FERTILITY AND STERILITY Copyright © 1977 The American Fertility Society

Vol. 28, No.6, June 1977 Printed in U.S.A.

SPONTANEOUS SPERMAGGLUTINATION IN EJACULATES FROM MEN WITH HEAD-TO-HEAD OR TAIL-TO-TAIL SPERMAGGLUTINATING ANTIBODIES IN SERUM

JAN FRffiERG, M.D.*t INGER TILLY-FRffiERG, M.D.*

Department of Obstetrics and Gynecology, University Hospital, Uppsala, and Department of Obtetrics and Gynecology, Lowenstromska Hospital, Upplands-Viisby, Sweden

Weak or sometimes up to moderate spermagglutination was encountered in ejaculates from a group of men without spermagglutinating antibodies in serum. A similar pattern of spermagglutination was seen in ejaculates from men with head-to-head (H-H) spermagglutinating antibodies in serum, even when a high titer (~1 :64) of antibodies was present. In contrast, men with high titers of tail-to-tail (T-T) spermagglutinating antibodies in serum showed very marked or complete spermagglutination in the ejaculates. The agglutination in ejaculates from men with low titers (~1:32) of T-T spermagglutinating antibodies in serum was much less conspicuous and equaled that in ejaculates from men without such antibodies. This indicates that only a high titer of T-T spermagglutinating antibodies is associated with marked or complete spermagglutination in ejaculates.

Dukes microscopic spermagglutination technique,13 It has been repeatedly reported that men with spermagglutinating antibodies in serum usually display varying degrees of spontaneous spermagglutination in their ejaculates. 7 , 10, 12, 14 However, the influence of the different types and titers of spermagglutinating antibodies on spontaneous spermagglutination in ejaculates has not been reported. In the present study the degree of spermagglutination in ejaculates from men with different titers of head-to-head or tail-to-tail spermagglutinating antibodies in serum was investigated. For comparison, the degree of spontaneous agglutination in ejaculates from men without such antibodies was also examined.

Human spermatozoa may be agglutinated by bacteria, viruses, mycoplasmas, and various physicochemical agents.1 9 Furthermore, human spermatozoa may be agglutinated in ejaculates from men with high sperm counts and may adhere to epithelial cells and cellular debris in ejaculates, forming aggregates. Another cause for spontaneous spermagglutination is the presence of spermagglutinating antibodies. Fjallbrant 10 found 21 cases with marked spontaneous agglutination among 263 infertile men. With the technique of Kibrick et aI.,l1 nine of these men were found to have sperm agglutinating antibodies. DeCooman et al,l2 studied 32 sera derived from subjects with marked spontaneous agglutination in their ejaculates and found 2 sera containing antibodies, as measured by means of the Franklin0

MATERIALS AND METHODS

Ejaculates from 243 male partners attending the Infertility Clinic in Uppsala were examined. One hundred and five subjects whose sera were known to contain spermagglutinating antibodies were included in group I. This group was further subdivided according to types of spermagglutinating antibodies. Group IA was composed of

Received May 4, 1976; revised September 2, 1976, December 13, 1976, and February 7, 1977; accepted February 9, 1977. *Present address: Department of Obstetrics and Gynecology, State University Hospital, Downstate Medical Center, Brooklyn, N. Y. 11203. tReprint requests: Jan Friberg, M.D., Department of Obstetrics and Gynecology, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, N. Y. 11203.

658

Vol. 28, No.6

659

SPONTANEOUS SPERMAGGLUTINATION IN EJACULATES

24 subjects with head-to-head (H-H) spermagglutinating antibodies and group IB was made up of 81 subjects with tail-to-tail (T-T) spermagglutinating antibodies in serum. Group II was composed of a consecutive series of 138 subjects whose sera and seminal fluid were devoid of spermagglutinating antibodies. The presence of spermagglutinating antibodies in serum or seminal fluid was examined by using the microagglutination technique described by Friberg.1s In this technique serum or seminal fluid was serially diluted 2-fold from 1:4, and 5-,..ti aliquots of these dilutions were applied on specially marked places under liquid paraffin oil in disposable microchambers (M,oller-Coates AS, Moss, Norway). One microliter of donor sperm of excellent quality (repeated samples showed 80 to 100 million sperm/ml, fewer than 30% morphologically abnormal cells, and a motility of 3 in a scale ranging from 0 to 3), diluted with Baker's solution 16 to 40 million sperm/ml, was applied to each 5-,.d sample. The chambers, with known positive and negative controls, were incubated at room temperature and after 4 hours the titer and type of spermagglutination were determined by using an inverted Zeiss microscope and x 60 to x 600 magnification. The agglutination was evaluated on a scale ranging from negative (-) to 3+. The highest dilution giving a 1+ agglutination was designated the agglutination titer. A sample was considered to contain spermagglutinating activity only if agglutination was observed after at least two dilutions. Thus, the lowest titer of sperm agglutinating activity reported was 1:8. Four semen samples from each subject were examined over a 3-month period. All of these samples showed a sperm concentration of more than 20 million/ml, fewer than 40% morphologically abnormal spermatozoa, and a motility grade of at least 2 in the scale ranging from 0 to 3. No attempt was made to exclude men with bacterial infections or increased cellular content in their ejaculates. After the initial series of examinations a new ejaculate was obtained by masturbation at the clinic after a requested 3 days of abstinence. After liquefaction (approximately 30 minutes after ejaculation), two 5-J.LI aliquots were taken from each ejaculate and applied under liquid paraffin oil in the same way as performed for antibody testing. After 1 hour's incubation the degree of spermagglutination was evaluated according to a scale ranging from negative to 4+ (Table 1).

TABLE 1. Evaluation of Spermagglutination Observed after Incubation of Aliquots of Human Ejaculates under Liquid Paraffin Oil for One Hour in Cytotox Boxes Degree of sperm agglutination

None (-) Weak (1+) Moderate (2+) Strong (3+ ) Complete (4+)

Evaluation

All spermatozoa free Most spermatozoa free, some scattered agglutinates Apparent agglutinates, majority of spermatozoa free Most spermatozoa agglutinated, some remaining free All spermatozoa agglutinated

Symbol in Figures 1 and 2



x

o

* +

RESULTS

Ejaculates from subjects without spermagglutinating antibodies (group II) generally displayed either weak or no spermagglutination. Only 6 patients had 2+ agglutination, whereas 132 had no agglutination or only a few scattered clumps of agglutinated spermatozoa. None of the subjects in this group showed strong (3+) or complete (4+) spermagglutination. Subjects with H-H spermagglutinating antibodies in serum (group IA) showed the same type of frequency distribution of spermagglutination in their ejaculates as did subjects without such antibodies. Of24 subjects, 1 had 2+ agglutination, whereas 9 had no agglutination and 14 had only a 1+ level, even though the titers of H-H spermaggl utinating antibodies in serum ranged from 1:8 to 1:128. The 2+ sperm agglutination was demonstrated in the ejaculate of a subject who had a 1:8 titer in his serum. The subjects in group IB displayed more pronounced spermagglutination in their ejaculates than did the men in group IA or group II. It was also apparent (Fig. 1) that men with a high titer (;?; 1:64) of T-T spermagglutinating antibodies in serum demonstrated a more marked spermagglutination than did men with low serum titers (~1:32). However, there was considerable overlapping. The subjects with strong (3+) or complete (4+) spermagglutination had serum titers of T-T spermagglutinating antibodies of 1:128 or more. Scoring the spermagglutination in ejaculates and comparing these results with both serum and seminal fluid titers ofT-T spermagglutinating antibodies revealed that men with serum antibodies, but without seminal fluid antibodies, showed no significant spermagglutination (Fig. 2). Strong or complete spermagglutination was generally found in ejaculates from men with a high serum titer of T-T spermagglutinating

FRIBERG AND TILLY-FRIBERG

660

June 1977

Titer of tail-to-tail sperm-agglutination antibodies in serum 1:8192 1:4096

*

++

1:2048

***

++

1:1024

0

****

++++

1: 512

000

**** * ****

++

1: 256 1: 128 1: 64 1: 32 1: 16 1:

8

,

..,

X

000

X

0000

+

0000 0

.

XXXX XXXX XX XXXX XX

,, ,

XXX

..

+++

1+

0

2+

3+

4+

Degree of sperm agglutination in correspond'ilg ejaculates

FIG.!' The degree of spermagglutination in ejaculates from subjects with tail-to-tail spermagglutinating antibodies in serum.

antibodies in association with the presence of such antibodies in the seminal fiuid. The types of spermagglutination observed in the ejaculates were similar in groups lA, IB, and II. Different parts of the spermatozoa were involved in the agglutinates: heads, middle pieces, and tails. This agglutination pattern is also known as mixed or tangled spermagglutination. No H-H spermagglutination was detected in any ejaculate studied. DISCUSSION

The microagglutination technique used in the present study to demonstrate the presence of spermagglutinating antibodies requires, as do both the Kibrick et al. ll and Franklin-Dukes l3 methods, motile sperm of good quality. However, the method has several advantages over both the Kibrick and Franklin-Dukes techniques. In the Franklin-Dukes technique, titration of serum is difficult. In the Kibrick technique the

type of spermagglutination cannot be detected. In the method used here, titration is simple and evaluation of the type of spermagglutination presents no difficulty. In addition, evaluation of the degree of agglutination is easy, and one single ejaculate can be used to test 200 to 300 dilutions. This advantage makes comparison among different serum or seminal fiuid samples more accurate. A comparison of this method and the Kibrick method has been published. 15 Agglutination of spermatozoa in ejaculates has usually been observed in a wet smear prepared during the routine examination of sperm samples. Before the wet smear is prepared, the sperm samples have often been shaken. Since agglutinated spermatozoa easily disintegrate during such mechanical agitation, agglutination is easily overlooked or its importance not fully recognized. 17 Therefore, it is important to allow ejaculates to remain undisturbed for some time before the degree of agglutination is evaluated. In the microchambers used in this investigation the

SPONTANEOUS SPERMAGGLUTINATION IN EJACULATES

Vol. 28, No.6

661

Ti ter of sperm-

agglutinating antibodies in serum. 1 :8192 1:4096

*

+

+

1:2048

*

+

*

*

++

1:1024

0

1:512

0

1:256

* + *

X 000

1:128

*X

+ 0

0

** +

+

* * **

+

**

+

+

*

+

* +

0

000

1:64 1:32

•00

0

0

1 :8

1:16

0

XXXXX XXXXX

... 0

1:16 1,:8

XXXX XXX

•• '-+fEll •••••

Neg.

I

1:32

1:64

1:128

1:256

1:512

Titer of tail-to-tail sperm-agglutinating antibodies in seminal fluid FIG. 2. The titer of tail-to-tail spermagglutinating antibodies in serum and seminal fluid and the degree of spontaneous spermagglutination in the corresponding ejaculates.

portions from the ejaculates could be kept undisturbed for a long time, but since no changes in degree of agglutination occurred after 1 hour we considered 1 hour of incubation time to be appropriate. Spermagglutination in the ejaculates was always of the mixed or "tangled" type, a finding consIstent with observations in most other studies. 10. 12. 18 Fjallbrant10 described one man who displayed both head-to-head and mixed spermagglutination in his ejaculate. However, this was not observed in any of the ejaculates we studied. Some authors have found a correlation between the serum concentration of spermagglutinating antibodies and the degree of spermagglutination in ejaculates,lo. 19.20 whereas others have been unable to confirm this. 21, 22 In the present study a correlation between the concentration of spermagglutinating antibodies and the degree of sperm-

agglutination was seen only for subjects with high titers of T-T sperm agglutinating antibodies in serum. The demonstration of strong or complete spermagglutination in the ejaculates handled and observed as described here strongly indicates that the subject has a high titer of serum T-T spermagglutinating antibodies. Agglutination in the ejaculates from men with H-H spermagglutinating antibodies in serum was of the same strength and frequency distribution as the degree of spermagglutination in the ejaculates from men devoid of antibodies. H-H spermagglutinating antibodies present in male sera apparently cannot penetrate to the seminal fluid,23 and H-H spermagglutination is normally not seen in ejaculates. It should also be noted that there is evidence that the H-H spermagglutinating antibodies found in male sera are of the immunoglobulin M type,24 and immunoglobulin M antibodies are normally not present in seminal

r FRIBERG AND TILLY-FRIBERG

662

fluid. 25 • 26 In view of these observations, it seems reasonable to assume that H-H spermagglutinating antibodies are not associated with the degree of spontaneous spermagglutination in ejaculates. REFERENCES 1. Rosenthal L: Spermagglutination by bacteria (abstr). Proc Soc Exp BioI Med 28:827, 1931 2. Buxton CL, Wong ASH: Spermicidal bacteria in the cervix as a cause of sterility. Am J Obstet Gynecol 64: 628, 1952 3. Chu HP: The agglutination of spermatozoa by viruses of influenza, mumps and Newcastle disease. Rome, VI Congr Int Microbiol m:131, 1953 4. Wilson L: Sperm agglutination due to autoantibodies. A new cause of sterility. Fertil Steril 7:262, 1956 5. Mann T: Biochemistry of Semen and of the Male Reproductive Tract. London, Methuen and Co Ltd, 1964, 493 p 6. Peleg BA, Ianconescu M: Sperm agglutination and sperm absorption due to myxo-viruses. Nature 211:1211, 1966 7. Riimke P: Clinical aspects of autoimmunity to spermatozoa in men. In Immunology and Reproduction, Edited by RG Edwards. London, International Planned Parenthood Federation, 1969, p 251 8. Taylor-Robinson D, Manchee RJ: Spermadsorption and spermagglutination by mycoplasmas. Nature 215:484, 1967 9. Hamerlynck JVTH: Cytotoxic and other auto-antibodies against spermatozoa in relation to infertility in the human male. Ph.D. thesis, University of Amsterdam, Amsterdam,1970 10. Fjallbrant B: Immunoagglutination of sperm in cases of sterility. Acta Obstet Gynecol Scand 44:474, 1965 11. Kibrick S, Belding DL, Merrill B: Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatin agglutination test. Fertil Steril 3:430, 1952

June 1977

12. DeCooman SE, Abdallah MA, Schirren C: Sperm autoagglutination. Clinical, morphological and immunological study. Andrologie 4:61,1972 13. Franklin RR, Dukes CD: Antispermatozoal antibodies and unexplained infertility. Am J Obstet Gynecol 89:6,1964 14. Riimke P: Autoimmunitat gegen Spermatozoen und Unfruchtbarkeit des Mannes. Andrologie 4:191, 1972 15. Friberg J: A simple and sensitive micro-method for demonsta~ion of sperm-agglutinating activity in serum from infertile men and women. Acta Obstet Gynecol Scand [Suppl] 36:21,1974 16. Baker JR: The spermicidal powers of chemical contraceptives. IV. More pure substances. J Hyg (Camb) 32:171, 1932 17. Wilson L: Sperm agglutinins in human semen and blood. Proc Soc Exp BioI Med 85:652, 1954 18. Williams WW: Autoagglutination of spermatozoa. Fertil Steril 21:222, 1970 19. ~iimke P: Autospermagglutinins. A cause of infertility In men. Ann NY Acad Sci 124:696, 1965 20. Riimke PW, Hellinga G: Autoantibodies against spermatozoa in sterile men. Am J Clin Pathol 32:357, 1959 21. Ansbacher R, Manarang-Pangan S, Srivannaboon S: Sperm antibodies in infertile couples. Fertil Steril 22:298, 1971 22. Bandhauer K: Immunoreaktionen bei Fertilitatsstorungen des Mannes. Urol Int 21:247,1966 23. Friberg J: Relation between sperm-agglutinating antibodies in serum and seminal fluid. Acta Obstet Gynecol Scand [Suppl) 36:73, 1974 24. Friberg J: Immunological studies on sperm-agglutinating sera from men. Acta Obstet Gynecol Scand [Suppl) 36:43,1974 25. Herrmann WP, Hermann G: Immunoelectrophoretic and chromatographic demonstration of IgG, IgA, and fragments of ')I-globulin in the human seminal fluid. Int J Fertil 14:211, 1969 26. Riimke P: The origin of immunoglobulins in semen. Clin Exp ImmunoI17:287, 1974

Spontaneous spermagglutination in ejaculates from men with head-to-head or tail-to-tail spermagglutinating antibodies in serum.

FERTILITY AND STERILITY Copyright © 1977 The American Fertility Society Vol. 28, No.6, June 1977 Printed in U.S.A. SPONTANEOUS SPERMAGGLUTINATION IN...
638KB Sizes 0 Downloads 0 Views