Antisperm antibodies in women: variability in antibody levels in serum, mucus, and peritoneal fluid

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1 FERTILITY AND STERILITY Copyright 1 The American Fertility Society Printed on acid free paper in U.S.A. Antisperm antibodies in women: variability in antibody levels in serum, mucus, and peritoneal fluid Judy E. Stern, Ph.D.*t:j: Peter M. Dixon, B.S. * Paul D. Manganiello, M.D.* Truls Brinck-Johnsen, Ph.D.t Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire Objective: To look for patterns of antisperm antibody expression in women by exploring the levels of antisperm antibodies in different body fluids. This was achieved by studying sequential serum samples from individual patients and by comparing the levels of antisperm antibodies in serum from a number of patients with the levels of antisperm antibodies in cervical mucus or peritoneal fluid (PF). Design: Prospective studies were performed on sequential serum samples within a menstrual cycle. Retrospective studies were done to compare antisperm antibodies in serum and mucus or PF. The immunobead assay was used to measure antisperm antibodies in these fluids. Setting: Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire. Patients: A random sample of patients undergoing evaluation for infertility. Results: The levels of antisperm antibodies in sera drawn from patients at different points in a menstrual cycle stimulated by the presence of exogenous hormones did not change during the follicular phase of the menstrual cycle. Also, in many samples, the antisperm antibody level in serum did not correlate with the antisperm antibody levels in mucus or PF. Conclusions: The data suggest that measurement of antisperm antibodies at a single point in time or from a single fluid is not sufficient when evaluating a woman for immunological infertility. The data also suggest that numerous and complex factors contribute to the expression of antisperm antibodies in women. Fertil Steril 1;58:5-8 Key Words: Antisperm antibodies, serum, mucus, peritoneal fluid, immunological infertility Antisperm antibodies are present in only a small percentage of sexually active women. As a subgroup of these women, female patients presenting with problems of infertility have a slightly higher incidence of antisperm antibodies (1). Numerous studies have shown that antisperm antibodies impair fertility in women and experimental animals (, 3) and that this impairment results from binding of the an- Received November 11, 11; revised and accepted July, 1. * Department of Obstetrics and Gynecology. t Department of Pathology. :j: Reprint requests: Judy Stern, Ph.D., Department of Obstetrics and Gynecology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire Department of Surgery. 5 Stern et al. Antisperm antibodies in women tibodies to the surface of sperm. Antisperm antibodies manifest their effects through decreased sperm movement (4), reduced rates of fertilization (5), and decreased rates of cell division in the early pre-embryo (5,6). Impairment of fertility in the presence of antisperm antibodies would theoretically be expected if vaginal, cervical, uterine, or oviductal secretions contained high levels of antisperm antibodies. Nevertheless, in women, antisperm antibodies are most commonly measured in serum in which the significance ofthese measurements is less clear. Confusion about the meaning of serum antisperm antibody levels is particularly apparent in light of several recent studies that suggest that antisperm antibodies measured in serum do not reflect the antisperm an-

2 tibodies found in reproductive tract secretions (7- ). At present, we have little understanding of the factors that influence antisperm antibodies in secretions of the female reproductive tract, nor do we understand why serum and reproductive tract secretions should express antisperm antibodies differently. Studies in experimental animals suggest that the reproductive tract is a part of the mucosal immune system (1, 11). Other studies suggest that the levels of antibody present in reproductive tract secretions depend on the experimental conditions used to induce antibody formation (1, 13). In these animal models, different immunization protocols and hormone levels are responsible for different patterns of antibody expression in reproductive tract fluids and serum. Our goal in this study was to look for patterns in the expression of antisperm antibodies in serum and other fluids that could in turn reflect different mechanisms for the generation of antisperm antibodies. To do this, we have compared the antisperm antibody levels in serum and mucus as well as the levels in serum and peritoneal fluids (PFs) from a number of patients. We have also evaluated sequential serum samples from three patients under changing hormonal conditions. MATERIALS AND METHODS Patient Population and Fluid Collection The patients studied were being seen for problems of infertility at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire. Serum and mucus samples were collected as part of the infertility evaluation. Peritoneal fluid was collected during routine laparoscopy. Mucus and PF samples were scrutinized for the presence of blood, and samples that contained obvious blood contamination were eliminated from consideration in the study. Most serum samples were collected randomly during different stages of the menstrual cycle. In one set of patients, however, the serum was collected daily and estradiol (E ) levels and antibody levels were determined in the same samples. Serum was collected into a 13 ml serum separator tube and was processed by centrifugation at 1, X g for 1 minutes. The supernatant was filtered through a.-jlm filter and heat inactivated at 6 C for 3 minutes. The serum was either assayed for antisperm antibodies immediately or stored frozen at - C until the assay could be performed. The as- says were performed within months of fluid collection. It was often not possible to draw mucus and serum or serum and PF samples at the same time. All of the mucus and serum samples were drawn within months of each other. Peritoneal fluid and serum samples were drawn within months of each other with several exceptions that are presented separately (see Results). Cervical mucus was collected at midcycle. In some cases, this was during a natural cycle and in other cases women were given ethinyl E (EE) (5 JLgjd for 15 days) to increase the amount of mucus available for collection. In still other patients, the mucus was collected after administration of human menopausal gonadotropin (hmg) ( to 4 ampules each containing 75 IUjd for 1 to 15 days) or clomiphene citrate (CC) (15 mgjd for 5 days). After collection, mucus samples were mixed with an equal volume of bromelain (Sigma Chemical Co, St. Louis, MO B5; mgjml) and allowed to liquefy at room temperature for 1 minutes. The mucusjbromelain mixture was spun at 1, X g for 5 minutes, and the supernatant fluid was separated from any cellular debris present in the pellet. The mucus was then heat inactivated at 6 C for 3 minutes and either assayed immediately or stored frozen at - C. Peritoneal fluid was collected by aspiration of fluid from the cul-de-sac during laparoscopy. The fluid was checked to ensure that it was free of obvious contamination with red blood cells and was spun at 1, X g for 5 minutes. The cell-free supernatant was filtered through a.-jlm filter. The fluid was heat inactivated and stored as described for serum and mucus. Assay of Antisperm Antibodies Antisperm antibodies were assayed using an indirect immunobead binding test (IBT) as described previously (14). Briefly, the procedure was as follows: JLL of the fluid sample were incubated at 37 C for 6 minutes with 5 JLL of an antisperm antibodynegative semen sample containing between and 4 X 1 6 motile sperm. After the incubation, the sample was washed three times at room temperature with 1 ml of Tyrode's balanced salt solution (Sigma Chemical Co T145) containing.3% bovine plasma albumin (BPA), as previously described by us (15). For the washes, the samples were spun at 1, X g for 5 minutes. The final pellet was resuspended in JLL of Tyrode's solution containing 5% BPA. Rabbit antihuman immunobeads (BioRad, Richmond, CA) directed against immunoglobulin (Ig)G- Stern et ai. Antisperm antibodies in women 51

3 class (no ) and IgA-class (no ) were used. Two hundred microliters of the immunobead suspension were washed once in 1 ml of Tyrode's balanced salt solution containing.3% BPA and then resuspended in ttl of Tyrode's solution containing 5.% BPA. For the assay, 5 ttl of the sperm suspension were mixed on a slide with 5 ttl of the immunobead suspension and incubated at room temperature in a moist chamber for 15 minutes. The percentage of motile sperm bound to beads was determined for each test (IgG or IgA) by counting 1 motile sperm per slide on each of two slides ( sperm total). The results were reported as the percentage of positive motile sperm. A further evaluation was made as to the location and intensity of bead binding on the head, midsection, or tail regions of the sperm. Any sperm showing head binding, even those with midsection and tail-bound beads were counted as "head bound." Sperm reported as tail bound showed tail binding only. We determined the variability present in the indirect IBT as run in our laboratory. Separate counts performed on the same day on the same sample from 1 randomly selected samples showed an intra-assay variability of 4.7 ±. out of 1 for the IgG assay, and.1 ±.6 out of 1 for the IgA assay. Interassay variability was determined by two methods. In the first method, variability was determined at several levels of antibody binding from standard samples run on 5 different days over a period of 6 months. Interassay variability for IgG was ± at 1% binding, 3.8 ± 1.5 at 8% binding, and 1. ±.6 at 5% binding. For IgA, the interassay variability was 11.4 ± 3.1 at 45% binding, 5.6 ± 1.6 at 15% binding, and 1.6 ±. at 5% binding. In the second method, five separate serum samples in the midrange (5% binding) of each assay were performed on separate days, and the variability was calculated by the mean of the differences in assay results. Interassay variability was. ± 4.6 for the IgG assay and 17 ± 6.4 for the IgA assay. Samples were counted without reference to previous results. They were counted by one of four different individuals. Assay ofe The levels of E were determined in sequential serum samples from several patients. Estradiol was measured using a highly specific radioimmunoassay kit from Diagnostic Products Inc. (Los Angeles, CA) as previously described (16). Statistics Comparisons of estrogen (E) and antisperm antibodies or of different antisperm antibody values were performed using least squares regression analysis on a computer-generated graphics program. Correlation coefficients (r) indicate the degree to which the points fit the regression line. Correlation coefficients can range between. (no fit) and 1. (perfect fit). Significance was determined using a two-sided test. RESULTS Dilution and Antisperm Antibody Binding Parameters In this study antisperm antibodies were measured using the IBT. Antisperm antibody values measured using this IBT assay were reported as a percentage of motile sperm bound to immunobeads. To test the significance of a positive antibody measurement, we first determined what would happen to antisperm antibody binding after dilution of a positive serum sample. Table 1 shows the results of dilution of two serum samples that, when undiluted, were strongly positive for both IgG and IgA antibodies. Dilution was increased to a concentration of 1/5. The percentage of antisperm antibody binding measured in the two samples shown in Table 1 decreased with increasing dilution. Nevertheless, a large increase in dilution was required to show a small percentage change in antisperm antibody binding. Thus a 1/5 dilution of sample resulted in only a % reduction in IgG-class antisperm an- Table 1 Antisperm Antibody Binding in Diluted Serum * Neat 1/5 Patient 1-IgG Total 1 1 Head 1 1 Tail Other Patient 1-IgA Total 65 4 Head 16 Tail 4 44 Other 3 Patient -IgG Total 1 1 Head 1 1 Tail Other Patient -IgA Total 1 Head 7 5 Tail 4 Other * Values are percents. Dilution 1/ / / Stern et al. Antisperm antibodies in women

4 A C E,.,., I } 8 I 8 6 l! 6 l! 6! "I "I i 4 i. i , DoyalCyc" Doy 1 Cyc" Day olcyclo " 16 B D F ! 1! 1! j j ----v j.,. 4.,. 4., , ,. 16 DoyolCyclo DoyalCy." o.yofcycle Figure 1 Serum E and antisperm antibody levels from three patients during the follicular stage of the menstrual cycle. Estrogen levels (.) from patients 1 (A), (C), and 3 (E) rose in response to hmg administration. The IgG-class antisperm antibodies () and IgA-class antisperm antibodies (+) did not vary during this phase of the cycle. Antisperm antibody levels were low in patient 1 (B), moderate in patient (D) and high in patient 3 (F). tibodies, whereas a 1/1 dilution resulted in a 4% reduction in IgA-class binding. In general, head binding decreased more precipitously than did tail binding. Sequential Serum Samples and Antisperm Antibody Values In most clinical settings, serum for antisperm antibodies is drawn at indiscriminate times during the menstrual cycle and may even be drawn after exogenous administration of hormones, such as hmg and CC. Data from animal models of the secretory immune system suggest that after immunization and subsequent antibody production by some routes, the antibody levels in serum change in response to hormonal stimulation (1). To investigate whether changes in serum levels of antisperm antibodies could be observed after augmentation of E levels in response to hmg, we obtained serum from three patients undergoing ovulation induction as part of an in vitro fertilization program. The chosen patients represented three levels of binding of IgGclass antisperm antibodies: low (%), middle (4%), and high (1%). Where IgG levels were high (1% in undiluted serum), antisperm antibody determinations were made at a 1/1 dilution, which is where changes in the percentage of head-bound an- tibodies began to occur. In this way, we hoped to detect fluctuations in the antisperm antibody levels more readily than if the serum were assayed at full strength. Serum from all of the days was assayed, and the results are shown in Figure 1. Estrogen levels increased significantly during hmg administration over 15 days of the cycle in all three patients. Although there was fluctuation in the levels of antisperm antibody binding from serum samples obtained on different days, the antisperm antibody levels did not correlate with changing E levels. This was the case for both IgG-class and IgA-class antibodies and either low or high antibody binding levels. The greatest changes were seen in the IgA levels of patient 3. When compared with the changes in E, however, these showed a regression coefficient of only.7, which does not suggest significant correlation (n = 1). The percentages of head and tail binding measured in the different samples similarly did not vary significantly with increasing E (not shown). Antisperm Antibodies in Serum A comparison of the levels of IgG-class and IgAclass antisperm antibodies in serum from 51 patients is shown in Figure. The patients presented were Stern et al. Antisperm antibodies in women 53

5 1, , :.... II a Serum IgG (%bound) sa Figure Scattergram showing serum levels of IgA-ciass and IgG-ciass antisperm antibodies from 51 patients. The line shown represents the best fit correlation between the points using least squares regression analysis; r =.58, which shows correlation (P <.5). seen at the clinic between August 188 and May 11, and they represent a group of individuals in whom both serum and mucus antisperm antibody values were obtained. Antisperm antibody measurements were made using the IBT method, and the results are reported as the percentage of motile sperm bound by immunobeads. As can be seen in Figure, the levels of both IgGclass antisperm antibodies and IgA-class antisperm antibodies ranged from % to 1%. Although a positive correlation could be seen between the levels of IgG-class and IgA-class antisperm antibodies (r =.58), the levels ofthese two classes of antibodies were not the same in all cases. In some patients, both IgG-class and IgA-class antisperm antibodies were strongly negative «% binding), and in others both were strongly positive (>75% binding). However, there were also patients in whom IgG-class antisperm antibodies were strongly positive and IgAclass antisperm antibodies were negative, and patients in whom IgA-class antisperm antibodies were positive and IgG-class antisperm antibodies were negative. Antisperm Antibodies in Cervical Mucus (CM) Figure 3 shows a comparison between the levels of antisperm antibodies in serum and mucus that were present in the same 51 patients shown in Figure. Figure 3A shows the levels of IgG-class antisperm antibodies in serum and mucus. Serum IgG-class antisperm antibodies ranged from % to 1%. In contrast, the levels of mucus IgG-class antisperm antibodies were, in general, either very low «% binding) or very high (>75% binding). This same pattern was repeated for IgA-class antisperm antibodies as shown in Figure 3B. Serum IgA-class antisperm antibodies varied from % to 1%, whereas mucus IgA-class antisperm antibodies were either very low or very high. In three of the four cases in which levels of mucus IgG-class antisperm antibodies were high, the levels of mucus IgA-class antisperm antibodies were high as well (not shown). The correlation coefficients for the lines that defined the best fit between serum and mucus values were.58 for IgG and.64 for IgA. These r values indicate an overall positive correlation; however, it should be noted that neither regression line is at a 45 angle, and therefore these do not show a 1:1 correlation. All samples were evaluated to determine whether the antibody binding was present on the head region, midsection, or tail region of the sperm. Where both the serum and the mucus were positive for antisperm antibodies, patterns of head and tail binding were similar in the two fluids. In other words, when antibody binding was present on the head region in the serum, head binding was also found in the mucus; when most of the serum binding was to the tail region, mucus samples presented mostly tail binding. According to our clinical protocol, mucus for antisperm antibodies may be drawn in natural cycles or in cycles stimulated by EE (5 j.lgjd for 15 days), hmg ( to 4 ampules per day for 1 to 15 days), or A 1 -; co 4 Q I: B Q Se-F"um IgG (.. bound) 1.' Serum IgA (.. bound) Figure 3 Scattergram comparing em and serum levels of antisperm antibodies from 51 patients. Depicted are mucus antisperm antibodies and serum antisperm antibodies for both IgGclass (A) and IgA-ciass (B) antibodies. The lines shown were generated by best fit least squares regression analysis. The correlation coefficients are: r =.58 for IgG-ciass antibodies (P <.5) and r =.64 for IgA-class antibodies (P <.5). 54 Stern et ai. Antisperm antibodies in women

6 CC (15 mgjd for 5 days). Previous data from other laboratories have suggested that the levels of mucus immunoglobulin are influenced by the stage of the menstrual cycle and the presence of steroid hormones (1). We therefore retrospectively evaluated the hormonal conditions under which mucus had been drawn for antisperm antibody determinations in these patients. The data revealed no pattern in mucus antisperm antibodies that could be seen on the basis of hormonal augmentation. Individuals from both the natural cycle and the augmentation categories had mucus antisperm antibody levels that were either as high as serum levels or well below those levels. Antisperm Antibodies in PF Peritoneal fluid was collected from a random sample of patients at laparoscopy, and the levels of antisperm antibodies present in this fluid were measured. At a later date, these values were compared with the serum levels of antisperm antibodies found in these same individuals. Data from 3 patients are presented in Tables and 3. Table shows 16 patients in whom serum and PF were drawn within a -month period. In some patients, PF was collected first. In others, serum was the first fluid collected. As described in Materials and Methods, any PF sample showing apparent contamination with red blood cells was eliminated and is not shown here. With two exceptions, the data show serum and PF Table Antisperm Antibodies in Serum and PF Samples Collected Within Months IgG Patient Serum PF Serum IgA % % * PF * * Indicates a difference of> 17 percentage points between serum andpf. Table 3 Antisperm Antibodies in Serum and PF Samples Collected at Over 7 Months * IgG Patient Serum PF Serum IgA % % t t t PF 4 5 6t * Samples were run within months of collection; comparisons were made retrospectively. t Indicates a difference of> 17 percentage points between serum and PF. values to be within 17 percentage points of each other, a difference that we consider to be nonsignificant and within limits of assay variability (see Materials and Methods). In the first exception, IgAclass antisperm antibodies were 43% positive in serum and 14% positive in PF. The PF was drawn 1 month before the serum in this patient. In the second exception, IgA-class antisperm antibody levels were 6% positive in serum and 31 % positive in PF. Samples were drawn 1 day apart. Table 3 shows data on a group of eight patients who had had serum and PF samples drawn 7 or more months apart. As seen in Table 3, antisperm antibodies in serum of these patients did not correlate in all cases with antisperm antibodies in PF. Most striking were two samples that had a negative serum value for IgG-class antibodies (4% and 1 % binding, respectively) while exhibiting a positive PF value for these antibodies (8% and 66%, respectively). In the first sample, the serum was drawn before the PF; in the second sample, the PF was drawn before the serum. We had the opportunity to remeasure the serum of the first of these patients 1 year after the laparoscopy in which results showed positive PF fluid binding. Her serum at this second measurement was 1% positive for IgG-class antisperm antibodies (data not shown). Another patient had IgG-class antisperm antibody binding of 33% in serum and 8% in PF (Table 3). In one other patient, IgA-class antisperm antibodies were measured at 41 % positive in serum and 6% in PF. Peritoneal fluids (lower percentage binding) were drawn before the serum in both of these patients. DISCUSSION In this study, we measured the levels of antisperm antibodies in serum, mucus, and PF from patients Stern et al. Antisperm antibodies in women 55

7 undergoing evaluations for infertility. We found little correlation between the levels of antisperm antibodies in these three fluids. In our initial studies, we performed serial dilutions on two antisperm antibody-positive serum samples and demonstrated that the percentage of antisperm antibody binding decreased very little after large increases in serum dilution. These data suggest that the IBT assay follows equilibrium dynamics in which the numbers of bound and free sperm are proportional to, not absolute values of, the concentrations of antibody present. The data also suggest that when the IBT is used to evaluate an undiluted serum sample, small differences in the magnitude of antibody binding can reflect much greater differences in the titer of antibody present in the serum sample. On the basis of this study, we believe that designation of samples merely as positive or negative for antisperm antibody binding using an arbitrary cutoff point of, for example, 5% would be inadequate to represent the variability within the samples. We have therefore reported all antisperm antibody values as the percentage of motile sperm cells bound to beads. From a clinical standpoint, the data also suggest that routine investigation of antisperm antibody levels in diluted serum could be advantageous when an undiluted serum sample is strongly positive. We are continuing these studies in an attempt to gain enough patient data to be able to recommend a standard dilution to be used in such cases. Our initial studies also showed that changing E concentrations did not affect the antisperm antibody levels in serum samples from the three patients studied. This evaluation was undertaken because, when measured in a clinical setting, serum for antisperm antibodies is often drawn at different stages of the menstrual cycle. Variability in reported serum antisperm antibody levels could result if changing hormone concentrations at different stages of the menstrual cycle influenced antisperm antibody levels in serum. Wira and Sandoe (1) have shown that in rats antibody levels in serum change in response to hormonal manipulation under certain conditions. These investigators performed Peyer's patch immunization with sheep red blood cells and demonstrated significant changes in the concentrations of specific IgG-class and IgA-class antibodies after administration of E In the present study, we evaluated serum samples drawn during the follicular stage of a stimulated menstrual cycle, and we determined that in the three patients studied no cyclic fluctuations in antisperm antibodies occurred during the follicular portion of the cycle. It is possible, however, that the changes in E levels in these patients were not as extensive as those in the animal model system. In the present study, we have shown that there is no direct correlation between antisperm antibodies in serum and antisperm antibodies in mucus. Previous studies have also demonstrated a lack of correlation between antisperm antibodies in serum, mucus, and uterine fluids. Shai et al. (7) examined antisperm antibodies in serum and CM using a reverse enzyme-linked immunosorbent assay. These authors identified individuals who were positive for serum antisperm antibodies and negative for antisperm antibodies in CM. These authors also found patients who were negative for antisperm antibodies in serum while positive for antisperm antibodies in mucus. Bronson (8) measured antisperm antibodies in serum, vaginal secretions, and uterotubal fluid and found IgA-class antisperm antibodies to be more prevalent in reproductive tract washings than in serum. A study by Clarke et al. () compared antisperm antibodies found in plasma with antisperm antibodies found in follicular fluid (FF) and showed that comparable levels of IgG-class antisperm antibodies and IgA-class antisperm antibodies could be measured in serum and FF while the levels of IgM were markedly lower in the FF than in the serum. One explanation for the lack of correlation between serum and mucus could reflect the origin of the antisperm antibodies present in the different fluids. Previous reports suggest that there is a large concentration of plasma cells in the cervix (17, 18). It is possible that when these plasma cells are present and secreting antisperm antibodies, the measured mucus levels are high, but when the plasma cells are absent the mucus levels are negative. By contrast, contributing factors to antisperm antibodies in serum include plasma cells secreting antibody from a variety of locations. In the present study, we observed few differences between levels of antisperm antibodies in serum and those in PF. Antisperm antibodies in PF and uterotubal fluid have previously been measured by Bronson (8). In this previous study, no differences were demonstrated between the levels of antisperm antibodies in serum and PF, although differences were seen between antisperm antibodies in serum and uterotubal fluid. Although PF is considered to be largely a serum transudate, the oviduct may contribute fluid to the peritoneal cavity, particularly during the ovulatory stages of the menstrual cycle (1). The oviduct contains immunoglobulin-positive cells (18) and may be capable of responding directly 56 Stern et al. Antisperm antibodies in women

8 to infectious agents (). It is possible, therefore, that at times the oviduct fluid does contribute to antisperm antibody concentrations in the PF. Findings in the present study suggest that when antisperm antibodies in PF differed from those in serum, some of the variability could be attributed to the length of time separating the collection of the serum and PF samples. Because four of seven patients in whom serum and PF were drawn at 7 months or more apart showed differences in binding, the data suggest that antibody levels may be more variable over time than has been previously suggested. One of the patients in this category showed a positive PF value followed months later by a negative serum value. One explanation for this finding is that the patient underwent a spontaneous reduction in antibody levels. Without additional cases of this nature, however, we more strongly favor the explanation that the difference in the fluid compartments accounts for the change in this case. Our data show that the levels of IgG-class and IgA-class antisperm antibodies do not necessarily correlate with one another in any of the fluids studied. In reproductive tract secretions, the differing levels of IgA-class and IgG-class antisperm antibodies may reflect differences in the levels of total IgA and total IgG found in different regions of the female reproductive tract. Data from other studies demonstrate that the Ig levels in reproductive tract secretions of women vary with the stage of the menstrual cycle and in response to exogenously administered sex steroids (1). Immunoglobulins in reproductive tract secretions of rats and mice also vary in response to E and progesterone administration, stage of the estrous cycle (1, ), decidualization (3), and pregnancy (4). The effects of the hormonal balance are influenced by whether the secretion studied is uterine fluid or vaginal fluid (1). In addition to variations in total Igs, IgA-class and IgG-class antisperm antibodies, which are specifically directed against a known antigen, have also been shown to vary in the reproductive tracts of study animals (1, 13, 5). This variation is in response to the route of immunization and the administration of sex hormones. In studies by Wira and Sandoe (1,5), for example, the levels of IgAclass as compared with those of IgG-class antibody after Peyer's patch immunization with sheep red blood cells were equal in the uterus but were significantly greater in vaginal and in salivary secretions (1). Using intraperitoneal immunization, the levels of IgG-class antibody were greater than those of IgAclass antibody in serum but not in uterine, vaginal, or salivary fluids. When serum from uterine-immunized animals was collected after treatment with E for 3 days, the levels of IgG-class antibody increased over those in saline-treated animals. No change was seen in the levels of IgA-class antibody when comparing E and saline treatment. Parr et a1. (13) have also demonstrated differences in IgAclass and IgG-class antibody titers when different routes of immunization were used in mice immunized with horse ferritin. In these studies, subcutaneous (SC) immunization followed by intravaginal boost produced more vaginal IgA-class antibody than did SC immunization alone. Levels of IgG-class antibodies did not differ in the two treatment groups (13). The differing levels of antisperm antibodies in serum, mucus, and PF shown in this study might, as in animal models, reflect differences in the conditions under which antisperm antibodies were generated. As yet, we know little about the mechanisms that result in the formation of antisperm antibodies in a small number of women. When antisperm antibody formation does occur in women, it is not clear that immune exposure occurs during sperm deposition in the reproductive tract. Alternate routes of immune exposure to sperm could include direct contact with blood (e.g., through cuts or abrasions in the vagina) or exposure of the mucosa of the digestive tract (e.g., during oral or anal intercourse). If, as in animals, antisperm antibody levels in women are influenced by the route of antigen presentation and the hormonal balance, the differing fluid patterns of antisperm antibodies seen in this study may suggest that routes of antigen presentation leading to antisperm antibody formation vary among different women. Our failure to find any consistent patterns in antisperm antibody levels between one fluid and another suggests that, in women, these factors are numerous and complex. Acknowledgments. The authors thank Ms. Alicia Green and Ms. Ellen Swain for their editorial assistance with this manuscript. The authors also thank Ms. Tina Nelson and Ms. Sarah Gibson for their excellent technical assistance. REFERENCES 1. Bronson RA. 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