Inhibition of sperm penetration through human zona pellucid a by antisperm antibodies*

Transcription

1 FERTILITY AND STERILITY Copyright 1986 The American Fertility Society Vol. 46, No.1, July 1986 Printed in U.s A. Inhibition of sperm penetration through human zona pellucid a by antisperm antibodies* Shinichi Tsukui, M.D. t Yoichi Noda, M.D., D.Med.Sci. Juri Yano, M.D., D.Med.Sci. Aisaku Fukuda, M.D. Takahide Mori, M.D., D.Med.Sci. Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Kyoto, Japan An in vitro penetration test using human spermatozoa, sera, and eggs stored in a highly concentrated salt solution was designed for examination of the effect of antisperm antibodies on the process of fertilization. Spermatozoa from a healthy fertile donor incubated in modified Biggers, Whiiten and Whittingham (BWW) medium containing 7.5% antisperm-antibody-negative serum, could penetrate through the zonae pellucidae of the stored eggs, but not when the spermatozoa from the same donor had been incubated in modified BWW medium containing 7.5% antispermantibody-positive serum. After the antisperm-antibody-positive serum was absorbed with washed spermatozoa, the sperm penetration was not blocked. Therefore, antisperm antibodies appear to block human sperm penetration through the human zona pellucida. Fertil Steril 46:92, 1986 Antisperm antibodies cause infertility by inhibiting the penetration of spermatozoa through the cervical mucus, which results in fewer spermatozoa reaching the ovum and fertilizing it.1-4 However, this mechanism is not the only cause of infertility in patients with antisperm antibodies, because intrauterine insemination is generally ineffective in achieving pregnancies in these women. Several investigations 5-7 have recently shown that antisperm antibodies are involved in Received July 23, 1985; revised and accepted February 27, *Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan ( , , ). treprint requests: Shinichi Tusukui, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, 54 Kawaharacho, Shogoin, Sakyo-ku, Kyoto, 606, Japan. 92 Tsukui et ai. Inhibition of sperm penetration sperm/egg interactions in animals and humans. Autoantibodies from vasectomized guinea pigs have inhibitory effects on acrosome reaction, sperm-zona binding, and sperm-ovum fusion. 5 Antisperm antibodies lower the penetrability of human spermatozoa through the zona-free hamster ova. 6 7 Moreover, isoantibodies and autoantibodies to human spermatozoa inhibit sperm attachment to the human zona pellucida. 8 However, the influence of antisperm antibodies on sperm penetrability through the zona pellucida has not yet been investigated. Indirect but important evidence for the blockade of human fertilization under the influence of antisperm antibodies recently reported is that pregnancy was established in a patient who had antisperm antibodies in her serum by the use of human cord serum or donor serum in the culture medium instead of the patient's own serum The present investigation was carried out for examination ofthe effects

2 of antisperm antibodies on in vitro penetrability of spermatozoa through the human zona pellucida, that was for clarification of the etiologic role of the antibodies at a site of fertilization other than at the site of the cervical mucus. MATERIALS AND METHODS SPERM IMMOBILIZATION TEST A total of 160 serum samples were obtained from women with infertility of different causes who had been treated at our infertility clinic from September 1983 to February Serum samples were also obtained from virgin donors as control sera. After inactivation of complements by heating at 56 C for 30 minutes, each serum was divided into several aliquots and stored at - 20 C until use for the following sperm immobilization test and zona penetration test. The sperm immobilizing antibodies (SIAs) in the serum were examined by the sperm immobilization test as described by Isojima et al. 11 A sperm suspension adjusted to the concentration of 40 x 10 6 spermlml was prepared from freshly ejaculated semen of a 28-year-old healthy donor with proven fertility. Twenty-five microliters of the washed sperm suspension and 50 J-Ll of guinea pig serum as the complement source were mixed with 250 III of each test serum or control serum, and the mixture was incubated at 32 C for 60 minutes. After incubation, the sperm motility was estimated under a microscope. As a control, inactivated virgin serum with no SIA activity was used in place of the test serum. The sperm immobilization value (SIV) was calculated by dividing the sperm motility in the control serum by that in the test serum. Reactions with an SIV of over 2.0 were regarded as positive. All SIApositive sera with infinite SIV were subjected to the following zona penetration test, whereas SIAnegative sera were used as control sera. ABSORPTION OF SERA For the absorption of SIA with washed spermatozoa, freshly ejaculated semen samples of good quality from fertile donors were subjected to spontaneous liquefaction for 30 minutes and were then centrifuged twice at 750 x g for 5 minutes. The sperm pellet was suspended in 1.0 ml of the test serum at the concentration of 120 to 200 x 10 6 spermlml and was further incubated at 37 C Vol. 46, No.1, July 1986 for 60 minutes under gentle shaking.11 The same procedure was repeated several times until SIA activity became undetectable in the absorbed serum. PREPARATION OF SPERM SUSPENSION Freshly ejaculated semen obtained from a 28-year-old healthy fertile donor was subjected to spontaneous liquefaction at room temperature for 30 minutes, divided into 0.5-ml aliquots, and transferred gently to the bottoms of three tubes containing 2 ml of modified BWW medium l2-14 (modified BWW, mm NaCI, 4.78 mm KCI, 1.71 mm CaCI2, 1.19 mm KH2P04, 1.19 mm MgS0 4e7H 20, mm NaHC0 3, 0.25 mm Naepyruvate, mm Naelactate, 5.56 mm glucose, 100 IU/ml K-penicillin-G, 50 Ilg/ml streptomycin sulfate) supplemented with SIA-negative, SIA-positive, or sperm-absorbed SIApositive serum at the concentration of 7.5% (vol/vol). These sera had been inactivated as described previously. Each tube was slanted at a 30-degree angle under 5% CO2 in air for 60 minutes; this procedure allowed spermatozoa to swim upward out of the seminal plasma into the modified BWW medium. 13 The upper modified BWW layers were then centrifuged for 5 minutes at 230 x g. The sperm pellets were resuspended in the serum-supplemented modified BWW medium with an adjusted concentration of 2 to 12 X 10 6 spermlml. ZONA PENETRATION TEST Follicular oocytes with cumulus cells were recovered by puncturing follicles or dissecting tissues of human ovaries obtained under informed consent from patients undergoing laparotomies for gynecologic indications. They were placed in 0.4 ml of modified Krebs-Ringer bicarbonate (KRB) medium (modified KRB, 94.6 mm NaCI, 4.78 mm KCI, 1.71 mm CaCI 2, 1.19 mm KH2P04, 1.19 mm MgS0 4, mm NaHC0 3, mm Naelactate, 0.5 mm Naepyruvate, 5.56 mm glucose, 4 mg/ml crystalline bovine serum albumin [Sigma Chemical Co., St. Louis, MO], 50 Ilg/ml streptomycin sulfate, and 75 Ilg/ml K-penicillin G) covered with paraffin oil in a plastic culture dish (10 x 35 mm, Nunc, Roskilde, Denmark) and were then cultured for 48 hours at 37 C under 5% CO 2 in air for maturation of the oocytes in vitro as described previously. 15 Immediately after termination of culture, morphologic details of the Tsukui et ai. Inhibition of sperm penetration 93

3 Table 1. Incidence of Sperm Immobilizing Antibodies in Sera from Infertile Women No. of No. of positive Percentage of Clinical diagnosis subjects reaction" positive reaction Unexplained Anovulation Tubal obstruction Uterine abnormality Sperm abnormality Total ascreened by the sperm immobilization test. Table 3. Absorption of Sperm Immobilizing Antibody Positive Sera with Washed Spermatozoa Times of Volume No. of washed SIV" after Case absorption of serum spermatozoa absorption ml x x x x x asperm immobilization value. ova were examined under a dissecting microscope, and those showing clear deformity of zonae pellucidae or severe cytoplasmic degeneration were discarded.14 Other ova, regardless of their maturational stage, were rinsed with modified KRB medium and transferred to 0.3 ml of a highly concentrated salt solution (0.5 M [NH4] 2S04, 1.0 M MgCI2, 0.1% Dextran, Nakarai Chemical Co., Kyoto, Japan), which had been placed at the center of a plastic culture dish.14 The surface of the medium was covered with paraffin oil, and the dish was stored at 4 C until use. Before the zona penetration test, stored ova were desalted overnight in a modified BWW medium and were thoroughly rinsed with the same medium. Aliquots (0.3 md of the three sperm suspensions were placed under paraffin oil in plastic culture dishes, and the desalted human ova were introduced into suspensions with one ovum per spot. To clarify the binding site of SIA, some ova were preincubated with SIA-positive serum without dilution for 60 minutes at 37 C and were introduced into the SIA-negative serum~supplemented sperm suspension after washing. After incubation for 24 hours at 37 C under 5% CO2 in Table 2. Sperm Immobilizing Antibody Titer Demonstrated in Sera from Infertile Women Case no. Age Clinical diagnosis SIV Tubal obstruction Unexplained co Sperm abnormality Tubal obstruction Unexplained Unexplained Unexplained Tubal obstruction co Unexplained Anovulation (Xl Unexplained 8.2 asperm immobilization value assessed by the sperm immobilization test. 94 Tsukui et al. Inhibition of sperm penetration air, the ova were washed three times with modified BWW medium and mounted on a slide glass. They were then examined under a Nomarski interference contrast microscope (Olympus IMT-2-21NR, Tokyo, Japan) for assessment of the sperm penetration into the perivitelline space through the zonae pellucidae. RESULTS SPERM IMMOBILIZATION TEST Eleven of the 160 serum samples showed positive reactions for the sperm immobilization test with an overall incidence of 6.9% for SIA (Table 1). The antibody titers calculated as SIV varied considerably from 2.2 to infinity, as shown in Table 2. ABSORPTION OF SERA Repeated absorption of SIA-positive sera from cases 104 and 112 with washed spermatozoa resulted in gradual reduction ofsia, and finally the SIV of these sera were reduced to 1.0, a value that can be regarded as SIA-negative (Table 3). ZONA PENETRATION TEST Spermatozoa incubated with SIA-negative serum were able to penetrate the zona pellucida with a high incidence at concentrations ranging from 2 to 12 X 10 6 sperm/ml (Fig. 1). As shown in Table 4, the numbers of penetrated spermatozoa in the perivitelline space ranged from 0 to 6. The same spermatozoa were also able to penetrate the zonae pellucidae that were preincubated for 60 minutes with SIA-positive serum. By contrast, no spermatozoa incubated with SIA-positive serum penetrated the zonae pellucidae. However, in such complement-free conditions, sperm motility

4 Figure 1 Zona Penetration Test. Spermatozoa incubated with SIAnegative serum could penetrate through the zona pellucida (A), but the penetrating ability of spermatozoa incubated with antibody-positive serum was completely blocked (B). Arrow indicates penetrated spermatozoon in the perivitelline space (x 400). was well maintained throughout the incubation period even in the SIA-positive serum-supplemented medium. There was also no remarkable difference between the sperm motility in SIAnegative serum supplemented medium and that in SIA-positive serum supplemented medium. When SIA-positive serum was absorbed with washed spermatozoa, the serum lost its ability to influence the 'sperm penetrability, and there was no remarkable difference in the penetrability between spermatozoa incubated with SIA-negative serum and that incubated with absorbed SIApositive serum. DISCUSSION Franklin and Dukes16 demonstrated sperm agglutinin in the sera of women whose infertility was otherwise unexplained. However, sperm agglutination tests have proved to be oflimited clinvol. 46, No.1, July 1986 ical usefulness because of the frequency of positive results in the control groups. 11, 16 On the other hand, the presence of SIAs in the sera of women more closely relates to unexplained infertilityy,17 The complement-dependent sperm immobilization test is widely used and is believed to be a reliable method. 4, 11, 18 According to Jones,18 the test was positive in 7.6% of the unexplained infertility cases. We obtained a similar incidence of positive reaction but associated with a relatively high incidence of tubal obstruction, presumably because of our criteria of diagnosing tubal obstruction, which include complete unilateral occlusion and also incomplete occlusion, such as narrow leakage on hysterosalpingography. To investigate spermlzona interaction, we used human zonae pellucidae stored in a highly concentrated salt solution at various stages of maturation. One of the merits of salt-stored zona pellucida is that penetrated spermatozoa in the perivitelline space can be easily distinguished from attached or binding spermatozoa on the surface of salt-stored zona pellucida because of enlargement of the perivitelline space. Another benefit is that we can perform experiments at any time because salt-stored zona pellucida is always available, and the biologic properties of human zona pellucida can be maintained in such a solution for several months, as previously reported. 14 Our results showed no correlation between zona penetrability and the maturational stage of ova and are in agreement with reports that no correlation was found between zona penetrability and the morphologic characteristics of the 00cytes The present study clearly demonstrates that sperm penetration into the perivitelline space through the human zona pellucida is inhibited in the presence of SIA-positive serum. Because this Table 4. Zona Penetration Test Supplemented serum Mean no. of No. of No. of penetrated experiments penetrated ova sperm % SIA-negative SIA-positive Absorbed SIApositive SIA-negativeC /9 (89) 3/3 (loo)b /3 (67) 1.5 0/11 (o)a asignificantly different from SIA-negative serum (X 2 < 0.002). bsignificantly different from SIA-positive serum (X2 = 8.7; P < 0.004). cova were pretreated by SIA-positive serum. 11.7; P Tsukui et ai. Inhibition of sperm penetration 95

5 inhibitory activity disappeared after withdrawal of SIA by absorbing SIA-positive serum with washed spermatozoa, it appears that the SIA in SIA-positiveserum are responsible for the inhibition. Because spermatozoa incubated with SIAnegative serum could penetrate through the zonae pellucidae that had beenpreincubated with SIA-positive serum, SIA-binding appears to be sperm-specific. However, the mechanism by which sperm penetration is blocked under the influence of SIA-positive serum is unknown. From the findings that antisperm antibodies can inhibit sperm-zone binding by interfering with acrosome reaction in vasectomized guineapigs5 and that only acrosome-reacted human spermatozoa can bind and penetrate the human zona pellucida,21 it is possible that SIA-positive serum interferes with capacitation and/or acrosome reaction in the human spermatozoa, leading to interference with sperm/zona interaction. Experiments are under way to test this hypothesis. It is reported that relatively high concentrations of immunoglobulins are present in the fallopian tube fluid but that full complement activity is almost absent Therefore, if the blockade of sperm penetration through the zona pellucida shown in this study occurs even in vivo as an additional mechanism to cause infertility in patients with SIA, in vitro fertilization and embryo transfer using human cord serum or donor serum should be indicated in these patients for achieving successful pregnancy. REFERENCES 1. Fjiillbrant B: Cervical mucus penetration by human spermatozoa treated with antispermatozoal antibodies from rabbit and man. Acta Obstet Gynecol Scand 48:71, Manarang-Pangan S, Behrman SJ: Spermatoxicity of immune sera in human infertility. Fertil Steril 22:145, Shulman S, Friedman MR: Antibodies to spermatozoa. V. Antibody activity in human cervical mucus. 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Fertil Steril 27:815, Gould JE, Overstreet JW, Yanagimachi H, Yanagimachi R, Katz DF, Hanson FW: What functions ofthe sperm cell are measured by in vitro fertilization of zona-free hamster eggs? Fertil Steril 40:344, Tauber PF, Wettich W, Nohlen M, Zaneveld LJD: Diffusable proteins of mucosa of the human cervix, uterus, and fallopian tubes: distribution and variations during the menstrual cycle. Am J Obstet Gynecol 151:1115, Ping WW: Sperm antibody activity in human fallopian tube fluid. Fertil Steril 32:681, Oliphant G, Cabot C, Ross P, Marta J:Control of the rabbit oviduct. BioI Reprod 31:205, 1984