Prognosis of fertility in women with unexplained infertility and sperm agglutinins in the serum

FERTILITY AND STERILITY Copyright < 1984 The American Fertility Society

Vol. 42, No.4, October 1984 Printed in U.S.A.

Prognosis of fertility in women with unexplained infertility and sperm agglutinins in the serum

Philip Rumke, M,D, * Cees N, M, Renckens, M.D. t Pieter D. Bezemer, Ph.D.:!: Nanda van Amstel t The Netherlands Cancer Institute, Academic Medical Center, University of Amsterdam, and Free University, Amsterdam, The Netherlands

From 1967 to 1973 serum samples of 1709 infertile women were tested for antispermatozoal antibodies with the gelatin agglutination test. In 110 cases sperm agglutinins were demonstrated in titers ranging from 4 to 1024. The clinical data of 99 couples were evaluable, and in 65 there appeared to be unexplained infertility. Out of these 65 women, 44 became pregnant in the posttesting follow-up period of 6 to 13.5 years (median, 10 years). With increasing titers there was a significant decrease in the probability of becoming pregnant. Of the women who became pregnant, in general, those with higher titers had to wait longer for their pregnancy than those with lower titers. Moreover, with increasing titers there was a tendency toward a longer duration of infertility before the serum was tested. This latter observation supports the contention that the agglutinin titer influences the fecundability rate. The titers of those becoming pregnant and those remaining infertile, however, showed considerable overlap. Favorable results in the postcoital test were associated with a better prognosis, but this was independent of the titers. Fertil Steril 42:561, 1984

Evidence is accumulating that infertility in women may be caused by a local immune response against spermatozoa in the genital tract, in particular in the cervix uteri. 1. 7 Controversy still exists as to the significance of systemically produced sperm antibodies, as measured in serum by means of agglutination techniques. When infertile women had sperm agglutinins in the serum, their later reproductive performance was not found to be worse than in women without Received February 6, 1984; revised and accepted June 22, 1984. *Reprint requests: Philip Rumke, Prof. Dr" Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. tDepartment of Obstetrics and Gynaecology, Academic Medical Center, University of Amsterdam. *Department of Medical Statistics, Free University, Amsterdam. Vol. 42, No.4, October 1984

circulating antibodies. 7 , 8 However, when the infertility lasted longer than 3 years, the fertility rate of women with agglutinins in the serum was significantly lower than that of women without agglutinins, and also the abortion rate was higher in these cases. 9 Further, it took a longer time to become pregnant when sperm-agglutinating antibodies were present, although the fertility rate was similar in women with and without agglutinins. 10 In one study, the probability of becoming pregnant was found to be much lower when the agglutination titer was 16 or higher. 6 In our laboratory, the sera of infertile men have been examined for the presence of sperm agglutinins using Kibrick's gelatin macro agglutination technique (GAT). It was found that the higher the serum titer, the smaller the probability that a man could impregnate his wife. 11 Since, to the end of 1973, we had a large number of serum Rumke et a!. Sperm agglutinins and infertility in women

561

samples from infertile women tested as well, we decided to test the hypothesis that the sperm agglutinin titer in the serum of women is also inversely related to the posttesting reproductive performance. Attention was paid not only to the probability of becoming pregnant but also to the time factor involved and the abortion rate. If the hypothesis is correct that the probability of becoming pregnant decreases with increasing titers, then the time elapsing before pregnancy occurs (if it occurs) will be longer with increasing titers. Women with low titers would be expected to take only slightly longer than normal to become pregnant: they may therefore never seek help for an infertility problem, and such women would have selected themselves out of the study. This point can best be illustrated with the following theoretical example: if it takes 5 years to become pregnant in the presence of a high titer and 2 years with a low titer, and if, independent of the titer, half of the women visit a doctor after attempting to become pregnant for 1 year while the other half of the population wait 3 years before seeking medical help, then for the women with the low titers, the mean waiting time before visiting the doctor is 1 year, since in theory all are pregnant by 3 years. The mean waiting time of the women with the high titers would be 2 years (50%,1 year; 50%, 3 years), and therefore it seems that these women wait longer before seeking help. An analysis of the pretesting waiting time, i.e., pretesting infertility duration, in relation to the titers is the more important, because a set of data is used that is not used in the computing of the probability of becoming pregnant after testing, or the time that it takes to become pregnant after serum testing. Such an analysis can thus provide an independent test of the hypothesis under investigation.

PATIENTS

In the 7-year period from 1967 to 1973, serum samples from 1709 female patients were tested at the request of many specialists in The Netherlands and Belgium. Of these, 110 patients (6.4%) were found to have sperm agglutinins in the serum with titers ranging from 4 to 1024. One patient was excluded from the study because supposed allergy to semen and not infertility was the indication for the examination. In all others the reason for serum testing was infertility, including one patient who suffered from habitual abortion. The clinical data were collected from the referring specialists. Ten cases were not evaluated for various reasons (incomplete infertility investigations in three, loss to follow-up within a few months after serum testing in two, and loss of case reports in five patients). In 65 of the remaining 99 patients the infertility was considered to be unexplained, since the husband was normospermic and did not have sperm agglutinins himself, and the patient had patent tubes and ovulatory menstrual cycles. Couples with poor postcoital tests were included. It should be noted that in the period concerned diagnostic laparoscopy had not been widely introduced, and few of the patients had undergone this procedure. These 65 patients form the sample of the target population of the present retrospective follow-up study. Follow-up data were collected from the referring specialists and additionally, with permission of these specialists, by directly contacting the patients. The period of follow-up for those remaining infertile was calculated from the time of attempting to achieve a conception, 9.5 to 22 years (median, 16 years); whereas, calculated from the time of serum testing, the follow-up period was 6 to 13.5 years (median, 10 years). STATISTICAL METHODS

MATERIALS AND METHODS SPERM ANTIBODY DETECTION

The GAT, as described earlier,!1 was used for sperm antibody detection. Various donors, but never male partners of the infertile couples, supplied the semen samples necessary for the test. Known positive and negative control sera were included in every test. Serum samples were screened in the dilutions 1:4 and 1:20 with two different donor semen samples. Twofold serial dilution steps were used to titrate positive sera. 562

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The estimated time of conception rather than the date of birth was considered as the end of the infertile period, regardless of the outcome of the pregnancy. With the life-table (actuarial) method 12, 13 the probabilities of pregnancy were compdted starting from the time of serum testing, whereas the monthly fecundability rates 13 were computed starting from the time of attempting to achieve a conception. The hypothesis that the probability of pregnancy decreases with increasing titer was tested with a chi-square test for linear trend. 12 Moreover, we Fertility and Sterility

Table 1. Incidence of Pregnancy in the Posttesting Perioda

Titer (210g titer) 4 (2) 8 (3) 16 (4) 32 (5) 64 (6) 128 (7) 256 (8) 512 (9) 1024 (10)

Total 14 20 15 10 3 1 0 0 2

65 Total Mean 210g titer 3.7

Percentage pregnant (95% conNo. of patients fidence limits) Pregnant Not pregnant

6 2 0

2 7 4 4 1 1

86 65 73 60 67 0

0

2

0

12

13 b 11 c

44 3.4

21 4.5 P < 0.01

(57-98) (41-85) (45-92) (26--88)

68

aConclusions: (1) There is decreasing probability of pregnancy with increasing titer (;. 64 together, P < 0.05). (2) The difference between mean 210g titers of those becoming .pregnant and those remaining infertile is statistically significant (P < 0.01). bOne patient aborted, and 2.5 years later she had a normal pregnancy. cIncluding three patients who aborted and did not have a normal pregnancy afterward.

used the tests of Yates,14 Spearman15 (with correction for ties), and Wilcoxon. 12 As usual, a 5% level of significance was chosen, one-sided because of the definite direction of the hypotheses.

RESULTS Table 1 shows the incidence of pregnancy in the posttesting period in relation to the sperm agglutinin titer. All pregnancies were achieved spontaneously and not as the result of any treatment. Taking titers;;. 64 together (because of the small numbers), we found that there is a decreasing probability of pregnancy with increasing titer (chi-square test for linear trend, P < 0.05). This also finds expression in a difference between the mean 2log titers for those becoming pregnant and those remaining infertile (test of Yates, P < 0.01). Confidence intervals for the pregnancy percentages are only given where appropriate because of the numbers. The corrected probabilities according to the life-table method and the monthly fecundability rates with confidence intervals are given in Table 2. The corrected probabilities show little difference from those directly computed, because in all but three cases (two with a titer of 8, and one with a titer of 16) the period of infertility after serum testing for those becoming pregnant was shorter than the shortest follow-up period Vol. 42, No.4, October 1984

after serum testing for those remaining infertile. The analysis of the pretesting waiting times is shown in Table 3, where the waiting periods are subdivided in years. As can be seen, there is indeed a tendency for a lower mean pretesting waiting time with the lower titers. Based on the waiting periods in months, this tendency was not statistically significant, although not far from it (test of Spearman, P = 0.10). However, when the waiting periods of those becoming pregnant are compared with those of the patients who remained infertile, there was a definite difference (test of Wilcoxon, P < 0.001). Those who became pregnant had a median pretesting waiting period of 45.5 months, and those who remained infertile had a median waiting period of 72 months. The difference is even apparent for the titers separately, e.g., for titer 8 (20 patients had this titer), the median pretesting period for those who became pregnant was 45 months, as compared with 73 months for those remaining infertile (P < 0.01). It must thus be concluded that there are factors of greater importance than the titers in determining the pregnancy rate. It was found that with increasing titers the period from serum testing to pregnancy becomes longer (test of Spearman, P < 0.05). The median periods for titers 4, 8, 16, 32, and> 64 were 13.5, 19,27,25.5, and 68 months, respectively. A relation between the period before and after serum testing was not found (test of Spearman, P > 0.10). Table 4 shows the outcome of the postcoital test (available in 57 of the 65 patients) in relation to the incidence of pregnancy. If the notations "negative" for no spermatozoa, "poor" for one to five spermatozoa, and "positive" for more than five spermatozoa per high-power field on endocervical mucus are scored as 0, 1, and 2, respectively, the Table 2. Probability ofPregnancy According to the Life-Table Methoda from the Time of Serum Testing, and Monthly Fecundability Rate from the Time of the Attempt to Achieve Pregnancy (Titers ;. 64 Excluded)

Titer

4 8 16 32

Percentage probability of pregnancy in 2.5 5 10 years after serum testing 71 50 47 40

79 50 60 60

86 69 75 60

Percentate monthly fecunda ility rate (approximate 95% confidence interval) 1.1 0.6 0.7 0.5

(0.5-1.8) (0.3-0.9) (0.3-1.1) (0.1-0.9)

aWith periods of 2.5 years and exact (i.e., up to 1 month) correction for "loss to follow-up."

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Table 3. Pretesting Waiting Time in Relation to the Titer' No. of patients with period to serum testing in yearsb

Titer

1-2

2-3

2 0 0 1 1

2 4 3 2 0

3 2 1 2 0

4

11

8

~1

4--5

5--6

6--7

7-8

>8

2 4 2 1 1

1 4 4 0 0

2 1 2 1 1

0 4 1 0 0

1 0 2 1 1

1 1 0 2 2

10

9

7

5

5

6

3--4

Total

Median time

14 20 15 10 6

39.5 51 51 35 79

mo

4 8 16 32 > 64 Total

aConciusion: no significant relation between titer and period expressed in months (P bUpper limit of the intervals included.

fertile patients score an average of 1.1, and the infertile patients, of 0.6. The difference is significant (test of Yates, P < 0.05) and points in the same direction in all titer groups (including the group with titers ~ 64), which means that this is likely to be a factor independent of the titer of the circulating sperm agglutinins. The abortion rate in our patient sample was low. Of the 44 women who conceived, 3 had one abortion and never became pregnant again. There was one patient who aborted once, before carrying her next pregnancy to term. Disregarding later pregnancies after the first one, we recorded four spontaneous abortions in 44 patients, indicating an abortion percentage of only 9% (95% confidence interval, 3% to 22%).

=

0.10).

ter serum testing for 6 to 13.5 years (median, 10 years). This number of patients is sufficiently large to investigate the significance of sperm agglutinins in the serum of infertile women. If sperm agglutinins have an influence on the reproductive performance, a dose-related effect can be expected to exist in the sense that women with the higher titers should have a lower probability of pregnancy than those with the lower titers. Furthermore, it can then also be expected that if pregnancy occurs, the time it takes before it occurs will be longer in women with higher titers. If this is so, it can be expected that more women with lower titers will become pregnant before they seek medical help than those with higher titers, and thus the mean waiting time before going to the doctor will be shorter in women with low titers than in those with the higher titers. This will lead to two "biases": (1) the probability of becoming pregnant is more likely to be underestimated with low titers, and (2) the mean pretesting waiting time will be shorter the lower the titer. Because clinical studies aimed at measuring the influence of a certain factor such as a sperm antibody upon fertility are difficult to design in a totally prospective manner, these biases have to be accepted. The second bias, however,

DISCUSSION

Reports on the prognostic significance of circulating sperm antibodies in infertile women are conflicting. Most reports deal with small numbers of patients, and the duration of follow-up is usually limited. The present study comprises 65 women with unexplained infertility and circulating sperm agglutinins in titers ranging from 4 to 1024 in whom the fertility state was followed af-

Table 4. Incidence of Pregnancy and Serum Titers Related to the Result of the Postcoital Test (57 Patients) Result of postcoital test (score) Negative a (0) Poor b (1) PositiveC (2) Mean score

No. of patients Total 22 18 17 0.9

Pregnant

Not pregnant

11 11

11

7 3

14 1.1

Titer range

Titer median

Mean 210g titer

4-1024 4-1024 4-128

16 8 8

4.26 3.21 3.83

0.6

P < 0.05 aNo motile spermatozoa seen. bOne to five motile spermatozoa per high-power field. cMore than five motile spermatozoa.

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can be tested and, iffound, would provide support for the proposition that sperm antibodies adversely affect fertility. The biases are of no importance when considering the predictive value of a certain titer in a certain patient. Because the aim was to investigate the relationship of the titer not only with the probability of becoming pregnant but also with the time it will take a woman to become pregnant, the calculated dates of conception rather than of childbirth were used to define the endpoint of the infertile periods. Those pregnancies known to end in abortion were recorded separately. The probability of becoming pregnant was computed starting from the time of serum testing, because it is at this moment that the question of the predictive value of the titer for the individual patient arises. On the other hand, the fecundability rate was computed starting from the month of attempting to achieve a pregnancy, because this rate can be considered as the reproductive capacity of the couple, Le., before as well as after serum testing. In this respect, we deviate from the otherwise excellent methodologic recommendations given by Cramer et al.,13 who computed the fecundability rate from the time of investigation rather than the time the couple attempted to achieve a pregnancy. The present analysis shows that the probability of becoming pregnant after serum testing decreases with increasing titers. Moreover, the mean agglutinin titer in women who became pregnant was found to be significantly lower than in those who remained infertile (Tables 1 and 2). However, there appears to be a considerable overlap in titers between the two groups, the decrease in probability and in fecundability rate is not impressive; and, moreover, the probability of 86% becoming pregnant in the titer 4 group seems to be higher than could be expected from a similar population of infertile women without sperm antibodies. Therefore, in spite of the inverse relationship between serum titer and pregnancy rate, it should be concluded that, contrary to the situation in males,l1 the titers have little predictive value for individual patients, possibly with the exception of the very high titers (~ 64), which, however, were rarely found. Because there was a tendency for shorter pretesting waiting times among women with the lower titers, compared with those with the higher titers (P = 0.10) (Table 3), there is some additional support for the contention that sperm antiVol. 42, No.4, October 1984

bodies in some way influence the reproductive performance. On the other hand, if the pretesting waiting periods of those becoming pregnant were compared with those remaining infertile, there was a significant difference. In all titer groups the pretesting waiting time was shorter for those becoming pregnant than those remaining infertile (e.g., in titer group 8, 45 and 72 months, respectively). It seems, therefore, likely that other factors, operating independently of the agglutinin titer, play an important role in determining whether a woman remains infertile or not. Such factors may well be reflected in negative or poor postcoital tests, which gave similar results in the different titer groups (Table 4). Another aim of the study was to investigate whether abortions could be related to the presence of the agglutinins, as has been suggested in other studies. 6 , 9 Four of 44 women aborted; the rate of 9% is not abnormal. Various explanations are possible for the marginal relationship between the titer of the sperm agglutinins in the serum and the fertility prognosis in women: (1) The GAT measure I!!. immunoglobulins but also other factors which are irrelevant to the reproductive performance. Although there is a general belief that such factors, e.g., the l3-macroglobulin described by Boettcher,16 are more often encountered in the tray agglutination test (TAT) than in the GAT,17 it is possible that low-titer agglutinins in the GAT, as, for instance, found in 4 of 70 aged nuns,18 are of nonimmunologic origin. In fact, the "proof" that the GAT measures immunoglobulin activity only comes from studies in which high-titer sera were investigated. 19 (2) Although antibodies against spermatozoa may well affect the potential for spermatozoa to reach the ovum and/or to adhere or penetrate the ovum, the antibodies in the serum may not reach the spermatozoa in sufficient amounts to do so. Since the albumin and IgG concentrations in cervical mucus are low, in the order of 1% of the serum concentration,20 it seems unlikely that antisperm antibodies would ever be concentrated sufficiently in the cervical mucus if antibodies in the mucus were derived entirely from the serum. Higher in the fallopian tubes serum proteins are likely to be present in much higher concentrations, but the question remains whether the transudation would be sufficient to reach critical concentrations. (3) Although there might be some relationship between the concentration of systemically and locally formed sperm antibodies

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in the cervix, the serum titer does not parallel the concentration in the cervical mucus. Some investigators4 ,6 have even claimed that sperm antibodies may be present locally without detectable serum antibody. (4) Ingerslev and Ingerslev 10 found that antibodies had disappeared at the second examination in 2 of 16 women who had initial titers of 32 and 64 in the TAT: both women had become pregnant. Although in the meantime we had changed our technique (replacing the GAT by the TAT), we noticed considerable decreases in titers in some instances where new serum samples could be obtained many years after the original testing. On the other hand, significant fluctuations were hardly seen by us when, in the period of original testing shortly after the first testing, new serum samples were investigated. (5) It is likely that only the very high titers (which occur rarely) reflect a more permanent isoimmune infertility problem. Whether this is due to antibodies in the serum or to those produced locally remains uncertain. If it is assumed that a kind of "dose-effect" relationship exists, it may well be that the relationship is not linear but follows a sigmoid distribution. Only the very high titers would then fit at the straight and upper part of the curve; whereas the lower titers, such as those up to 32 in our study, forming the vast majority of the observed titers, would still all occur in the nearly horizontal lower part of the curve. The present study does not show a strong correlation between the titer of sperm agglutinins in the serum and the reproductive performance after serum testing. More attention should be paid to the detection of complement-dependent immobilizing (or cytotoxic) antibodies, as recommended years ago by Isojima et al. 21 Their presence may merely indicate that agglutinins are present in high titers,17 but it is possible that the complement-dependent antibodies have a higher biologic relevance than the agglutinating antibodies. Of utmost importance remains the testing of cervical mucus, because it is in this fluid that locally formed sperm antibodies may affect the migration of spermatozoa.

REFERENCES 1. Shulman S, Friedman MR: Antibodies to spermatozoa. V.

Antibody activity in human cervical mucus. Am J Obstet Gynecol 122:101, 1975

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2. Sudo N, Shulman S, Stone ML: Antibodies to spermatozoa. IX. Spermagglutination phenomenon in cervical mucus in vitro: a possible cause of infertility. Am J Obstet Gynecol 129:360, 1977 3. Kremer J, Jager S, van Slochteren-Draaisma T: The "unexplained" poor postcoital test. Int J Fertil 23:277, 1978 4. Moghissi KS, Sacco AG, Borin K: Immunologic infertility. I. Cervical mucus antibodies and postcoital test. Am J Obstet Gynecol 136:941, 1980 5. Ingerslev HJ: Antibodies against spermatozoal surfacemembrane antigens. Acta Obstet Gynecol Scand (SuppD 100:1,1981 6. Menge AC, Medley NE, Mangione CM, Dietrich JW: The incidence and influence of antisperm antibodies in infertile human couples on sperm-cervical mucus interactions and subsequent fertility. Fertil Steril 38:439, 1982 7. Lehmann F, Stripling K, Budel B, Krebs D, Masson D: Spermantibodies in infertile women: incidence and clinical significance. In The Uterine Cervix in Reproduction, Edited by V Insler, G Bettendorf. Stuttgart, Georg Thieme Publishers, 1977, p 204 8. Ansbacher R, Keung-Yeung K, Behrman SJ: Clinical significance of sperm antibodies in infertile couples. Fertil Steril 24:305, 1973 9. Jones WR: The use of antibodies developed by infertile women to identify relevant antigens. In Immunological Approaches to Fertility, Edited by E Diczfalusy. Stockholm, Karolinska Institutet, 1974, p 376 10. Ingerslev HJ, Ingerslev M: Clinical findings in infertile women with circulating antibodies against spermatozoa. Fertil Steril 33:514, 1980 11. RUmke P, Van Amstel N, Messer EN, Bezemer PD: Prognosis of fertility of men with sperm agglutinins in the serum. Fertil Steril 25:393, 1974 12. Armitage P: Statistical Methods In Medical Research. Oxford, Blackwell Scientific Publications, 1971 13. Cramer DW, Walker AM, Schiff I: Statistical methods in evaluating the outcome of infertility therapy. Fertil Steril 32:80,1979 14. Yates F: The analysis of contingency tables with groupings based on quantitative characters. Biometrika 35: 176,1948 15. Kendall MG: Rank Correlation Methods, Fourth edition. London, Griffin, 1970 16. Boettcher B: The molecular nature of spermagglutinins and sperm antibodies in human sera. J Reprod Fertil (SuppD 21:151, 1974 17. Rose NR, Hjort T, Riimke P, Harper MJK, Vyazov 0 (Eds): Techniques for detection of iso- and auto-antibodies to human spermatozoa: based on a Workshop held at the Institute of Medical Microbiology, University of Aarhus, Denmark, July 15 to 19, 1974, and sponsored by the WHO Expanded Programme of Research, Development and Research Training in Human Reproduction, International Union of Immunological Societies and Danish Medical Research Council. Clin Exp ImmunoI23:175, 1976 18. Hamerlynck JVTH: Cytotoxic and Other Auto-Antibodies against Spermatozoa in Relation to Infertility in the Human Male. Thesis, University of Amsterdam, 1970 19. Ingerslev HJ, Hjort T, Linnet L: Immunoglobulin classes of human sperm antibodies: immuno-affinity chromatographic analysis, with special attention to agglutinating and immobilizing activity of IgG and IgM. J Clin Lab Immunol 2:239, 1979

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20. Schumacher GFB: Humoral immune factors in the female reproductive tract and their changes during the cycle. In Immunological Aspects of Infertility and Fertility Regulation, Edited by GFB Schumacher, DS Dhindsa. New York, Elsevier-North Holland, 1980, p 93

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21. Isojima S, Tsuchiya K, Koyama K, Tanaka C, Naka 0, Adachi H: Further studies on sperm-immobilizing antibody found in sera of unexplained cases of sterility in women. Am J Obstet Gynecol 119:199, 1972

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