Clinic of Dermatology, Department of Andrology, Clinical Training Center of the European Academy of Andrology, Philipp University, Deutschhausstraße 9, D-35033 Marburg, Germany
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Abstract |
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Key words: antisperm antibody/donor spermatozoa/intra-, inter-assay variation/mixed antiglobulin reaction
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Introduction |
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The test most commonly used is the mixed antiglobulin reaction (MAR), which was firstly described by Jager et al. (1978). A standardization was recommended by the World Health Organization task force group (WHO, 1987). The MAR test may be performed using one of two modifications: a direct or an indirect variation. In the direct MAR test, the antibodies attached to motile spermatozoa are detected. The indirect MAR test is used if no spermatozoa or no motile spermatozoa can be found in the semen. In this case the soluble sperm antibodies in seminal plasma are detected by using healthy donor spermatozoa as the antigen.
Each laboratory performs the indirect test using different donor spermatozoa, and even in one laboratory different donors are used for the test. The comparability of the results is therefore poor. This is not only a problem of structure and process quality, but individual interactions between seminal antisperm antibodies and donor sperm antigen structure seem to influence the test results. Focus was placed on antibodies in the seminal plasma rather than sera because sperm antibodies in sera are not thought to be related to infertility.
In this study we systematically investigated the influences of donor spermatozoa and antibody sources upon the indirect MAR test and resultant differences in intra- and inter-assay variation.
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Materials and methods |
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Donor sperm preparation:
Spermatozoa from five donors, obtained by masturbation after 36 days' abstinence, showing normal sperm parameters according to the WHO recommendations (WHO, 1987) and the absence of sperm antibodies, were isolated by a swim-up preparation. Firstly, 1 ml of semen was diluted 1:5 with in-vitro fertilization (IVF) buffer (MediCult a/s, Copenhagen, Denmark) and thereafter centrifuged at 300 g at room temperature for 10 min. The sperm pellets were resuspended in 5 ml IVF buffer and washed again. Two ml of IVF medium were then carefully placed above the final pellet, after which the tube was inclined at an angle of 45° and incubated for 1 h at 37°C. The supernatant containing the enriched fraction of motile spermatozoa was aspirated and centrifuged again for 10 min at 500 g at room temperature. Finally, the cell concentration was adjusted to 20 x 106 ml1 with IVF medium.
Direct (SpermMar®) test for sperm antibodies
Prior to the experiments, the donor ejaculates were tested with direct SpermMar® test to exclude the presence of sperm antibodies in the donor seminal plasma. For the detection of sperm antibody, the reagent kit SpermMar® IgG Test from FertiPro N.V. (Sint-Martens-Latem, Belgium) was used. The test was performed according to technical data given by the manufacturer. Briefly, the direct SpermMAR® test was performed on a microscopic slide by mixing 10 µl of fresh semen and 10 µl suspension, containing latex particles coated with human immunoglobulin (IgG). In the next step, 10 µl of monospecific antihuman IgG antiserum anti Fab'-fragments were added. A cover-slip was applied to the mixture and after 3 min 100 motile spermatozoa were evaluated. The percentage of spermatozoa with attached latex particles represented the test result.
Indirect MAR test
The soluble antibodies of the patients' seminal plasma samples were tested using the indirect MAR test (Hinting et al., 1988). Firstly, 100 µl seminal plasma were diluted 1:4 with 300 µl IVF buffer; 100 µl of this dilution was then carefully mixed with 100 µl of the donor sperm suspension and incubated for 30 min at 37°C. The mixture was then processed as in the direct MAR test (see above).
Intra-assay variation
The indirect MAR test assay was run seven times with seven sperm antibody positive samples of seminal plasma using identical donor spermatozoa, originating from one individual ejaculate. Control seminal plasma samples without sperm antibodies were assayed under the same conditions.
Inter-assay variation
Day-to-day variability of donor samples
Four seminal plasma samples containing antibodies and three control samples were diluted at 1:4 with IVF buffer and tested five times using spermatozoa from one donor over a period of 5 weeks. Each test was assayed in triplicate.
Comparison between different donors
Seminal plasma samples of eight patients containing different antibody concentrations and four control samples were diluted at 1:4 with IVF buffer and were tested with five different donor spermatozoa. Each test was assayed in triplicate.
Statistical methods
The multivariate tests were performed using the SPSS 6.1.3 for windows programme (SPSS Inc., Chicago, IL, USA). Tables were carried out with EXCEL 7.0 (Microsoft).
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Results |
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Discussion |
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By repeating the tests using identical antigens and antibodies, internal comparisons can be performed. As shown in our study, the intra-assay variation of the MAR test is low and independent of individual donors.
However, in the calculation of inter-assay variations two dimensions have to be considered. The first dimension is represented by the use of serial ejaculates from one individual donor at different times. This test condition leads to high variations of the indirect MAR test results, making it doubtful that this comparison is useful for quality control.
The second dimension of inter-assay variation is the application of different donor sperm samples to the patient's antibody source. This would be unavoidable if external comparisons between different laboratories were to be made. In our study, we found that the donor sperm samples strongly influence the results of the indirect MAR test. Although the `positive' or `negative' test results showed a relationship between the different donors, the results could be different for a given patient sample according to the donor. The variation of values was significantly greater than the intra-assay variation or the inter-assay variations using a single donor providing serial ejaculates. Therefore, from a biological standpoint, as well as a statistical one, the results are entirely satisfactory: small variation when using the same donor and much larger, interactive variation when using different donors.
The differences obtained in the indirect MAR test results (Table III) using different donors may lead to distinct clinical treatments of infertile couples. In the literature, the usual recommendation for couples with a positive MAR test is to perform intracytoplasmic sperm injection (ICSI). However it was indicated (Ombelet et al., 1997
) that intrauterine insemination (IUI) yielded unexpectedly high pregnancy rates in patients with long-standing infertility due to sperm surface antibodies, even if the MAR test results were higher than 50%. It was reported (Abshagen et al., 1998
) that the rate of spontaneous pregnancy among women whose partners had sperm antibody concentrations of <50% was good, while ICSI should be recommended to patients with concentrations of >90%.
It was already assumed (Hellström et al., 1987) that spermatozoa from different donors might differ significantly in their reaction to identical sperm antibody sources. Our study supports the suggestion that there are specific interactions between the antigenic epitopes of the donor spermatozoa and the patient's antibodies. These differences may be due to minor variations of sperm membrane proteins between individual men, as was demonstrated by means of two-dimensional gel electrophoresis (Naaby-Hansen et al., 1997
). On the other hand, the differences may be caused by sperm populations of different degrees of maturity, capacitation, and acrosome reaction, during which processes different surface antigens are expressed (Lenzi et al., 1997
).
As we do not have standardized human sperm donors, the standardization of the indirect MAR test and external quality control is not possible at this time. The high inter-assay variation of the indirect MAR test will therefore give misleading results.
In order to establish external quality assessment and to improve the precision and comparability of different tests, a pilot programme was started to compare sperm antibody detection methods in 20 laboratories (Matson, 1995). Each laboratory screened aliquots of the same samples. As a result of this study, the detection of sperm antibodies by the tray agglutination, gelatin agglutination and indirect immunobead tests was found to discriminate between positive and negative samples, but the inter-laboratory variation was large. Several publications have compared the different methods, but the techniques varied widely. Thus no general conclusion regarding the comparability of methods may be drawn. Even the particular antibody assays are insufficiently standardized regarding specificity, sensitivity and objectivity (Collins et al., 1993
).
The development of a reliable and reproducible method to detect sperm antibodies in seminal plasma or other human fluids is urgently needed. It has also been noted (Dondero et al., 1997) that there is no universally accepted method for sperm antibody detection. To reach this goal, it will be necessary to isolate intact sperm surface antigens in order to guarantee the presence of the whole immunologically active structure.
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Notes |
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References |
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Collins, J.A., Burrows, E.A., Yeo, J. and Young Lai, E.V. (1993) Frequency and predictive value of antisperm antibodies among infertile couples. Hum. Reprod., 8, 592598.[Abstract]
Dondero, F., Gandini, L., Lombardo, F. et al. (1997) Antisperm antibody detection: 1. Methods and standard protocol. Am. J. Reprod. Immunol., 38, 218223.[ISI][Medline]
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Hinting, A., Vermeulen, L. and Comhaire, F. (1988) The indirect mixed antiglobulin reaction using a commercially available kit for the detection of antisperm antibodies in serum. Fertil. Steril., 49, 10391044.[ISI][Medline]
Jager, S., Kremer, J. and van Slochteren-Draaisma, T. (1978) A simple method of screening for antisperm antibodies in human male. Detection of spermatozoal surface IgG with the direct mixed antiglobulin reaction carried out on untreated fresh human semen. Int. J. Fertil., 23, 1221.[ISI][Medline]
Lenzi, A., Gandini, L., Lombardo, F. et al. (1997) Antisperm antibody detection: 2. Clinical, biological, and statistical correlation between methods. Am. J. Reprod. Immunol., 38, 224230.[ISI][Medline]
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Naaby-Hansen, S., Flickinger, C.J. and Herr, J.C. (1997) Two-dimensional gel electrophoretic analysis of vectorially labelled surface proteins of human spermatozoa. Biol. Reprod., 56, 771787.[Abstract]
Ombelet, W., Vandeput, H., Janssen, M. et al. (1997) Treatment of male infertility due to sperm surface antibodies: IUF or IVF. Hum. Reprod., 12, 11651170.[ISI][Medline]
World Health Organization (1987) WHO Laboratory Manual for the Examination of Human Semen and Semen CervicalMucus Interaction. Cambridge University Press, Cambridge.
Submitted on December 4, 1998; accepted on April 1, 1999.