The Relationship of Circulating Antisperm Antibodies to Sperm Surface Antibodies in Infertile Men

0022-5347 /88/1405-1039$02.00 /0 Vol. 140 1 November

THE JOURNAL OF UROLOGY

Printed in U.S.A.

Copyright© 1988 by The Williams & VVilkins Co.

THE RELATIONSHIP OF CIRCULATING ANTISPERM ANTIBODIES TO SPERM SURFACE ANTIBODIES IN INFERTILE MEN WAYNE J. G. HELLSTROM, JAMES W. OVERSTREET,* STEVEN J. SAMUELS ERNEST L. LEWIS

AND

From the Departments of Urology and Obstetrics and Gynecology, School of Medicine, University of California, Davis, California

ABSTRACT

The correlation between the amount and location of antisperm antibody binding to the sperm surface and the level measured in the serum has not been previously reported. Hence, the value and limitations of screening blood sera from men with suspected immunologic infertility are not currently known. In this study 70 paired sera and semen samples were assayed by the immunobead test (IBT). A screening protocol for blood sera was constructed to be 100% sensitive for detecting semen specimens with 20% or more of sperm binding IgG or IgA immunobeads. The specificity of this screening protocol was determined to be 79%. Serum IgA was not a good predictor of IgA on the sperm surface. The true positive predictive rate for antisperm antibodies on the sperm surface using circulating antisperm antibodies as a screening assay was estimated to be as low as 35%. There was little correlation between the site of immunobead binding following passive antibody transfer from patients' sera to donor sperm and the site of naturally occurring antibodies on the patients' sperm surface. Although direct assessment of antibodies on the sperm surface is preferred, these data suggest that serum IgG alone can be used as a sensitive screening assay for antisperm antibodies in men. A positive screen dictates that a direct assay on semen should be performed. (J. Ural., 140: 1039-1044, 1988). There is a strong association between involuntary infertility and the occurrence of antisperm antibodies in the male and/or the female. 1 • 2 Currently there is no clear understanding of the mechanisms by which antibodies may impair fertility. The lack of reliable, standardized testing protocols for antisperm antibodies has contributed to the uncertainty of the magnitude and importance of immunologic phenomena in human reproductive failure. 3 The interpretation of previous clinical research as well as current testing protocols must take into account the fact that the amount of antibody measured systemically may not reflect the level present in the reproductive tract fluids.4· 5 In men with suspected immunologic infertility there is an uncertain relationship between the amount of antibody measured in the blood serum or seminal plasma and the amount of antibody bound to the sperm surface. Practically, we may assume that only those antibodies which are bound to the sperm surface are clinically significant and have the ability to interfere with the sperm transport or fertilization processes. 6 Of the many assays that are available for measuring antisperm antibodies, we believe that the immunobead rosette test (IBT) is the most informative. 7 This assay identifies the class of immunoglobulin (lg) involved (G, A, or M), and the location of antibodies on the sperm surface. The results of the IBT are usually reported as the proportion of motile (living) sperm binding immunobeads. 8 It is this reliance upon maintenance of sperm motility that lin1its the procedure to fresh semen rather than previously frozen sperm. The IBT can be used to identify Accepted for publication May 13, 1988. *Requests for reprints: Dept. of Obstetrics and Gynecology, Div. of Reproductive Biology and Medicine, University of California at Davis School of Medicine, Davis, CA 95616. Supported by NIH Research Grant HD15149 and an AUA Scholarship. Awarded First Prize for Clinical Research at the 83rd annual meeting of the AUA, June 3-7, 1988, Boston, Massachusetts.

native antibodies that are bound to the sperm surface (direct IBT); or the presence of antibodies in body fluids (seminal plasma, serum, cervical mucus, or follicular fluid) may be determined by incubating the fluid with donor sperm and subsequently testing these sperm for surface antibodies (indirect IBT). It is generally acknowledged that the direct IBT method (rather than the indirect IBT) for measuring antibodies on the surface of the patient's sperm is the preferred approach for workup of the infertile male. However, the IBT is currently available in a limited number of clinical laboratories. It may be difficult to schedule a great number of patients from remote locations to give semen specimens. Logistically, it is easier to send a serum sample by mail, store the samples in a freezer, and at a convenient time perform a number of assays concurrently. In this way a test may be repeated or samples may be compared in the same assay with specimens obtained later in the patient's diagnostic workup or therapy. Yet, the value and limitations of screening men for antisperm antibodies by tests on blood sera are not currently known. There are few reports in the literature addressing the relationship of serum antisperm antibody levels with antibody levels in semen 9 • 10 and there are a limited number of published studies using the IBT to correlate systemic antibody levels to the presence and binding location of antibody on sperm surfaces.11· 12 The IBT provides information on immunoglobulin class and on the anatomical location of sperm surface antibodies that is not provided by other classical tests. This information may have clinical value for cases of immunologic infertility in assessing the prognosis for fertility and in choosing the therapeutic approach. The relationship of these characteristics of circulating antibodies to those of the sperm surface antibodies is also unknown and is of scientific and clinical interest. For this study our aim was to characterize the circulating antisperm antibodies (indirect IBT) of infertile men and to compare these to the native antibodies on sperm surfaces

1039

1040

HELLSTROM AND ASSOCIATES

(direct IBT). In practical terms, we sought to determine the sensitivity, specificity and predictive value of a serum screening protocol in identifying a man as having clinically significant amounts of antisperm antibodies on his sperm cells. We also sought to understand whether any of the prognostic information provided by the data on regional binding of antibodies to the sperm surface could be obtained by indirect tests with blood sera. MATERIALS AND METHODS

Patients and methods. During the interval from March 1984 to July 1987, a total of 330 serum samples were obtained from men being investigated for immunologic infertility and were assayed for the presence of antisperm antibodies by the indirect IBT. During the same interval, 250 patients provided semen specimens which were assayed for antibodies with the direct IBT. A total of 70 patients had paired semen and serum samples assayed within two to three weeks. These patients who received paired tests were referred by physicians who requested both tests. No known information on antisperm antibody status was shown prior to either test. IBT tests were performed in accordance with published methods. 3 •8 Briefly, in the indirect method, normal donor semen (without evidence of antisperm antibodies) was diluted with a Hepes buffered saline (HBS). 13 The sperm were washed twice from the seminal plasma by dilution and centrifugation at 800 g for six minutes. Following washing, the sperm suspension was adjusted to 50 x 106 motile sperm per ml. A 0.4 ml. aliquot of the patient's serum was diluted with 1.2 ml. of medium and combined with 0.1 ml. of adjusted sperm suspension. Following 30 minutes of incubation at 37C the sperm were washed three times by successive dilution and centrifugation before being resuspended for testing. For the direct method, semen was obtained from the patient after three days of abstinence. The sperm were washed twice and resuspended in HBS to a concentration of 10 to 20 million motile sperm per ml. In both the direct and indirect methods, suspensions of immunobeads (G, A, and M) (Biorad, Richmond, California) were prepared in HBS at a concentration of six mgm/ml. A 0.05 ml. aliquot of each immunobead suspension was added to 0.005 ml. of the sperm suspension being tested. Following incubation for 10 minutes at 37C, a 0.01 ml. aliquot of the sperm and bead suspension was transferred to a microscope slide for counting at 400 X magnification. A total of 100 motile sperm on each slide were scored for the percentage of sperm binding beads, and the location of bound beads (head, midpiece, tail, or entire sperm). Bead binding frequency, location of bead binding, and type of immunobead were tabulated for the patient population assayed by one or both of the IBT's (direct or indirect). Descriptive statistics included XY plots of the percentage of sperm binding beads in each assay and cross tabulation frequencies. Relationship of circulating antibodies to sperm surface antibodies. The IBT results on the paired samples of serum (indirect test) and semen (direct test) were inspected to pick the group of samples positive for antibodies in the direct test. For this purpose, a positive direct test was one in which 20% or more of the motile sperm bound immunobeads. 8 The percent of samples positive by the more conservative definition of =::::50% sperm bead binding14 was also tabulated. In the group of patients with positive tests for antisperm antibodies on the sperm surface, the results of the paired serum tests were examined to determine the lowest value for the percent of donor sperm with immunobeads after passive antibody transfer. A tentative prediction rule was then formulated as follows: declare a serum sample "positive", if the percent of sperm binding beads in the indirect test exceeded this lowest value. This cutoff was then revised downward to take account of possible error in determining a minimum value based on the

testing protocol. By definition, 100% of samples in which the direct test was positive would have a paired positive serum. Phrased in the language of prediction rules, 15 the cutoff was chosen so that the test for predicting the presence of sperm surface antibodies from a test on serum would have 100% sensitivity. At the same time, the specificity would be maximized. Naturally, such a rule is optimized for our data, so that neither the same 100% sensitivity nor maximum specificity would be expected in new samples. Two initial screening rules were then examined: 1) prediction of sperm surface lgG from serum lgG; 2) prediction of sperm surface lgA from serum lgA. The lack of success of the second rule had led us to consider two more rules based on serum lgG: 3) prediction of sperm surface lgA from serum lgG and 4) prediction of sperm surface lgG and/or lgA from serum lgG (antisperm antibodies in general). Finally the screening rules were reviewed in light of sample variability. To estimate the relative frequency of occurrence of immunoglobulins lgG and lgA on seminal sperm (either alone or in combination), we examined the larger set of 250 direct IBT evaluations of sperm surface antibodies. The frequencies of antibody binding to the sperm surface were tabulated in five categories based on the percent of sperm binding beads: 0%, 1-19%, 20-49%, 50-89% and 90-100%.The joint distribution of lgG and lgA on the sperm surface was evaluated in a cross-tabulation. The "predictive value positive" of a screening rule (true positives among apparent positives as predicted by indirect test values (figure 1)) will depend on the prevalence of positive direct tests in the population to which the rule will be applied. 16 This prevalence rate was determined for the series of 250 patients with direct IBT tests and the false-positive rate was estimated. Based on a series of likely assumptions about test sensitivity, specificity, and population prevalence, the predictive value positive for a screening rule based on the findings of antisperm antibodies in serum was calculated. Similarity of semen and serum binding patterns. The similarity of bead binding patterns to sperm in the direct test and in the indirect test was evaluated by Pearson correlations of the total percent of sperm binding beads in the two tests and of the corresponding percentages of sperm binding beads to specific sites. The analysis was confined to those pairs which had some bead binding in both tests to avoid the heavy weighting that would be given to the majority of samples with no bead binding in either test. Data entry and handling. The binding results were abstracted

ANTIBODIES IN BLOOD

+ ANTIBODIES

+

A

B

C

D

ON SPERM

Sensitivity = B/(A+ B) X 100% Specificity= C/(C+D) X 100% Predictive Value Positive= B/(B+D) X 100% Fm. 1. Rules for prediction of positive test for antisperm antibodies on sperm surface (direct test) based on results of screening tests for antisperm antibodies in serum (indirect test).

.l~-,.J\JTISPERf/1 -'\NTIBODI.ES

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from books into dBas,~ II files on a m1cn,cc,m·1Ju1ter AH analyses were carried out with the SAS statistical µa,&,~~t on a VAX 11/750 microcomputer. 15 RESULTS

Prediction of semen JgG serum IgG. Of the 70 paired samples, 32 had no detectable serum IgG or sperm surface IgG by IBT. Figure 2 is a plot of the percentage of sperm binding immunobeads in the direct test vs. the percentage of donor sperm binding immunobeads in the indirect test for the remaining 38 pairs. Eighteen semen samples showed some evidence of IgG on the sperm surface and of these, nine had more than 20% of the sperm binding immunobeads. The lowest serum IBT value for IgG in these nine samples was 28% of sperm binding beads. The sampling standard error for estimating a fraction of 28% of 100 sperm bound is about 4.5%. Subtracting three standard errors for safety from 28% leads to a suggested prediction cutpoint in the serum test of 15% or more of sperm binding beads. A rule based on this cutpoint would have 100% sensitivity in our data and a specificity of 79% (figure 3). Prediction of sperm surface IgA from serum IgA. Of the 70 paired samples, 36 (51 %) had no detectable serum IgA or sperm surface IgA by IBT. Only three semen samples had more than 20% of sperm binding IgA immunobeads, the percentages of donor sperm binding IgA beads after antibody transfer from the paired sera were 42%, 4% and 50%. The number of observations is too small fo:r any definite conclusions, but it appears that the indirect IBT assay using IgA immunobeads does not reflect accurately the presence of IgA antibodies on the sperm surface. Discounting the 4 % value and the same prediction rule for serum IgA as for IgG (15%), the sensitivity would be 67% and the specificity would be 91 % (figure 3). Prediction of sperm surface IgG and/or IgA from serum Iga The serum samples that corresponded to the three semen samples with 20% or more sperm binding IgA immunobeads were among the nine serum samples nAmt'"'" for IgG binding. Thus identification of serum samples with 15% or more sperm binding IgG immunobeads in the indirect test will also identify 100% of these IgA positives in semm. In our paired data the same entries as in figure 3 would with a of 100% and a specificity of 79%. Comparison of bead binding patterns on seminal sperm us. the

1041

8E}v1EN AND BLOOD SERA

pattern bead j-yc,nc•rnw to donor sperm. correlations of binding patterns on sperm with binding patterns on donor sperm following exposure to patient sera are shown in table L The only large spermserum correlations for IgG binding were for the total percent of sperm binding beads to any region (0.57) and the percent of sperm binding beads to the tail only (0.45). Among 34 pairs with any IgA bead the only significant sperm-serum correlations were those for the percent of sperm binding beads to any region of sperm (0.40) and the percent with bead binding

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FIG. 2. Relationship of immunobead binding to patients' sperm in direct test versus immunobead binding to donor sperm after passive transfer of antibodies from patients'serum. Thirty-two paired samples had no detectable IgG on sperm surface or in serum and are not included in figure.

FIG. 3, Prediction of IgG and IgA on sperm surface from serum levels of IgG and IgA respectively. Sensitivity and specificity of test for each immunoglobulin class are shown based on positive value of ?:15% sperm bead binding in serum test. L Correlations by antibody class between the percent of sperm in patients' semen with immunobead binding to specific sites and the corresponding values for donor sperm after antibody transfer from the patients' blood sera

TABLE

Regional Bead Binding To To To To To

any region head midpiece tail entire sperm

Correlation of Serum Ig to Sperm Surface lg IgG (N = 38)

0.57** 0.16 0.11

0.45** 0.36**

(N

IgA = 34)

0.40* 0.32 -0.09 0.25 0.94**

Only pairs with positive (non-zero) sperm bead binding in semen were included.

* p < .05. ** p < .01.

1042

HELLSTROM AND ASSOCIATES

to the entire sperm (0.94). Even correlations as large as r = 0.50 correspond to a percent of variation explained (r-squared) of only 25%. The general lack of correlation for the site specific binding patterns is not surprising, in light of the poor linear relationships shown in figure 2 for the total percent of sperm binding immunobeads. Binding patterns of immunobeads on seminal sperm from 250 men with suspected immunologic infertility. The results of IBT tests for lgG on seminal sperm are shown in table 2. Forty-four samples (17.6%) had 20% or more sperm binding beads and 36 (14.4%) had 50% or more sperm with binding. The site-specific binding profiles in table 2 show that binding patterns to the tail largely paralleled and were responsible for the overall binding patterns. Over 13% of samples had greater than 20% of sperm binding beads to the tail only. The binding profiles for lgA immunobeads on seminal sperm are shown in table 3. Sixty samples (24%) showed some lgA immunobead binding to sperm; 25 (10%) showed greater than 20% of sperm binding beads, while nine (3.6%) showed 50% or more binding beads. As with lgG immunobeads, the majority of samples with lgA beads bound were those with bead binding to the sperm tails. Figure 4 shows the cross tabulation of lgG and lgA immunobead binding for the semen specimens from 250 patients. Fifty semen specimens had significant sperm binding of lgA or lgG immunobeads (:=: 20% sperm bound); 25 of these (50%) were positive for lgG alone, six (12%) were positive for lgA alone and 19 (38%) had significant levels of both lgG and lgA bead binding. For the six semen samples positive for lgA alone, the corresponding values for lgG bead binding were 2%, 4%, 9%, 11 %, 16% and 17%. In one case 14% of seminal sperm bound lgM immunobeads. This test was not repeated, so the possibility of artifact cannot be ruled out. However, concurrent binding of lgG and lgA immunobeads beads (10% and 14% respectively) was also observed in this specimen. Binding patterns of immunobeads on donor sperm following passive antibody transfer from the sera of 330 men with suspected immunologic infertility. The distribution of serum samples ac-

cording to the percent of sperm binding lgA immunobeads and the location of bead binding following passive antibody transfer are shown in table 4. Only 35 serum samples (10.6%) resulted in more than 20% of donor sperm with lgA immunobead binding, while 7 (2.1%) resulted in 50% or more of sperm with beads bound. Only bead binding to the sperm tail was very prevalent, with 22 (6.7%) of the samples resulting in more than 20% bead binding to the tail. In contrast, antisperm lgG appeared to be much more prevalent in serum (table 5). Of the 330 samples, 115 (34.8%) resulted in more than 20% bead binding to donor sperm and 99 (30%) resulted in more than 50% bead binding. Seventytwo cases (20.3%) had over 20% of sperm with lgG immunobead binding to the tail, showing that as with lgA, this was the predominant site of binding. DISTRIBUTION OF SEMEN SPECIMENS ACCORDING TO PERCENT OF SPERM BINDING lgG IMMUNOBEADS (/)

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FIG. 4. Cross tabulation of lgG and lgA immunobead binding to seminal sperm of 250 patients with suspected immunologic infertility. Percentages of total 250 semen specimens are given in parentheses. 4. Distribution of serum specimens according to the percent of donor sperm binding IgA immunobeads at specific sites after passive antibody transfer

TABLE

Distribution of semen specimens according to the percent of patient sperm binding IgG immunobeads at specific sites

TABLE 2.

Percent of Sperm Binding lgG lmmunobeads

Regional Bead Binding To any region To head To midpiece To tail To entire sperm

N % N % N % N % N %

0

1-19

179 (71.6) 205 (82.0) 233 (93.2) 183 (73.2) 215 (86.0)

27 (10.8) 35 (14.0) 17 (6.8) 34 (13.6) 18 (7.2)

20-49

50-89

90-100

8 (3.2) 10 (4.0) 0 (0.0) 18 (7.2) 6 (2.4)

24 (9.6) 0 (0.0) 0 (0.0) 12 (4.8) 9 (3.6)

12 (4.8) 0 (0.0) 0 (0.0) 3 (1.2) 2 (0.8)

Percent of Sperm Binding lgA lmmunobeads

Regional Bead Binding To any region To head To midpiece To tail To entire sperm

0

N % N % N % N % N %

200 (60.6) 266 (80.6) 313 (94.8) 206 (62.4) 301 (91.2)

1-19 95 (28.8) 60 (18.2) 16 (4.8) 5 (1.5) 25 (7.3)

20-49

50-89

90-100

28 (8.5) 4 (1.2) 1 (0.3) 22 (6.6) 3 (0.9)

5 (1.5) 0 (0.0) 0 (0.0) 2 (0.6) 2 (0.6)

2 (0.6) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

Percentages of the total of 330 serum specimens are given in parentheses.

Percentages of the total of 250 ejaculates are given in parentheses.

5. Distribution of serum samples according to the percent of donor sperm binding IgG immunobeads at specific sites after passive antibody transfer TABLE

Distribution of semen specimens according to the percent of patient sperm binding IgA immunobeads at specific sites

TABLE 3.

Percent of Sperm Binding lgA Immunobeads

Regional Bead Binding To any region To head To midpiece To tail To entire sperm

0

N % N % N % N % N %

190 (76.0) 226 (90.4) 239 (95.6) 191 (76.4) 231 (92.4)

1-19 35 (13.2) 19 (7.0) 11 (4.4) 42 (16.8) 15 (6.0)

20-49

50-89

90-100

16 (6.4) 5 (2.0) 0 (0.0) 14 (5.6) 2 (0.8)

8 (3.2) 0 (0.0) 0 (0.0) 3 (1.2) 2 (0.8)

1 (0.4) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

Percentages of the total of 250 ejaculates are given in parentheses.

Percent of Sperm Binding lgG Immunobeads

Regional Bead Binding To any region To head To midpiece To tail To entire sperm

0

N % N % N % N % N %

162 (49.1) 237 (71.8) 296 (89.7) 169 (51.2) 219 (66.5)

1-19 53 (16.1) 79 (23.9) 33 (10.0) 89 (28.5) 37 (11.5)

20-49

50-89

90-100

16 (4.8) 12 (3.6) 1 (0.3) 43 (11.5) 27 (8.2)

31 (9.4) 2 (0.6) 0 (0.0) 23 (7.0) 30 (9.1)

68 (20.6) 0 (0.0) 0 (0.0) 6 (1.8) 17 (4.8)

Percentages of the total of 330 serum specimens are given in parentheses.

1043

ANTISPERM ANTIBODIES IN SEMEN AND BLOOD SERA DISCUSSION

Significant advances have been made in the diagnosis of male immunologic infertility during the last two decades. Early assays often relied upon nonspecific end~oi1:1ts such as s~e:m agglutination and for the most part were limited to determmmg circulating antibody titers. 1 • 16- 19 In contrast, the IBT allows us to assay directly for the presence of antibodies on the surface of motile sperm. . . . Previous reports have cited an uncertam relat10nship between circulating and sperm-surface antibodies. Using a radiolabelled antiglobulin assay, Haas documented in 104 consecutive infertile men 16 with lgG binding to their sperm surface, but only two with igG in their blood. 10 High background noise due to nonspecific attachment of radiolabelled rabbit lgG necessitated excluding 9% of these patients from consideration. This assay is not in wide use, and no other groups have duplicated these studies. Generally, most authorities have concurr~d that total lg levels in semen are in the order of one magmtude lower than those recorded in blood. 11 Using the immunobead test, researchers have noted that 20% of patients with sperm-reactive antibodies in blood were without antibody binding to their sperm surface. 12 Conversely, 15% found to have sperm-surface antibodies had none in their serum. 12 This phenomenon has been attrib~ted to the local secretion of lgA in the reproductive tract. The presence of circulating antisperm ant~bodies indicates _a humoral immune response to sperm cell antigens. The magmtude of the antigenic stimulus and the co-events which may be required to precipitate an immunologic respons_e to sperm_ cells are unclear. It is logical to assume that only antisperm antibodies that reach the sperm surface can interfere with sperm function. 6 • 20 It is known that the presence of circulating antibodies does not necessarily imply their presence within reproductive tract secretions'· 5 • 21 and in the case of semen even the antibodies in seminal plasma may not be representative of those on the sperm surface. 22 However, there may be valid clinical or logistical reasons to test for antisperm antib?dies in blood serum. In tertiary centers it may be more convement for the patient and for the physician to screen f~r im~unolog~c infertility early in the workup by testing for circulatmg ant~sperm antibodies, providing the sensitivity of t~~ assay is adequate to avoid false-negative results: When positiv~ results are obtained, the direct test can be earned out at the time of a subsequent visit preceded by appropriate instructions to the patient. Many physicians providing primary or secondary care may not have access to the IBT locally, necessitating shipment of blood sera to a reference laboratory for initial testing. Based on the results of this study, it appears that a screening protocol for circulating antisperm antibodies of the lgG class can be nearly 100% sensitive for predicting sperm surface antibodies of that class. The data are inadequate for firm conclusions to be drawn on the sensitivity of serum lgA in predicting seminal lgA but the possibility o_f local se_cretio~ of this immunoglobulin class by the reproductive tract is consi~tent with the apparent reduced sensitivity of blood levels m predicting the occurrence of these antibodies on the sperm surface. 2° For routine screening of blood serum there seems to be little advantage in testing for lgA since the presence of circulating IgG is quite effective in identifying men with sperm surface lgA. The reason for this success is the likelihood that sperm surface lgA will be found in association with lgG. 5• 21 In the group of 50 semen samples containing antisperm antibodies on the sperm surface, more than 75% of the specimens with IgA were also positive for lgG (>20% sperm bi~ding b~ads)._ In the majority of these lgA positive semen specimens m which fewer than 20% of the sperm bound lgG immunobeads, there was still evidence of low levels of IgG on the sperm surface. In our large data set on patient sera, about_35~ o~ samples resulted in more than 20% of donor sperm bmding immunobeads. What percent of these sera can be expected to be "true

positive," that is, be paired with a semen sample having 20% or more of the sperm binding IgA or lgG beads? We have attempted to answer this question with several different assumptions: sensitivity, 100% (as in our data) and 95% (allowing for error in the choice of our cut off point); specificity, 75% (similar to our data) and 65%; and prevalence of positive semen samples, 20% (as in our 250 cross-sectional samples) and 15%. These calculations are shown in table 6. Under the worst (but realistic) conditions of test sensitivity of 95%, specificity of 65%, and prevalence of true positives in semen of 15%, only about 32% of patient semen samples tested because of a "positive" value in the sera would be positive (table 6). Even with 100% test sensitivity, the percent of antibody positive serum samples that would lead to a positive test in semen would not exceed 50%. These calculations underscore the necessity of testing the semen directly for antibodies whenever a "positive" serum value is obtained. In conclusion, our data suggest that tests of blood serum for antisperm antibodies of the lgG cla3s can serve as a sensitive screen for male immunologic infertility. We recommend that whenever IBT results on blood serum result in more than 15% of sperm binding beads, that the findings be confirmed by a direct test for antisperm antibodies on the sperm surface. Tests for circulating antisperm lgG will identify most men with antisperm antibodies on the sperm surface. Tests for antibodies of the lgA class in sera do not appear to be warranted. In contrast to the serum tests, there is clinical value in testing for lgA on the sperm surface since the presence of this immunoglobulin class appears to imply a poorer prognosis for male fertility. 23 • 24 There appears to be little value in testing either the blood serum or semen of infertile men for antisperm antibodies of the lgM class. The sites of antibody binding on donor sperm after passive transfer from patients' sera were not well correlated with the location of native antibodies on the patients' sperm. The poor correlation may be due in part to the different sources of sperm being used in the two assays. 9 Since the site of antibody bindi~g as well as the immunoglobulin class appears to have prognostic value in predicting fertility, 3•6 these findings provide additional motivation for direct assessment of antibodies on the sperm surface whenever possible. 6. Percentage of patients with antisperm antibodies (IgG and/ or IgA) in blood serum (predicted positives) that can be expected to have antibodies on the surface of their sperm cells (true positives)

TABLE

Sensitivity of IBT Test on Serum (SN) %

Specificity of IBT Test on Serum (SP)

100 100 100 100 95 95 95 95

75 75 65 65 75 75 65 65

%

Prevalence of Patients with Antibodies on Sperm Surface (F)

True Positive Predicted Positive

(TP)*

%

%

20 15 20 15 20 15 20

50 41 42

40 40

15

32

34 49

SN x F * TP = SN x F + (1 - SP) (1 - F)

REFERENCES

1. Rumke, P.H. and Hellinga, G.: Autoantibodies against spermatozoa in sterile men. Am. J. Clin. Path., 32: 357, 1959. 2. Rumke, P. H., Van Amstel, N., Messer, E. N. and Benzemer, P. D.: Prognosis of fertility of men with sperm agglutinins in the serum. Fertil. Steril., 25: 393, 1974. 3. Bronson, R., Cooper, G. and Rosenfeld, A.: Membrane-bound sperm-specific antibodies: their role in infertility. In: Bioregulators of Reproduction. Edited by H. Vogel and G. Jagiello. New York, Academic Press, p. 521, 1981.

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HELLSTROM AND ASSOCIATES

4. Boettcher, B.: Antigens of the male tract. J. Reprod. Fertil. (Suppl.), 18: 77, 1973. 5. Rumke, P. H.: The origin of immunoglobulins in semen. Clin. Exp. Immunol. 17: 287, 1974. 6. Bronson, R., Cooper, G., Rosenfeld, D.: Sperm antibodies: their role in infertility. Fertil. Steril., 42: 171, 1984. 7. Bronson, R., Cooper, G., Hjort, T., Ing, R., Jones, W.R., Wang, S. X., Mathur, S., Williamson, H. 0., Rust, P. F., Fudenberg, H. H., Mettler, L., Czuppon, A. B. and Sudo, N.: Antisperm antibodies detected by agglutination, immobilization, microcytotoxicity, and immunobead-binding assays. J. Reprod. Immunol., 8: 279, 1985. 8. Clarke, G. N., Elliott, P. J. and Smaila, C.: Detection of sperm antibodies in semen using the immunobead test: a survey of 813 consecutive patients. Am. J. Reprod. Immunol., 7: 118, 1985. 9. Haas, G. G., Weiss-Wik, R. and Wolf, D. P.: Identification of antisperm antibodies on sperm of infertile men. Fertil. Steril., 38: 54, 1982. 10. Haas, G. G., Schrieber, A. D. and Blacso, L.: The incidence of sperm-associated immunoglobulin and C3 , the third component of complement, in infertile men. Fertil. Steril., 39: 542, 1983. 11. Bronson, R. A., Cooper, G. W. and Rosenfeld, D. L.: Factors affecting the population of the female reproductive tract by spermatozoa: their diagnosis and treatment. Sem. in Reprod. Endocrin., 4: 371, 1986. 12. Pavia, C. S., Stites, D. P. and Bronson, R. A.: Reproductive immunology. In: Basic and Clinical Immunology. Edited by Stites, D. P., Stobo, J. D. and Wells, J. V., Lange Publishers, (6th Edition) p. 619, 1987. 13. Hellstrom, W. J. G., Overstreet, J. W., Moore, S. M., Samuels, S. J., Chang, R. J. and Lewis, E. L.: Antisperm antibodies bind with different patterns to sperm of different men. J. Urol., 138: 895, 1987.

14. Ayvaliotis, B., Bronson, R., Rosenfeld, D. and Cooper, G.: Conception rates in couples where autoimmunity to sperm is detected. Fertil. Steril. 43: 739, 1985. 15. Fletcher, R.H., Fletcher, S. W. and Wagner, E. H.: Diagnostic test. In: Clinical Epidemiology: The Essentials. Baltimore, Williams & Wilkins Co., p. 41, 1982. 16. Franklin, R.R. and Dukes, C. D.: Antispermatozoal antibody and unexplained infertility. Am. J. Obstet. Gynecol., 89: 6, 1964. 17. Kibrick, S., Belding, D. L. and Merrill, B.: Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil. Steril., 3: 430, 1952. 18. Shulman, S.: Immunologic barriers to fertility. Obstet. Gynecol. Surv., 27: 553, 1972. 19. Friberg, J.: A simple and sensitive micro-method for demonstration of sperm-agglutinating activity in serum from infertile men and women. Acta Obstet. Gynecol. Scand. (Suppl.), 36: 21, 1974. 20. Jager, S., Kremer, J. and Van Slochteren-Draaisma, T.: A simple method of screening for antisperm antibodies in the human male. Detection of spermatozoa! surface IgG with the direct mixed antiglobulin reaction carried out on untreated fresh human semen. Int. J. Fertil., 23: 12, 1978. 21. Uehling, D. T.: Secretory IgA in seminal fluid. Fertil. Steril., 22: 769, 1971. 22. Bronson, R. A., Cooper, G. W. and Rosenfeld, D. L.: Seminal fluid antisperm antibodies do not reflect those present on the sperm surface. Fertil. Steril., 48: 505, 1987. 23. Parslow, J.M., Royle, M. G., Kingscott, M. M. B., Wallace, D. M. A. and Hendry, W. F.: The effects of sperm antibodies on fertility after vasectomy reversal. Am. J. Reprod. Immunol., 3: 28, 1983. 24. Parslow, J. M., Poulton, T. A., Besser, G. M. and Hendry, W. F.: The clinical relevance of classes of immunoglobulins on spermatozoa from infertile and vasovasostomized males. Fertil. Steril., 43: 621, 1985.