Macrophage migration inhibitory factor in female sterility L.

METTLER

D.

SCHIRWANI

Kiel,

West

Germany,

and

Teheran,

Iran

Cellular sensitization against spermatozoa as antigen was studied in 21 fertile and 41 infertile female subjects. In addition, 20 virgin girls and a group of 11 women presenting positive spermagglutinating (n = 8) and immobilizing (n = 3) antibodies were investigated. Cellular sensitization was expressed in terms of macrophage migration inhibitory activity (MIF-reaction). For this screening program washed and pooled spermatozoa (WPS) and were used as antigens. In 52 to 72 per cent of all sonicated spermatozoa solutions (SSS) cases, including wjas encountered. sensitization. The need the results

primary and secondary sterility of unexplained Women with humoral sperm antibodies

Only for the achieved

the group identification indicate

Materials

Received Revised Accepted Reprint Obstetrics 23 Kiel,

11 I of the

for

publication

May

8, 1974.

May

MIF-reaction a cellular

German

December

and

as

methods

The cases selected for these studies were assigned to four categories. Group 1 was made up of 21 mostly multiparous fertile female volunteers. Group 2 comprised 41 women attending our sterility clinic. In this group there were 27 cases of primary sterility : Subgroup a, obstructive causes (seven cases) diagnosed by tubal insufflation, pelviscopy, chromotubation, hysteroscopy, or laparotomy; Subgroup b, endocrinologic disturbances (five cases) ; and Subgroup c, unexplained reasons (15 cases). The remaining 14 cases of secondary sterility comprised three with organic, three with endocrinologic, and eight with unexplained causes of sterility. Group 3 was made up of 11 sterile women presenting positive humoral antibodies against spermatozoa. Of this group, eight revealed spermagglutinating and three sperm-immobilizing antibodies. Group 4 covered 20 virgin girls between 9 and 16 years of age. The migration inhibitory factor (MIF) was detected according to a modification described by Bendixen and Serborg.2 Leukocyte concentrates obtained from cubital vein blood were placed into microhematocrit capillaries and allowed to migrate into a fluid culture medium (TC-199). Areas

From the Department of Obstetrics and Gynecology, University of Kiel FRG (Director: Prof. Dr. K. Semm), and Department of Obstetrics and Gynecology, Royal Pahlavi Hospital. by SFB

a positive

of virgin girls demonstrated in no case a positive MIF-reaction. of the specific fertility-diminishing antigens is stressed a cellular sensitization in all cases that had sperm contact.

T H E R E I s growing acceptance that a considerable number of female sterility cases are caused by female sensitization to sperm antigens.‘! I43 Ii The occurrence of humoral sperm-agglutinating and sperm-immobilizing antibodies has been repeatedly demonstrated and regarded as further proof for the possible antigenicity of sperm components.G, ‘a lo The cellular immunity has not been sufficiently studied in connection with the female immune response against sperm antigenic factors. No reports exist on macrophage migration inhibitory activity in cases of female sterility. This is the subject of the present preliminary communication.

Supported Foundation.

cause,

always reuealed

Research 4, 1973.

10, 1974.

requests: Dr. L. Mettler, Department of and Gynecology, University of Kiel, Hegewischstr. 4, West Germany.

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Schirwani

covered by the fan of migrating cells out of the capillary cpenings spread into the culture medium were measured planimetrically after 20 hours of incubation at 37’ C. MIF activity was expressed in terms of the ensuing MIF index: MIF

index average

=

n&ration area in the presence of antigen average migration area without antigen

Only values within a range of 0.3 to 0.8 were considered as a positive reaction. The antigens from washed and pooled intact spermatozoa (WPS) were suspended in culture medium and added to capillaries containing leukocyte concentrates. The spermatozoa count was adjusted to 3 x lo’, 6 x lo’, 12 x 107, and 18 x 10’ cells per milliliter. The leukocyte count was doubled in cases where WPS was added directly into the capillaries to compensate for dilution. Streptomycin and penicillin were added to every antigen sample in a concentration of 100 /*g per milliliter each. In addition, sonicated spermatozoa solutions (SSS) were obtained by ultrasonic treatment (3 x 10 seconds at 0’ C.) of washed and pooled spermatozoa suspended in distilled water ( 1 : 1, v/v). Following centrifugation (1,000 x g at 4’ C.) the aqueous supernatant was oxygen dried and redissolved in a culture medium with a concentration of 0.9 mg. per milliliter. These samples were applied in dilutions of 1: 10, 1: 100, and 1: 1,000. Furthermore, lipid-soluble extracts in alcohol, ether, acetone, and chloroform of sonicated spermatozoa were prepared and applied in the same manner as indicated above. Each antigen sample was tested six times. Only results with a standard deviation below 10 per cent were evaluated. Results Leukocyte migration was observed in fan-shaped areas around the opening of the capillaries inserted into the culture medium. By using a microscope with an adapted projector, planimetrical evaluation of the well-defined areas could easily be performed. Out of 124 samples 13 had to be excluded due to technical deficiencies (Fig. 1) . Group 1. In the group of fertile women, 10 out of 17 (58 per cent) cases, applying SSS, and 13 out of 21 (62 per cent), applying WPS as antigen, revealed a positive MIF reaction. Group 2. In the group of sterile women, 22 out of

32 (69 per cent), using SSS? and 29 out of ll ( 71 per cent‘), using WPS as antigen, reacted positively. When the inean \,alucs for MIF indices of Groups 1 and 2 were compared, no statistically sicnificant differences were detected. ln the subgroups of Group 2, positive reactions were found as follows. In Subgroup a : applying SSS, in four out of seven cases; applying WPS as antigen, in three out of seven. In Subgroup b: applying SSS, in four out of five cases; applying WPS as antigen, in two out of four. In Subgroup c: applying SSS, in 11 out of 15 cases; applying WPS as antigen, in 10 out of 1 I. Women with secondary sterility presented positive reactions in Subgroup a under the application of SSS in two out of two cases, under WPS in three out of three cases; in Subgroup b under SSS in two out of three cases, under WPS in two out of three cases; in Subgroup c under SSS in six out of eight cases and under WPS in four out of six cases. Group 3. In this group all 11 patients with spermagglutinating (n = 8) and sperm-immobilizing (n = 3) serum activity revealed a cellular sensitization against SSS and WPS. Group 4. No positive migration inhibitory reaction was encountered under the application of WPS or SSS as antigens. Comment Identification of the specific fertility-diminishing sperm antigens has not yet been achieved.‘l It can be hardly expected that the same sperm fragment would act as antigen in different cases. Since the specific immunogenic components of spermatozoa are still obscure, the application of a complex antigen is unavoidable in screening studies. As seminal plasma components do not seem to induce a fertility-diminishing immune reaction,l’, *” seminal plasma was not used as an antigen in the present studies. Additionally it is known that an active antigen released by accessory sex glands-the socalled sperm-coating antigen (SCA), or scaferrinfirmly accumulates upon the surface of spermatozoa, resisting simple washings.lG To demonstrate cellular sensitization to spermatozoa Tyler and co-worker?” used the uptake of a protein precursor. They stated that in a group of infertile women a higher incidence of cellular immune response is found than in a fertile control group. Their results show a certain similarity to the positive results on humoral sperm antibody production detected by various investigators.‘, 4, ti, lR

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121 1

Macrophage

m -7 LFig. 1. Macrophage columns), cases of columns) subdivided endocrinologic, and

inhibitory

factor

119

i i

migration inhibitory indices of fertile and sterile primary sterility (middle six columns), and secondary into three groups according to the cause of sterility: (c) unexplained. For further explanation, see the text.

In males with humoral antibodies against spermatozoa no cellular sensitization could be found when measured either by cytologic evaluation of lymphocyte transformation” or by the incorporation rate of labeled thymidine or uridine.?! I5 When a microlymphocyte transformation technique was used, sperm-immunized female rabbits presented a positive cellular sensitization.12 To our knowledge, the macrophage migration inhibitory factor has not been used to date to test cellular immune reactions against spermatozoa. We are well aware of the fact that the migration inhibitory factor expresses only one part of lymphocyte reactivity, but as it is very sensitive-a migration inhibition occurs even at very low antigen concentrations-and as it covers the entire lymphocyte population, we consider it a valuable tool for gaining insight into cellular stimulation against spermatozoa. Recent studies have well established that MIF is closely associated with immunity of the cellular type.3’ ‘3 I8 Concerning our results, we did not find a statis-

migration

females (left sterility (right (a) mechanical,

four six (b)

tically significant different cellular immune reaction, particularly in cases of unexplained primary or secondary sterility as compared to the fertile cases. In contrast, a mean macrophage migration inhibitory rate of approximately 0.5 could be encountered in all investigated groups except in Group 4. This stresses the fact that a cellular immune reaction to spermatozoa is induced in all females having had repeated sperm contact. The statistically significant difference between Groups 1 and 4 must be explained by the fact that Group 4 has never been exposed to sperm antigens. Even if in the current studies no significant difference was found between Groups 1 and 2, the possibility of such a difference cannot be excluded once specific fertility-diminishing antigens are available. Furthermore the possibility has to be indicated that merely quantitative differences in MIF activities can result in fertility or sterility. These results being contradictory to findings concerning the humoral sperm antibody production in fertile females, where we never found a positive

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humoral immune reaction, raised the question of the existence of cellular and humoral immunity to spermatozoa in a group of girls between 13 and 16 years of age (Group 4), who had had no sperm contact. The 20 girls investigated did not demonstrate any positive humoral or cellular immune reaction to spermatozoa. Such a negative control system appears to be of extreme importance in testing the efficiency of the applied method. As a great disadvantage to all techniques dealing with cellular sensitization against sperm antigens, it must be mentioned that no proper positive control is available, which is mainly due to the lack of knowledge of specific fertility-diminishing antigens. Considering the connection between humoral antibody production and cellular sensitization (Group 3), it could be demonstrated that all cases of posi-

tive humoral sperm antibodies were accompanied by a cellular sensitization to spermatozoa, applying WPS and SSS as antigens. So far no clinically relevant conclusions, considering immunologic causes of sterility, can be drawn from positive cellular reactions. Sperm contact seems to induce a positive MIF reaction in fertile and sterile women. Only the identification of specific. fertility-diminishing antigens would enable a proper evaluation. An appropriate explanation of the results hitherto gained is made difficult by the gap in our knowledge of the role of the lymphatic system in reproductive physiology. We acknowledge with thanks the expert technical sistance of Birgitt Meseck-Selchow.

as-

REFERENCES

Behrman, S. I.: Harper Hosp. Bull. 24: 147, 1966. Bendixen, G., and Seborg, M.: Dan. Med. Bull. 16: 1, 1969. David, J. R., and David, R. R.: Progr. Allergy 16: 300, 1972. Edwards, R. G.: Immunology and Reproduction, I,ondon, 1966, International Planned Parenthood Federation. El Alfi, 0. S., and Bassili, F.: J. Reprod. Fertil 21: 23, 1970. Franklin, R. R., and Dukes, C. D.: AM. J, OBSTET. GYNECOL. 84: 6, 1964. Gordon, H. L., Barsales, D. B., Westermann, E. I,., and Mumford, D. M.: J. Ural. 105: 863, 1971. Harris. I. E.: In Proceedines Fifth Leukocvte Culture Conference, June 25-27, Ottawa 1970, New York, 1970, Academic Press, Inc. 9. Isojima, S., Li, T. S., and Ashitaka, Y.: AM. J. OBSTET. GYNECOL. 101: 677, 1968. 10. Kibrick, S., Belding, D. I,., and Merrill, B.: Fertil. Steril. 3: 430, 1952. I

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11. Li, T. S., and Behrman, S. J.: Fartil. Steril. 21: 565, 1970. 12. Masson, D., Kauther, K.-D., Krebs, D., and Lehmann, F.: In First International Congress of Immunology in Obstetrics and Gynaecology, June 6-9, Padua, 1973. 13. Mettler, L., und Scheidel, P.: Schl.-Hoist. Xrztebl. 4: 193, 1973. 14. Mettler, I,.: Diagnostik 7: 1, 1974. 15. Mumford, D. M., Barsales, D. B., Ball, K. D., and Gordon, H. I,.: J. Ural. 105: 858, 1971. 16. Roberts. T. K.. and Boettcher. B.: I. Renrod. Fertil. 18: 347, 1969.’ 17. Shulman, S.: Obstet. Gynecol. Surv. 27: 8, 1972. 18. Stasny, P., Cooke, T. D., and Ziff, M.: Clin. Exp. Immunoi. 14: 141, 1973. 19. Tyler, A., Tyler, E., and Denny, P. C.: Fertil. Steril. 18: 153, 1967. 20. Weil, A. J. and Rodenburg, J. M.: Proc. Sot. Exp. Biol. Med. 131: 1040, 1960. /

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Macrophage migration inhibitory factor in female sterility.

Cellular sensitization against spermatozoa as antigen was studied in 21 fertile and 41 infertile female subjects. In addition, 20 virgin girls and a g...
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