Immunologic Infertility—Fact or Fiction?

;O])ERN Edward WallaCh, M.D. Associate Editor FERTILITY AND STERILITY Copyright

©

Vol. 33, No.6, June 1980

Printed in U.SA.

1980 The American Fertility Society

IMMUNOLOGIC INFERTILITY-FACT OR FICTION?

WARREN R. JONES, M.D., PH.D., F.R.C.O.G., F.AuST.C.O.G. Department of Obstetrics and Gynaecology, Flinders Medical Centre, Adelaide, Australia

tending over many decades relating to the immunogenicity of seminal components in experimental models and to the antifertility effects of immunity to sperm in many animal species. These accumulated data have given substance to the possibility that immunity to sperm may operate in nature as a cause of human infertility. Animal data have been complemented by experiments, both contrived and natural, in humans. The male genital tract has been subjected to a number of insults (including vasectomy), and these procedures can provoke autoimmunity to sperm. In the human female it is of more than passing interest that the literature contains reference to at least 12 studies of immunologic contraception in women carried out between 40 and 50 years ago. In some of these reports, infertility and humoral antibody formation were achieved by repeated injections of semen without the use of immunologic adjuvants. Again, this approach suggests that such effects may be provoked in nature.

Immunologic infertility-fact or fiction? Why do we have to ask this question? Simply because the subject has been bedeviled over the years by confusion, contradictions, unjustifiable claims, and untidy methodology. This has led to understandable skepticism among both immunologists and our own clinical colleagues about the role of immunologic factors in human infertility. Let me state at the outset that I believe that immunologic infertility is fact. What is necessary now is to advance the field with impeccable attention to the methods used both in the laboratory and in the structuring and interpretation of clinical studies. Only by critically assessing published data, by applying valid tests in controlled studies, and by gaining knowledge about the natural reproductive history of individuals with immunologic factors can we restore scientific respect for our efforts and avoid false claims for diagnostic methods and results of therapy. In reviewing this topic my comments will be confined almost entirely to immunologic infertility in the female with only sporadic excursions into the male side. Theoretically, immunologic factors may operate at several steps in the human reproductive process (Fig. 1), since the gametes and the fertilized egg, as well as the hormones, tissues, and other secretions associated with the genital tract, are potentially antigenic and capable of eliciting an immune response. This review deals only with immunity to semen and to the ovum. Representative reviews of the entire field can be found in the writings of Katsh,l Menge,2 Shulman,3 Rumke and Hekman,4 Jones,5 and Beer and Neaves,6 and in relevant chapters by Diczfalusy,7 Scott and Jones,s Boettcher,9 Edidin and Johnson,lo Insler and Bettendorf,l1 and Jones. l2

ANTIGENS OF SEMEN

Human semen is an antigenic nightmare. Seminal plasma contains a vast array of antigens, many of which are common to other tissues. Those that are unique to seminal plasma have an uncertain relevance to the induction of immune infertility. The intrinsic cell surface antigens of the spermatozoon itself are of more importance in this context. With the use of antibody localization techniques, the spermatozoon has been shown to possess intrinsic antigens on the acrosome, midpiece, and tail, some of which may provoke immunologic infertility. The significance of sperm-coating antigens derived from seminal plasma is uncertain. They may provide a degree of immunologic protection by masking the possibly more important immunogenicity of intrinsic sperm surface antigens. On

EXPERIMENTAL IMMUNITY TO SPERM

There is a vast background of investigation ex577

578

JONES HYPOTHAlAMUS

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FIG. 1. The human reproductive process leading up to implantation showing the stages at which immunologic influences might operate.

the other hand, sperm-coating antigens themselves may-theoretically, at least-provoke immunity in the female, but the clinical significance of this response requires further study. Sperm enzymes also contribute to the antigenicity of sperm. Hyaluronidase, acrosin, and lactic acid dehydrogenase-X (LDH-X) have been studied extensively, but of these only the last enzyme shows any evidence of an association with immunologic infertility. Alloantigens such as HL-A and ABO blood group substances are present on the sperm surface but appear to have no relevance to infertility.

June 1980 ically competent cells. These cells phagocytose spermatozoa and process their antigens for recognition by the host's immune defenses. The operation of these mechanisms may also be influenced by several factors: (1) Large numbers of biologically redundant sperm are introduced into the vagina. It is possible that only a genetically selected minority gain access to the fallopian tube and that these are immunologically different from the residual majority. (2) Sperm are phagocytosed by somatic cells of the uterine cervix as well as by macrophages, and semen is chemotactically attractive to macrophages and neutrophils. (3) Other foreign antigens gaining access to the genital tract may have adjuvant effects. For example, immune responses to sperm antigens may be exaggerated in the presence of vaginal infections. (4) Lymphocytes in semen may contribute to the induction in the female of sterilization to histocompatibility antigens of the male partner. (5) Factors may be present which modify the immune response to sperm. These include prostaglandin E, which is immunosuppressive, and various glycoprotein fractions of seminal plasma which also have immunosuppressive properties. In addition, seminal plasma exerts a strong anticomplementary effect, at least part of which may be due to precipitins directed against complement components. Isoimmune responses to sperm in the female may be antibody- or cell-mediated, each of which may have a predominantly local rather than a systemic effect. Humoral antisperm antibodies have been detected by a variety of techniques in .,. -,,,

In nature, the introduction of sperm into the female genital tract may provoke an isoimmune response. The afferent and efferent limbs of this response are depicted in Figure 2. In the human female, the genital tract, particularly the uterus, is well endowed with immunolog-

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ISOIMMUNIZATION IN THE HUMAN FEMALE AGAINST SPERM ANTIGENS

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FIG. 2. Immunologic pathways involved in isoimmunization to antigens of semen. Antigens are processed by macrophages and presented to the reticuloendothelial system in the afferent pathway. The efferent responses may be both local and systemic and involve antibody formation and cell-mediated immunity.

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IMMUNOLOGIC INFERTIUTY

infertile women, and their clinical significance is discussed below. Experimental studies in humans involving chemical and microbial antigens indicate that the effector pathways oflocal immunization in the female genital tract mediate both systemic and local cellular immune responses. These include transplantation immunity and immunologic memory with secondary responses and local hypersensitivity reactions. There is also clinical evidence showing that coitally induced antigens may in rare cases provoke systemic and local acute hypersensitivity reactions. An awareness of the immunologic characteristics of genital tract secretions is of central importance to our understanding of immune mechanisms in female infertility. The uterine cervix appears to be the major site of secretory immunologic activity in the female genital tract. The uterine endometrium and the fallopian tubes may also participate to a limited extent, but the vagina is probably inactive. Local (intravaginal) immunization with experimental antigens results in a specific antibody response in the cervical mucus. Usually an antigenic stimulus to a mucus membrane results in the formation of both local and circulating antibodies, but in some circumstances antigen may not reach the circulation in immunogenic quantities and therefore a systemic response may not occur. The immunologic characteristics of the cervix and its secretions have been the subject of several reviews. 13-17 The cervix is adequately equipped with plasma cells, and cervical mucus contains immunoglobulin-at least part of which is locally secreted. This can be demonstrated by finding secretory immunoglobulin A (SIgA) as well as serum immunoglobulin A (lgA) and immunoglobulin G (lgG) in the mucus. SIgA is formed locally at mucus surfaces and is composed of two serum IgA molecules and a nonimmunoglobulin peptide called secretory component (SC). Immunoglobulin and C3 complement levels in cervical mucus vary during the menstrual cycle, being lowest at the time of ovulation. Total complement levels are low in cervical mucus but require further study. Specific antibodies may also appear in oviductal and follicular fluids. IgG, IgA, and SC can be demonstrated in tubal epithelium, and IgG and IgA are present in tubal fluid in concentrations 10% and 20%, respectively, of levels in serum. C3 but not total complement activity is detectable in tubal fluid. The potential ubiquity of sperm antibodies is also suggested by the presence of IgG and IgA in

579

follicular fluid and the demonstration of specific antibodies in this fluid in patients with circulating antisperm antibodies. The potential importance of follicular fluid in the mediation of local immunity in the reproductive tract is further enhanced by evidence that it contains a respectable representation of complement cascade components. In summary, therefore, the ubiquity and potential importance oflocal antibodies, both transudated and secreted, makes the determination of the clinical significance of circulating antibodies a particularly difficult and confusing exercise. THE INVESTIGATION OF IMMUNOLOGIC INFERTIUTY

A bewildering variety of methods has been used to investigate the possible operation of immunologic factors in infertility. This array reflects a generally held concern about the immunologic validity, interpretation, and standardization of many of the tests. In addition, there has been considerable confusion regarding the clinical significance of some of the commonly used methods for the detection of circulating antisperm antibodies. This in turn has placed a major emphasis on the development and application of tests for local immunity to sperm. Certainly, many ofthe problems ofstandardization have been overcome by a collaborative program sponsored by the World Health Organization which has given rise to definite reviews of methodology for antisperm antibody detection. 18-2o Beer and Neaves6 have also provided a detailed critical review of antisperm antibody tests. Since there is no epidemiologic or serologic evidence that the HL-A or ABO systems are involved in immunologic infertility, donor sperm may be used as an antigen source in antisperm antibody testing. The reactions, therefore, are tissue-specific rather than individual-specific. Sperm M icroagglutination. The agglutination of sperm may be observed microscopically on slides or in the wells of a microchamber. Personal experience and that of many other workers indicate a perplexing variation in the incidence of sperm microagglutination in the blood of both infertile and control subjects. Several reasons may account for this variation and should dictate caution in the immunologic and clinical interpretation of this test. Human sperm may agglutinate in the presence of microbial agents, fungi, chemicals, steroids, and even homologous serum. Technical variations in quantitation and the omission of appropriate control experiments have tended to

580

JONES

exaggerate the effect of these nonspecific factors in many published studies. The relevance of micro agglutination tests to immunologic infertility has also been questioned by the demonstration that the activity may be in the j3-g1obulin fraction rather than the "i-globulin fraction. Furthermore, head-to-head spermagglutination can be induced by test serum containing sex steroids (progesterone and testosterone) of endogenous or exogenous origin; this activity resides in the steroid-binding j3-lipoprotein fraction. These findings explain many of the vagaries of the sperm microagglutination test in different groups of patients, including the occurrence of positive tests in pregnancy sera. Notwithstanding these considerations, there are some sera exhibiting strong antisperm activity in other immunologic tests which show positive sperm microagglutination due to the presence of IgG and IgM antibodies. It has also been a consistent finding that positive activity in female sera tends to be of the head-to-head type, and that in male sera of the tail-to-tail type. This suggestion of a divergence of antibody specificity in males and females is reflected in the results of other tests for both circulatory and local antisperm antibodies (see below). It should be stressed that virtually nothing is known about the precise antigen-antibody systems involved in the various patterns of spermagglutination. Resolution of this problem will await the isolation and characterization of relevant sperm membrane antigens. Gelatin Agglutination. In this test, motile spermatozoa are suspended in a gelatin medium and the serum or fluid under test is added in serial dilution to small test tubes containing the sperm suspensions. Agglutination is then observed macroscopically. This method has been used extensively in the investigation of infertile males, but its use in females has been limited. One reason might be that head-to-head spermagglutination, which appears to be mediated more commonly by antibodies developed in the female, is not readily detected by the gelatin technique. Although there is no doubt that gelatin agglutination activity resides in the IgG ~nd IgM fractions, the method requires careful control and interpretation under consistent laboratory conditions. Failure to recognize these requirements may account for the disturbingly high incidence of positive tests in unmarried and fertile control groups in some reports. Sperm Immobilization. Complement-dependent sperm immobilization forms the basis of a simple reproducible test for sperm antibodies. The interaction of antibody molecules with sperm anti-

June 1980 gens activates the complement system and disrupts the permeability and integrity of the cell membrane of the sperm acrosome and midpiece. The ultimate effect on the sperm can be microscopically detected as a loss of motility followed by cell death. Sperm-immobilizing activity has been shown to reside in the IgG and IgM factions of positive sera and appears to provide the most reliable method of detecting humoral antisperm activity with possible clinical significance. Sera with immobilizing activity usually show spermagglutinating activity, but the converse does not apply. The sperm immobilization test is the method of choice for screening females for serum antibodies and may also be applied to cervical mucus. It appears to exhibit relatively low titer activity in male sera which are strongly positive in gelatin agglutination. It is simpler to perform, however, and may be used as a screening test on blood to detect male subjects requiring further investigation for seminal plasma antibodies. For this purpose, the gelatin agglutination test appears to have a clear advantage in sensitivity over the immobilization test. Spermatozoa used in immobilization tests must be obtained fresh from ejaculates of high quality. Serum used as a source of complement should also be obtained fresh and stored frozen in small aliquots. Sera from humans, guinea pigs, and rabbits appear to be equally suitable sources of complement, but activity must be checked and titrated periodically. Several sperm-immobilization techniques have been devised. Most are based on the initial method described by Isojima (see Rose et a1. 20 ), but when large numbers of sera are to be tested, a microtechnique based on the principle of lymphocyte tissue typing21 has certain advantages, particularly in ease of titration. The test end-point may be extended from sperm immobilization to cytotoxicity by the use of dye exclusion; however, this step appears neither to improve the validity nor the sensitivity of the method. Immunofluorescence Techniques. The sensitive indirect immunofluorescent antibody technique has been applied to the study of antisperm antibodies. Early unsatisfactory results were obtained by using unfixed spermatozoa as substrate, but subsequent refinements in technique 22-notably the use of methanol fixation of spermatozoal smears-have led to widespread use of the test in the investigation of infertility. The method involves three basic steps. The antigenic substrate is prepared by making an air-dried

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IMMUNOLOGIC INFERTILITY

and fixed smear of spermatozoa. The smear is then exposed successively to test serum (or cervical mucus or seminal plasma) and to fluorescencelabeled antibody against immunoglobulins. Antigen-antibody reactions between serum and reproductive tract fluids and sperm cells may be visualized and localized by microscopy and their surface representation related to the anatomy of the spermatozoon. The staining patterns obtained on fixed sperm involve subsurface antigens, which are presumably less important than surface antigens in the provocation of antibodies with potential clinical significance. This fact is borne out by the generally poor correlation of immunofluorescent sperm antibodies with fertility status and with antibodies detected by immobilization and gelatin agglutination techniques. The immunofluorescence technique presents a number of difficulties in the present context. Background fluorescence and unevenness of staining on individual spermatozoa cannot be avoided. Staining patterns vary with the quality ofthe substrate semen, and there is usually a heterogeneity of staining in which up to 30% of the spermatozoa fail to exhibit the predominant pattern. A particular problem is that the animal sera from which the fluorescent conjugates are prepared often contain small amounts of antibodies reactive against spermatozoa of other species. To overcome this problem it is important to use conjugates of such a strength that this potential source of unwanted staining can be diluted out. Nonspecific staining is common and dictates the careful use of control slides. Despite precautions, the high incidence and ubiquitous nature of weakly reactive antibodies has caused problems at test serum titers less than 1:16. Specificity can be reasonably assured by using F(ab) preparations of antibodies to exclude nonspecific Fc binding, but the complexity of such an approach is not compatible with the use of the method as a routine screening test. Weak-staining reactions of uncertain significance are often seen with undiluted serum, and positive results should be reported only on titers greater than 1:16. Some regions of the sperm such as the equatorial segment, neck, and midpiece are common sites of nonspecific staining patterns. Antisperm antibodies in blood react in the immunofluorescence technique mainly against antigens in the acrosome and tail. Acrosome staining occurs with both IgM and IgG antibodies, and staining of the main tail piece appears to be almost exclusively due to the presence ofIgG. Staining of the tail end-piece is occasionally observed and is

581

due to the presence of IgM antibodies. The treatment of normal human spermatozoa with trypsin in the presence of dithiothreitol causes swelling ofthe head and exposes DNA and nuclear proteins. Some of the sera of infertile or postvasectomy males with high titers of spermagglutinating antibodies contain IgG antibodies which localize in the immunofluorescence test on the swollen sperm heads. This reaction is rare in female sera and may reflect autoimmunity to somatic nuclear antigens. These antibodies appear to be directed against a strongly basic human protein, protamine. Methodology and Antibodies in Cervical Mucus. Largely because oflogistic difficulties in collection and technical processing problems, cervical mucus has remained somewhat neglected in the investigation of immunologic infertility. More recently, however, the realization that sperm antibodies may appear in the mucus in the absence of readily detectable serum levels or may be locally secreted in the SIgA class has led to attempts to establish routine methods for their measurement. In general terms, cervical mucus is most easily and appropriately collected at midcycle. For the specific purpose of sperm antibody measurement, the time of collection is less important, since there tends to be an inverse relationship between the amount of cervical mucus produced and its immunoglobulin concentration. Various techniques have been devised to prepare cervical mucus for sperm antibody testing: (1) ultracentrifugation to produce a watery supernatant (this tends to exclude a large proportion of the antibodies); (2) liquefaction with bromelin, a proteolytic enzyme (the introduction of such an agent into a test system involving sperm surface antigens is not advisable); (3) extraction with a physiologic buffer followed by centrifugation to produce an antibody-containing supernatant. In my laboratory, mucus is aspirated into a syringe with 0.5 ml of phosphate-buffered saline at pH 7.6. After storage at 4° C for 24 hours, it is liquefied by repeated passage through a 25-gauge needle. It may be stored at - 20° C in this state before centrifugation for 5 minutes at 12,000 rpm . in a microfuge. The resultant supernatant contains the immunoglobulin fraction and is suitable for use in a sperm microimmobilization method. This appears to be the most suitable test for use on cervical mucus. Both the indirect immunofluorescence test and sperm microagglutination test have proved unreliable and lacking in correlation with infertility.

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Antibodies in Seminal Plasma. Sperm antibodies in the IgG and IgA class may appear in seminal plasma when they are present in relatively high titer in serum. When high titers are present in seminal plasma they are usually due to a significant contribution from locally secreted (SIgA) ~tibodies. When head-to-head agglutinins are present in serum, being mostly IgM antibodies, these are unlikely to appear in seminal plasma. Tail-to-tail (IgG) antibodies appear in semen but only when serum titers are greater than 1:64. The direct measurement of seminal plasma antibodies may be helpful, but serum titers can usually be used to predict the likely local situation about which additional information can be obtained from the sperm-cervical mucus contact test (see below). The Need for New Methodology. There remains an urgent need for the development ofradioimmunoassays for the precise detection and quantitation of antibodies to specific sperm antigens. Several investigators have moved toward this need with the development of radiolabeling methods involving whole sperm cells. Sung et al. 23 have used radiolabeled actinomycin D in a microassay for sperm cytotoxicity, and its value has been confirmed by Boettcher and Boettcher. 24 Another potential test system involves radiolabeled Staphylococcus protein A binding to antigen-antibody complexes. 25 Another approach yet to be fully developed involves the use of the in vitro fertilization systems to assess the biologic effects of antisperm antibodies. Such tests may utilize human sperm and zona-free rodent eggs or an isologous system using human oocytes. Cell-Mediated Immunity (CMI). The in vitro assessment of CMI to spermatozoal or testicular antigens has proved fraught with technical and interpretational difficulties. There is little doubt that CMI may be part ofthe autoimmune response to genital tract disruption in the male. In the female, however, there is little evidence that CMI

to sperm is a necessary accompaniment of immunologic infertility. The final answer awaits improvement in techniques, particularly the preparation of purified, soluble sperm antigens for use in lymphocyte stimulation and leukocyte migration inhibition tests. Sperm-Cervical Mucus Contact Test (SCMC). This test was devised by Kremer and J ager26 to demonstrate the presence of local antibodies in either partner. A positive result indicating antisperm antibodies in either semen or cervical mucus (or both) is associated with characteristic shaking movements of the sperm when they come into contact with cervical mucus on a slide. It was originally hypothesized that the phenomenon was mediated by "stickiness" of the Fc fragment oflgA antibody molecules which adhered to the glycoprotein micelles of cervical mucus. It now appears that the "shaking" phenomenon can be mediated by F(ab)2 fragments of antisperm antibodies in both IgA and IgG classes. Nonetheless, the SCMC test is a valuable means of detecting local antibodies and is also suitable for crossover testing. CLINICAL SIGNIFICANCE OF ANTISPERM ANTIBODIES

In the Female. The complement-dependent sperm immobilization test appears to be a reliable index of immunologic infertility in the female. In our review 14 of 539 women with "unexplained" infertility, this test was positive in 7.6%. A prospective evaluation of reproductive patterns in women with positive tests was undertaken to compare the significance of sperm immobilization with the immunologic suspect sperm microagglutination test. A form of treatment was undertaken by a small group of women, but most were not offered therapy for several reasons. In order to assess subsequent fertility in untreated women with positive tests and in those without antibodies, questionnaires were sent to 430 consecutive patients who had been investi-

TABLE 1. Reproductive Performance Subsequent to Antisperm Antibody Testing in 391 Infertile Couplesa Infertility category

Mean age (range)

Duration of infertility

No. of patients

Mean yr

Unexpl!lined (primary and secondary) Organic Minor Major

Range

No. of patients who became pregnant

yr

28.5 (20-45)

281

3.2

1-15

142 (50.5%)

28.4 (22--37) 29.7 (18-39)

51

3.5

1-9

29 (56.9%)

59

4.1

1-13

14 (23.7%)

aFollow-up period 12 to 73 months, mean 45 months.

IMMUNOLOGIC INFERTILITY

Vol. 33, No.6

TABLE 2. Reproductive Performance Subsequent to Investigation in 332 Infertile Couples a Spenn antibody test in female partner

Microagglutination Negative Positive Immobilization Negative Positive

No. of patients

No. of patients who became pregnant

274 58

147 (53.6%) 24 (41.4%)

309 23

163 (52.8%) 8 (34.8%)

aprimary and secondary unexplained infertility, 281 patients; minor organic abnormalities, 51 patients.

gated between 1 and 6 years previously. Replies were received from 391 patients, a return rate of 91 %; details of these patients are shown in Table l. The mean follow-up period following investigation was 45 months (range 12 to 73 months). So that a more meaningful analysis might be made of the clinical significance of antisperm antibodies, a group of patients with major organic lesions was excluded and the data were analyzed in 332 women with unexplained infertility or minor organic abnormalities (Table 2). Although pregnancy rates were higher in those patients with negative tests as compared with those with positive tests, none of the differences approached statistical significance. In the face of this lack of clear evidence of an association of antisperm antibodies with subsequent infertility, a subgroup of 156 patients with relatively long-term infertility was analyzed (Table 3). These were women with unexplained and minor organic infertility of 3 or more years' duration at the time of testing. In this group, lower pregnancy rates were found in patients with positive tests as compared with those with negative tests. These results indicate that the clinical significance of these tests may be restricted to those patients with relatively long-term infertility of an unexplained nature. A superior clinical significance of sperm immobilization over sperm microagglutination was evident when serum titers were analyzed in selected patients (Table 4). The advantages of sperm immobilization as a screening test in women for circulating antisperm antibodies as compared with both microagglutination and gelatin agglutination have now been stressed by many workers. The indirect immunofluorescence test appears to correlate with infertility when performed at appropriate serum dilutions by certain experienced workers. Nonetheless, there is an undercurrent of concern about the value of this method as a screening test for antisperm antibodies in both

583

men and women. Certainly there is a poor correlation with other screening tests, which is not surprising in view of the different antigen-antibody systems involved. Another major source of confusion has stemmed from the demonstration by Tung and co-workers27 • 28 that naturally occurring antibodies giving homogeneous immunofluorescence staining of the sperm acrosome are present in up to 60% of normal adults. This finding presumably reflects exposure to foreign antigens, including ubiquitous microorganisms, which provokes antibodies cross-reactive with spermatozoa. Yet another source of complexity is the fact that antibodies against smooth muscle cross-react with the contractile protein, actin, in the postacrosomal region of the sperm. Anti-smooth muscle antibodies are relatively common in adults and are present in high incidence in infertile women both with and without sperm antibodies. Data such as these explain at least some of the confusion surrounding the incidence and significance of circulating antisperm antibodies detected by immunofluorescence techniques. The application of antisperm antibody testing to cervical mucus should become an integral part of the investigation of immunologic infertility. The sperm immobilization test appears suitable for this purpose, although long-term follow-up studies are necessary to elucidate its ultimate clinical significance in this situation. The value to be obtained from antibody testing of cervical mucus hinges on the incidence of local secretory activity in the absence of a positive serum test. Data from experimental model systems suggest that it is extremely difficult to provoke local immunity in the female without a concurrent systemic response; there is some evidence, however, that such concurrent response does not occur in nature, and this provides the rationale for cervical mucus testing. On the other hand, serum antisperm antibodies may on occasion be present in the absence of activity in cervical mucus and may exert an antifertility effect by transudation into the reproductive tract at a higher level. It is necessary therefore to TABLE 3. Subsequent Fertility in 156 Couples Previously Infertile for Three or More Years Spenn antibody test in female partner

Microagglutination Negative Positive Immobilization Negative Positive

No. of patients

No. of patients who became pregnant

124 32

55 (44.4%) 12 (37.5%)

141 15

64 (45.4%) 2 (13.3%)

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TABLE 4. Reproductive Performance Subsequent to

Sperm Antibody Testing in Twenty Couples with Unexplained Infertilitya Sperm antibody test in female partner

Microagglutination

Immobilization

No. of test results

No. of patients who became pregnant

Negative Undiluted positive >1:16

2 10

0 3

8

2

Negative Undiluted positive >1:16

6 9

3 2

5

0

Result

aMean follow-up period 3 years, range 1 to 6 years.

test both blood and cervical mucus to obtain a comprehensive profile. A search for naturally occurring antibodies against spermatozoal hyaluronidase and acrosin has produced confusing results, and, as pointed out previously, these antigens appear to have little relevance to infertility. On the other hand, isoantibodies to LDH-X have been demonstrated in infertile women using antigen from both mouse and human sources. In the Male. Many large groups of infertile males have been investigated for the presence of circulating antisperm antibodies using the gelatin agglutination test. Positive results are obtained in 3% to 8% of infertile men, as compared with up to 2% in fertile males. Rumke et a1. 29 studied the reproductive history of 254 men, originally infertile, who had been tested for gelatin agglutinating sperm antibodies between 2 and 16 years previously. In those with normal sperm counts initially there was a close positive correlation between high serum titers of antibodies and continuing infertility; low titers were compatible with fertility. High-titer gelatin agglutinins or sperm-immobilizing activity in serum usually indicate the concurrent presence of antibodies in seminal plasma. Immunofluorescence antibody tests in men show a poor correlation with fertility status and with other tests of antisperm antibodies. There are two exceptions to this generalization: an autoantibody directed against sperm heads swollen by treatment with dithiothreitol and trypsin 29 and an antibody described by Tung28 which causes speckled acrosomal fluorescence; both seem to reflect true autoimmunity and correlate with the infertile status. The possible presence of occult genital tract pathology (including infection) in men with antisperm antibodies must be considered. Notwith-

standing this, in infertile males without demonstrable organic lesions, there is a close association between sperm antibodies in serum and the presence in semen of agglutinated or sluggish spermatozoa with a poor capacity for cervical mucus penetration. It is likely that, in men who remain fertile in the presence of circulating antibodies, the antibodies fail to enter the seminal plasma in sufficient concentration to impair spermatozoal efficiency. INVESTIGATION GUIDELINES

On the basis of my comments on methodology and the clinical significance of antisperm antibody tests, several guidelines can be proposed to underpin the investigation of immunologic infertility. 1. The sperm immobilization method is a suitable screening test for circulating complementfixing antibodies in males and females and may also be applied to cervical mucus testing. 2. The SCMC test can be used to detect local antisperm immunity with possible biologic significance. By "crossover" testing using donor sperm and cervical mucus, the method may discriminate the site of antibody activity, whether in the male or the female. 3. The gelatin agglutination test is suitable for use in males, particularly on seminal plasma, but requires careful interpretation. 4. Locally secreted (SIgA) antibodies may not be detected in cervical mucus and seminal plasma by conventional tests for circulating antisperm antibodies. 5. The sperm microagglutination test is unreliable and should be abandoned. 6. Antisperm antibody tests based on indirect immunofluorescence microscopy have no place in the routine investigation of infertility but may have value in the characterization of antigenantibody reactions for research purposes. ASPECTS OF TREATMENT

Attempts to treat immunologic infertility have proved disappointing and the benefits derived from the detection of a presumptive immunologic etiology have been somewhat intangible. The results of immunologic investigations may provide both doctor and patient with the largely academic satisfaction of finding "something wrong"; ifproperly performed and interpreted they may form the basis of a helpful prognosis. In the Female. Over the years there have been enthusiastic claims for the success of occlusion

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IMMUNOLOGIC INFERTILITY

therapy (male partner using condoms for 6 to 9 months) in women with evidence of an immunologic basis for their otherwise unexplained infertility. However, the results of occlusion therapy compare unfavorably with the outcome in patients with circulating antisperm antibodies who remain untreated. The somewhat more rational selection of patients for this treatment on the basis of local immunity demonstrable in cervical mucus might lead to improved results. It must be stressed, however, that local immunity to sperm is not confined to the cervix, and the "block" may occur at a higher level. Immunosuppressive therapy with corticosteroids has been attempted and has led to anecdotal reports of lowered antibody levels and the occurrence of pregnancies. The use of high-dose immunosuppressive treatment for a non-lifethreatening disorder on tenuous theoretical grounds may be difficult to justify. In women with evidence oflocal antisperm antibodies in their cervical mucus, the intrauterine insemination of semen has an attractive theoretical basis. Several workers have attempted this approach, and pregnancy rates between 20% and 30% have been reported. Using this technique I have achieved four pregnancies in seven patients with clear evidence of antisperm antibodies in their cervical mucus. However, controlled studies are required to establish the validity of this approach. Also with this technique, as in other treatment methods in the female, it is important to attempt to distinguish therapeutic success from pregnancies occurring during one of the nadirs of the sperm antibody titer, which have been recorded in my long-term studies and those of Isojima. 30 In the Male. Therapy with adrenocorticotropic hormone or corticosteroids has proved disappointing in men with circulating antisperm antibodies. However, there is some evidence that this treatment may have a place in immunologic aspermatogenesis where cellular immunity is operative and where irreversible testicular changes have not occurred. Spermatogenetic suppression with longterm testosterone treatment has also been attempted by several workers, but the results have been unconvincing. Another approach has been adopted in which sperm from males with immunologic infertility are washed several times in a physiologic buffer supplemented with 5% to 10% human serum or albumin. The washed spermatozoa are then inseminated into the cervical canal or uterine cavity

585

of the female partner. Good-quality semen is an essential starting point of this procedure. Pregnancies have been reported, but as yet there is no clear evidence of a specific therapeutic benefit from this method. There is an important need for prospective controlled assessment of the therapeutic maneuvers currently in use to establish whether they offer any advantage over nature. This is all the more important when one considers the generally good prognosis in "unexplained" infertility. The other major problems in the field which confuse our understanding of immunologic infertility and make treatment prospects disappointing are the role of cell-mediated immunity and the precise significance and distribution of local antibodymediated immunity, especially in the female. IMMUNITY TO ZONA PELLUCIDA

No current review of immunologic infertility would be complete without some reference to the possible role of immunity to zona pellucida. 31 -33 The zona contains cell-specific antigens which may be involved in immunologic reactions related to infertility. Heterologous antibodies raised against zona pellucida coat its outer surface, block sperm attachment and fertilization, and prevent its dissolution by enzymes. This reaction is mostly species-specific; however, there is cross-reactivity of antibodies to primate and porcine zona. Isolated porcine zonae can therefore be used in test systems for the detection of anti-zona antibodies in humans. Methods used include the indirect immunofluorescence antibody technique and the ability of antibodies to coat the zona and alter its lightscattering properties. These tests are difficult to control and interpret, and many of the data obtained to date are open to methodologic criticism. In addition, anti-zona antibodies may cross-react with homologous and heterologous erythrocyte antigens so that an appropriate absorption must precede any assay procedure. Using the current methods, autoantibodies to zona pellucida have been demonstrated in the blood of a proportion of infertile women 34 ; however, they have also been found in fertile and perimenopausal subjects. Clearly there are many problems to be resolved in the assessment of the significance of autoimmunity to zona pellucida. At least some of these problems should be overcome when zona antigens are isolated and characterized and when specific radioimmunoassays are developed.

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June 1980 18. Boettcher B, Hjort T, Rumke P, Shulman S, Vyazov V: Auto- and iso-antibodies to antigens of the human reproductive system. Acta Pathol Microbiol Scand Sect C [Suppl 258],1977 19. Boettcher B, I-fjort T, Rumke P, Shulman S, Vyazov 0: Auto- and iso-antibodies to antigens of the human reproductive system. Clin Exp ImmunoI30:173, 1977 20. Rose NR, Hjort T, RumkeP, Harper MJK, Vyazov 0: Techniques for the detection of iso- and auto-antibodies to human spermatozoa. Clin Exp Immunol 23:175, 1976 21. Husted S, Hjort T: Microtechniques for simultaneous determination of immobilising and cytotoxic sperm antibodies. Clin Exp Immunol 22:256, 1975 22. I-fjort T, Hansen KB: Immunofluorescent studies on human spermatozoa. I. The detectiori of different spermatozoal antibodies and their occurrence in normal and infertile women. Clin Exp Immunol 8:9, 1971 23. Sung JS, Shijuya H, Black DD, Mumford DM: A radioimmunoassay for cytotoxic antibody to human spermatozoa. Clin Exp Immunol 27:469, 1977 24. Boettcher B, Boettcher MJ: Spermatoxic antibodies in sera from infertile couples determined with H3-actinomycin D. Proceedings of the Fourth Symposium on Immunology of Reproduction, Varna, Bulgaria, 1978. Sofia, Bulgarian Academic Scientific Press. In press 25. Han LB, Tung KS: A quantitative assay for antibodies to surface antigens of guinea pig testicular cells and spermatozoa. BioI Reprod 21:99, 1979 26. Kremer J, Jager S: The sperm cervical mucus contact test: a preliminary report. Fertil Steril 27:335, 1976 27. Tung KSK, Cook WK, Terestia A, McCarty TA, Robitaille R: Human sperm antigens and anti-sperm antibodies. II. Age-related incidence of anti-sperm antibodies. Clin Exp Immunol 25:73, 1976 28. Tung KSK: Human sperm antigens and anti-sperm antibodies. III. Studies on acrosomal antigens. Clin Exp Immunol 24:292, 1976 29. Rumke P, Van Amstel N, Messer EN, Bezemer PD: Prognosis of fertility of men with sperm agglutinins in serum. Fertil Steril 25:393, 1974 30. Isojima S: Personal communication 31. Kolk AHJ, Samuel T, Rumke P: Auto-antigens of human spermatozoa. Clin Exp ImmunoI16:63, 1974 32. Shivers CA, Dunbar BS: Autoantibodies to zona pellucida: a possible cause for infertility in women. Science 197:1082 1972 ' 33. Sacco AG: Antigenic cross-reactivity between human and pig zona pellucida. BioI Reprod 16:164, 1977 34. Mori T, Nishimoto T, Kitigawa M, Noda Y, Nishimura T, Oikawa T: Possible presence of auto-antibodies to zona pellucida in infertile women. Experientia 34:797, 1978

Received December 6, 1979. Presented at the Thirty-Fifth Annual Meeting of The American Fertility Society February 3 to 7 1979 S Franclsco, Cahf. ' " an Rep~int requests: Dr. .warren R. Jones, Department of Obstetrics and Gynaecology, Flinders Medical Centre Bedford Park Adelalde, South Austraha 5042. ' , Aye~st Lect~re.