Mixed erythrocyte-spermatozoa antiglobulin reaction (MAR test) for IgA antisperm antibodies in subfertile males*

Vol. 37, No. 1, January 1982 Printed in U.SA.

FERTILITY AND STERILITY Copyright c 1982 The American Fertility Society

Mixed erythrocyte-spermatozoa antiglobulin reaction (MAR test) for IgA antisperm antibodies in subfertile males*

William F. Hendry, Ch.M., F.R.C.S.t Jitka Stedronska, S.M.L.S.O. Richard A. Lake, B.Sc. · Seminology Laboratory and Fertility Clinic, Chelsea Hospital for Women, London, England

A mixed antiglobulin reaction (MAR test), using antibody-coated red cells and spermatozoa, has been used to detect the presence of either lgA or IgG on the surface of the spermatozoa. Group 0 Rh-positive red cells were sensitized with serum containing anti-D, which was partly lgA, though mainly IgG. Spermatozoa were added to the sensitized red cells and tested either with anti-lgA or anti-IgG. MAR (lgA) test results were negative or doubtful in 42 (44%) of94 samples with positive or strongly positive MAR (lgG) tests. Positive MAR (lgA) tests showed a highly significant correlation with the presence of antisperm antibodies in seminal plasma. Significantly impaired sperm penetration of cervical mucus was demonstrated for 15 patients, 12 of whom had positive MAR (lgA) tests, whereas good sperm penetration was observed for 5 patients with negative or dubious MAR (lgA) tests; all 20 patients had strongly positive MAR (lgG) tests and positive serum antisperm antibody tests. Fertil Steril 37:108, 1982

Initially we evaluated the direct MAR test for lgG antibodies as a routine addition to the analysis of 775 semen samples from 557 husbands of infertile marriages attending our hospital. The test was applicable to 664 samples (86%) from 463 patients (83%). Antisperm antibodies were also looked for by the gelatin agglutination test (GAT) in serum samples from 213 patients. Positive results for the MAR test were obtained in 29 (85%) of 34 patients with antisperm antibodies in serum, including 27 positive results (93%) in 29 patients with antisperm antibody titers of more than 32. A negative MAR test was found in 174 (97%) of 179 patients without antisperm antibodies in serum. We thus confirmed that this was a valuable addition to seminal analysis as a screening test for the presence of antisperm antibodies. However, since two patients with strongly positive MAR tests and low serum GAT titers (4 and 16) produced pregnancies without treatment, we concluded that estimation of serum and seminal plasma titers was essential for assessment of the likely effects of the antisperm antibodies on the patient's fertility. 5 Since then, we have developed

The mixed antiglobulin reaction (MAR test) was developed for the detection of platelet antibodies1 and was subsequently modified to demonstrate antibodies on spermatozoa. 2 Jager et al. 3 showed that a direct MAR test for lgG was a simple and effective method of screening for antisperm antibodies in semen samples from men. Kremer and Jager4 also showed that it could be used to define the type of antibody on the spermatozoa: IgG antibodies were always present when the serum antibody test was positive, but lgA was detected on the spermatozoa only when antibodies were present in seminal plasma, and only the latter finding correlated well with impaired sperm penetration of cervical mucus.

Received May 1, 1981; revised and accepted September 9, 1981. *Supported by the Royal College of Obstetricians and Gynaecologists, London, England. tReprint requests: Mr. William F. Hendry, Chelsea Hospital for Women, Dovehouse Street, London, SW3 6LT, England.

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a direct MAR test for IgA antibodies to see whether such a test would provide a better correlation with the existence of seminal plasma antibodies and with impaired sperm-cervical mucus penetration than the MAR test for IgG. MATERIALS AND METHODS

The presence or absence of IgA antibodies on spermatozoa was defined by the direct MAR test in 104 semen samples with counts of at least 1 million spermatozoa per milliliter, provided by 51 untreated subfertile males with positive MAR tests for IgG. In 34 cases the MAR tests for IgA were repeated on two or more occasions. The quantity of antisperm antibody in seminal plasma was measured in serial dilution by the gelatin agglutination test (GAT) 6 in all cases, and by tray agglutination test (TAT) 7 in 49 cases. The serum antibody titer was also defined in 4 7 cases by GAT, TAT, and sperm immobilization tests. 8 In 20 couples, the patients' spermatozoa were tested against preovulatory cervical mucus obtained with a 1-ml tuberculin syringe from the wife and from donors of proven fertility, comparing the behavior of patients' spermatozoa with those obtained from fertile donors. A drop of cervical mucus was examined for appearance, pH, spinnbarkeit, ferning, and quantity. If the mucus was unsatisfactory, it was discarded. If it was normal ovulatory mucus, a drop was placed on a glass slide and flattened with a coverslip. Semen was applied at the edge of the coverslip, and the slide was placed in an incubator at 37° C and examined after 30 minutes. Penetration was assessed as follows: 0, no penetration; 1, penetration not exceeding 2 mm into peripheral mucus; 2, sperm moving throughout the mucus. Movement within the mucus was assessed as positive if there was forward progression of the sperm or negative if the spermatozoa were immobile or shaking. 9 Semen samples were collected at home or in the hospital after 3 days abstinence and delivered to the seminology laboratory within 4 hours of production; volume, sperm count, and percentage of motility were recorded. The MAR tests were done as described in our previous paper, 5 modified as follows for the addition of IgA testing: group 0, Rh-positive red blood cells were washed three times in Alsever's solution and resuspended to a hematocrit of 50%. To one part red cell suspension was added two parts 1-in-5 dilution of serum containing anti-D, which was partly IgA, although mainly IgG (serum

Avgh., supplied by Professor P. L. Mollison). The red cells and serum were incubated at 37° C for 30 minutes. The cells were then washed three times again in Alsever's solution, and resuspended to a hematocrit of between 5% and 10%, and stored until required for use in small aliquots at 4° C. One drop of fresh semen was placed on a microscope slide with one drop of sensitized red cell suspension and one drop of undiluted monospecific anti-human antiserum for either IgG or IgA (Behring Diagnostics, Somerville, N. J.). The three drops were thoroughly mixed, and the reaction was read within 10 minutes. No interpretation was made unless agglutination of the red blood cells was observed. The test was read as negative (0) if no motile mixed agglutinates were seen and freely swimming spermatozoa could be observed between the clumps of agglutinated red cells. If motile mixed agglutinates were observed, the reaction was graded as follows: doubtful ( ± ), only an occasional mixed agglutinate seen, with less than 10% of the motile spermatozoa involved with adhering red blood cells (this result is interpreted as negative); positive ( + + ), 10% to 90% of the motile spermatozoa attached to the erythrocytes; strongly positive ( + + + ), more than 90% of the motile spermatozoa incorporated into mixed agglutinates. RESULTS

The MAR (IgA) test was applicable to all except one of the semen samples suitable for IgG testing, although the reaction with IgA was slower and less easy to read than the IgG reaction; furthermore, the spermatozoa tended to lose motility about 10 minutes after addition of the anti-IgA, and so the reading had to be completed by this time. In the one exceptional case, the spermatozoa were caught in large clumps, and a satisfactory conclusion could not be reached in the IgA test. The results of the MAR (IgA) test appeared to be reasonably consistent (Table 1). In 34 patients who had two or more tests, the first and second results were comparable in 30 instances (taking negative and doubtful together and comparing them with positive and strongly positive, we found x2 = 22.61, P < 0.001). The MAR (IgA) tests were negative or doubtful in 42 (44%) of 94 samples with positive or strongly positive MAR tests for IgG (Table 2). The results of the MAR (IgA) tests are shown correlated with the presence of seminal plasma antibodies, defined by TAT in Table 3 and by GAT

110

Table 1. Correlation Between Results of First and Second MAR (lgA) Tests in 34 Subfertile Men Repeat MAR agA) test First MAR (lgA) test - - - - - - - - - - - Negative/doubtful Positive or strongly positive

Negative/doubtful Positive/strongly positive

17

2

2

13

DISCUSSION

The accurate diagnosis of immunologic infertility has presented a number of problems, first in Table 2. Correlation Between Results of MAR Tests for IgG and lgA in 104 Samples from 51 Subfertile Men MAR agA) test MAR agG) test Negative Doubtful Positive

4

13

8 4 21

Strongly positive

2 4

16

Table 3. Results of MAR (lgA) Tests Related to the Presence of Antisperm Antibodies in Seminal Plasma Detected in Tray Agglutination Tests of 49 Patients Seminal plasma antiMAR agA) test IIJM'l'lll antibody titer - - - - - - - - - - - (TAT) Negative/doubtful Positive/strongly positive

0 ;;..4

in Table 4. There was a good correlation with the results of both tests, which was more significant with TAT (x2 = 15.732, P < 0.001) than with GAT (x2 = 5.649, 0.02 > P > 0.01). This was largely due to the increase in the number of patients with positive MAR (IgA) tests who were found to have seminal plasma antibodies with the more sensitive TAT test. The 20 patients who had sperm-cervical mucus penetration tests done are detailed in Table 5, and these results are summarized in Tables 6 and 7. It may be seen that good penetration of cervical mucus was only seen in the absence of antisperm antibodies from the seminal plasma and with a negative or dubious MAR (IgA) test. In contrast, good and poor penetration of mucus was seen with positive serum antibody tests and with positive MAR (IgG) tests. However, in three cases with dubious MAR (IgA) tests, penetration of cervical mucus was poor, and two of these patients had antisperm antibodies in seminal plasma. Overall, there is evidently a strong association between the results of the MAR (IgA) test and the ability of spermatozoa to penetrate cervical mucus. The probability of results shown in Table 7 having been obtained by chance is calculated to be 1 in 277 by Fisher's exact method for 2 x 2 tables. On the other hand, there is evidently no correlation between the results of the MAR (IgG test) and cervical mucus penetration.

Negative Doubtful Positive Strongly positive

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HENDRY ET AL.

32

20 8

3 18

detection of patients who may be affected, and second in discrimination between significantly positive results and results that, for practical purposes, can be ignored. Many tests for the presence of antisperm antibodies rely on the induction of agglutination in donor spermatozoa-a reaction that may occur spontaneously with certain donors and may occur with certain test sera apparently due to nonimmunologic causes. These difficulties have led to some confusion surrounding this diagnosis, which have been summarized in a review. 10 The MAR test has the advantage that it tests for a specific immunologic reaction between spermatozoa and sensitized red blood cells, and it tests directly for the presence of antibodies on the surface of the patients' spermatozoa. It appears to be very sensitive when used for the detection of IgG on spermatozoa. Thus the MAR (lgG) test is useful as a screening test, but one must recognize that antibody titers must be defined for an assessment of their significance. The MAR (IgA) test was slower and more difficult to read but might add refinement in diagnosis when used with the test for IgG. In the past, serum has generally been used for antisperm antibody tests since it is relatively plentiful and easy to handle. However, Kremer and Jager4 have demonstrated clearly that what impairs fertility is the presence of antisperm antibodies in the genital secretions predominantly as IgA, and not their existence in serum as IgG. This conclusion was derived from the observation that only IgA antibodies caused spermatozoa to react with cervical mucus, impeding penetration Table 4. Results of MAR (lgA) Tests Related to the Presence of Antisperm Antibodies in Seminal Plasma Detected by Gelatin Agglutination Tests in 51 Patients Seminal plasma antiIIJM'l'lll antibody titer (GAT)

MAR agA) test Negative/doubtful

18 9

Positive/strongly positive

8

16

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Table 5. Results of Antisperm Antibody Tests in Serum and Seminal Plasma, MAR Tests for IgG and lgA, and Sperm/Cervical Mucus Penetration Tests in 20 Untreated Men Sperm/cervical" mucus penetration

Serum antisperm antibody titerb

Seminal plasma antibody titer

MAR test"

Patient

1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18 19 20

Husband

Donor

GAT

SIT

TAT

GAT

10 1111110 110 110 2± 22± 2± 2-

2+ N'F 2+ 2+ 2+

512 512 256 128 128 128 128 128 128

128 16 0 32 16 16 16

2048 128 2048 128 NT 256 16 32 1024 1024 128 128

0 16 16 8 16 0 0 0 16 4 0 8 16 0 0 0 0 NT 0 0

NT

2+ 2+ 2+ 2+ 2+ NT 2+ 2+ 2+ 2+ 2+ 2+ 2± 2+

64

64 64 32 32 32 128 128 32 32 16

NT

64 16 16 16 0 4 4 0 8 NT 0 0

64

16 128 128 64

8 16 8

TAT

64 NT

8 8 16 4 0 8 64 NT 0 64 256 8 64 0 0 0 0 0

lgG

IgA

+++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++

++ +++ ± +++ ++ +++ ± +++ ++ +++ ++ ± ++ ++ +++ ± ± ± ±

"Sperm penetration: 0 = none; 1 = peripheral; 2 = central. Sperm motility: - = poor, shaking; + = progressive. bGAT = gelatin agglutination test; SIT = sperm immobilization test; TAT = tray agglutination test. cMAR test results: - = negative; ± = doubtful; + + = positive; + + + = strongly positive.
and causing the shaking phenomenon. Earlier work by Fjallbrant11 in men with antisperm antibodies had shown a close correlation between the ability of spermatozoa to penetrate cervical mucus and subsequent fertility. More recently, evidence from vasectomy reversal patients has confirmed that the presence or absence of antisperm antibody in seminal plasma is the critical factor in determining whether or not pregnancy occurs in the spouse. 12 The comparative studies reported in this paper showed that there was a good correlation between a positive result on MAR (lgA) testing and the presence of antisperm antibodies on seminal plasma, especially when the sensitive tray agglutination test was used. The correlation was not perfect, however, which may indicate that much of the antibody is absorbed onto the spermatozoa in some cases, or the MAR (lgA) test may be some-

what insensitive in other cases. Similarly, the correlation between positive MAR (lgA) test results and impaired sperm penetration of cervical mucus was good, although, once again, not perfect. In two cases, a dubious result on MAR (lgA) testing probably should have given a positive result, since antibodies were present in seminal plasma and sperm penetration was impaired. Quantitation of the MAR test is difficult, especially with lgA testing; however, we believe that IgA is the correct class of antibody to be assayed in these patients, rather than the lgG antibodies which were identified by Haas et al. 13 MAR testing cannot be used in patients with severe oligozoospermia, asthenospermia, or azoospermia, and it is difficult to quantitate accurately. For these reasons we believe that it should be one of a battery of tests for antisperm antibodies, which can appear in very diverse forms (Table 5).

Table 6. Analysis ofResults of Sperm/Cervical Mucus Penetration Tests, Comparing Husbands' and Fertile Donors' Spermatozoa, Related to Antisperm Antibodies in Serum and Results of the MAR (lgG) Test Sperm/cervical mucus MAR test (IgG)" penetration• Serum antisperm antibodies• No. of p a t i e n t s - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Husband sperm Donor sperm GAT-positive SIT-positive TAT-positive Negative/ Positive/strong(;;. 32) (;;. 4) (;;. 32) doubtful ly positive

15

1-

5

2±

"See footnotes to Table 5.

2+ 2+

15/15 4/5

12/14 114

12/14 2/5

15 5

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HENDRY ET AL.

Table 7. Analysis of Results of Sperm/Cervical Mucus Penetration Tests, Comparing Husbands' and Fertile Donors' Spermatozoa, Related to Antisperm Antibodies in Seminal Plasma and Results of the MAR (lgA) Test No. of patients

Sperm/cervical mucus penetration• Husband sperm

15 5

Donor sperm

12±

2+ 2+

Seminal plasma antisperm antibodies"

MAR test (lgA)"

GAT-positive

TAT-positive

(;. 4)

(;. 4)

Negative/ doubtful

6/12 2/3 0/4

9/10 2/3 0/5

3 5

Positive/strongly positive 12

aSee footnotes to Table 5.

The MAR (lgG) test seems to be ideal as a screening test for antibodies-quick, sensitive, andresponsive; however, one should perform the MAR (lgA) test as well for all patients with a positive IgG reaction to define the class of antibody present and thus assess the likely effect of the antibodies on fertility. Titers of antibody should be defined, both in serum and seminal plasma, so that the quantity of antibody is known and can be remeasured at intervals during treatment. Preliminary observations suggest that the MAR (lgA) test may be a very useful method for monitoring response to treatment with steroids, 14· 16 which may be of great importance in the selection of the minimum effective dose, and in adjustment of the timing of intermittent high-dose therapy in relation to the anticipated date of the wife's ovulation. Acknowledgments. We should like to thank Professor P. L. Mollison, F.R.S., for suggesting the use of a serum containing IgA anti-D for the preparation of IgA-coated red cells and for supplying a suitable serum. We are grateful toM. Phillipou and Nicky Day, B.Sc., for technical assistance, and to Miss Anthea Minchom for preparation of the manuscript. REFERENCES 1. Coombs RRA, Marks J, Bedford D: Specific mixed agglutination: mixed erythrocyte-platelet antiglobulin reaction for the detection of platelet antibodies. Br J Haematol 2:84, 1956 2. Coombs RRA, Riimke P, Edwards RG: Immunoglobulin classes reactive with spermatozoa in the serum and seminal plasma of vasectomised and infertile men. In the Second International Symposium on Immunology of Reproduction, Edited by K Bratanov. Sofia, Bulgarian Academy of Science Press, 1973; p 354 3. Jager S, Kremer J, van Slochteren-Draaisma T: A simple method of screening for antisperm antibodies .in the human male. Int J Fertil 23:12, 1978

4. Kremer J, Jager S: Characteristics of anti-spermatozoal antibodies responsible for the shaking phenomenon with special regard to immunoglobulin class and antigen-reactive sites. Int J Androl 3:143, 1980 5. Hendry WF, Stedronska J: Mixed erythrocyte-spermatozoa antiglobulin reaction (MAR Test) for the detection of antibodies against spermatozoa in infertile males. J Obstet Gynaecol 1:59, 1980 6. Kibrick S, Belding DL, Merrill B: Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 3:430, 1952 7. Friberg J: A simple and sensitive micro method for demonstration of sperm agglutinating antibodies from infertile men and women. Acta Obstet Gynecol Scand [Suppl] 36:21, 1974 8. Isojima S, Li TS, Ashitaka Y: Immunologic analysis of sperm-immobilizing factor found in sera of women with unexplained sterility. AmJ Obstet Gynecol101:677, 1968 9. Morgan H, Stedronska J, Hendry WF, Chamberlain GYP, Dewhurst CJ: Sperm/cervical mucus crossed hostility testing and antisperm antibodies in the husband. Lancet 1:1228, 1977 10. Jones WR: Immunologic infertility-fact or fiction? Fertil Steril 33:577, 1980 11. Fjallbrant B: Interrelation between high levels of sperm antibodies, reduced penetration of cervical mucus by spermatozoa, and sterility in men. Acta Obstet Gynecol Scand 47:102, 1968 12. Linnet L, Hjort T, Fogh-Andersen P: Association between failure to impregnate after vasovasostomy and sperm agglutinins in serum. Lancet 1:117, 1981 13. Haas GG, Cines DB, Schreiber AD: Immunologic infertility: identification of patients with antisperm antibody. N Engl J Med 303:722, 1980 14. Shulman S: Treatment of immune male infertility with methylprednisolone. Lancet 2:1243, 1976 15. Hendry WF, Stedronska J, Hughes L, Cameron KM, Pugh RCB: Steroid treatment of male subfertility caused by antisperm antibodies, Lancet 2:498, 1979 16. Hendry WF, Stedronska J, Parslow J, Hughes L: The results of intermittent high dose steroid therapy for male infertility due to antisperm antibodies. Fertil Steril 36:351, 1981

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