Antisperm antibodies detection by flow cytometry is affected by aggregation of antigen–antibody complexes on the surface of spermatozoa*

Marina A. Nikolaeva1, Vladimir I. Kulakov, Irina V. Korotkova, Elena L. Golubeva, Diana V. Kuyavskaya and Gennady T. Sukhikh

Laboratory of Clinical Immunology, Russian Scientific Centre for Obstetrics, Gynecology and Perinatology, Moscow, Russia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Flow cytometry (FCM) analysis of live antibody-coated spermatozoa subjected to immunofluorescence staining (FCM test) is considered an objective method for the quantitative detection of antisperm antibodies (ASA). But the cross-linking of cell surface antigen (Ag) with bivalent antibodies and/or antigen-antibody (Ag–Ab) complexes with second antibodies may induce the reorganization of surface components (patching and capping) and result in their shedding from the sperm surface. The present study estimates the relationship between aggregation of Ag–Ab complexes on the sperm surface and the results of indirect FCM test. Swim-up spermatozoa of normozoospermic men were incubated with ASA-positive sera from infertile patients and with second antibodies fluorescein isothiocyanate (FITC)-labelled goat anti-human IgG polyclonal antiserum under different conditions and then analysed by FCM and fluorescence microscopy. It was shown that low temperature, cytochalasin B, excess or lack of the primary and/or secondary antibodies and sperm fixation by paraformaldehyde may inhibit aggregation and shedding of Ag–Ab complexes and dramatically increase ASA quantity determined on the sperm surface. However, inhibition of aggregation on the live sperm surface was observed only in a minority of ASA-positive samples and was poorly reproducible using semen of different donors. A high probability of Ag–Ab complex shedding from the sperm surface during experimental manipulation limits the use of indirect FCM test for quantitative ASA determination.

Key words: antigen antibody complex/antisperm antibodies/flow cytometry/human spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The concept of antisperm antibodies (ASA) in fertilization is based on their presence in serum and different secretions of the human reproductive tract. However, although some researchers have pointed to the prevalence of ASA in infertile patients (Bronson et al., 1984Go; Clarke et al., 1985Go; Isojima, 1988Go; Bronson, 1999Go), other authors revealed ASA in a relatively high percentage of both fertile and infertile men and women (Crister et al., 1989Go; Eggert-Kruse et al., 1989Go; Windt et al., 1989Go; Collins et al., 1993Go; Tomlinson et al., 1993Go). Thus, the role of ASA in reproduction remains debatable.

The conflicting notions about the significance of antisperm immunity in fertilization may be connected either with the methodological problems (Collins et al., 1993Go) and (or) with the problem of adequacy of methods used for ASA detection (Helmerhorst et al., 1999Go). 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., 1988Go; Hjort, 1999Go; Mahmoud and Comhaire, 2000Go).

Flow cytometry (FCM) is the other prospective method permitting quantitative estimation of ASA on the surface of live spermatozoa (Haas and Cunningham, 1984Go; Sinton et al., 1991Go; Räsänen et al., 1992Go, 1994Go, 1996Go; Nikolaeva et al., 1993Go, 1997Go; Ke et al., 1995Go; Nicholson et al., 1997Go). 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., 1981Go; Zanchetta et al., 1982Go), fluorescein-conjugated antiglobulin assay (Hjort and Hansen, 1971Go; Tung et al., 1976Go) and radiolabelled antiglobulin assay (Haas et al., 1980Go). 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 Ag–Ab complexes by second antibodies can cause aggregation of Ag–Ab complexes into patches and caps; these phenomena have been observed in many cell types (Taylor et al., 1971Go; Bretscher and Raff, 1975Go). 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, 1971Go; Cross and Moore, 1990Go; Haas et al., 1991Go). It was shown that capping may be accompanied by shedding of Ag–Ab complexes from the sperm surface and that it gives a misleading picture of regions to which antibodies were directed (Trimmer and Vacquier, 1988Go; Cross and Moore, 1990Go; Shaha, 1994Go).

Thus, it should not be excluded that the processes of antigen interaction with ASA and/or Ag–Ab complexes with second antibodies can result in a significant change both to the distribution and to the quantity of Ag–Ab complexes on the sperm surface. The aim of this study was to estimate the effect of the Ag–Ab complex aggregation on the results of ASA detection by FCM.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specimen collection and preparation
Fresh semen samples were collected by masturbation from healthy donors after 3–4 days of sexual abstinence and analysed immediately after liquefaction according to the World Health Organization criteria (WHO, 1992). Only specimens with counts >60x106/ml, motility rate >50% and MAR percentage = 0% were used (n = 29).

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., 1978Go; Hinting et al., 1988Go) 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 2–3 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 4GoGo) for 30 min and then washed twice in PBS.



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Figure 1. Results of (A, B) FCM and (C) combination of phase-contrast and fluorescence microscopy analysis of antibody-coated spermatozoa after their incubation with (A) non-immune FITC-labelled goat IgG (negative control) or (B, C) FITC-anti-HIgG. Incubations of spermatozoa with ASA-positive serum and antibody-coated spermatozoa with FITC-anti-HIgG were performed at 23°C and sperm count 10x106/ml. (A, B) Multiparameter plots with the x axis the green (FITC-anti-HIgG) fluorescence, the y axis the red (propidium iodide, PI) fluorescence, and the z axis the number of cells. Fluorescence data were collected in logarithmic mode. (C) relatively large clusters and/or small spots of green fluorescence were localized at different regions of tail and/or head of live spermatozoa and PI-stained dead spermatozoa. Original magnification x630.

 


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Figure 4. The effect of the dilution of ASA-positive serum and/or FITC-anti-HIgG on the results of indirect FCM test. Spermatozoa were incubated with ASA-positive serum or M199-BSA-0.5% (control) and with FITC-anti-HIgG, then washed once before FCM analysis. All manipulations were performed at 4°C and sperm count 1x106/ml. Flow cytometric plots of the green (FITC-anti-HIgG) versus the red (PI) log fluorescence intensities are shown.

 
Second antibody labelling
Antibody-coated spermatozoa and control sample were adjusted to the cell concentration 1x106/ml and 10x106/ml in 25 µl of PBS at 4° or 23°C fluorescein isothiocyanate (FITC)-labelled goat anti-human IgG polyclonal antiserum (FITC-anti-HIgG) (Sigma) was added to each sample at saturating concentration or at different dilution, as indicated in Figure 4Go. Saturating concentration of FITC-anti-HIgG was determined by FCM using three paraformaldehyde-fixed swim-up spermatozoa after their incubation with two-fold diluted ASA-positive serum as described below. Antibody-coated spermatozoa (10x106 spermatozoa/ml) were incubated with FITC-anti-HIgG at different dilutions at 23°C. The saturation was achieved at 1:50 dilution when the percentage of ASA-positive spermatozoa and their fluorescence intensity was maximal. Some antibody-coated sperm samples were incubated also with non-immune FITC-labelled goat IgG. All mixtures were incubated at 4°C or 23°C for 15 min, then washed twice (or once, as indicated in Figure 4Go) with PBS, resuspended in 200 µl of PBS, stained with propidium iodide (PI) (20 µg/ml) for distinguishing living spermatozoa (Evenson et al., 1982Go) and analysed by FCM.

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 6GoGo). 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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Figure 6. Results of indirect FCM test after different incubation periods of antibody-coated spermatozoa with FITC-anti-HIgG. Antibody-coated spermatozoa were incubated for (A, C) 30 s and (B, D) 5 min with FITC-anti-HIgG at sperm count (A, B) 1x106/ml and (C, D) 10x106/ml at 23°C and washed twice before FCM analysis. Multiparameter plots with the x axis the green (FITC-anti-HIgG) fluorescence, the y axis the red (PI) fluorescence, and the z axis the number of cells. Antibody load expressed as peak channel number of fluorescence intensity.

 
Fluorescence microscopy
Fluorescence and phase-contrast microscopy (Leitz Laborlux S®; Leica, Wetzlar, Germany) was used to determine the location of sperm-bound fluorescence.

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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The typical results of FCM and microscopy analysis of swim-up spermatozoa after their incubation in ASA-positive serum from an infertile man and with FITC-anti-HIgG are shown in Figure 1Go. The subpopulation of live antibody-coated spermatozoa, the subpopulation of live spermatozoa without ASA on their surface and dead spermatozoa were detected using FCM (Figure 1BGo). The number of dead cells significantly increased after incubation of antibody-coated spermatozoa with FITC-anti-HIgG. The fluorescent clusters, the size, number and localization of which varied considerably, were detected on the surface of live and dead antibody-coated spermatozoa (Figure 1CGo).

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 2Go, Table IGo). There were significant differences in the results of ASA detection in one serum under different conditions (Figures 2–4GoGoGo). 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 2–178) regardless of the incubation conditions (Figures 2 and 3AGoGo). 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 3BGoGo). Increase of temperature to 23°C or sperm concentration to 10x106/ml resulted in reduction of sperm antibody load in these samples by 87.3–97.1% (Figures 2 and 3C, DGoGo). 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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Figure 2. Effect of temperature and sperm concentration on the results of indirect FCM test. Results of FCM analysis of antibody-coated spermatozoa ({circ},{square}) from nine donors when one ASA-positive serum was used and (•,{square}) from one donor when six ASA-positive sera (including the ASA-positive serum {square} used for the nine donors) were used. Antibody-coated spermatozoa were incubated with FITC-anti-HIgG at sperm count 1x106/ml and 10x106/ml and at 4°C and 23°C.

 

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Table I. Results of FCM analysis of antibody-coated spermatozoa after their incubation with FITC-anti-HIgG at different temperatures and different sperm concentrations. Values are shown as mean ± SD
 


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Figure 3. Detection of ASA in one serum using different modifications of indirect FCM test and spermatozoa from different donors. Antibody-coated spermatozoa of (A) donor 1 and (B–D) donor 2 were incubated with FITC-anti-HIgG at (A, B) 4°C and sperm count 1x106/ml, (C) 4°C and sperm count 10x106/ml, (D) 23°C and sperm count 1x106/ml. E, F show the result from swim-up spermatozoa of donor 3 which were preincubated (F) with or (E) without cytochalasin B. Paraformaldehyde-fixed spermatozoa of donor 4 (G, H) and donor 5 (I, J) were incubated with (G, I) M199-BSA-1.5% or (H, J) ASA-positive serum. Flow cytometric plots are shown of the green (FITC-anti-HIgG) versus the red (PI) log fluorescence intensities. Vertical lines indicate thresholds of positive and negative immunofluorescent staining.

 
The positive correlation of both the FCM% and the sperm antibody load with the percentage of live spermatozoa was revealed under the same conditions (Table IIGo).


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Table II. The correlation between the parameters of FCM analysis of antibody-coated spermatozoaa after their incubation with FITC-anti-HIgG at different temperatures and different sperm concentrations
 
In one of five samples a very high level of fluorescence intensity (peak channel = 3398) was seen after pre-incubation of swim-up spermatozoa with cytochalasin B before interaction with ASA-positive serum (Figure 3E, FGo). In the other four samples there was no effect of cytochalasin B on sperm antibody load (data not shown).

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 Ag–Ab complex aggregation. The high percentage (89.1–95.5%) 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 3HGo). The fluorescence intensity of the spermatozoa was much lower after their incubation with ASA-negative serum (range of peak channel was 5–21). In three semen samples, both in control samples and after incubation of spermatozoa with ASA-positive serum 15.5–48.5% live spermatozoa with high fluorescence intensity were detected (Figure 3I, JGo). 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 4Go). 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 Ag–Ab complexes on the sperm surface and sperm antibody load was estimated (Figure 5Go). 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 5aGo). The pattern on the head and tail of dead fixed spermatozoa with high level of fluorescence intensity was uniform (Figure 5bGo). On the surface of live spermatozoa with high fluorescence intensity there was diffuse labelling which was not entirely uniform (Figure 5c,dGo). 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 5eGoGo).



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Figure 5. Immunofluorescence patterns of (a, c–e) live and (b) paraformaldehyde-fixed dead antibody-coated spermatozoa with (a) very high (b–d) high and (e) low fluorescence intensity. Original magnification x630.

 
Flow cytometry parameters were estimated after different incubation periods of antibody-coated spermatozoa with FITC-anti-HIgG (Figure 6Go). The maximal intensity of Ag–Ab complex shedding was detected after 30 s of incubation at low sperm concentration (1x106spermatozoa/ml) (Figure 6AGo)—the FCM% and sperm antibody load as measured by peak channel was much lower than those at higher sperm concentration.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The detection of ASA on the surface of live spermatozoa by FCM is considered to be a significant method allowing high quality laboratory assays for immunological infertility diagnostics and estimation of treatment efficiency (Haas and Cunningham, 1984Go; Räsänen et al., 1992Go, 1994Go; Nikolaeva et al., 1993Go). Comparison of indirect MAR test and indirect FCM test shows a higher sensitivity of MAR test and a higher specificity of FCM test. Thus, Räsänen et al. (1996) obtained positive results with indirect MAR test in nine of 11 (82%) IBT-positive semen samples with a high proportion of IgG positive spermatozoa (71%), whereas the FCM test result was positive only in five of 11 (45%) samples with a very low proportion of IgG positive spermatozoa (13%). A 95% false-positive rate of ASA detection in serum of fertile donors by MAR test compared with FCM test has been demonstrated (Peters and Coulam, 1992Go). Our previous study has indicated the high sensitivity (100%) of indirect FCM test compared with indirect MAR test, but the proportion of FCM-positive spermatozoa varied from 45 to 93% for serum samples where indirect MAR test gave 100% (Nikolaeva et al., 1993Go).

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, 1971Go; Cross and Moore, 1990Go; Haas et al., 1991Go). The disappearance of the cap may be due to endocytosis and subsequent degradation and/or shedding of Ag–Ab complexes, as reported for many types of cells (Matsuo et al., 1985Go; Caldwell et al., 1986Go). However, endocytosis in mature spermatozoa is unlikely but there are data on shedding of Ag–Ab complexes from the sperm surface in rat, sea urchin and rabbit (Gaunt et al., 1983Go; Trimmer and Vacquier, 1988Go; Shaha, 1994Go). 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 Ag–Ab 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 Ag–Ab 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, 1990Go).

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, 1990Go). Probably the independence of Ag–Ab 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 Ag–Ab 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., 1983Go). 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 Ag–Ab 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 Ag–Ab 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 Ag–Ab 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, 1988Go; Shekarriz et al., 1995aGo). A high level of ROS generation is associated with sperm membrane damage through spontaneous lipid peroxidation (Storey, 1997Go) and may enhance surface receptor shedding in some types of cells (Philippova et al., 1994Go; Hino et al., 1999Go).

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., 1990Go; Harrison et al., 1998Go) and/or acrosome reaction induced with second antibodies (Nikolaeva et al., 1998Go).

So the relatively low sperm antibody load detected by indirect FCM test (Sinton et al., 1991Go; Ke et al., 1995Go; Nicholson et al., 1997Go) or by direct FCM test (Nikolaeva et al., 1993Go) may be due to the experimental procedures. It is interesting that Räsänen et al. (1992, see Figure 2BGo) 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 Ag–Ab 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 Ag–Ab complexes on the cell surface also occurs within 1–2 min after cross-linking of sperm surface antigens with antibodies (Trimmer and Vacquier, 1988Go). Probably, the stimulation of shedding and antibody-coated sperm damage may be observed in the correspondence between Ag–Ab 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 7Go). 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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Figure 7. Hypothetical relationship between aggregation of Ag–Ab complexes on the sperm surface and results of FCM analysis. (Top) the probable process of cell death and of Ag–Ab complex shedding from the spermatozoa while washed at different stages of aggregation. Dead spermatozoa were shown by hatching. (Bottom) the flow cytometric plots of the green (FITC-anti-HIgG) versus the red (PI) log fluorescence intensities showing the pattern of sperm heterogeneity. The detection of live antibody-coated spermatozoa with very high or high fluorescence intensity (subpopulations a and b) testify to the absence of aggregation (patching or capping) during washing procedures. The beginning of aggregation makes spermatozoa more vulnerable to damage. Therefore, coincidence of capping (or patching) with sperm washing results in partial or complete shedding of Ag–Ab complexes and/or membrane damage and detection of live and dead spermatozoa with low fluorescence intensity (subpopulations c–f). If the aggregation is completed by patch or cap formation, membrane resistance to manipulation is again observed and spermatozoa become non-permeable for PI, by a decrease of shedding intensity during sperm washing and a corresponding increase of fluorescence intensity (subpopulations b and g).

 
The decrease of antibody-coated sperm concentration was shown to enhance the probability of false-negative results of indirect FCM test. These results are in accordance with the data obtained by Haas and Cunningham (1984) and might well be conditioned by the inhibition of Ag–Ab complex aggregation with enhancing of antibody/antigen ratio and/or decrease of ROS level with lowering of sperm concentration (De Lamirande and Gagnon, 1995Go; Shekarriz et al., 1995bGo). This may lead to coincidence of aggregation with washing and shedding intensification.

The manipulations with ASA-positive spermatozoa during MAR test are minimized and there is a small possibility of Ag–Ab complex shedding that enhances the sensitivity of MAR test. However, Gould et al. (1994) have shown that spermatozoa–immunobead 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., 1991Go). Our data have shown fluorescence of homogeneously distributed Ag–Ab 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 Ag–Ab complexes. The absence of Ag–Ab 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 Ag–Ab 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 load—the FCM parameters used both in experimental researches and for diagnostics of immunological infertility and efficacy of immunological infertility treatment—depend on the aggregation of Ag–Ab 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 Ag–Ab 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.


    Acknowledgments
 
We are grateful to Anton Krutskikh for his assistance in preparation of this article.


    Notes
 
1 To whom correspondence should be addressed at: Laboratory of Clinical Immunology, Russian Scientific Centre for Obstetrics, Gynecology and Perinatology, 117815, Oparin 4, Moscow, Russia. E-mail: anikom{at}online.ru Back

* Presented at the International Symposium on Male Infertility and Assisted Reproduction, Genk, Belgium, April 22–25, 1998, and at the European Meeting of Immunology and Reproduction, Rome, Italy, October, 28–29, 1999. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 9, 2000; accepted on September 8, 2000.