AUTOANTIBODIES TO ZONA PELLUCIDA IN INFERTILE AND AGED WOMEN*

Transcription

1 FERTILITY AND STERILITY Copyright ' 1980 The American Fertility Society Vol. 34, No.6, December 1980 Prinred in U.SA. AUTOANTIBODIES TO ZONA PELLUCIDA IN INFERTILE AND AGED WOMEN* TOSHIHUMI NISHIMOTO, M.D.t TAKAHIDE MORl, M.D., D.MED.ScI. ICHIRO YAMADA, M.D. TOSHIO NISHIMURA, M.D., D.MED.ScI. Department of Obstetrics and Gynecology, School of Medicine, Kyoto University, Kyoto, Japan A total of 735 human serum samples were tested against isolated porcine ova for detection of autoantibodies to zonae pellucidae by the specific method reported previously using indirect immunofluorescence. Positive fluorescence was found in 13 of 175 samples (7.4%) from patients with unexplained infertility, in 2 of 122 samples (1.6%) from patients with proven etiology of infertility, but in none of 141 samples. from age-matched control subjects (fertile women and men). There was an age difference in the incidence of positive reactions: 4 of 156 samples (2.6%) from women over 40 years old were positive in contrast to only 1 of 159 samples (0.6%) from women under 40 years old. Although autoantibodies to zonae pellucidae may be produced during the aging process, the observed high incidence in unexplained infertility suggests an etiologic association of the antibody with the disease when produced in younger women. Fertil Steril34:552, 1980 On the basis of recent findings in reproductive immunobiology ofzonae pellucidae in animal species, the existence of autoantibodies to a specific antigen(s) of the structure in infertile women was proposed as a tempting hypothesis to explain an aspect of the etiology of unexplained infertility. 1, 2 This concept was later dismissed because of widespread anti-zona pellucida activity as detected by immunofluorescence among sera from infertile and fertile human male and female subjects. 3 However, we have confirmed the presence of this kind of antibody by developing a method for the specific detection of autoantibodies to human zonae that involves removing hemagglutinins which are normally present in' human sera and which are reactive with blood group substances on the surface of zonae. 4 It has recently been reported Received July 15, 1980; accepted August 13, *Supported in part by grants from the Ministry of Health and Welfare and from the Ministry of Education (Grant ). treprint requests: Toshihumi Nishimoto, M.D., Department of Obstetrics and Gynecology, School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan. 552 that sera from old female mice contain a factor which impairs fertilization and produces immunofluorescence on zonae, suggesting the presence of autoantibodies to this structure. 5 It seems of interest to ascertain how often this kind of autoantibody can be detected among various categories of infertility and to determine whether the antibodies occur during the physiologic process of aging in women. MATERIALS AND METHODS Serum Samples and Absorption Procedures. Serum samples were collected from 297 infertile women between the ages of23 and 39: 175 patients had unexplained infertility; 65 had ovulatory failure; 44 had infertility of proven etiology, including tubal obstruction or sperm abnormality; and 13 were habitual aborters. A control group of 141 serum samples from normal volunteer subjects of proven fertility who were age-matched to the infertile women was provided by 71 nonpregnant women, 39 pregnant women, and 31 normal men. A separate group of 315 serum samples was

2 Vol. 34, No.6 AUTOANTIBODIES TO ZONA PELLUCIDA IN INFERTILE AND AGED WOMEN 553 TABLE 1. Incidence of Positive Immunofluorescence as Tested against Porcine Zonae in Sera from Infertile Patients and Age-Matched Control Subjects No. of No. of ~ Positive Clinical diagnosis sera tested positive reactions reactions Infertile women Unexplained Anovulation 65 1a 1.5 Tubal obstruction and/or sperm abnormality 44 1b 2.3 Habitual abortion Total Control subjects Nonpregnant women Pregnant women Normal men Total aa 31-year-old nulliparous woman with ovulation failure; she had been infertile since her marriage 7 years previously. b A -year-old one-gravid woman with tubal obstruction; she had been infertile since delivery of her child 7 years previously. provided to the study by informed, nonpregnant women of teenage through the 60s. Sera were kept frozen at - 20 C until use and were inactivated by heating at 56 C for minutes prior to testing. Each serum sample showing a positive zona immunofluorescence or zona precipitate was further absorbed with 2000 porcine ova/0.1 ml of serum (usually three times until the positive reaction on porcine zonae disappeared). The serum absorbed with porcine ova was tested for anti-zona activity against human zona. Examination of Anti-Zona Activities. Anti-zona activities of the test sera were examined by immunofluorescence and light scattering of zona precipitates. The method described in our previous report 4 was followed for the detection of specific immunofluorescence against porcine and human zonae. Briefly stated, cumulus-encased follicular ova were collected by puncture from porcine ovaries l 6 or by aspirating the follicles of human ovaries obtained at laparotomy for uterine malignancies. The ova were stripped of the surrounding cumulus cells mechanically.6 For the indirect immunofluorescence test, porcine or human ova were incubated with heteroagglutinin- or isoagglutinin-absorbed sera for minutes at room temperature, washed thoroughly with several changes of 0.01 M phosphate-buffered saline (PBS), ph 7.4, and incubated further for 10 minutes with rabbit antiserum to human immunoglobulin (Ig) G, M, and A, and light chains labeled with fluorescent isothiocyanate (Dakopatts AlS, Copenhagen) in appropriate dilutions which had been determined as not giving nonspecific positive reaction' by direct staining with the conjugates. It was found necessary to dilute the original conjugate SO-fold with PBS for porcine zonae and 20-fold for human zonae. Ova were washed with PBS and examined under a fluorescence microscope (Olympus BH-RFL). The titer of the anti-zona activities was expressed by the highest dilution that gave a positive fluorescence. For the light-scatteringtest,7 ova were incubated in agglutinin-absorbed sera at room temperature for minutes, washed thoroughly in PBS, and examined for the presence of precipitate on the outer surface of the zonae as viewed by a strong light-scattering layer under dark-fieldillumination. RESULTS Distribution of Positive Immunofluorescence in Sera from Women with Various Categories oflnfettility and Increasing Age. As is shown in Table 1, TABLE 2. Incidence of Positive Immunofluorescence as Tested against Procine Zonae in Sera from Control Woman among Various Age Groups No. of No.ofposi- % Posi Age sera tested tive reactions tive reactions ", la b " 3.8 ; Total a A 24-year-old unmarried woman with regular menstrual cycles. bone patient was a 43-year-old, two-parous woman with irregular menstruation; the other was a 49-year-old. twoparous woman with irregular menstruation. COne patient was a 52-year-old, three-parous woman with irregular menstruation; the other was a 53-year-old, oneparous woman who had undergone menopause at age 52.

3 554 December 1980 NISHIMOTO ET AL. TABLE 3. Titration of Anti-Zona Activity by Immunofluorescence Subject Infertile patients. Y.O. Y.H. K.K. M.S. C.K. T. Y. K.M. F. K. Y.S. S. K. K. N. Y.T. H. Y. K. T. Y.K. Control subjects F.O. K.N. Y.M. K.A. F.O. Age Titer" athe highest dilution of a test serum that gave positive immunofluorescence on porcine zonae. positive reactions were found in 13 of 175 serum samples (7.4%) from patients with unexplained infertility, whereas positive reactions were found in 1 of 65 (1.5%) and 1 of 44 (2.3%) serum samples from patients with ovulatory failure and patients with tubal and/or sperm abnormality, respectively. No positive reaction was found in sera of patients with habitual abortion or in sera from agematched control subjects (nonpregnant women, pregnant women, and normal men, all of whom were proven fertile). As is shown in Table 2, positive reactions were recorded in 5 of 315 (1.6%) control women of varying age, 4 of whom were over 40 years old. The only positive reaction among women under 40 was found in serum from a 24-year-old unmarried woman. As is shown in Table 3, positive antizona activities in serum samples from 15 infertile women showed varying titers between and, whereas the activities of serum samples from 5 control subjects of different age groups remained relatively low with titers of. Interrelation between the Anti-ZonaActivities qn Porcine and Human Zonae. Each of the infertile sera showing positive immunofluorescence on porcine zonae produced a precipitate on porcine zonae. The positive immunofluorescence and zona precipitate were clearly reproduced on huma zonae. However, these anti-zona activities on human zonae disappeared after absorption of the serum with porcine ova (Fig. 1). A similar finding was confirmed for sera with positive anti-zona activities from control subjects of various age groups (Fig. 2). DISCUSSION The results of the present study with a large number of serum samples from subjects in comprehensive categories tested by the specific method developed in our laboratory4 has further indicated that positive anti-zona activities are predominantly detected in sera from female patients with unexplained infertility and not found in sera from agematched control subjects of proven fertility. The results are quite different from those in a recent report in which positive anti-zona activities were detected even in sera from fertile female and male subjects. 3 It is evident that absorption with porcine red blood cells is mandatory for specific detection of anti-zona pellucida activities in human sera, as long as porcine zonae are used as targets, 4 because of the presence of blood group substances on porcine zonae. However, it is not clear whether (and how completely) human heteroagglutinins, which interfere with specific detection of autoantibodies to the structure, are removed in the latter FIG. 1. Human ova with positive immunofluorescence (upper left) and a positive precipitation reaction (lower left) on the surface of the zona pellucida; the ova were exposed to serum from a 35-year-old patient with unexplained infertility after absorption of the serum with human AB erythrocytes. The ova demonstrate negative immunofluorescence (upper right) and a negative precipitation reaction (lower right) following exposure to the same serum but after absorption with human AB erythrocytes and porcine ova sequentially.

4 Vol. 34, No.6 AUTOANTIBODIES TO ZONA PELLUCIDA IN INFERTILE AND AGED WOMEN 555 FIG. 2. Human ova with positive immunofluorescence (upper left) and a positive precipitation reaction (lower left) on the surface of the zona pellucida; the ova were exposed to serum from a 49-year-old woman after absorption of the serum with human AB erythrocytes. The ova demonstrate negative immunofluorescence (upper right) and a negative precipitation reaction (lower right) following exposure to the same serum but after absorption with human AB erythrocytes and porcine ova sequentially. study. The difference in the incidences of positive immunofluorescence against porcine zonae between the two studies may well be derived from the difference in the extent of absorption of heteroagglutinins. With the exception of two cases, the association of positive immunofluorescence against zonae in samples from patients with unexplained infertility may signify the involvement of autoantibodies to zonae in the development of unexplained infertility either by blocking fertilization8 or by inhibiting implantation of fertilized eggs, 9 as demonstrated originally in hamsters. In spite of the strong antigenic potency of the human zona pellucida as confirmed in heteroimmune systems,1o, 11 the titer of the autoantibodies was relatively low as assessed by immunofluorescence, probably because of the weakened antigenicity in autoimmunization, as has been observed in isoimmunization of mice 12 and rats.13 However, the low titer of immunofluorescence could not be neglected as a possible cause of infertility, because fluorescencepositive sera produce precipitates on human zonae which have been shown to be an indication of fertilization-blocking activity for antibodies to mouse14 and porcine zonae.15 To resolve this question, further investigation using human in vitro fertilization is now in progress in our laboratory. The etiologic significance of the positive antizona activity observed in the sera of three young women cannot as yet be estimated, because two of them have obvious causes for their infertility and the remaining woman not yet married. It would be of interest to follow these patients with regard to future fertility after the known factors are treated or after marriage. On the other hand, the positive anti-zona activities detected in the sera of older women, even though the incidence and titers were relatively low, may suggest the appearance of autoantibodies to the zona pellucida structure with advancing age, as has recently been reported in mice.5 Specific autoantibodies to the brain cells has also been demonstrated in serum of old mice. 16 Furthermore, autoantibody formation is a feature of aging even in humans, as evidenced by high incidences of antinuclear and rheumatoid factors in aged people.17 Even if the production of antizona autoantibodies is a physiologic process of age dependency, autoantibodies could be produced in younger women who have an intrinsic immune factor(s) that sensitizes them to the zona antigen(s) much more easily than in average women, leading to the development of unexplained infertility. The disappearance of anti-zona activities after rigorous absorption of the positive sera with a large number of porcine ova may signify that the antibody is directed against antigen(s) common to porcine and human zonae,l, 6, 11 and not against antigen(s) specific to human zonae as detected on heteroimmunization with human ova. 11 This mechanism would provide additional evidence for the utilizability of porcine zona antigen(s) in human immunocontraception,18 because the dose of antigen required to increase the antibody titer sufficiently to produce an effective block to fertilization in vivo could be minimized. REFERENCES 1. Shivers CA, Dumbar BS: Autoantibodies to zona pellucida: a possible cause for infertility in women. Science 197: 1082, Mori T, Nishimoto T, Kitagawa M, Noda Y, Nishimura T, Oikawa T: Possible presence of autoantibodies to zonae pellucidae in infertile women. Experientia 34:797, Sacco AG, Moghissi KS: Anti-zona pellucida activity in human sera. Fertil Steril 31:503, Mori T, Nishimoto T, Kohda H, Takai I, Nishimura T, Oikawa T: A method for specific detection of autoanti-

5 556 NISHIMOTO ET AL. December 1980 bodies to the zona pellucida in infertile women. Fertil Steril :67, Tsunoda Y, Chang MC: The suppressive effect of sera from old female mice on in vitro fertilization and blastocyst development. BioI Reprod 20:355, Sacco AG: Antigenic cross-reactivity between human and pig zona pellucida. BioI Reprod 16:164, Oikawa T, Yanagimachi R: Block of hamster fertilization by anti-ovary antibody. J Reprod Fertil 45:487, Shivers CA, Dudkeiwicz AB, Franklin LE, Fussell EN: Inhibition of sperm-egg interaction by specific antibody. Science 178:1211, Dudkiewicz AB, Noske IG, Shivers CA: Inhibition of implantation in the golden hamster by zona-precipitating antibody. Fertil Steril 26:686, Sacco AG: Antigenicity of the human zona pellucida. BioI Reprod 16:158, Takai I, Mori T, Noda Y, Nishimura T: Heteroimmunization with isolated human ova. J Reprod Fertil. In press, Gwatkin RL, Williams DT, Carlo DJ: Immunization of mice with heat-solubilized hamster zonae: production of anti-zona antibody and inhibition offertility. Fertil Steril 28:871, Tsunoda Y, Chang MC: Reproduction in rats and mice isoimmunized with homogenates of ovary or testis with epididymis, or sperm suspensions. J Reprod Fertil 46:379, Tsunoda Y, SugieT, MoriJ: Quantitative determination of titers of anti-zona serum. J Exp Zool 207:315, Sacco AG: Inhibition of fertility in mice by passive immunization with antibodies to isolated zonae pellucidae. J Reprod Fertil 56:533, Nandy K, Fritz RB, Threatt J: Specificity of brain-reactive antibodies in serum of old mice. J Gerontol :269, Talal N: Tolerance and autoimmunity. In Clinical Immunology, Vol 1, Edited by CW Parker. Philadelphia, WB Saunders Co, 1980, p Shivers CA: Immunological interference with fertilization. In Immunological Approaches to Fertility Control, Edited by E Diczfalusy. Stockholm, Karolinska Institute, 1974, p 233