Human anti-luteinizing hormone-releasing hormone antibodies in patients treated with synthetic luteinizing hormone-releasing hormone *

Human anti-luteinizing hormone-releasing hormone antibodies in patients treated with synthetic luteinizing hormone-releasing hormone *

FERTILITY AND STERILITY Copyright © 1985 The American Fertility Society Vol. 43, No.5, May 1985 Printed in U.SA. Human anti-luteinizing hormone-rele...

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FERTILITY AND STERILITY Copyright © 1985 The American Fertility Society

Vol. 43, No.5, May 1985 Printed in U.SA.

Human anti-luteinizing hormone-releasing hormone antibodies in patients treated with synthetic luteinizing hormone-releasing hormone*

Janice L. Meakin, B.Sc.t+ Edward J. Keogh, M.B.B.S., F.R.A.C.P., Ph.D.t§ Clayton E. Martin, B.Sc., Ph.D.11 Reproductive Medicine Research Institute, Queen Elizabeth II Medical Centre, Nedlands, Western Australia, Australia

Since human luteinizing hormone-releasing hormone (LH-RH) was characterized and synthesized,1, 2 it has been available commercially and has achieved widespread use. LH-RH is used for diagnostic and therapeutic purposes in hypogonadotropic hypogonadism, infertility, delayed puberty, and cryptorchidism. Two instances of antibody reaction to synthetic LH-RH in men with isolated gonadotropin deficiency have been reported. 3 ,4 The patients received 0.25 to 2 mg LH-RH once daily for 10 months 3 or 1 mg LH-RH three times daily for 3 months,4 administered subcutaneously. In both men, the initial clinical improvement was followed by refractoriness, and one 4 developed an urticarial reaction. They both had immunoglobulin G (IgG) antibodies, and the patient with an

Received October 15, 1984; revised and accepted January 7, 1985. *Supported by the National Health and Medical Research Council of Australia, Ayerst Australia, TVW Telethon Foundation, and the King Edward Memorial Hospital Research Foundation. tReproductive Medicine Research Institute. :j:Department of Endocrinology and Diabetes. §Reprint requests: Dr. E. J. Keogh, Reproductive Medicine Research Institute, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, Australia. IIDepartment of Clinical Biochemistry. Vol. 43, No.5, May 1985

urticarial reaction also had immunoglobulin E (IgE) antibodies to LH-RH. We have observed urticarial reactions in 5 of 163 patients treated with synthetic LH-RH and failure of hormone response in 26 patients, and 11 patients have become refractory to the initial dose of LH-RH. We report here the IgG antibody reactions to LH-RH treatment in a series of 163 patients and IgE antibody reactions in a subset of this series. MATERIALS AND METHODS PATIENTS

Twenty-two women with hypothalamic amenorrhea or polycystic ovarian disease and infertility, 4 men with hypogonadotropic hypogonadism, and 137 boys with cryptorchidism or delayed puberty were treated with pulsatile LH-RH (HRF Gonadorelin, Ayerst Laboratories, Parramatta, New South Wales, Australia; 50 to 360 f.Lg/day, at 60, 90, or 120 minute pulse intervals) administered subcutaneously for 3 weeks to 9 months (mean duration, 2 months). Blood samples were taken, at least 30 minutes after a bolus ofLH-RH, during the week before LH-RH therapy was stopped, and serum was stored at - 20°C. Three untreated subjects and a pooled serum sample were used as controls. Meakin et aI_ Communications-in-brief



Table 1. 125I_LH_RH Binding in Patients' Serum


Ten microliters of the patient's serum was incubated overnight (16 hours) at room temperature (18° to 23°C) with 100 f.LI of 125I_LH_RH (New England Nuclear, North Ryde, New South Wales, Australia; about 20,000 cpm) and 190 f.LI of 0.25% bovine serum albumin in 0.01 M phosphate-buffered saline (PBS-BSA), pH 7.4, with and without 10 f.Lg unlabeled LH-RH. One hundred microliters of antiserum (diluted 1:2 with PBS-BSA) to human IgG, raised in goats (Lawrence Laboratories, Boyanup, Western Australia), and 600 f.LI of 25% aqueous polyethylene glycol were added prior to incubation for 40 minutes at room temperature. The tubes were centrifuged for 20 minutes at 3000 rpm at 4°C, and the supernatant was aspirated. The pellet was washed with 1 ml PBS-BSA and recentrifuged. After aspiration the pellet was counted in a gamma counter. SERUM IMMUNOGLOBULIN E BINDING OF LUTEINIZING HORMONE-RELEASING HORMONE

With the radioimmunoadsorbent method of Ceska et al.,5 50 f.LI of serum from the patients in whom urticaria developed during LH-RH treatment and those with positive serum IgG binding ofLH-RH was incubated with LH-RH-impregnated cellulose disks. After washing, the disks were incubated with 125I-anti-(Fc)-IgE (Pharmacia Fine Chemicals, Uppsala, Sweden), and the fraction bound to the serum IgE/LH-RH/cellulose was measured in a gamma counter.

RESULTS Five of 163 patients (3%) had specific binding to IgG of 125I_LH_RH > 0.4%. Nonspecific binding (in the presence of 10 f.Lg of unlabeled LH-RH) was < 2% in all patients. The results of patients who had specific binding of LH-RH to IgG antibodies are shown in Table 1. The control serum did not bind 125I_LH_RH to IgG, and neither did serum from the start of treatment from two of the patients (1 and 3) in whom IgG antibodies developed. Patient 1 had oligospermia and normal luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T). He was treated for 3 months with no change in sperm count or LH, FSH, or T concentrations but had an urticarial response. This was shown to be due to the LH-RH 812

Meakin et aI. Communications-in-brief

1 2 3 4 5

Total binding

+ 10 fLg LH-RH

Specific binding


Age yr





28 12 27 2.5 32

13.2 8.0 4.8 8.2 2.3

1.6 1.7 1.7 1.3 0.9

11.6 6.3 3.1 6.9 1.4

when a 200-mm2 wheal and a 3500-mm2 flare developed at the site of injection of 0.2 f.Lg LH-RH in 0.5 ml of saline. There was no reaction to the vehicle. There was no significant increase in IgE antibodies to LH-RH as assessed by the radioimmunoadsorbent test. Patient 2 had bilateral cryptorchidism and treatment for 11 weeks elicited neither a hormonal (LH, FSH, or T) nor a clinical (descent of testes) response. Patient 3 was treated for 5 months for secondary amenorrhea, ovulating four times without conceiving. After a 10-month interval, she returned for treatment; but because she had no increase in estrogen or follicle growth within 3 weeks, the LH -RH dose was increased. The dose was increased again for the same reason, and the pulse interval was changed from 60 to 90 minutes before ovulation occurred without conception. Patient 4 had bilateral cryptorchidism and failed to respond clinically (descent of testes) or hormonally (LH, FSH, and T) to treatment for 8 weeks. Patient 5 had hypo gonadotropic hypogonadism with anosmia and ovulated and conceived on 1 month of treatment. After a 2-year interval, she desired a second child. Three weeks of LH-RH failed to increase estrogen concentration or follicle growth, and so the dose of LH-RH was doubled. She ovulated and conceived on the second ovulatory cycle but miscarried at 6 weeks' gestation. Three patients who had urticarial reactions to LH-RH, which necessitated cessation of therapy, did not have IgG antibodies to LH-RH detected. One of these patients received a test dose of 0.2 f.Lg LH-RH in saline, and a 100-mm2 wheal and a 1600-mm2 flare developed. Five patients with urticaria (one with IgG antibodies) and four other patients with IgG antibodies to LH-RH did not have a positive serum IgE antibody response. We did not have a positive control subject. Fertility and Sterility


There was marked diversity in the clinical histories and hormonal and clinical responses to LHRH among the five patients in whom IgG antibodies developed. No recurring clinical pattern has enabled prediction of such a response. Three patients did not show the appropriate clinical response, and the two anovulatory women were both in their second treatment courses when they required increased doses ofLH-RH to induce ovulation. Teleologically, and based on the previous reports in the literature, it was thought that only patients with a congenital deficiency of LH-RH would develop antibodies to exogenous LH-RH. However, two ofthe five patients described (1 and 3) went through puberty spontaneously, indicating the presence of endogenous LH-RH. Patient 3 probably had a relative deficiency ofLH-RH when she presented with hypogonadotropic hypogonadism and amenorrhea. These data do not support the hypothesis that a lack of endogenous LH-RH is a prerequisite for LH-RH antibody formation. The formation of antibodies to the small molecule LH-RH may be related to the mode of its delivery. The subclinical chronic inflammation at the tip of the scalp vein needle may have led to the complexing ofLH-RH to larger proteins, thus potentiating its antigenicity. The failure to detect antibodies in most of the patients treated in this manner argues against this mechanism. Because this form of medical therapy is becoming more widespread, the prevalence rate of antibody formation of 3% could represent a serious limitation in its use. SUMMARY

One hundred sixty-three patients who were given synthetic LH-RH therapeutically under-

Vol. 43, No.5, May 1985

went monitoring of serum IgG anti-LH-RH antibodies. Five of the patients showed specific binding to antibodies. Development of anti-LH-RH antibodies was not limited to those patients with a congenital deficiency of LH-RH. Urticarial responses occurred in four patients, only one of whom had IgG antibodies. Patients who had IgG antibodies or an urticarial response underwent monitoring of their serum IgE anti-LH-RH antibodies, but none had a positive binding response. The refractory state which has been reported in patients in whom similar antibodies to LH-RH develop was not invariably observed among these patients. Acknowledgments. We thank Mr. Peter Kay, Mr. Malcolm Sears, and Dr. Peter Hollingsworth, of the Department. of Immunology, Queen Elizabeth II Medical Centre, for their advice. REFERENCES 1. Matsuo H, Nair RM, Arimura A, Schally AV: Synthesis of





the porcine LH- and FSH-releasing hormone by the solid phase method. Biochem Biophys Res Commun 45:882, 1971 Monahan M, Rivier J, Burgus R, Amoss M, Blackwell R, Vale W, Guillemin R: Synthese totale par phase solide d'un decapeptide qui stimule la secretion des gonadotropines hypophysaires LH et FSH. C R Acad Sci [D] (Paris) 273:508, 1971 Brown GM, Van Loon GR, Hummel BCW, Grota LJ, Arimura A, Schally AV: Chracteristics of antibody produced during chronic treatment with LHRH. J Clin Endocrinol Metab 44:784, 1977 Lindner J, McNeil LW, Marney S, Conway M, Rivier J, Vale W, Rabin D: Characterization of human anti-luteinizing hormone-releasing hormone (LRH) antibodies in the serum of a patient with isolated gonadotropin deficiency treated with synthetic LRH. J Clin Endocrinol Metab 52:267, 1981 Ceska M, Eriksson R, Varga JM: Radioimmunosorbent assay of allergens. J Allergy Clin Immunol 49:1,1972

Meakin et aI. Communications-in-brief