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ANDROLOGIA

23, 3 6 7 - 3 7 1 ( 1 9 9 1 )

ACCEPTED: AUGUST14, 1 9 9 1

Immunomagnetic separation of antibody-labelledfrom antibody-free human spermatozoa as a treatment for immunologic infertility. A preliminary report P. Vigan6, F. M. Fusi, C. Brigante, M. Busacca and M. Vignali Key words. Spermatozoa

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immunomagnetic separation

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immunologic infertility

Summary. A method is described where superparamagnetic polymer microspheres coated with monoclonal antibodies are used to isolate antibodylabelled from antibody-free spermatozoa in male autoimmune infertility. Autoimmune sperm samples or antibody-free spermatozoa adsorbed with antisperm-antibodies from sera were incubated with microspheres coated with a specific monoclonal antibody to murine immunoglobulins, after their preincubation with mouse anti-human IgG and IgA. Using a magnet, the microsphere-labelled spermatozoa were separated from the samples. Immunobead binding was performed before and after the treatment in order to detect changes in the percentage of antibody-bound spermatozoa. After the immunomagnetic separation, approximately 50 yo of the IgA-labelled spermatozoa was isolated while no difference was demonstrated when antisperm antibodies of IgG class were involved. The evaluation of sperm motility and membrane integrity after treatment seemed to indicate that the technique did not have any relevant effect on sperm characteristics. The fact that only a partial success in separation of IgA-bound spermatozoa and no success for IgG-labelled sperm was obtained indicates that the method needs to be improved before its clinical utilization might be postulated.

111 Dept. of Obstetrics and Gynecology, University of Milano, Milano, Italy.

Correspondence: Dr Paola Viganb, I11 Department ofobstetrics and Gynecology, H. San Raffaele, Via Olgettina 60, 1-20132 Milano, Italy.

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human.

Introduction Antisperm antibodies (ASAs) detected in sera or seminal plasma of infertile men have been demonstrated to cause agglutination and immobilization of spermatozoa and to impair their ability to penetrate human cervical mucus (Rumke et al., 1974; Jager et al., 1981; Bronson et al., 1984 b). In addition, they can interfere with fertilization: ASAs have been shown to either promote or inhibit zonafree hamster eggs penetration by human spermatozoa (Bronson et al., 1981; Alexander, 1984; Haas et al., 1985). In human egg fertilization in vitro, sera containing both IgG or IgA class ASAs may have an inhibitory effect and inhibition seems to be caused predominantly by IgA class antibodies or alternatively by a synergistic effect (Bronson et al., 1982; Clarke et a]., 1985 a; Clarke et al., 1986; Tusuki et al., 1986). ASAs reduce the possibility of achieving a pregnancy but do not always prevent it: in fact the chance offathering children, in couples in which husbands possess antisperm antibodies, usually varies with the level of autoimmunity to sperm (Ayvaliotis et al., 1985). The best method for ASAs detection is the immunobead binding (Clarke et al., 1985 b; Alexander & Anderson, 1987), which directly assesses the percentage of antibody-coated spermatozoa, the regional specificy and the class of bound antibodies. Ayvaliotis et al. (1985) showed that patients with more than 50 percent of sperm immunobead binding had a significantly lower spontaneous pregnancy rate than those with less than 50 percent binding. Anyway, neither the lowest limits of significant immunobead binding nor the exact role of IgG versus IgA antibodies is currently known, depending probably upon the sperm antigens involved. Although progress has been made in the diagnosis of immunologic infertility, there are a number of unresolved problems related to its treatment. Different clinical regimens have been pro-

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posed based mainly upon immunosuppressive substances, for instance corticosteroids or cyclosporine A (Shulman & Shulman, 1982; Bouloux et al., 1986; Scarselli et al., 1987). The effectiveness of these treatments is controversial and generally the results are not completely satisfying. An alternative possibility might be the separation of antibody-free spermatozoa from the antibody-labelled ones in cases in which not all the spermatozoa are antibody-coated. Different methods have been proposed as the immunobead IgG and IgA incubation to adsorb antibody in the seminal plasma, followed by a swimup procedure into overlaying buffer, or the treatment of sperm by passage through a column consisting ofdextran beads (Kiser et al., 1987). All these techniques have been almost completely unsuccessful. Recently an immunomagnetic isolation of fractions of cells subsets directly from blood has been described (Brinchmann et al., 1988), Superparamagnetic polymer microspheres coated with monoclonal antibodies were used for the direct and quick quantification of the absolute number of cells of various lymphocyte subpopulations. Blood samples were incubated with microspheres coated with a subset-specific monoclonal antibody. Using a magnet the microsphere-rosetting cells were isolated and washed. With monoclonal antibodies specific for CD2, CD4, CD8 and CD15, reproducible absolute counts of the corresponding lymphocyte subsets were obtained. The aim of this work is to describe a similar method where superparamagnetic polymer microspheres coated with monoclonal antibodies are used to isolate antibody-labelled from antibody-free spermatozoa. O n this basis immunobead binding of spermatozoa was performed before and after the treatment with magnetic microspheres, in order to evaluate the percentage of sperm with autoantibodies. In addition, we evaluated the effects of this technique on seminal parameters. We asked whether isolation of such antibody-bound sperm would lead to a suspension of antibody-free spermatozoa useful of in vitro or in vivo insemination.

Materials and methods Sperm and semen samples From the patients attending the Andrology Laboratory of I11 Dept. of Obstetrics and Gynecology, University of Milan, we selected 11 men with autoimmunity to spermatozoa detected direct and indirect immunobead binding. Semen from four of them and serum from the other seven was utilized for our experiments. Semen samples and sera, when incubated with antibody-free sperm, showed a range of immunobead binding varying between 30

to 80 yo.Antibody-free spermatozoa for the experiments were obtained from seven healthy donors whose specimens were normal following the WHO (1980) criteria and free from antisperm antibodies, as detected by immunobead binding.

Immunobead binding Immunobead binding of antibody-labelled spermatozoa was performed as previously described (Busacca et al., 1989). Briefly, rabbit anti-human IgG or IgA immunobeads (BioRad, Richmond, CA, USA) were washed three times in Hank's balanced salt solution (HBSS, Flow, Opera, Milan, Italy) containing 3 mg ml-' human serum albumin (HSA, Irvine Scientific, Irvine, CA, USA) at 1800 x g for 5 min and resuspended to a 10 mg ml-' concentration. For the evaluation of ASAs directly bound to spermatozoa, 0.5 ml of sperm were washed with HBSS + 3 mg ml-I HSA, by centrifugation at 300 x g for 5 min. Then, spermatozoa were resuspended in HBSS containing 30 mg ml-' HSA to a concentration of 5 x 106/ml. Approximately 10 pl of this suspension was placed on a slide, mixed with 50 pl of beads and incubated in a moist chamber for 5 min. Thereafter, evaluation was performed within 10 min by counting at least 100 motile spermatozoa under phase contrast optics. If the number of spermatozoa carrying beads exceeded 20 yo, the test was estimated as positive. For the indirect assay 100 yl of 5 x 106/ml antibody-free spermatozoa, prepared as previously described, were coincubated with human serum at the 1:6 dilution for 60 min at 37 "C in 5 yo GO, in air. The test was then performed as described for the direct assay.

Magnetic beads Autoimmune sperm samples, or antibody-free spermatozoa adsorbed with ASAs from sera, were washed twice in Ham's F-10 and resuspended at a 2 x 1OG/mlconcentration. Spermatozoa were divided into aliquots for subsequent exposure to magnetic beads. The steps shown in the flow chart of Figure 1 were followed for the separation of ASAbound spermatozoa. Magnetic microspheres conjugated with goat anti-mouse IgG (Dynabeads, Oxoid, Hampshire, UK) were washed three times in Phosphate Buffer Saline (PBS, Flow, Opera, Milan, Italy) containing 0.1 yo bovine serum albumine (BSA, Sigma Chemical, St. Louis, MO, USA). Mouse monoclonal antibody to human IgG and IgA was added to the microspheres at a 2 pg mg-' beads concentration and incubated for 24 h at 4 OC, gently shaking. Successively each tube was exposed for 1 or 2 min to a magnet which determine the adhesion of the beads to the inner surface of the tube near to the magnet surface, and the supernaANDROLOGIA 23, 367-371 (1991)

IMMUNOMAGNETIC SEPARATION

tant was removed by decanting. The magnetic beads were washed twice in PBS 0.1 oio BSA with the same method and resuspended in the initial volume, in order to eliminate the unbound mouse antibody. Thereafter, washed microspheres, preincubated with monoclonal antibody, were added to each sperm sample at a ratio of Dynabeads/spermatozoa 40/1 for 1 h at room temperature, gently shaking. After incubation, the beads were washed using the magnet and each surnatant, containing spermatozoa unbound to microspheres, were tested by immunobead binding to detect changes in the percentage of antibody-labelled sperm. Finally, samples were checked to compare sperm motility and swelling ability versus other aliquots of the same sperm not exposed to Dynabeads. Hypoosmotic swelling (HOS) test The swelling of membrane intact spermatozoa was performed by adding 1 ml of hypoosmotic solution (150 mOsm, 7.35 g sodium citrate 2H,O and 13.51 g fructose in 1 000 ml distilled H,O) to 0.1 ml of liquified sperm. After 30 min incubation at 37"C, part of the sample was examined under phase con-

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trast microscope. At least 100 spermatozoa were examined in order to calculate the percentage of swollen sperm. Sta tistical analysis Statistical significances between the mean values were analyzed by Student's paired t-test.

Results The presence of ASAs in samples previously found to be positive was confirmed by mean of immunobead binding (Table 1 ) . The motility and the HOS test of free spermatozoa in the surnatants were unmodified after the treatment with Dynabeads antiIgG or antiIgA respect to another aliquot of each semen (Table 2). After magnetic separation of Dynabeads, beads were eluited and checked for the presence of adhering spermatozoa. Following immunomagnetic separation of IgA, a great number of IgA-bound spermatozoa was found to be successfully eliminated (Fig. 2), as confirmed by the statistically significant decrease of ASA-bound sper-

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ti \ C

1 Figures 1 and 2.

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1 d

(1) Magnetic separation of antibody-labelled from antibody-free spermatozoa: (a) Tube containing antibody-free and antibody-bound spermatozoa; (b) magnetic beads covered with anti-mouse antibodies and preincubated with mouse anti-human Ig antibody are added to the tube and bind to antibody-labelled spermatozoa; (c) a magnet separates ASA-bound spermatozoa by attraction of magnetic beads; (d) only ASA-free sperm last in the supernatant. (2) Binding of superparamagnetic polymer microspheres to antibody-labelled human spermatozoa after separation from ASA-free sperm by means of a magnet. ANDROLOGIA 23, 367-371 (1991)

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trary, very little spermatozoa were demonstrated to be separated by Dynabeads when IgG-ASA were involved, as confirmed by the absence of a difference in the percentage of ASA-labelled sperm in the bead-free surnatant (Table 3).

matozoa in the bead-free sample (Table 1). The mean percentage of IgA-bound spermatozoa after immunomagnetic elimination of antibody-labelled sperm was 27.4 f 20.05, versus 43.1 & 2 1.4 of the untreated control (P < 0.05; Table 3). O n the con-

Discussion

Table 1. Comparison of the percentage of antibody-labelled spermatozoa in ASA positive samples before and after immunomagnetic separation, as detected by immunobead binding Percentage of antibody-bound spermatozoa” Pre-treatment IgA IgG

Post-treatment IgA IgG

50 17 12 N.D.”

87 62 35 93

45 4 4 N.D.”

44 32 40 48 90 48 50

32 72 22 94 70 73 88

23 0 35 28 57 28 50

Seminal fluid identification

R.R. M.V. C.B. A.L.

66 80 40 90

Serum identification 1 2 3 4 5 6 7 a

40 74 50 I00 63 72 81

Each value represents the percentage obtained by counting at least 100 sperm per sample N.D., not detected

Table 2. Percentage of motile and swollen spermatozoa before and after treatment with Dynabeadsa Pre-treatment

Motility 21.1 f 3.575 HOS test 40.8 f 4.142

Post-treatment with Dynabead anti-IgG

Post-treatment with Dynabead anti-IgA

21.8 f 4.009” 46.1 f 6.125”

23.7 f 4.282“ 41.3 f 4.813“

amean f SEM ” P > 0.05, NS vs correspondent pre-treatment values

Table 3. Percentage of antibody-bound spermatozoa before and after immunomagnetic separation, as detected

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Percentage of antibody-bound spermatozoa” Pre-treatment Post-treatment %A IgGh Igk 66.2 f 7.750 43.1 & 6.455 68.7 f 5.868 27.4 f 6.047

bP > 0.05, NS vs correspondent pre-treatment values ? P < 0.05 vs correspondent pre-treatment values

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The importance of ASAs as a cause of infertility has been repeately confirmed (Bronson et al., 1984 a). Lower conception rates were demonstrated in partners of men with elevated percentage of labelled spermatozoa than in those without antibodies (Ayvaliotis et al., 1985). Different regimens have been used in the treatment of immunologic infertility such as immunosuppressive therapy or inhibitors of spermiogenesis (Jones, 1976; Shulman & Shulman, 1982; Bouloux et al., 1986; Scarselli et al., 1987). Corticosteroid and cyclosporine A are immunosuppressive agents utilized in the management of autoimmune diseases and to avoid transplant rejection. Such therapies produce adverse effects and are not acceptable to all patients. Furthermore, the timing of therapy and the optimum dose have not yet been established. Treatments which result in an inhibition of spermiogenesis, such as administration of testosterone, were utilized to eliminate for a long period the expression of sperm antigens, in order to achieve a desensitization. However, this approach appeared to be difficultly acceptable by infertile patients because of the delay in attempts of conceiving, and, since no evidence demonstrated its usefulness, it is no more utilized. An alternate approach is to treat semen samples in order to detach antibodies from sperm, or to select antibody-free from antibody-labelled spermatozoa to be utilized for assisted fertilizations. All methods to eliminate antibodies from the sperm surface have failed, because of the high affinity of ASAs for their antigens. It would seem likely that sperm fertilizing potential for assisted fertilization techniques in men with ASAs could be enhanced by the removal of antibodybound spermatozoa from the whole semen when not all spermatozoa are ASA-labelled. For this purpose, it has been suggested the use of a swim-up technique, in association or not to a IgG or IgA-linked immunobead separation which should have resulted in the recovery of motile ASA-free spermatozoa but this treatment was demonstrated to be unsuccessful too (Kiser et al., 1987). The quantification of lymphocyte subsets in blood by means of superparamagnetic monosized microspheres coated with monoclonal antibodies specific for the various lymphocyte subsets, suggested us the magnetic isolation of antibody-coated from antibody-free spermatozoa. Separation of lymphocyte subpopulations is an important routine test in clinANDROLOGIA 23, 367-371 (1991)

IMMUNOMAGNETIC SEPARATION

ical immunology: for this reason the method utilized must be simple, fast and give reproducible results. The immunomagnetic selection compares favourably with other methods for quantification of lymphocyte subpopulations. More than 97 yo of the cells of each subset are isolated and the isolated cell subsets appear to be more than 99 yo pure. O n this basis we supposed it would be useful for the clinical management of autoimmune infertility to utilize the immunoparamagnetic separation. In these preliminary experiments, we selected a group of 4 men with autoimmune infertility and transferred antisperm IgA and IgG antibodies from 7 sera to antibody-free spermatozoa of normal donors. Approximately 50 yo of the IgA-bound spermatozoa was isolated from the semen after immunomagnetic separation. O n the contrary, no IgG-bound spermatozoa were separated. Such results are probably related to differences in affinity for immunoglobulins of our detection method. IgA have a larger size than IgG and form bigger immunocomplexes with antiIg, so they are probably strong enough to resist to magnetic separation. The use of smaller magnetic beads and of high affinity anti-human antibodies might probably lead to an improvement of the technique, mainly for IgG-bound spermatozoa, and allow the separation of ASA-free sperm. Although sperm motility and membrane integrity are not damaged by magnetic separation, before the clinical utility of this method, further studies will be needed to determine whether spermatozoa, after exposure to magnetic strengths, maintain their physiological characteristics, such as the ability of undergoing capacitation and acrosome reaction, binding zona pellucida and fusing with the oolemma, or whether they are damaged and become unable to fertilize. In conclusion, our preliminary results indicate that the hypothesis of a magnetic separation of ASA-free from ASA-labelled spermatozoa should be developed in order to improve its results and to test its efficacy in obtaining spermatozoa with a good fertilizing ability. However, the technique might become in the future a useful device in the treatment of autoimmune infertility when only part of the spermatozoa are ASA-labelled, mainly thought that no other really efficacious method is actually available.

References Alexander NJ (1984) Antibodies to human spermatozoa impede sperm penetration of cervical mucus or hamster eggs. Fertil Steril 41:433438 Alexander NJ, Anderson DJ (1987) Immunology ofsemen. Fertil Steril 47:192-205 Ayvaliotis B, Bronson RA, Cooper GW, Rosenfeld DL (1985) Conception rates in couples where autoimmunity to sperm is detected. Fertil Steril 43:739-742 ANDROLOGIA 23, 367-371 (1991)

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Brinchmann JE, Vartdal F, Gaudernack G, Markussen G, Funderud s, Ugelstad J, Thorsby E (1988) Direct immunomagnetic quantification of lymphocyte subsets in blood. Clin Exp Immunol 7 1: 182-186 Bronson RA, Cooper GW, Rosenfeld DL (1981) Ability of antibody-bound human sperm to penetrate zona-free hamster ova in vitro. Fertil Steril 36:778-783 Bronson RA, Cooper GW, Rosenfeld DL (1982) Sperm specific isoantibodies and autoantibodies inhibit the binding of human sperm to the human zona pellucida. Fertil Steril 38:724-729 Bronson RA, Cooper GW, Rosenfeld DL (1984a) Sperm antibodies: their role in infertility. Fertil Steril 42:171-183 Bronson RA, Cooper GW, Rosenfeld DL (1984 b) Autoimmunity to spermatozoa: effect on sperm penetration of cervical mucus as reflected by postcoital testing. Fertil Steril 45:609614 Bouloux PMG, Wass JAH, Parslow JM, Hendry WF, Besser GM (1986) Effect of cyclosporin A in male autoimmune infertility. Fertil Steril 46:81-84 Busacca M, Fusi F, Brigante C, Doldi N, Smid M, Viganb P (1989) Evaluation of antisperm antibodies in infertile couples with immunobead test: prevalence and prognostic value. Acta Eur Fertil 20:77-82 Clarke GN, Lopata A, McBain JC, Baker HW, Johnston W I (1985a) Effect of sperm antibodies in males on human in vitro fertilization (IVF). Am J Reprod Immunol Microbiol 8:6266 Clarke GN, Elliot PJ, Smaila C (1985 b) Detection of sperm antibodies in semen using the immunobead test: a survey of 813 consecutive patients. Am J Reprod Immunol Microbiol 711 18-123 Clarke GN, Lopata A, Johnston WIH (1986) Effect of sperm antibodies in females on human in vitro fertilization. Fertil Steril 46:435-441 Haas GG, Sokoloski JE, Wolf DP (1985) The interfering effect of human IgG antisperm antibodies on human sperm penetration ofzona-free hamster egg. Am J Reprod Immunol 1:4& 43 Jager S, Kremer J, Kuiken J, Van Slocteren-Draaisma T, Mulder de Wilde-Janssen, IW (1981) Induction of shaking phenomenon by pretreatment of spermatozoa with sera containing antispermatozoal antibodies. Fertil Steril 36:784-791 Jones WR (1976) Immunological aspects of infertility. In: Immunology of Human Reproduction. Scott JS, Jones WR (eds) Academic Press, London, pp 375-394 Kiser GC, Alexander NJ, Fuchs EF, Fulgham DL (1987) In vitro immune absorption of antibodies with immunobead-rise, immunomagnetic, and immunocolumn separation techniques. Fertil Steril 47:466474 Rumke Ph, Van Amstel N, Messer EN, Bezemer PD (1974) Prognosis of fertility of men with sperm agglutinins in the serum. Fertil Steril 25:393 Scarselli G, Livi C, Emmi L, Chelo E, Noci I, Pellegrini S (1987) Effect of corticosteroid therapy in immunological infertility: a preliminary report and an alternative hypothesis. Am J Reprod Immunol Microbiol 15:57--60 Shulman JF, Shulman S (1982) Methylprednisolone treatment of immunologic infertility in the male. Fertil Steril 38:591-599 Tusuki S,Noda Y, Yano J, Fukuda A, Mori T (1986) Inhibition of sperm penetration through human zona pellucida by antisperm antibodies. Fertil Steril 46:92-96 World Health Organization (1980) Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Belsey MA, Eliasson R, Gallegos AJ, Moghissi KS, Paulsen CA, Prasad MRN (eds) Press Concern, Singapore, pp 9 4 3

Immunomagnetic separation of antibody-labelled from antibody-free human spermatozoa as a treatment for immunologic infertility. A preliminary report.

A method is described where superparamagnetic polymer microspheres coated with monoclonal antibodies are used to isolate antibody-labelled from antibo...
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