Capacitated sperm cells react with different types of antisperm antibodies than fresh ejaculated sperm*

Transcription

1 FERTILITY AND STERILITY Copyright Q 1992 The American Fertility Society Vol. 57, No.2, February 1992 Printed on acid-free paper in U.S.A. Capacitated sperm cells react with different types of antisperm antibodies than fresh ejaculated sperm* Ehud J. Margalioth, M.D.t:\: George W. Cooper, Ph.D. Frances H. Taney, M.D. Gerald M. Scholl, M.D. David L. Rosenfeld, M.D. Bikur Cholim Hospital, Jerusalem, Israel, and North Shore University Hospital, Cornell University Medical College, Manhasset, New York Objective: To determine if sera of some women have antibodies against capacitated but not freshly ejaculated sperm. Design: The sera of 66 women undergoing in vitro fertilization (IVF) were tested for sperm antibodies after 1 hour and 18 hours of sperm incubation in the maternal sera. Subsequently, 5 sera were tested with capacitated versus noncapacitated sperm cells. Setting: The study was carried out in a university hospital department. Patients, Participants: The patients were 66 consecutive couples undergoing IVF. Interventions: Sera and semen that were taken for routine tests as part of the IVF procedures were used. Main Outcome Measures: A case with IVF failure associated with late appearance of sperm antibodies prompted us to study the detection of sperm antibodies after 1 hour and 18 hours incubation. Results: Of 37 cases negative for sperm antibodies after 1 hour incubation, 7 demonstrated high levels of antibodies after 18 hours incubation. In 21 of 23 cases with low or intermediate levels of antibodies after 1 hour incubation, significantly higher levels (P < 0.05) of antibodies were found after 18 hours. Different and higher levels of sperm antibodies were observed in five sera after incubation of 1 hour with capacitated sperm as compared with noncapacitated controls. Conclusions: Major antigenic differences may exist between capacitated and noncapacitated sperm. In some women sperm antibodies are reactive against capacitated sperm only. This has no certain clinical significance but may explain certain cases of IVF failure, unexplained infertility, and part of the variation in sperm antibodies testing methods. Fertil Steril 1992;57:393-8 Key Words: Sperm antibodies, capacitation, in vitro fertilization Isoimmunity to spermatozoa is generally considered a relative rather than an absolute cause of infertility in women. 1-2 In part, this may be because Received May 1, 1990; revised and accepted October 22, * Presented at the 45th Annual Meeting of The American Fertility Society, San Francisco, California, November 13 to 16, t Reprint requests: Ehud Margalioth, M.D., Department of Obstetrics and Gynecology, Bikur Cholim Hospital, 5 Strauss Street, Post Office Box 492, Jerusalem 91004, Israel. :j: Department of Obstetrics and Gynecology Bikur Cholim Hospital. Human Reproduction Division, North Shore University Hospital, Cornell University Medical Center. of the fact that the procedures used to assay antisperm antibodies do not reflect the conditions in vivo during which antibodies can influence the character of the sperm surface. This is seen in recent studies of the effect sperm antibodies have on in vitro fertilization (IVF) when female sera are used to supplement media. 3-5 Recently, in one of our IVF cases, we could not achieve fertilization of a patient's oocytes with her husband's sperm cell, although no sperm antibodies were detected in her serum. Yet, examining the sperm cells after 18 hours of spermegg incubation (in medium containing 15% of the wife's serum), most cells were antibody-positive in Vol. 57, No.2, February 1992 Margalioth et al. Antibodies to capacitated sperm cells 393

2 r three different immunoglobulin (Ig) classes (IgA 45%, IgG 66%, IgM 95%). This finding prompted us to routinely screen all female sera for sperm antibodies by comparing 1- versus 18-hour incubations in 15% serum-supplemented Ham's F-10 medium (GIBCO, Grand Island, NY), to determine if some women have circulating sperm antibodies to antigens expressed on capacitated rather than freshly isolated spermatozoa. The aim of the present study was to investigate this hypothesis and to examine the prevalence and extent of sperm antibodies to capacitated sperm versus freshly ejaculated sperm in the sera of women undergoing IVF procedures. Serum Collection MATERIALS AND METHODS Sera were collected from 66 women undergoing IVF. In all cases, serum was obtained during the early follicular phase of the menstrual cycle. The sera were inactivated at 56 C for 30 minutes. These sera were used for preparing IVF media only if sperm antibodies were not detected on sperm cells incubated for 18 hours in Ham's F-10 with 15% of the serum. Control serum negative for sperm antibodies was collected from fertile women who volunteered to donate blood. These sera were also obtained during the early follicular phase of the menstrual cycle and inactivated at 56 C for 30 minutes. These sera were considered negative for sperm antibodies only if antibodies were not detected on donor sperm cells incubated for 18 hours in Ham's F-lO with 15% ofthe serum. Sperm Preparation Semen specimens were collected by masturbation into a sterile plastic cup. After liquefication, 0.5-mL aliquots were placed at the bottom of cupped sterile plastic test tubes and covered with 2 ml of Ham's F -10 medium supplemented with 2.1 mg/ml sodium bicarbonate (S8875; Sigma, St. Louis, MO), mg/ml streptomycin sulfate (S6501; Sigma), and mg/ml penicillin-g (PEN-NA; Sigma). The tubes were incubated for 60 to 90 minutes at 37 C in an atmosphere of 5% CO 2 in air. The top 1.8 ml of all tubes were collected, pooled, and centrifuged at 200 X g for 6 minutes. The pellet was washed and resuspended in medium at a concentration of 10 X 10 6 motile sperm cells/ml. Immunobead Binding Assays Testing for sperm antibodies in female serum was performed by immunobead binding assay as described by Bronson et al. 6 The assay was performed with immunobeads, polyacrylamide spheres of 5 to 10 JLm diameter with covalently bound rabbit antibodies to the constant domain of human IgA (a-chain specific), IgG (T-chain specific), or IgM (JL-chain specific); (anti-iga, cat. no ; anti IgG, cat. no ; anti-igm, cat. no ; Bio-Rad Laboratories, Richmond, CA). Lyophilized anti-iga, anti-igg, or anti-igm immunobeads were washed once by centrifugation (800 X g for 8 minutes) with Dulbecco's phosphate-buffered saline (PBS; GIBCO Laboratories) and were resuspended at 5 mg/ml in sterile PBS containing 10 mg/ml bovine serum albumin (BSA, fraction V; Calbiochern, La Jolla, CA). Semen Immunobead Binding Testing Semen was kept at room temperature (RT) for 1 hour after ejaculation. After adequate sperm number and motility (~1 X 10 6 motile sperm) for testing were ensured by semen analysis, sperm were washed once by centrifugation (800 X g for 8 minutes) in PBS containing BSA, 5 mg/ml (BSA/PBS), resuspended, and washed in BSA/PBS three times with a Beckman Microfuge B (Beckman Instruments Palo Alto, CA; 8 seconds/spin). After the third wash: the sperm pellet was resuspended in BSA/PBS at 10 to 20 X 10 6 sperm/ml. Five microliters of this sperm suspension was added to 50 JLm of well-mixed immunobead suspension. A drop of the mixture was placed on a glass slide, covered with a coverslip, and kept at RT for 5 minutes to allow immunobead binding to occur. The reaction was observed and counted with phase-contrast microscopy at X200. Two counts of 100 motile sperm for immunobead binding were made on each preparation. The region of sperm surface to which antibody was bound and the percent of motile sperm cells bound were determined for each of three isotypes of immunoglobulin (IgA, IgG, IgM). Serum Immunobead Binding Assay Each woman's serum was tested for antisperm antibodies using her husband's sperm, unless these were found positive for IgA, IgG, or IgM antibodies in the direct immunobead binding test. In those cases, sperm from a donor (A+ blood type), found to be negative for sperm antibodies by immunobead binding, was used. Spermatozoa were selected for 394 Margalioth et ai. Antibodies to capacitated sperm cells Fertility and Sterility

3 motility with the use of a swim-up method in which semen (containing ;::::2 X 10 6 motile sperm) was overlaid with BSA/PBS in a test tube and incubated for 1 hour at 37 C. Motile spermatozoa were collected from the top of the test tube into 0.5 to 1.0 ml, making the serum dilution 15%, and the preparation was incubated for 1 hour at 37 C. The spermatozoa were then washed and tested for surfacebound sperm antibodies by immunobead binding, as described previously for semen. In all experiments, direct immunobead binding testing was performed on every sperm sample after 18 hours of incubation. Sera was considered negative for sperm antibodies if direct immunobead binding testing after 18 hours of incubation was <5%. Capacitation Capacitation was obtained by overnight incubation of post swim-up motile sperm cells in 15% (volj vol) serum in Ham's F-10 medium, or in Ham's F lo with 30 mg/ml human serum albumin (HSA, A-9511; Sigma) tested negative for sperm antibodies). To evaluate capacitation, the zona-free hamster egg sperm penetration assay 7 was performed on three sperm antibodies-negative sperm samples. Each sample was divided into three groups of sperm cells and incubated overnight at RT in Ham's F-10: (1) without any protein addition, (2) with 15% serum, or (3) with 30 mg/ml HSA. After 18 hours' inc~bation, all samples were washed in Biggers, WhItten, and Wittingham mediums and incubated with zona-free hamster eggs for 3 hours. Sperm cells incubated in Ham's F-lO alone had a mean penetration of 3% (range 0% to 10%). In contrast, those incubated with 15% serum had a mean penetration of 89% (range 68% to 100%), and the three samples with HSA had a mean penetration of 86% (range 79% to 100%). Thus we defined capacitated versus noncapacitated sperm cells. Because motile sperm cells could be preserved overnight without any protein source only at RT and not at 37 C, we examined similar aliquots of sperm cells incubated at RT and at 37 C in sperm antibodies-positive serum. No difference in the direct immunobead binding testing was seen after incubation at RT versus incubation at 37 C. Neither was any difference found at indirect immunobead binding testing when sperm cells incubated overnight with sperm antibodies-negative serum at RT versus at 37 C were tested after 1 hour incubation in sperm antibodies-positive serum. Statistical analysis was performed using the paired t-test. RESULTS The sera of 66 women undergoing IVF were examined for the presence of sperm antibodies. After 1 hour of sperm-serum incubation, 37 women did not have any sperm head-directed sperm antibodies in their sera. Fifteen women had low levels (6% to 30%),8 had intermediate levels (31% to 70%), and 6 had high levels of head-directed sperm antibodies (>70%) (Table 1). After 18 hours of sperm-serum incubation, 7 of the 37 1-hour negative sera had up to 72% head-directed sperm antibodies, whereas 30 remained negative (Table 2, cases 1 to 7). All 15 with low sperm antibodies levels had significantly elevated levels, although the difference varied among cases and among different isotypes (Table 2, cases 8 to 22). Of the 8 cases with intermediate sperm antibodies, 5 cases had significantly increased sperm antibodies activity after 18 hours of incubation (Table 2, cases 23 to 27). Of the 6 sera with high levels of IgA, IgG, or IgM sperm antibodies, these levels persisted for 18 hours of incubation, and additional high levels of other isotypes were found in 2 cases (IgA from 22% to 71% and IgM from 56% to 94%). To investigate if the elevated antisperm antibody titer was directly correlated with the duration of the incubation period, the following tests were performed. First, equal amounts of sperm cells were incubated for 18 hours in a capacitating medium (Ham's F-lO containing 15% serum known to be negative for antisperm antibodies) versus in a noncapacitating medium (Ham's F-10 only). On the following day, (after performing direct immunobead binding testing on all samples), sperm from each treatment were used to test five different sera by indirect immunobead binding assay. Sperm antibodies levels in all five sera were higher when tested with capacitated sperm versus noncapacitated sperm (Table 3). Table 1 Antisperm Antibodies (lga/igg/igm) Testing of 66 Maternal Sera for IVF: 1 Hour Incubation at 37 C of Husbands' Sperm in 15% Serum in Ham's F-10 Medium Head immunobead No. of cases Rank binding (n = 66) Women %' % Negative o to Low 6 to Intermediate 31 to High 71 to a Number of sperm bound to immunobeads out of 100 sperm Vol. 57, No.2, February 1992 Margalioth et al. Antibodies to capacitated sperm cells 395

4 r Table 2 Head-Directed Antisperm Antibodies Detected in Female Sera After 1 Hour Versus 18 Hours of Sperm Incubation Head-directed immunobead binding IgAa IgGa IgM b Patient 1h 18 h 1h 18 h 1h 18 h a 1 hour versus 18 hours, P < b 1 hour versus 18 hours, P < C Number of sperm cells bound to immunobeads out of 100 %' Another test was performed using four aliquots of the same serum. One was incubated with fresh ejaculated sperm for 1 hour, a second was incubated for 1 hour with noncapacitated sperm (sperm that were incubated in Ham's F-lO alone for 18 hours); the third aliquot was incubated for 1 hour with capacitated sperm cells (sperm incubated for 18 hours in Ham's F-I0 supplemented with 15% serum). The fourth aliquot was incubated with sperm cells for 18 hours continuously. The differences in the antisperm antibody levels between the first and the second aliquot (Table 4) demonstrate the antigenic change of sperm during 18 hours of incubation in a noncapacitating medium. The change is relatively small (IgA, 5% versus 16%; IgG, 22% versus 28%; and IgM, 21 % versus 24 %). The difference between the second and the third aliquots demonstrates the antigenic change induced by capacitation only (lga, 16% versus 50%; IgG, 28% versus 78%; and IgM, 24% versus 64%). The difference between the third and the fourth aliquot demonstrates the additive binding over incubation time (IgA, 50% versus 73%; IgG, 78% versus 91%; and IgM, 64% versus 65%). It is obvious that capacitation induces the most significant change. Essentially similar differences were seen using five other selected sera. To rule out the possibility that the observed changes were because of other effects of serum than capacitation, identical sperm samples were examined after overnight incubation in Ham's F-I0 with (1) control serum, free of sperm antibodies; (2) 30 mg/ml HSA; or (3) without any protein. Higher levels of head-directed sperm antibodies were found when sperm cells were incubated overnight with either HSA (IgA, 50%; IgG, 54%; IgM, 21%) or serum (IgA, 53%; IgG, 64%; IgM, 20%), compared with sperm antibodies levels detected by sperm cells incubated overnight in Ham's F-lO medium only (IgA, 7%; IgG, 9%; IgM, 7%). No statistical difference was noted between the results of sperm antibodies testing using sperm incubated in serum versus sperm incubated with albumin. DISCUSSION The present study demonstrates that 7 of 66 sera that were negative for antisperm antibodies after incubation for 1 hour with sperm cells were positive for antisperm antibodies after 18 hours of incubation. Of the 29 cases in which some levels of headdirected antisperm antibodies were detected after 1 hour of incubation, most had significantly increased levels of antibodies bound to the sperm after 18 hours. We have found that this increase was mainly because of changes of the sperm cells rather than an additive phenomen over time. Capacitated sperm detected higher levels of antibodies in 32 different sera. Thus, our study suggests that capacitated sperm absorb serum antibodies different from those absorbed by freshly ejaculated sperm. This might be because of exposition of new sperm surface antigen during the capacitating procedure. Yanagimachi 9 states that capacitation is a series of changes that involve removal or alteration of substances absorbed on, or integrated in, the sperm plasma membrane. These changes may alter some antigens on the sperm surface. Okabe et al. lo,ll have demonstrated that during the capacitation process, in murine spermatozoa, some antigens disappear, 396 Margalioth et al. Antibodies to capacitated sperm cells Fertility and Sterility

5 Table 3 Head-Directed Antisperm Antibodies Detected After Incubating Capacitated Versus Noncapacitated Sperm for 1 Hour in Aliquots From the Same Serum Head-directed immunobead binding IgA" IgG b IgM Patient N oncapacitated' Capacitated d N oncapacitated Capacitated N oncapacitated Capacitated %' a Noncapacitated versus capacitated, P < b Noncapacitated versus capacitated, P < 'Donor sperm incubated for 18 hours in Ham's F-10 medium alone. d Donor sperm incubated for 18 hours in Ham's F-10 medium containing 15% antibody negative serum. e Number of sperm cells bound to immunobeads out of 100 whereas others appear and are detectable on the sperm surface only after capacitation in vitro. They concluded that during capacitation some antigens of the sperm plasma membrane are removed or altered, whereas others that are hidden or masked in fresh epididymal spermatozoa are exposed. O'Rand,12,13 studying the modification of the sperm plasma membrane during capacitation, found in the rabbit that after in vivo capacitation a certain sperm surface membrane glycoprotein antigen was not exhibited. This antigen was always found on epididymal and ejaculated spermatozoa. Our study indicates that similar changes occur in human sperm during capacitation. Our work demonstrates that some women have antisperm antibodies only to capacitated sperm and Table 4 Head-Directed Antisperm Antibodies Detected on Freshly Isolated Sperm; on Noncapacitated; Capacitated Sperm After 1 Hour Exposure; and on Sperm After 18 Hours in Serum With Ham's F-lO 1 h incubation Fresh sperm Noncapacitated sperm 18 hr F-10 alone Capacitated sperm 18 hr F-10 + negative serum 18 h incubation F-10 + positive serum Head-directed immunobead binding IgA IgG IgM %" a Number of sperm cells bound to immunobeads out of 100 that the usual method of examining antisperm antibodies to freshly ejaculated sperm may not detect these antibodies. This phenomenon provides an explanation for part of the different results obtained when detecting antisperm antibodies by different laboratories. It seems that varying degrees of capacitated sperm may detect different levels of antisperm antibodies in the same serum. The method. of processing the sperm may affect the results of the antibody testing in different laboratories. It is assumed that antibodies to freshly ejaculated sperm have an important role in prohibiting sperm entry through the cervical mucus. Therefore in certain cases of unexplained infertility the female reproductive tract may contain antibodies to capacitated sperm. These antibodies have not yet been detected by the usual methodology of testing for sperm antibodies. It is suggested that capacitation or acrosome reaction results in exposure of different antigenic components, causing antisperm antibodies in the uterus, tube, or peritoneal cavity, to inhibit fertilization. This has been shown in experimental animal models.14 Our observations may apply to failure in IVF because of low or failed fertilization. We suggest that sera of women used for preparing IVF medium should be screened for antibodies against capacitated as well as freshly ejaculated sperm. If routine screening is not feasible, we suggest that in cases of low fertilization or failed fertilization, antisperm antibody levels to capacitated sperm should be tested and if present, replacement of the maternal serum by donor or cord serum is highly recommended. The data of this study advocate the use of capacitated sperm in addition to freshly ejaculated sperm while testing (with any method) for antisperm antibodies. Vol. 57, No.2, February 1992 Margalioth et al. Antibodies to capacitated sperm cells 397

6 r i! Acknowledgment. We are grateful to Miriam Almagor, Ph.D., Director of Fertility Laboratory, Bikur Cholim Hospital, Jerusalem Israel, for reviewing the manuscript. REFERENCES 1. Bronson RA, Cooper GW, Rosenfeld DL: Factors effecting the population of the female reproductive tract by spermatozoa: their diagnosis and treatment. Sem Reprod Endocrinol 4:371, Bronson RA, Cooper G, Rosenfeld D: Sperm antibodies: their role in infertility. Fertil Steril 42:171, Clarke GN, Hyne RV, Plessis YI, Johnston WIH: Sperm antibodies and human in vitro fertilization. Fertil Steril 49: 1018, Margalioth EJ, Cooper GW, Taney FH, Kvapil G, Scholl GM, Rosenfeld DL: Immunological infertility and IVF. (Abstr.) Presented at the Sixth World Congress of IVF, Jerusalem, Israel, April 2 to 7, Published by the World Congress of IVF, in the Abstracts Book, 1989, p Clarke GN: Sperm antibodies and human fertilization. Am J Reprod Immunol 17:65, Bronson RA, Cooper GW, Rosenfeld DL: Sperm-specific isoantibodies and autoantibodies inhibit the binding of human sperm to the human zona pellucida. Fertil Steril 38:724, Margalioth EJ, Feinmesser M, Navot D, Mordel N, Bronson RA: The long-term predictive value of the zona-free hamster ova sperm penetration assay. Fertil Steril 52:490, Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JD Daniel. San Francisco, Freeman, 1971, p Yanagimachi R: Mammalian Fertilization. In The Physiology of Reproduction, Edited by E Knobil, J Neil. New York, Raven Press, Ltd., 1988, p Okabe M, Takada K, Adachi T, Kohama Y, Miura T, Aonuma S: Studies on sperm capacitation using monoclonal antibody-disappearance of an antigen from the anterior part of mouse sperm head. J Pharmacol Dyn 9:55, Okabe M, Takada K, Adachi T, Kohama Y, Miura T: Inconsistent reactivity of an anti-sperm monoclonal antibody and its relationship to sperm capacitation. J Reprod Immunol 9: 67, O'Rand MG: Restriction of a sperm surface antigen's mobility during capacitation. Dev Bioi 55:260, O'Rand MG: Modification of the sperm membrane during capacitation. Ann NY Acad Sci 383:392, Menge AC, Naz RK: Immunological reactions involving sperm cells and pre implantation embryos. Am J Reprod Immunol 18:17, Margalioth et al. Antibodies to capacitated sperm cells Fertility and Sterility