Laboratory of Clinical Immunology, Russian Scientific Centre for Obstetrics, Gynecology and Perinatology, Moscow, Russia
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Abstract |
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Key words: antigen antibody complex/antisperm antibodies/flow cytometry/human spermatozoa
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Introduction |
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The conflicting notions about the significance of antisperm immunity in fertilization may be connected either with the methodological problems (Collins et al., 1993) and (or) with the problem of adequacy of methods used for ASA detection (Helmerhorst et al., 1999
). One of the main characteristics of antisperm immunity widely used both in diagnostics of immunological infertility and in experimental research is the presence of ASA on the surface of live spermatozoa detected by MAR (mixed agglutination reaction) test (Hinting et al., 1988
; Hjort, 1999
; Mahmoud and Comhaire, 2000
).
Flow cytometry (FCM) is the other prospective method permitting quantitative estimation of ASA on the surface of live spermatozoa (Haas and Cunningham, 1984; Sinton et al., 1991
; Räsänen et al., 1992
, 1994
, 1996
; Nikolaeva et al., 1993
, 1997
; Ke et al., 1995
; Nicholson et al., 1997
). The possibility of detecting ASA on the surface of living cells is considered a significant advantage of the MAR and FCM tests and both avoid the disadvantages of methods where fixed spermatozoa are used, e.g. ELISA (Ackerman et al., 1981
; Zanchetta et al., 1982
), fluorescein-conjugated antiglobulin assay (Hjort and Hansen, 1971
; Tung et al., 1976
) and radiolabelled antiglobulin assay (Haas et al., 1980
). Fixation of spermatozoa may lead to nonspecific binding of IgG, detection of intracellular antigens, denaturation of sperm antigens or membrane damage resulting in false-positive or false-negative results.
However, use of live cells for ASA detection can also compromise the interpretation of results obtained. It is known that cross-linking of surface antigens (Ag) by multivalent antibodies (Ab) or AgAb complexes by second antibodies can cause aggregation of AgAb complexes into patches and caps; these phenomena have been observed in many cell types (Taylor et al., 1971; Bretscher and Raff, 1975
). The processes of capping that occur during interaction between ASA bound to human spermatozoa and second antibodies has been described in several reports (Hjort and Hansen, 1971
; Cross and Moore, 1990
; Haas et al., 1991
). It was shown that capping may be accompanied by shedding of AgAb complexes from the sperm surface and that it gives a misleading picture of regions to which antibodies were directed (Trimmer and Vacquier, 1988
; Cross and Moore, 1990
; Shaha, 1994
).
Thus, it should not be excluded that the processes of antigen interaction with ASA and/or AgAb complexes with second antibodies can result in a significant change both to the distribution and to the quantity of AgAb complexes on the sperm surface. The aim of this study was to estimate the effect of the AgAb complex aggregation on the results of ASA detection by FCM.
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Materials and methods |
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Blood samples (5 ml) without anticoagulant were taken from six hitherto infertile men with previously confirmed male autoimmunity to spermatozoa and from a healthy donor. The blood was allowed to clot and then was centrifuged. The serum was divided into 100 µl aliquots and kept at 20°C until use. Before use, all the sera were heat inactivated at 56°C for 30 min.
Mixed agglutination reaction test
The direct MAR test (Jager et al., 1978; Hinting et al., 1988
) was performed by mixing on a microscope slide one drop (approximately 10 µl in volume) of fresh semen, one drop of latex particles coated with IgG and one drop of antiserum from rabbit immunized against human IgG (SpermMar Kit®; FertiPro, Aalter, Belgium). The reactions were examined by phase contrast microscopy at x400, and the percentage of motile spermatozoa carrying one or more latex particles was determined by the scoring of 100 motile spermatozoa. The results were read after 23 min and again after 10 min. The indirect MAR test was performed after incubation of 25 µl of ASA-negative donor semen swim-up fraction (>20x106 cells/ml) with 25 µl of complement-inactivated patient serum. The suspension was incubated for 30 min at room temperature, washed twice in phosphate-buffered saline (PBS), pH 7.4 and analysed.
Sperm labelling with IgG antisperm antibodies
Motile spermatozoa were provided by swim-up procedure and then mixed with diluted (1:2) ASA-positive sera (indirect MAR% = 100%) and with medium 199 (Sigma, St Louis, MO, USA) containing 1.5% bovine serum albumin (M199-BSA-1.5%) (negative control). Antisperm antibody-negative serum diluted two-fold (indirect MAR% = 0%) was used in some experiments. All the mixtures were incubated at 4°C and at cell concentration 10x106/ml (unless otherwise indicated in Figures 1 and 4) for 30 min and then washed twice in PBS.
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Incubations with cytochalasin B
Swim-up spermatozoa were incubated overnight in M199-BSA-1.5% with or without cytochalasin B (40 mmol/l) (Sigma) at sperm count 10x106/ml. Then sperm samples (1x106/ml) were incubated with diluted (1:2) ASA-positive serum or M199-BSA-1.5% and labelled with FITC-anti-HIgG. All manipulations were performed at 23°C.
Incubations with sodium azide
Antibody-coated spermatozoa were incubated at 4°C or 23°C with FITC-anti-HIgG (1x106 cells/ml and 10x106 cells/ml) in the presence of metabolic inhibitor sodium azide (4 mg/ml) (Sigma). Control tubes did not contain sodium azide.
Labelling of fixed spermatozoa
Swim-up spermatozoa of eight donors were fixed in 4% paraformaldehyde for 15 min, washed twice in PBS, incubated with M199-BSA-0.5% for 30 min and then incubated with two-fold diluted ASA-positive and ASA-negative sera and with M199-BSA-1.5% (negative control). All manipulations were performed at 23°C and sperm count 10x106/ml. All the sperm samples were incubated with FITC-anti-HIgG, as mentioned above, and some of them were incubated with non-immune FITC-labelled goat anti-human IgG (Sigma).
Flow cytometric analysis
All the samples were analysed by Bryte® (Bio Rad; Microscience Ltd, Hemel Hempstead, UK) or Facscan® (Becton Dickinson, Immunocytometry Systems, CA, USA) (results presented in Figures 1 and 6). A gate was set on dot plot distributions of forward versus 90° light scatter to exclude debris and clumps from the analysis. Fluorescence data of 5000 spermatozoa were collected with logarithmic amplification for green fluorescence specific for antibody staining and for red fluorescence specific for dead sperm staining. Negative controls were included in all experiments and showed a background fluorescence of virtually zero. The percentage of positive spermatozoa among live spermatozoa (FCM%) was determined by subtraction of background fluorescence from each histogram. To assess the fluorescence intensity that reflects ASA quantity on the surface of spermatozoa (sperm antibody load), the peak channel number associated with the highest number of fluorescent cells was determined. The inter- and intra-assay variation of the percentage of dead spermatozoa was <3%, and those of FCM% was <8%. The intra-assay variation of the fluorescence intensity was <10% and interassay variation was <24%.
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Statistical analysis
Comparison between the groups was made using the Wilcoxon matched pairs signed rank sum test. The relationship between sperm viability, FCM% and sperm antibody load was determined using the Spearman rank correlation. The level of significance was set at P < 0.05.
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Results |
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The concentration of antibody-coated spermatozoa incubated with FITC-anti-HIgG and the temperature of incubation were found to have influence on the parameters of FCM analysis (Figure 2, Table I
). There were significant differences in the results of ASA detection in one serum under different conditions (Figures 24
). Sperm antibody load in seven of nine samples of antibody-coated spermatozoa obtained from different donors was comparatively low (range of peak channel fluorescence intensity was 2178) regardless of the incubation conditions (Figures 2 and 3A
). In two samples after incubation of antibody-coated spermatozoa (1x106 sperm count/ml) with FITC-anti-HIgG at 4°C, fluorescence of high intensity was detected (peak channel = 673 and 875) (Figures 2 and 3B
). Increase of temperature to 23°C or sperm concentration to 10x106/ml resulted in reduction of sperm antibody load in these samples by 87.397.1% (Figures 2 and 3C, D
). The FCM% following the incubation of spermatozoa from nine semen samples with ASA-negative serum did not exceed 3% under any conditions.
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Incubation of antibody-coated spermatozoa with FITC-anti-HIgG in the presence of sodium azide at different temperatures and sperm concentrations had no influence on the results of the FCM test (data not shown).
Fixation of sperm samples with paraformaldehyde before ASA-positive serum addition was used to avoid AgAb complex aggregation. The high percentage (89.195.5%) of dead spermatozoa with high fluorescence intensity (peak channel ranged from 723 to 835) was estimated after incubation of fixed cells of five semen samples with ASA-positive serum (Figure 3H). The fluorescence intensity of the spermatozoa was much lower after their incubation with ASA-negative serum (range of peak channel was 521). In three semen samples, both in control samples and after incubation of spermatozoa with ASA-positive serum 15.548.5% live spermatozoa with high fluorescence intensity were detected (Figure 3I, J
). When fixed spermatozoa from these samples were incubated with non-immune FITC-IgG, the FCM results were negative.
The parameters of FCM analysis dramatically changed at different dilutions of ASA-positive serum and/or FITC-anti-HIgG (Figure 4). Both raising and lowering dilutions of first and/or second antibodies resulted in increase of FCM% and sperm antibody load. Sperm viability was decreased dramatically at 1:8 ASA-positive serum dilution and 1:50 secondary antibodies dilution. Under such conditions the maximal sperm antibody load (peak channel = 2206) was shown.
The relationship between distribution of AgAb complexes on the sperm surface and sperm antibody load was estimated (Figure 5). The head of live spermatozoa with a very high fluorescence intensity was uniformly labelled and a spotted type of fluorescence was detected on the tail (Figure 5a
). The pattern on the head and tail of dead fixed spermatozoa with high level of fluorescence intensity was uniform (Figure 5b
). On the surface of live spermatozoa with high fluorescence intensity there was diffuse labelling which was not entirely uniform (Figure 5c,d
). Fluorescent clusters, the size, number and localization of which varied considerably, were detected on the surface of live and dead antibody-coated spermatozoa with relatively low fluorescence intensity (Figures 1C and 5e
).
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Discussion |
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The present study shows that false-negative results of indirect FCM test may be due to disappearance of ASA from the sperm surface after binding of ASA with antigens. It is known that interaction of ASA with human sperm surface antigens and/or complexes with second antibodies can cause aggregation of cell surface molecules into patches and caps (Hjort and Hansen, 1971; Cross and Moore, 1990
; Haas et al., 1991
). The disappearance of the cap may be due to endocytosis and subsequent degradation and/or shedding of AgAb complexes, as reported for many types of cells (Matsuo et al., 1985
; Caldwell et al., 1986
). However, endocytosis in mature spermatozoa is unlikely but there are data on shedding of AgAb complexes from the sperm surface in rat, sea urchin and rabbit (Gaunt et al., 1983
; Trimmer and Vacquier, 1988
; Shaha, 1994
). The present study has revealed the typical changes of fluorescence pattern from homogeneous distribution to the cap formation through patches followed by decrease of sperm antibody load, and seems to testify to the AgAb complex shedding. It should be noted that small fluorescent particles, probably the shed complexes in some samples, were detected both by fluorescent microscopy and FCM (data not shown). Thus, shedding is the most probable mechanism that leads to removal (partial or complete) of AgAb complex from the cell surface and to false negative results of FCM test. The concentration of fluorochromes to a small area of sperm surface also decreased fluorescence intensity detected by cytometer (Chanh and Alderete, 1990
).
It has been shown that the human spermatozoon is a highly polarized cell with a surface membrane which may be divided into some functionally, structurally and biochemically distinct domains (Bearer and Friend, 1990). Probably the independence of AgAb complex aggregation and shedding in these domains can determine the simultaneous presence of caps and patches of different localization on the sperm surface detected by fluorescence microscopy.
The inhibition of AgAb complex aggregation was attempted by some methods. Like immunoprecipitation reactions, antibody-induced aggregation of sperm surface antigens requires lattice formation, and is critically dependent upon antibody concentrations (Gaunt et al., 1983). The present study shows that increase of FCM% and sperm antibody load can take place in cases of both excess and lack of ASA and/or second antibodies. Therefore, the optimal dilution of immunoreagents which inhibits aggregation and shedding of AgAb complexes and maximally enhances FCM test sensitivity is determined by sperm surface antigen expression and ASA titre in serum. It should be noted that these dilutions are not universal for different semen and sera samples.
The temperature decrease and use of cytoskeleton disrupting agent cytochalasin B resulted in AgAb complex aggregation and shedding inhibition only in a minority of sperm samples. We did not detect shedding inhibition with the use of metabolic inhibitor sodium azide. However, these data do not give definite evidence concerning either cytoskeleton independence or energy independence of aggregation because in some samples shedding can also take place in the case of aggregation inhibition.
Stimulation of AgAb complex shedding may take place during FCM analysis and may result from a mechanical influence on the cells during washing procedures (resuspension and centrifugation) and/or during spermatozoa moving in liquid stream under high pressure. The washing procedure also causes intensification of reactive oxygen species (ROS) generation (Aitken and Clarkson, 1988; Shekarriz et al., 1995a
). A high level of ROS generation is associated with sperm membrane damage through spontaneous lipid peroxidation (Storey, 1997
) and may enhance surface receptor shedding in some types of cells (Philippova et al., 1994
; Hino et al., 1999
).
It should not be excluded also that appearance of ASA-negative spermatozoa might be due to the spontaneous acrosome reaction of antibody-coated spermatozoa (Lansford et al., 1990; Harrison et al., 1998
) and/or acrosome reaction induced with second antibodies (Nikolaeva et al., 1998
).
So the relatively low sperm antibody load detected by indirect FCM test (Sinton et al., 1991; Ke et al., 1995
; Nicholson et al., 1997
) or by direct FCM test (Nikolaeva et al., 1993
) may be due to the experimental procedures. It is interesting that Räsänen et al. (1992, see Figure 2B
) has demonstrated a subpopulation of ASA-positive spermatozoa with very high fluorescence intensity using FCM for ASA detection in native semen. A relatively high concentration of second antibodies, the single washing procedure and the short duration of centrifugation used by the authors appear to inhibit the aggregation and/or shedding of AgAb complexes.
The significant positive correlation between both the FCM% and sperm antibody load with the percentage of live spermatozoa may demonstrate the relationship between intensity of surface complex shedding and the sensitivity of spermatozoa to damage. The maximal shedding intensity was demonstrated at the shortest incubation time of antibody-coated spermatozoa with second antibodies. The clustering of AgAb complexes on the cell surface also occurs within 12 min after cross-linking of sperm surface antigens with antibodies (Trimmer and Vacquier, 1988). Probably, the stimulation of shedding and antibody-coated sperm damage may be observed in the correspondence between AgAb complex aggregation and sperm washing. Significant heterogeneity of spermatozoa as regards the viability and amount of ASA on the sperm surface may indicate either varying resistance of antibody-coated spermatozoa at different stages of aggregation to experimental manipulation, or the different ability of antibody-coated spermatozoa to react to washing by aggregation (Figure 7
). Thus, when aggregation coincides with washing of antibody-coated spermatozoa, or when washing induces aggregation, there is a possibility that false negative results will be obtained by FCM test. Positive results of FCM test can be obtained in cases when aggregation is completely blocked and cannot be stimulated by washing or when the aggregation processes are finished.
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The manipulations with ASA-positive spermatozoa during MAR test are minimized and there is a small possibility of AgAb complex shedding that enhances the sensitivity of MAR test. However, Gould et al. (1994) have shown that spermatozoaimmunobead co-incubation during the detection of ASA by MAR test results in decrease of the number of spermatozoa bound to immunobeads. Thus, it should not be excluded that shedding can also take place during detection of ASA in some sperm samples by MAR test.
To exclude the effect of the surface molecules, lateral rearrangement on FCM test results, the paraformaldehyde-fixed cells were used. Previously it has been shown that paraformaldehyde fixation increases the postfixation binding of immunoglobulin to the sperm surface (Sinton et al., 1991). Our data have shown fluorescence of homogeneously distributed AgAb complexes on the fixed sperm surface to be in reality much higher than that of live spermatozoa with caps on their surface but equal to that of live ASA-positive spermatozoa with patching of AgAb complexes. The absence of AgAb complex aggregation on the fixed sperm surface as well as the low level of unspecific binding of immunoglobulin makes the fixed spermatozoa a more acceptable model for ASA determination by indirect FCM test than native spermatozoa. However sperm antibody load of fixed spermatozoa was much lower than that of native uniformly labelled spermatozoa. It is possible that disruptive effects of fixation can include removal or destruction of some epitopes of surface antigens. So the use of fixed cells can also impose a definite limitation on the interpretation of obtained results.
In some cases IgG were detected on the surface of live MAR-negative spermatozoa after their fixation with paraformaldehyde. It should not be excluded that the pattern of AgAb complex distribution on the sperm surface might determine the MAR test results. Perhaps the presence of homogeneously distributed immunoglobulin on the cell surface results in false-negative results of MAR test and promotes the preservation of vitality after fixation in these sperm samples. Obviously, further researches on the effect of aggregation concerning MAR test results are necessary.
Thus, percentage of antibody-coated spermatozoa and sperm antibody loadthe FCM parameters used both in experimental researches and for diagnostics of immunological infertility and efficacy of immunological infertility treatmentdepend on the aggregation of AgAb complexes, which, in turn, may be determined by cell factors (antigens and/or metabolic characteristics of spermatozoa) as well as by the ratio of immunoreagents (surface antigens, ASA and second antibodies). There is no linear correlation between the quantity of ASA on the surface of live, unfixed spermatozoa and in the tested serum. So the results of indirect FCM test should not be used for quantitative ASA estimation without excluding the post-binding changes of ASA quantity on the live sperm surface. The FCM can be very useful for further detailed analysis of the relationships between the AgAb complex aggregation and shedding, acrosome reaction and ROS that might be important for the development of adequate quantitative assay of ASA in serum and secretions of fertile and infertile patients.
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Acknowledgments |
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Notes |
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* Presented at the International Symposium on Male Infertility and Assisted Reproduction, Genk, Belgium, April 2225, 1998, and at the European Meeting of Immunology and Reproduction, Rome, Italy, October, 2829, 1999.
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Submitted on March 9, 2000; accepted on September 8, 2000.