2 =.48 60r b) 60 y = x , r2 = E Q. 9l 20 ;! Log Chemiluminescence Log Chemiluminescence 40 Q. 30 I 20 c) y = x , r2 =.401 d) y = 57.47x , r2 =.574 Figure 3 The stimulation...j u of reactive oxygen species production by human spermato 9l > 30 zoa by the 12-myristate, 13- acetate phorbol ester () 20 was associated with a highly ;!. significant decline in sperm movement, including such criteria as (A) percentage motility, 10 (B) percentage progres sive motility, (C) VAP, and (D)VCL. Log Chemiluminescence Log Chemiluminescence 40 spermatozoa and the extent to which sperm movement was impaired, giving correlation coefficients of r = for percentage motility, r = for progressive motility, r = for VAP, r = for VCL, and r = for VSL (Fig. 3). Despite this close relationship between the magnitude of the phorbol ester-induced chemiluminescent signals and impaired sperm motility in these leukocytefree samples, the potential to generate such responses was not correlated with the fertilization rates recorded in the concurrent IVF cycle. DISCUSSION The data obtained in this study suggest that the use of chemiluminescence procedures to detect the presence of leukocytes in human sperm suspensions may be of significant importance in the context of IVF therapy. The measurement of induced chemiluminescence has been shown to give such an excellent correlation with leukocyte numbers that it obviates the need to perform laborious manual cell counts (12, 14). In clinical terms, the speed of the chemiluminescence technique means that an assessment of leukocyte contamination can be carried out during the sperm preparation procedure and additional steps can be taken to complete the removal of the leukocytes should the test prove positive. This contrasts with the immunocytochemical detection of leukocytes using anti -CD45 monoclonal antibodies (12), which takes several hours to perform and can give only a retrospective indication of leukocyte contamination. In addition to the speed and convenience of the test, it gives a functional assessment of the level of leukocyte contamination, because it provides information on the capacity of these cells to generate reactive oxygen species, which are known to be potentially damaging to both gametes and embryos (1-4, 17, 18). The inherent sensitivity of chemiluminescent methods is also a strong point in their favor because the levels of leukocyte contamination observed in such Percoll-purified material is extremely low (4, 12). In view of the inherent advantages of -induced chemiluminescence in detecting leukocyte contamination, the present study set out to examine the diagnostic value of this test in an IVF-ET setting. Krausz et al. Reactive oxygen species and IVF 603

6 Despite the paucity of leukocytes in such sperm suspensions, there was a correlation between the intensity of the signals generated by these cells and the functional competence of the spermatozoa, judging from their impaired motility and poor capacity to fertilize human ova in vitro. These data are presented with the caveat that the number of -positive samples recorded in this study was low and the correlations observed will have to be confirmed with larger groups of patients. Nevertheless, the negative relationship between leukocyte contamination and sperm function is consistent with the known susceptibility of human spermatozoa to oxidative stress (2, 3) and the practical implications ofthese results are such that they should be of immediate interest to IVF-ET specialists. The mechanism by which contaminating leukocytes impair sperm function is suggested by the correlation observed between the -induced chemiluminescent signals and the basal, spontaneous levels of reactive oxygen species generation in these sperm suspensions. Thus, the leukocytic infiltration associated with the -positive samples was associated with a significantly elevated level of spontaneous reactive oxygen species generation compared with the -negative samples as indicated in Figure 1E. To appreciate the magnitude of this difference in basal levels of reactive oxygen species generation, it should be stressed that these data have been presented as the log of the chemiluminescence counts in Figure 1. If the untransformed values were considered, the mean chemiluminescence values in the -positive samples (3.8 X 10 6 counts/min) were seen to be 10-fold higher than the -negative samples (0.3 X 10 6 counts/min). This considerable difference in spontaneous reactive oxygen species generation readily could account for the reduced fertilizing potential observed in the presence of leukocytes. It is well established that human spermatozoa are very susceptible to oxidative stress and inverse correlations exist between the degree of peroxidative damage sustained by these cells and their capacity for movement and oocyte fusion (2, 19). Moreover, coincubation of human spermatozoa with leukocytes has been shown to reduce sperm motility as a consequence of oxidative damage that could be prevented by the concomitant presence of catalase and dimethylsulfoxide (20). The conclusion that low levels of leukocyte contamination can impair the fertilizing potential of human spermatozoa as a consequence of oxidative damage has clear implications for the design of IVF protocols that might be used to treat such patients. The negative impact of oxidative stress might be addressed by the development of antioxidant-supplemented IVF culture media and/or the selective removal of contaminating leukocytes using antibody-coated paramagnetic beads or ferrofluids (14). A second potential source of oxidative stress in the sperm suspensions used for IVF is the spermatozoa themselves. To assess the potential of the spermatozoa for reactive oxygen species generation, leukocyte-free, -negative samples were stimulated with phorbol ester. A majority of the spermatozoa treated in this way generated reactive oxygen species and this activity was correlated with the quality of sperm movement in the original sperm preparations, as reflected by the significant negative associations with beat cross frequency and straightness. Much more dramatic correlations were observed when the movement characteristics of the spermatozoa were examined once they had been exposed to phorbol ester. Under these circumstances there was an extremely close correlation between the magnitude of the free radical response given by the spermatozoa and the suppression of sperm movement. Although the production of toxic oxygen metabolites by human spermatozoa is associated with a loss of sperm function (4), the potential to express this activity was not correlated with the fertilization rates observed in the present study. These results are an expression of the fact that, within this set of patients, the spermatozoa, while having the potential to generate reactive oxygen species on exposure to phorbol ester, were not expressing this activity spontaneously. It was only when they were stimulated with phorbol ester that good evidence for a free radical-mediated loss of sperm function was obtained. In conclusion, these results emphasize the benefits of monitoring reactive oxygen species production by human sperm suspensions prepared for IVF therapy. From the observations made in this study it would appear that oxidant generation by contaminating leucocytes is a significant factor in limiting the fertilizing potential of human spermatozoa in an IVF context. In contrast, the intrinsic ability of human spermatozoa to generate reactive oxygen species after stimulation with phorbol ester did not appear to reflect the capacity of these cells for fertilization. Whether sperm pathologies associated with the chronic generation of damaging oxidants (21) would reveal a closer correlation between phorbol ester-induced chemiluminescence and fertilization 604 Krausz et al. Reactive oxygen species and IVF Fertility and Sterility

7 in vitro will have to examined in future studies. Whatever the source of the reactive oxygen species spontaneously generated by human sperm suspensions, the results obtained in this study emphasize the importance of counteracting the availability and activity of these molecules in the development of IVF protocols designed to meet the special problems set by male factor infertility patients. REFERENCES 1. MacLeod J. The role of oxygen in the metabolism and motility of human spermatozoa. Am J Physiol 1943;138: Jones R, Mann T, Sherins RJ. Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil Steril1979;31: 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: Aitken RJ, Buckingham D, West K, Wu FC, Zikopoulos K, Richardson DW. On the contribution of leucocytes and spermatozoa to the high levels of reactive oxygen species recorded in the ejaculates of oligozoospermic patients. J Reprod Fertil1992;94: Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl1989;9: Aitken RJ, Irvine DS, Wu FC. Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol 1991;164: Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. J Androl 1987;8: Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steril 1992; 57: de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa: I. effects on the motility of intact spermatozoa and on sperm axonemes. J Androl1992;13: de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa: II. Depletion of adenosine-triphos- phate (ATP) plays an important role in the inhibition of sperm motility. J Androl 1992;13: Aitken R J, Buckingham D, Harkiss D. Use of a xanthine oxidase oxidant generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil1993;97: Aitken RJ, West KM. Analysis of the relationship between reactive oxygen species production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. Int J Androl 1990;13: Kessopoulou E, Tomlinson MJ, Barratt CLR, Bolton AE, Cooke ID. Origin of reactive oxygen species in semen; spermatozoa or leukocytes? J Reprod Fertil1992;94: Krausz C, West K, Buckingham D, Aitken RJ. Development of a technique for monitoring the contamination of human semen samples with leucocytes. Fertil Steril 1992;57: Tan S-L, Kingsland C, Campbell S, Mills C, Bradfield J, Alexander N, et a!. The long protocol of administration of gonadotropin-releasing hormone agonist is superior to the short protocol for ovarian stimulation for in vitro fertilization. Fertil Steril 1992;57: Aitken RJ, Buckingham DW, West KM. Reactive oxygen species and human spermatozoa; analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence. J Cell Physiol 1992;151: Nasr-Isfahani M, Johnson M, Aitken RJ. The effect of iron and iron chelators on the in vitro block to development of the mouse preimplantation embryo: BTT6 a new medium for improved culture of mouse embryo in vitro. Hum Rep rod 1990;5: Nasr-Isfahani M, Aitken RJ, Johnson MH. The measurement of H 20 2 levels in preimplantation embryos from blocking and non-blocking strains of mice. Development 1990;109: Aitken RJ, Harkiss D, Buckingham D. Relationship between iron-catalysed lipid peroxidation potential and human sperm function. J Reprod Fertil1993;98: Kovalski NN, de Lamirande E, Gagnon C. Reactive oxygen species generated by human neutrophils inhibit sperm motility: protective effect of seminal plasma and scavengers. Fertil Steril1992;58: Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FCW. Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl 1989;10: Krausz et al. Reactive oxygen species and IVF 605