Effect of different procedures of semen preparation on antibody-coated spermatozoa and immunological infertility

Vol. 52, No.6, December 1989

FERTILITY AND STERILITY

Printed on acid-free paper in U.S.A.

Copyright <> 1989 The American Fertility Society

Effect of different procedures of semen preparation on antibody-coated spermatozoa and immunological infertility Aucky Hinting, M.D.* Lutgart Vermeulen, Ir.Biochem.t Ines Goethalst

Marc Dhont, M.D., Ph.D.:!: Frank Comhaire, M.D., Ph.D.t§

State University Hospita~ Ghent, Belgium

To assess whether procedures of semen preparation can reduce the proportion of antibody-coated spermatozoa, semen samples with positive direct mixed antiglobulin reaction (MAR) were washed in media supplemented with 10% or 50% fetal cord serum (FCS). Washing reduced the MAR to a negative level, but the MAR was identical to that in the native semen when spermatozoa were resuspended in serum-free medium. Donor spermatozoa, recovered after swim-up in media supplemented with 10% or 50% FCS or after passage through a column with 7.5% human serum albumin (HSA), were incubated in serum samples with both agglutinating and cytotoxic antisperm antibodies. Cytotoxic activity was significantly reduced against sperm filtered over the albumin column. Fertil Steril52:1022, 1989

Antisperm antibodies (ASA) have been reported to cause infertility in 8% of couples consulting for failure to conceive after at least 12 months of exposure to the risk of pregnancy. Antisperm antibodies bind to the spermatozoa and prevent their migration through the female reproductive tract. 1 They may also interfere with gamete interactions involved in fertilization 2 by inhibiting sperm binding to or penetration of the zona pellucida. 3 Until now, no treatment has convincingly proven effective against male immune infertility.' Sperm washing and intrauterine insemination have been attempted with variable success.5 ,6 Other techniques of assisted reproduction, such as in vitro fertilization (lVF), gamete intrafallopian transfer (GIFT), or zygote intrafallopian transfer (ZIFT), may constitute possible treatments for

Received December 27, 1988; revised and accepted June 28, 1989. .. On leave of absence from the Department of Biomedics, Faculty of Medicine, Airlangga University, Surabaya, Indonesia. t Department of Internal Medicine. =!: Department of Gynecology. § Reprint requests: Frank Comhaire, M.D., Ph.D., Department of Internal Medicine, Section of Endocrinology, State University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium.

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couples with immune infertility. Indeed, some pregnancies have been reported after IVF using ASA-coated spermatozoa. 7- 10 In the present study, it was evaluated whether particular procedures of semen preparation can reduce the proportion of spermatozoa coated with antibodies or increase sperm resistance against the agglutinating or cytotoxic activities of ASA.

MATERIALS AND METHODS Experimental Design

The effect of sperm washing on the proportion of motile spermatozoa coated with ASA was investigated in the first experiment. Ten semen samples known to react strongly in the direct mixed antiglobulin reaction (MAR) for immunoglobulin G (IgG) (SpermMAR; Ortho Diagnostic Systems, Beerse, Belgium) were washed and allowed to swim up in Earle's medium (Earle's Balanced Salts; Flow Laboratories, Irvine, Scotland), which was supplemented with either 10%11 or 50%12 fetal cord serum (FCS). Mixed antiglobulin reaction tests were repeated on the spermatozoa recovered in the upper layer. Next, the spermatozoa were resuspended in Fertility and Sterility

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diluted spermatozoa suspension on top of a column containing 1.5 mL of medium supplemented with 7.5% HSA. After 60 to 90 minutes' incubation at 37°C, the lower 0.5 mL of the column was removed and centrifuged at 200 X g for 10 minutes to concentrate the spermatozoa.

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serum-free medium, and the SpermMAR was repeated (Fig. 1). In the second and third experiments, respectively, it was investigated whether particular procedures of sperm preparation could protect the spermatozoa against the agglutinating, cytotoxic effects of ASA. Ten sera with strong agglutinating activity in the tray agglutination test (TAT)13 and cytotoxic effect in the adenosine triphosphate release cytotoxicity test (ARCT)14 were used as a source of ASA. Donor spermatozoa were prepared by swim-up in Earle's medium, supplemented with either 10% or 50% FCS, or by passing through a column with 7.5% human serum albumin (HSA).15 Spermatozoa recovered by swim-up in nonsupplemented medium were used as controls. The recovered spermatozoa were incubated with the ASAcontaining serum, and the agglutinating and cytotoxic activities were assessed by repeated TAT and ARCT (Fig. 2). Procedures of Semen Preparation

In the first experiment, sperm washing was performed by twofold centrifugation at 200 X g for 10 minutes at room temperature. 11,12 The final pellet was overlayed with 1 to 2 mL of Earle's medium, and the upper interface was recovered after 30 to 60 minutes of incubation in a 5% CO2 gas mixture at 37°C. In the second and third experiments, the swimup procedure was performed by placing 0.5 mL of semen beneath 2 mL of medium in culture tubes. After incubation for 30 to 60 minutes in 5% CO2 at 37°C,11,16 the upper layer was gently aspirated to recover the spermatozoa. The albumin column preparation was performed by layering 0.5 mL of Vol. 52, No.6, December 1989

Direct MAR Test

The direct MAR test was performed by mixing the following on a microscope slide: 10 ILL of semen, one drop oflatex particles coated with IgG, and one' drop of antiserum from rabbit against human IgG (SpermMAR kit). Reading was performed after 2 to 3 minutes under the microscope. The percentage of motile spermatozoa with latex particles attached was calculated. A sample was considered positive if ;;::40% of the motile spermatozoa reacted with the particles. 17 TAT

The TAT was performed as described by Friberg13 and included dilutions of 1/8 to 1/1,024 of complement-inactivated serum. Aliquots of 5 ILL of each dilution were transferred to a microchamber tray under mineral oil, and 1 ILL of a suspension of washed donor spermatozoa (concentration, 40 million/mL) was added. The presence of agglutination was evaluated in an inversion microscope after 2 hours' incubation at 37°C. The titer was determined as the highest serum dilution causing agglutination. A titer of 1/32 or more was considered evidence of sperm-agglutinating activity. IS

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ARCT

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The ARCT was performed using a simplification14 of the procedure described by Suominen et aL 19 Fifty microliters of complement-inactivated serum diluted 1/4 in Earle's balanced solution, 20 #t L of guinea pig complement (ORAY Behring, Marburg, FRG), and 50 #tL of washed donor sperm suspension with concentration of 20 million/mL were incubated at 37°C for 90 minutes. In parallel, a control mixture was incubated containing 1/4 diluted, complement-inactivated serum and sperm suspension, to which inactivated guinea pig complement was added. To measure the ATP concentration, 100 #tL of the previous preparations were added to 100 #tL of nucleotide-releasing substance (Lumac BV, Landgraaf, The Netherlands) and 300 #tL of Tris-acetate-ethylenediaminetetraacetic acid buffer. Next, 100 #tL of ATP-monitoring reagent (LKB-Wallac, Turku, Finland) was added, and light emission was measured in a luminometer (LKB-Wallac, 1250 Luminometer). The ARC index was calculated. This index is the quotient of the ATP content of the control preparation, containing inactivated complement, divided by the ATP content of the parallel preparation containing active complement. An ARC index of ~2 was considered evidence for sperm cytotoxic antibodyactivityY

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Figure 4 The agglutinin titers (top panel) and adenosine triphosphate release cytotoxicity (ARC) index (lower panel) against sperm recovered after different preparation procedures compared with the control values. FCS, fetal cord serum; *, P < 0.01 (Wilcoxon's rank sum test).

Statistical Analysis

Statistical analysis included Student's t-test and Wilcoxon's rank sum test as indicated. RESULTS Effects of Sperm Washing on the Proportion of Antibody-coated Spermatozoa

The proportion of spermatozoa reacting positively in the direct SpermMAR test in the native

semen and after sperm preparation in experiment 1 is shown in Figure 3. The percent MAR with spermatozoa prepared in medium supplemented with 10% and 50% FCS was significantly reduced (Student's t-test; P < 0.001), reaching a negative leveL After the spermatozoa had been resuspended in serum-free medium, the results were identical to those observed in the native semen. Effect on the Agglutinating Activity of ASA

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Figure 3 Proportion of spermatozoa reacting positively in the direct SpermMAR test in the native semen and after sperm preparation in experiment 1 (mean and range). FCS, fetal cord serum; *, P < 0.001 (Student's t-test).

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The agglutinin titers against spermatozoa recovered after different preparation procedures and the control values are shown in Figure 4. Because the reproducibility of the TAT is one titer step,20 reduction of the agglutinin titer was considered significant if it was at least two dilution steps lower than the control value. There was no reduction of the agglutinating activity against spermatozoa prepared in medium supplemented with 10% FCS. A significant reduction of agglutinating activity was recorded in 3 out of 10 samples containing spermatozoa prepared in medium supplemented with 50% FCS or filtered over an albumin column. Fertility and Sterility

Effect on the Cytotoxic Activity of ASA

The ARC index against sperm recovered after different preparation procedures and the control values are shown in Figure 4. There was no significant reduction of the cytotoxic effect against spermatozoa prepared by swim-up in media supplemented with either 10% or 50% FCS, but the cytotoxic effect was significantly reduced against spermatozoa prepared over an albumin column (Wilcoxon's rank sum test; P < 0.01).

DISCUSSION

In vitro manipulation of semen, followed by intrauterine insemination or IVF, has been advocated for the treatment for male immune infertility.5,12 It has been speculated that ASA present on the spermatozoa could be removed by repeated washing. The use of a medium supplemented with 50% FCS has been proposed for this purpose. 12 Our study demonstrates that neither washing nor supplementation with FCS reduces antibody coating of spermatozoa. This finding is in agreement with a report by Haas and co-workers,21 who have used radiolabeled antiglobulin techniques for ASA quantification. A significant reduction of the mixed antiglobulin reaction occurs when spermatozoa are suspended in serum-containing medium, but the mixed antiglobulin reaction is identical to that in the native sample after serum is removed. Hence reduction to negative level of the MAR test is not due to a reduction of sperm-bound antibodies, but to the presence of serum, which contains such an important amount of IgG as to block the MAR. The persistence of agglutination after in vitro transfer of antisperm antibodies to spermatozoa in experiments 2 and 3 underscores the high affinity of ASA for spermatozoa. It also demonstrates that high concentrations of FCS or human albumin do not interfere with the agglutinating activity. A tendency toward reduction of agglutinating activity against spermatozoa prepared in medium supplemented with 50% FCS or on an albumin column is observed in 3 out of 10 preparations. This may relate to the improved sperm motility observed after this type of sperm preparation rather than to a real decrease of the ASA effect. The cytotoxic effect of ASA is not reduced against spermatozoa prepared in medium supplemented with 10% or 50% FCS, contradicting suggestions that some immunosuppressive factors may be present in FCS, counteracting the effect of Vol. 52, No.6, December 1989

ASA.22 However, the cytotoxic effect was significantly decreased against spermatozoa prepared over an albumin column. This may result from the fact that a high concentration of albumin stabilizes the sperm membrane23 or reduces complement activity. The present study provides evidence that the procedures of semen preparation that are usually employed during IVF or GIFT neither reduce antibody binding to spermatozoa nor suppress the agglutinating or cytotoxic activity of ASA. The detrimental effect of ASA on sperm-fertilizing capacity depends only partly on the agglutinating effect and the inhibition of sperm transport, which can both be overcome by intrauterine insemination of washed spermatozoa. The cytotoxic effect of ASA is, however, activated by the complement present in the secretions of the female reproductive tract. The latter can be overcome by IVF, provided sperm-oocyte interaction occurs in medium free of complement, which is attained by heat inactivating the sera followed by filtration over a Millipore filter (Sterivex GV; Millipore, Bedford, MA). When these procedures are used, normal fertilization rates have, indeed, been recorded in couples with infertility due to the presence of cytotoxic antibodies in the male partner. 1O,24,25 Further studies are needed to evaluate the possible benefit of sperm preparation over albumin columns for clinical applications. REFERENCES 1. Jager S, Kremer J, Van Slochteren-Draaisma T: Presence

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9. Mandelbaum SL, Diamond MP, DeCherney AH: Relationship of antisperm antibodies to oocyte fertilization and in vitro fertilization-embryo transfer. Fertil Steril 47:644, 1987 10. Hinting A, Vermeulen L, Comhaire F, Dhont M: Submitted 11. Berger T, Marrs RP, Moyer DL: Comparison of techniques for selection of motile spermatozoa. Fertil Steril 43:268, 1985 12. Cohen J, Edwards R, Fehilly C, Fishel S, Hewitt J, Purdy J, Rowland G, Steptoe P, Webster J: In vitro fertilization: a treatment for male infertility. Fertil Steril43:422, 1985 13. Friberg J: Immunoglobulin concentration in serum and seminal fluid from men with and without sperm-agglutinating antibodies. Am J Obstet Gynecol136:671, 1980 14. Hinting A, Vermeulen L, Comhaire F: Evaluation of a simplified adenosine triphosphate release eytotoxicity test for the detection of sperm antibodies in serum. J Reprod Immunol13:123, 1988 15. Aitken RJ: Andrology and semen preparation for IVF. In In Vitro Fertilization, Past-Present-Future, Edited by S Fishel, EM Symonds. Oxford, IRL Press, 1986, p 89 16. Harris SJ, Milligan MP, Masson GM, Dennis KJ: Improved separation of motile sperm in asthenospermia and its application to artificial insemination homologous (AIH). Fertil Steril 36:219, 1981 17. Comhaire FH, Hinting A, Vermeulen L, Schoonjans F,

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Goethals I: Evaluation of the direct and indirect mixed antiglobulin reaction with latex particles for the diagnosis of immunological infertility. Int J Androl11:37, 1987 18. Rumke P, Van Amstel N, Messer EN, Bezemer PD: Prognosis of fertility of men with sperm agglutinins in the serum. Fertil Steril25:393, 1974 19. Suominen JJO, Multamaki S, Djupsund BM: A new method for measurement of cytotoxic antibodies to human spermatozoa. Arch Androl4:257, 1980 20. Hjort T: Detection of antisperm antibodies. In Immunology of the Male Reproductive System, Edited by PE Bigazzi. New York, Marcel Dekker, 1987, p 53 21. Haas GG, D'C~z OJ, Denum BM: Effect of repeated washing on sperm-bound immunoglobulin G. J Androl 9:190, 1988 22. Jacoby DR, Olding LB, Oldstone MB: Immunological regulation of fetal maternal imbalance. Adv Immunol 35:157, 1984 23. Cholewa JA, Whitten WK: Development of two-cell mouse embryos in the absence of a fixed-nitrogen source. J Reprod Fertil22:553, 1970 24. Mathur S, Mathur RS, Holtz GL, Tsai CC, Rust PF, Williamson HO: Cytotoxic sperm antibodies and in vitro fertilization of mature oocytes: a preliminary report. J In Vitro Fert Embryo Transfer 4:177,1987 25. Hinting A, Comhaire F, Vermeulen L, Vermeulen A, Dhont M, Vandekerckhove D: Submitted

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