Prognosis of Fertility of Men with Sperm Agglutinins in the Serum*

Vol. 25, No.5, May 1974

FERTILITY AND STERILITY

Copyright

©

1974 The American Fertility Society

Printed in U.S.A.

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PROGNOSIS OF FERTILITY OF MEN WITH SPERM AGGLUTININS IN THE SERUM* PH. RUMKE, NAND A VAN AMSTEL, E. N. MESSER,

AND

P. D. BEZEMER

The Netherlands Cancer Institute, Department of Immunology, Sarphatistraat 108, Amsterdam, The Netherlands, and Free University, Department of Medical Statistics, Amsterdam, The Netherlands

Autoantibodies against spermatozoa can be present in blood as well as in seminal plasma of infertile men. 1 • 4 Occasionally, sperm agglutinins also have been found in serum of fertile men. 5 ,6 It may be assumed that in fertile men the sperm agglutinins do not reach the spermatozoa. The presence of the autoantibodies in the seminal fluid depends on local production of antibodies in the genital tract and on diffusion of antibodies from the blood to the semen. This diffusion of antibodies was found when blood plasma proteins such as albumin and IgG were observed to be present in normal semen in concentrations of about 1%.7 However, since IgM has generally not been found in semen and sperm antibodies sometimes appeared to belong to this type, one may presume that in such cases fertility is not hindered by the presence of sperm agglutinins. Presumably, the importance of diffusable IgG sperm agglutinins versus nondiffusable IgM antibodies and versus the local production of sperm antibodies in the genital tract will depend on the degree of a negative relation between the serum sperm agglutinin titer and the probability of becoming fertile in the years following serum testing. The main purpose of this paper is to establish whether the serum sperm agglutinin titer count is negatively correlated Received September 5, 1973. *Supported by a grant (to E.N.M.) from the World Health Organization.

with the probability of becoming fertile. Such a reverse relation in patients with otherwise normal semen would demonstrate the importance of autoantibodies against spermatozoa as a cause of infertili ty. MATERIALS AND METHODS

The macro-agglutination technique of Kibrick et al 8 was used for sperm agglutinin detection. Many different donors supplied the semen samples necessary for the test. Serum samples were routinely tested in the dilutions 1: 4 and 1: 20 in saline with at least two different donor sperm samples. Positive samples were titrated by twofold dilution steps. Patients. Since 1954, serum samples of infertile men were sent to us by many different specialists in the Netherlands and Belgium. Several thousand samples have been tested since then. In 1970, an investigation was started on 254 patients who (between 1954 and the end of 1968) were found to have sperm agglutinins in the serum with titers ranging from 4 to over 1,024. Since it was to be expected that not only the sperm agglutinin titers but also the sperm qualities were important for prognosis, the patients were divided into four groups according to their sperm counts. For this purpose the data of the referring specialists were used. If semen was investigated more than once, qualification was based on the majority of the

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TABLE 1. Incidence of Fertility as Related to Serum Sperm Agglutinin Titer and Sperm

Concentration

Azoospermia

No. of

Extreme oligospermia a No. of men who No. of became men fertile studied

Oligospenniab No. of men who No. of became men fertile studied

Normospennia c

No. of men who became fertile

No. of men studied

Total No. of men who became fertile

No. of men studied

22 21 15 31 32 42 33 30 24 4 254

Spenn agglutinin titer

men who

became fertile

No. of men studied

4 8 16 32 64 128 256 512 1024 >1024

0 0 0 0 0 2 0 0 0 0

5 4 3 10 8 10 3 7 7 1

0 0 1 0 0 0 0 0 0 0

2 2 1 2 4 0 1 5 3 1

0 0 0 1 1 0 1 0 0 0

3 4 3 5 3 5 9 1 4 1

5 6 4 3 4 2 3 3 0 0

8 14 17 27 20 17 10 1

5 6 5 4 5 4 4 3 0 0

2

58

1

21

3

38

30

137

36

Total

12 11

"Fewer than 3.106 spermatozoa per ml. b3 to 20.10 6 spermatozoa per ml. "More than 20.10 6 spermatozoa per ml.

semen analyses. The arbitrarily chosen groups were: azoospermia, extreme oligospermia (fewer than 3.10 6 spermatozoa per ml) oligospermia (3 to 20.106 spermatozoa per ml), and normospermia (more than 20.10 6 spermatozoa per ml). The recording of other sperm qualities, such as motility, was in general not well enough standardized to be useful for the study. Noted also were the date of marriage (or the first date of conception attempts), the first serum testing, and the birth of the child(ren). Statistical analysis. Each statistical test was performed at a two-sided level of significance equal to 0.05. For details of the statistical procedures used, see Armitage 9 and De Jonge. 10 For the statistical analyses of sperm counts, the aforementioned sperm count categories were quantified as 1, 2, 3, and 4, respectively. RESULTS

I ncidence of patients becoming fertile after serum testing. Table 1 shows the incidence of fertility in relation to sperm agglutinin titers among men with azoo-

spermia, extreme oligospermia, oligospermia, and normospermia. Of the 36 of 254 who became fertile, 30 belonged to the group of 137 men with normal sperm counts. As would be expected, the sperm counts were higher for the men who became fertile than for the men who remained infertile (test of Yates, P < 0.001). Also, the mean 2log titer is lower for those who became fertile than for those who remained infertile; this is the case for the total group as well as for the normospermic men only (test of Yates, P < 0.001). It indicates a reverse relation between titer and probability of becoming fertile. No relation between sperm count and titer could be demonstra ted (test for independence) . The probabilities were computed for normospermic men only because of the low numbers of fathers in the other categories. The actuarial method 9 ,1l was applied to account for the different periods between serum testing and the evaluation date. Table 2 shows the computed probabilities with the approximate 95% confidence limits per titer and per low, medium, and

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SPERM AGGLUTININS AND FERTILITY PROGNOSIS

TABLE 2. Probability for Infertile Normospermic Men with Different Serum Sperm Agglutinin Titers to Become Fertile Within 5,10, and 15 Years After Serum Testing Percent probability of becoming fertile within' Titer

No. of patients

4 8 16 32 64 128 256 512 1024 >1024

12 11 8 14 17 27 20 17 10 1

25 22 25 7 6 4 5 6

43 57 39 14 26 8 15 22

43 78 70 36 26 8 28 22

4, 8, 16 32,64,128 >128

31 58 48

24 (8 - 39) 5 (0 - 11) 4 (0 -10)

46 (27 - 65) 15 (5 - 25) 13 (2 - 24)

61 (39 - 84) 19 (7 - 32) 18(4-31)

10 years

5 years

(0 - 50) (0 - 49) (0 - 55) (0 - 21) (0-17) (0 - 11) (0 - 15) (0 - 17) o (0 - 26) o (0 - 95)

(14 - 72) (24 - 90) (4 - 74) (0 - 33) (4-48) (0 - 19) (0 - 36) (0 - 44) o (0 - 26) o (0 - 95)

15 years

(14 - 72) (44 - 100) (24 - 100) (0 - 75) (4 - 48) (0 - 19) (0 - 56) (0 - 44) o (0 - 26) o (0 - 95)

"Approximate 95% confidence limits in parentheses.

high category. From titer 32 on, the probability seems to be considerably lower than in the category of lower titers. It can be concluded from this table that a reverse relation between titer and probability of becoming fertile exists. We are uncertain about the shape of the curve showing the relation between 2log titer and the probability of becoming fertile. A satisfactory fit was not reached with either a straight line or an S-shaped curve. This may have been due to the relatively low numbers; as a result, the estirpated probabilities are rather inaccurate (Table 2). The results indicate that a relation between the titer and the time interval to conception for the fertile subjects might be expected. However, we have not investigated such a relationship because a calculation would be too inaccurate to be useful for prognosis. It was expected that the longer an infertility had lasted, the lower the probability of becoming fertile would be. This was tested for the men who became fertile by establishing a relation between the time interval of "waiting to go to the doctor" (ie, the time between attempts at conception and serum testing) and the

time interval of "waiting for the child" (ie, the time between attempts at conception and childbirth). Indeed, this relation was found (Spearman's rank correlation test, P < 0.01). Re-examination of serum of men who became fertile. The question arose whether the fathers had become fertile due to a spontaneous fall of the sperm agglutinin titer or in spite of the presence of serum sperm agglutinins. Therefore, new serum was collected and tested again. It was possible to retest sperm of 22 of the 36 fathers. The results are shown in Figure 1. In 19 of the 22, there was no difference between the first and the last determination greater than two twofold dilution steps. Most relevant was the observation that of the three patients with titers of 512 (two at the end of 1958, and one at the end of 1966), two fathered two children each, in spite of titers of 1,024 in 1970. The third one apparently lost his sperm agglutinins nearly completely; his titer decreased to 4 in 1971. If differences of two dilution steps and less are taken as insignificant (see Discussion), only three patients showed a change in titer. As mentioned, one declined from 512 to

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PH. RUMKE ET AL

May 1974

1024 512 256 128 64

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32 16

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1954 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 FIG. 1. Titers before and after fatherhood of infertile men with serum sperm agglutinins who became fertile.

4, and the two others increased from 8 to 256 and from 64 to 1,024. Thus, it may be concluded that infertile men with serum sperm agglutinins can eventually become fertile in spite of the persisting presence of these autoantibodies. DISCUSSION

It should be stressed that our study was retrospective. Although the possibility of a bias should therefore be considered, we feel it is unlikely that a bias substantially affected the outcome of the study. The main finding of this study was the demonstration of a reverse relationship between the serum sperm agglutination titer and the probability of becoming fertile for normospermic men. It is conceivable that the higher the serum titer is, the more sperm agglutinins that may diffuse into the seminal compartment where they hinder spermatozoa from penetrating the cervical mucus. Also, the degree of local production in the genital tract

may be related to the systemic production of antibodies. This explains then the relation between serum titer and remaining infertility. An explanation for the exceptions (ie, why men became fertile in spite of persisting high serum titers) could not be obtained from this study. In view of the fact that both IgG and IgM type serum sperm antibodies have been found to exist12 - 14 and in view of the fact that IgG and IgM have not been found in general to be present in seminal plasma, it can be postulated that sperm agglutinins of the IgM type did not diffuse into the seminal compartment and that therefore the exceptions were men with IgM sperm agglutinins. Another explanation for these exceptions could be that in spite of the presence of sperm agglutinins in seminal plasma, some spermatozoa could escape from being sensitized. Hansen and Hjortt 5 showed with the indirect fluorescent antibody technique that not all spermatozoa stained to the same extent with certain iso-antisera from infertile women. Apparently in one ejaculate, spermatozoa

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Vol. 25, No.5

SPERM AGGLUTININS AND FERTILITY PROGNOSIS

differed in antigenic expression. It is, however, unknown whether spermatozoa lacking antigenic expression still have the capacity to fertilize. Whether the probability of becoming fertile for the low titer group (titers 4, B, and 16) was the same as for the general population of men attending infertility clinics was not studied. However, in a subsequent study of 133 men in whom both the serum and the seminal plasma titers were determined, we found that of the 23 men with serum titers of 4, B, and 16, only three had detectable sperm agglutinins in the seminal plasma. 16 Therefore, it is conceivable that these low serum titers are generally of no clinical importance. It was also found in an earlier study that in a group of 416 fertile men, sperm agglutinins occurred in four men, but never in men with titers higher than 16. 3 Thus, at that time sera were arbitrarily considered to be meaningfully positive only when the titer was 32 or higher. The present study shows some justification for that arbitrarily chosen threshold, because the greatest decrease of the probability of becoming fertile occurred between the titers 16 and 32 (Table 2). One may even doubt whether sperm agglutinins in low titers represent true sperm antibodies. In a study of 70 elderly nuns, HamerlynckI7 found five sera containing low titered sperm agglutinins. Since it is not conceivable that they were true sperm antibodies (ie, induced by sperm resorption), it seems reasonable to assume that in general, low titered sperm agglutinins are not necessarily of true sperm antibody nature. Whether they are still antibodies induced by substances cross-reacting with the spermatozoal membrane, or non-y-globulin factors, has yet to be studied. With another agglutination technique, it was shown by Boettcher and coworkersl8 ,19 that sperm agglutination can be caused by molecules other

397

than antibodies, that is, by a ~-macro­ globulin. Although this ~-globulin did not interfere in our macro-agglutination test, Boettcher et al observed the possibility of "false positivity." It !night seem surprising that two azoospermic patients became fertile. However, these patients were treated in some way and became oligospermic. And of course, for obvious reasons, it could never be fully excluded that a "fertile" man was not a natural father, although information in that respect was gained as much as possible from general practitioners, specialists, and patients. At least, fathership due to homologous artificial insemination was excluded. The results of the study in which the fathers were re-examined sometimes even 16 years after the first determination show that in 19 of the 22 cases the titer was still about the same. Differences of one or two twofold dilution steps have not been regarded as significant because there is always some variation dependent on the donor sperm sample with which the titration is performed. Also, there is some variability due to difficulties in endpoint reading and in the interpretation of weak agglutination reactions. Since the tests and readings before June 1966 were performed by Ph. R. or by a former technical assistant and thereafter by N.v.A., differences in subjective readings undoubtedly have to be taken into account. The extent of difference all these factors can cause between the two readings is unknown. Two twofold dilution steps seem a reasonable upper limit. A surprisingly low number of patients (3 of 22) were found to have after so many years a titer difference of at least four twofold dilution steps. And only in one of them was there a titer fall suggesting that spontaneous disappearance of sperm agglutinins may rarely occur. In conclusion, this study confirms that sperm agglutinins detected with the

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PH. RUMKE ET AL

macro-agglutination technique in serum of men do playa role in the pathogenesis of male infertility.

5.

6.

SUMMARY

The fertility state of 254 infertile men with serum sperm agglutinin titers ranging from 4 to over 1,024, and detected with Kibrick's macroscopic agglutination technique between 1954 and 1968, was available by the middle of 1970. Thirty-six had become fertile in the years following serum testing. As expected, the probability of becoming fertile appeared to be closely related with the sperm quality (30 of the 36 fathers belonged to the group of 137 normospermic men). On the other hand, a reverse relation appeared to exist with the sperm agglutination titers (with titers of 16 or lower, 16 of 58 men, with titers of 32, 64 and 128, 13 of 105 men, and with titers of 256 or higher, 7 of 91 men had become fertile). The probabilities for the normospermic men to become fertile in the 5, 10, or 15 years following serum testing were computed again and showed a decrease with increasing titer. Retesting of serum from 22 fathers showed that becoming fertile occurred in general iIi spite of the persistence of the sperm agglutinins.

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Fjiillbrant B: Sperm antibodies and sterility in men. Acta Obstet Gynecol Scand Vol XLVII, Suppl4, 1968 Rumke P: The presence of sperm antibodies in the serum of two patients with oligospermia. Vox Sang 4:135,1954 Rumke P, Hellinga G: Autoantibodies against spermatozoa in sterile men. Am J Clin PathoI32:357, 1959 Wilson L: Spermagglutinins in human semen and blood. Proc Soc Exp BioI Med 85:652,1954

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Fjiillbrant B: Fertility in a man autoimmunized to spermatozoa. J Reprod Fertil 14: 143, 1967 Phadke AM: Fate of spermatozoa in case of obstructive azoospermia and after ligation of vas deferens in man. J Reprod Fertil 7: 1, 1964 Rumke P: The origin of immunoglobulins in semen. Clin Exp Immunol (In press) Kilbrick S, Belding DL, Merrill B: Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 3: 430, 1952 Armitage P: Statistical Methods in Medical Research. Oxford and Edinburgh, Blackwell Scientific Publications, 1971 De Jonge H: Inleiding tot de Medische Statistiek. Vol I and II, Nederlands Instituut voor Praeventieve Geneeskunde, Leiden, 1963 and 1964 Berkson J, Gage RP: Calculation of survival rates for cancer. Proc Staff Meet Mayo Clin 25: 270, 1950 Boettcher B, Kay DJ, Rumke P, et al: Human sera containing immunoglobulin and non-immunoglobulin spermagglutinins. BioI Reprod 5:236, 1971 Hansen KB: Immunofluorescent studies on human spermatozoa. III. Immunoglobulin classes of human spermatozoal antibodies. Clin Exp Immunol 10: 243, 1972 Jones WR, Ing RMY: Immunofluorescent studies of human spermatozoa. J Reprod Fertil 32: 357, 1973 Hansen KB, Hjort T: Immunofluorescent studies on human spermatozoa. II. Characterization of spermatozoal antigens and their occurrence in spermatozoa from the male partners of infertile couples. Clin Exp Immunol 9: 21, 1972 Rumke P, Van Amstel N: Unpublished data Hamerlynck JVTH: Cytotoxic and other auto-antibodies against spermatozoa in relation to infertility in the human male. Thesis, University of Amsterdam, 1970 Boettcher B, Hay J, Kay DJ, et al: Spermagglutinating activity in some human sera. Int J Fertill5: 143, 1970 Boettcher B, Kay DJ: Fractionation of a human spermagglutinating serum. Nature 223:737,1969