IMMUNE INFERTILITY AND NEW APPROACHES TO TREATMENT*t

Transcription

1 FERTILITY AND STERILITY Copyright 1978 The American Fertility Society Vol. 29, No.3, March 1978 Printed in U.s.A. IMMUNE INFERTILITY AND NEW APPROACHES TO TREATMENT*t SIDNEY SHULMAN, PH,D.t BARBARA HARLIN PHYLLIS DAVIS J, VICTOR REYNIAK, M,D, Sperm Antibody Laboratory, Departments of Microbiology and Obstetrics and Gynecology, New York Medical College, New York, New York A number of patients who have involuntary infertility show sperm antibodies in the blood serum, as detected by two quite different methods of spermagglutination. These techniques are the Kibrick method (gelatin agglutination test) and the F-D method (tube-slide agglutination test). By the former technique, the sera of 18% of women and 9% of men from infertile couples were found to be positive; by the latter technique, the sera of 15% of women and 5% of men were positive. Such cases are termed "immunologic infertility." In an effort to develop new methods for the treatment of infertility, two procedures were explored. One is a sperm washing insemination method in which fresh semen from a man with the antibody is centrifuged, resuspended in an albumin solution, and then used for insemination. The second is an immunosuppression method, using methylprednisolone at a dose of 96 mglday for 7 days. A striking decrease in antibody level was seen in some cases, with ensuing pregnancy. The success rates for these methods have been of the order of 14% to 22%. It is now widely accepted that the infertility problem for certain couples is due to sperm antibody.1.5 This immune response can occur in either the man or the woman. For a complete evaluation of this factor, one would need several kinds of information about the nature of the antibody: (1) the specificity against the antigenic determinant on the sperm cell and the localization of this determinant, (2) the class of the immunoglobulin, (3) the titer or other measure of its concentration, Received April 21, 1977; revised July 25, 1977, and October 13, 1977; accepted October 18, *Supported by Contract N01-HD from the National Institute of Child Health and Human Development, National Institutes of Health, and the Sperm Antibody Laboratory Fund. tpresented at the Ninth World Congress on Fertility and Sterility and the Thirty-Third Annual Meeting of The American Fertility Society, April 12 to 16, 1977, Miami Beach, Fla, :j:reprint requests: Sidney Shulman, Ph.D" Sperm Antibody Laboratory, New York Medical Center, Flower and Fifth Avenue Hospitals, Fifth Avenue and 106th Street, New York, N. y, 10029, and (4) its relative strength in the genital secretions and in the serum. Some of these characteristics can be determined, especially if we avail ourselves of the several different techniques for detection of sperm antibody, since we know that each procedure does respond better to a distinctive kind of antibody. We and others have tried to demonstrate the significance of the different activities, generally by means of comparisons of frequency of occurrence in large infertile and fertile populations.6-8 Finally, we have tried to develop newer methods for the removal of these antibodies, so that the patient will receive successful treatment for the infertility problem. This report describes some results for each of these efforts. METHODS Our laboratory studies have involved obtaining blood serum from each of the two partners in each couple for determination of sperm antibody 309

2 310 SHULMAN ET AL. March 1978 levels. This antibody detection has been achieved by either of two techniques of spermagglutination, based on the Kibrick technique 9 and the Franklin-Dukes technique,io as we have modified them. These methods, termed by us Kibrick and F-D, respectively, have been discussed in detail elsewhere 1, 2, "; alternative names have been proposed. l1 A total of 409 couples were studied for these antibodies; occasionally a partner did not provide serum, and occasionally a couple was not tested by both methods. The methods are described only briefly. The Kibrick Method (the Gelatin Agglutination Test) of Sperm Antibody Detection. The mixtures of serum (at a 1:4 dilution) and semen are put into Kibrick tubes (3 mm in diameter and 3 cm in length) and incubated at 37 C for 2 hours. Those sera that are found to be positive are tested again, in a dilution series. Results are expressed as follows: 4 means a titer of 1:4; <4 means there was no reaction at the 1:4 dilution, hence a negative result; ~4 means a positive result at a 1:4 dilution but no titrated value; larger numbers (8, 16, 32, etc.) are expressed analogously. We considered a serum to be positive only if it shows a titer of at least 1:4. The F-D Method (the Tube-Slide Agglutination Test) of Sperm Antibody Detection. After incubation of the serum-semen mixture at 37 C for 2 hours, single drops are put on slides and examined under the microscope. The sperm cells in each of 12 high-power fields are counted, noting only the motile cells and counting the number of free cells and the number of cells in clumps. The number of clumped cells is divided by the total number of cells, giving a percentage. We consider that values for this percentage that are less than 10 are of no significance; that is, the serum is positive only if it gives a percentage of more than 10. All values above 10% are considered to be essentially the same; that is, we have never attempted to distinguish the results by any variation in these values. RESULTS Frequencies of Sperm Antibody Occurrence The results of diagnostic testing of a large infertile population (about 400 couples) are summarized in Table 1. The results are quite different, according to whether women or men were tested and according to which technigue was used. We had shown in 1971 that any individual serum that was positive by these techniques was usually positive by either of these two methods, but not by the other.12 Only a few positive sera were positive by both methods. We did not know the reason for this at the time, but in the past year it has become rather certain that this difference reflects a distinction between antibodies to the head or the tail of the sperm cell. For the collected 400 couples reported here, the sera of the women showed positive results of 18% by the Kibrick method and 15% by the F-D method. In the sera of the men, there were positive results of 9% by the Kibrick method and 5% by the F-D method. We have also studied some fertile populations; these have not shown a complete absence of this antibody activity, but the incidence is much lower in the infertile (female) population.s Table 2 shows the current thinking as to which antibody specificities are revealed by these two procedures. 13 In most cases, a positive Kibrick result means that there is a tail-to-tail agglutinating antibody; in contrast, a positive F-D result means that there is a head-to-head agglutinating antibody. Treatment of Immunologic Infertility The traditional treatment for a woman with antibody has been the condom treatment; this regimen of using condoms at every coitus may have to be maintained for 6 to 12 months. 1, 2 We insist, for our cases, that the woman come back for testing every 3 months. We compare all of the serum samples obtained from her in simultaneous titrations. The success rate, in terms of pregnancies, is thought to be about 50%.1, 14 However, this method cannot be applied to cases where the man has the antibody. We have developed two new methods for cases in which men have sperm antibody activity; the second of the two methods can also be attempted for women if one does not wish to use the condom regimen. The treatment group included 32 couples. The Sperm Washing Insemination Method. The first of our two new methods is called the sperm washing insemination method, or SWIM. It is based on the rationale that the antibody that is present in the man's seminal plasma can be washed away. The procedure is as follows: We request that the couple visit us on 2 successive days in each menstrual cycle, choosing the 1st day according to the woman's basal body temperature chart and previous history, so that we try to see the

3 Vol. 29, No. 3 IMMUNE INFERTILITY AND NEW APPROACHES TO TREATMENT 311 TABLE 1. Positive Results in Serum Samples from Infertile Couples Female Male F -D (tube-slide aggl utination) method F-D (tube-slide Total no. of patients No_ positive % Positive couple on the day of ovulation, or the day before. Sometimes three or four visits in a cycle are necessary. On each visit, semen is obtained from the husband as quickly as possible after masturbation and samples are removed for initial sperm count and motility estimation. The semen is then diluted 4-fold with the suspending medium and centrifuged at 2000 rpm for 5 minutes. The soft pellet is suspended in the diluted volume, using the same suspending medium, and centrifuged again. This washing process is done for a total of three times. The final pellet is suspended in about 0.5 ml of the medium in order to end with about 1.0 ml of product, which again is evaluated for sperm count and motility estimation. In this procedure, the medium used is a sterile 4% solution of human serum albumin. In this manner, the motility of the spermatozoa is maintained with no decrease. The sperm count per milliliter is often increased. The final product is inseminated in intrauterine, or at least intracervical, fashion. (We should note that this technique is not comparable to inseminating an unmodified semen sample, since the prostaglandin content is largely removed; uterine cramping has not occurred in these women.) A plastic-coated tampon is used for a 6-hour period, since the product has a very low viscosity. In the first group of couples, for those who tried the. procedure for at least two cycles, there has been one success in seven couples. The data for this couple are shown in Table 3. The Immunosuppression Method. The second of our two new methods is called the immunosuppression method, or 1M. It is based on the rationale that some degree of suppression of circulating antibody can be achieved by the use of TABLE 2. Comparative Specificities and Sensitivities of Two Agglutination Methods for Sperm Antibody Detection" Specificity H-H T-T + ++ F-D (tube-slide +++ "The specificity is indicated as H-H (head-to-head) or T-T (tail-to-tail) agglutinating antibody. The sensitivity is indicated, in semiquantitative fashion, as ranging in intensity from +++ (very strong) to + (weak) and - (negative). corticosteroids. However, it must be emphasized that, according to newer knowledge in immunology, we must expect to use very high doses of the steroids in order to achieve any appreciable suppression in the human species. Accordingly, we have introduced the concept of using the drug methylprednisolone (MedroD, at a dose of 96 mg/ day, for 7 days. We ask for blood samples and also for samples of semen or cervical mucus at frequent intervals, such as (for blood and semen) weeks 1, 2, 3, 4, 6, and 8, after the 1st day the drug is administered. In our first case, with good collection of samples, we were able to demonstrate a considerable decrease in the antibody level in both the serum and seminal plasma over a period of 3 weeks, and the wife became pregnant in the next cycle. 15 The baby has been born and is apparently quite normal. In some cases, there was a decrease in antibody level, but no pregnancy; we have recommended that the drug be taken again in such cases. In some other cases, we have seen no decrease in antibody level; in such cases, we do not recommend repetition of the drug. In one couple, pregnancy occurred about 3 months after the man took the medication. Such a delayed result may be reasonable, since we have determined in some cases that, once the antibody level goes down, it may stay down for several months. By the end of 1976, 4 pregnancies had occurred in the wives of 18 men who took the steroid, that is, a success rate of about 22%, and 1 pregnancy had occurred in 7 women who took the steroid, that is, a success rate of about 14%. We believe TABLE 3. Sperm Suspension Characteristics in the Sperm Washing Insemination Method (Mr. B) Day 12 Day 13 Initial Final Initial Final findings findings findings findings Cycle 1 Volume (ml) Motility (%) Count (x 106/ml) ClumpslLPF" Cycle 2 Volume (ml) Motility (%) Count (x 106/ml) ClumpslLPF <1 2 "LPF, Low-power field.

4 312 TABLE 4. Medrol Medication in the Immunosuppression Method (Mr. A) Serum levels of Seminal plasma levels Weeks Sperm count sperm antibody of sperm antibody (Kibrickl (Kibrickl x 10'Imi (±) <32 4 (±) that both of these values are quite encouraging, inasmuch as these couples had been trying for several years to achieve pregnancy. Since the beginning of 1977, about a dozen more couples have been taking this medication, but it is too early to evaluate this latter group. In Table 4, the results of steroid medication in one man are shown-in particular, the various levels of the sperm count, as well as the decreasing level of sperm antibody, in both the serum and the seminal plasma. One should note that the sperm count did not really decrease. Notwithstanding the fears that some investigators have expressed for steroid medication, the sperm count remained essentially the same. We also asked the women who took the steroid medication to maintain their basal body temperature chart and noted that the curves were biphasic and were quite similar to those recorded prior to treatment; thus, this medication did not disturb ovulation. DISCUSSION We have presented only the findings in serum, although we have also conducted studies on cervical mucus. Some of our observations on sperm antibody in cervical mucus have been presented elsewhere In couples with involuntary infertility, there may be a sperm antibody factor in either partner. This problem may occur regardless of whether the infertility is labeled "unexplained" or "organic"; hence, we have preferred to designate such cases as "immunologic infertility." If a diagnostic study for this antibody activity is to be made, certain principles of testing and of clinical correlation must be applied.8 Adherence to these principles will give reliable and meaningful determinations of whether there is an immunologic problem for the couple. In a large infertile population, 9% of men do have sperm antibody, as tested by the Kibrick method; the sera of 5% of the group are positive SHULMAN ET AL. March 1978 by the F-D method. This means that almost 14% will be positive, since only rarely is a serum sample positive by both methods. In this way, we can also differentiate between two quite different types of sperm antibody, that is, a sperm head antibody and a sperm tail antibody. The former type is detected by the F-D method; the latter type is detected by the Kibrick method. For the women in a large infertile population, the sera of 18% are positive by the Kibrick method and the sera of 15% are positive by the F -D method. Thus, about 30% will be positive by either method. Again, we can differentiate between sperm head antibody (F-D) and sperm tail antibody (Kibrick). If the couple has shown a poor postcoital test, they should certainly be tested for sperm antibody, since there is a high probability-although not 100%-that the test will be positive. However, even with a good postcoital test result, the chance of finding sperm antibody is not totally excluded. 19 The possibilities for treatment of patients with these immunologic problems are limited, although the picture is not totally hopeless. We have two new methods of treatment. One method actually involves treatment of the ejaculated semen, followed by insemination with it. One problem with the SWIM procedure may be the difficulty of maintaining a high enough motility while adequately removing the antibody from the seminal plasma; the use of a 4% serum albumin solution has worked very well. A second problem is that of intervening quickly enough to prevent appreciable interaction between the dissolved antibody and the sperm cells. Perhaps the procedure will have to be modified in some cases to remove antibody that has very quickly attached itself to the sperm cells. A third problem is that of demonstrating that sperm antibody activity actually occurs in the semen, either in the seminal plasma or on the surface of the spermatozoa. We have made very limited progress in this direction and further studies are needed, especially to determine whether the antibody has already coated the sperm cell by the time the washing process is carried out. In our efforts to employ immunosuppression, it is considered that the use of corticosteroids can have an immunosuppressive effect only ifthey are given in doses of about 80 or 100 mg/day for at least 7 days. In an earlier study, Butler and Rossen20 provided evidence that the decreased levels of immunoglobulin G in consequence of this steroid regimen were probably due to increased

5 Vol. 29, No.3 IMMUNE INFERTILITY AND NEW APPROACHES TO TREATMENT 313 catabolism of immunoglobulin G during drug administration and to decreased synthesis for a time afterward. These details are discussed further elsewhere,21 along with a more complete description of one of our early cases. We have had several successes by this approach. In some cases, the antibody level did not decrease. In others, it decreased but no pregnancy ensued; in some of these cases we are recommending a repeated regimen of the medication, and these patients are being studied further. Third, in some cases, antibody levels decreased and pregnancy did ensue, sometimes in 1 month, sometimes in 3 months. Apparent success rates of about 14% in treated women and about 22% in treated men have been achieved. Very recently, another claim of success with a corticosteroid method has been published.22 A large, controlled series of cases is now needed for the critical evaluation of both ofthese possible new treatment procedures. REFERENCES 1. Shulman S: Reproduction and Antibody Response. Cleveland, CRC Press, Shulman S: Immunologic barriers to fertility. Obstet Gynecol Survey 27:553, Hekman A, Rumke P: Auto- and iso-immunity against spermatozoa. In Textbook of Immunopathology, Second Edition, Edited by PA Miescher, HJ Muller-Eberhard. New York, Grune & Stratton, 1976, p Rumke P, Hekman A: Auto- and isoimmunity to sperm in infertility. Clin Endocrinol Metabol 4:473, Johnson MH, Hekman A, Rumke P: The male and female genital tracts in allergic disease. In Clinical Aspects of Immunology, Third Edition, Edited by PGH Gell, RRA Coombs, PJ Lachmann. Oxford, Blackwell, 1975, p Rumke P: Autoantibodies against spermatozoa in infertile men: some unsolved problems. In Proceedings of the First International Congress on Immunology in Obstetrics and Gynaecology, Padua, Italy, 1973, Edited by A Centaro, N Caretti. Amsterdam, Excerpta Medica, 1974, p Fjallbrant B: Incidence of sperm antibodies in males with regard to age. In Proceedings of the First International Congress on Immunology in Obstetrics and Gynaecology, Padua, Italy, 1973, Edited by A Centaro, N Carretti. Amsterdam, Excerpta Medica, 1974, p Shulman S, Jackson H, Stone ML: Antibodies to spermatozoa. VI. Comparative studies of spermagglutination activity in groups of infertile and fertile women. Am J Obstet Gynecol 123:139, Kibrick S, Belding DL, Merrill B: Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 3:430, Franklin RR, Dukes CD: Antispermatozoal antibody and unexplained infertility. Am J Obstet Gynecol 89:6, Rose NR, Hjort T, Rumke P, Harper MJK, Vyazov 0: Techniques for the detection of iso- and auto-antibodies to human spermatozoa. Clin Exp Immunol 23:175, Shulman S, Shulman JF: Spermagglutinating activity in man and guinea pig. Fertil Steril 22:663, Boettcher B, Hjort T, Rumke P, Shulman S, Vyazov OE: Auto- and iso-antibodies to antigens of the human reproductive system. Results of an international comparative study. Clin Exp Immunol 30:173, Masson D, Lehmann F, Breckwoldt M, Krebs D: Sperma Antikiirper als miigliche Ursache einer ungeklarten Sterilitat. Geburtshilfe Frauenheilkd 30:103, Shulman S: Treatment of immune male infertility with methylprednisolone. Lancet 2:1243, Shulman S, Friedman MR: Antibodies to spermatozoa. V. Antibody activity in human cervical mucus. Am J Obstet Gynecol 122:101, Shulman S: Sperm antibodies in serum of men and women and in cervical mucus. In Proceedings of the Eighth World Congress on Fertility and Sterility, Buenos Aires, Argentina, Amsterdam, Excepta Medica, 1975, p Sudo N, Shulman S, Stone ML: Antibodies to spermatozoa. IX. Sperm agglutination phenomena in cervical mucus in vitro. Am J Obstet GynecoI129:360, Telang M, Reyniak JV, Shulman S: Antibodies to spermatozoa. VIII. Correlation of sperm antibody activity with post-coital tests in infertile couples. Int J Fertil. In press, Butler WT, Rossen RD: Effects of corticosteroids on immunity in man. I. Decreased serum IgG concentration caused by 3 to 5 days of high doses of methylprednisolone. J Clin Invest 52:2629, Shulman S, Harlin B, Davis P: The successful treatment of a case of immune infertility by a new method. Urology. In press, De Almeida M, Soufir JC: Corticosteroid therapy for male autoimmune infertility. Lancet 2:815, 1977