Modulation of luteinizing hormone secretion by estrogens in patients with Reifenstein’s syndrome*

FERTILITY AND STERILITY

Vol. 52, No.2, August 1989

Printed in U.S.A.

Copyright I[J 1989 The American Fertility Society

Modulation of luteinizing hormone secretion by estrogens in patients with Reifenstein's syndrome* Martha Medina, M.D. Gladys Castorena, M.D. Joaquin Herrera, M.Sc.

Jose A. Bermudez, M.D. Arturo Zarate, M.D. t

Endocrine Research Unit, Instituto Mexicano del Seguro Social, Mexico D.F., Mexico

The role of estrogens on gonadotropin secretion was assessed in two siblings with incomplete virilization syndrome type I due to partial androgen insensitivity (Reinfenstein's syndrome). Serum levels ofluteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured before and after 100 f.lg LH-releasing hormone (RH) intravenous (IV) stimulation, as well as during long-term clomiphene citrate (CC) administration. Serum testosterone (T) and estrogens were determined before and during daily administration of human chronic gonadotropin (hCG) and also during the CC treatment. Basal levels of LH were elevated in both patients: 12.5 ± 1.1 mIU in patient A and 19.8 ± 1.8 mIU/ml in patient B. Conversely, FSH levels were within normal limits. Administration of LH -RH in both subjects induced a rise in LH levels, while FSH concentration showed no increase. The CC administration resulted in a significant (P < 0.005) increment in serum LH levels without changes in FSH concentration. An important increase of serum T and estradiol (E 2 ) levels was noted during CC administration; thus, in patient A those levels augmented from 20 to 48 ng/ml for T and from 78 to 220 pg/ml for E 2 ; and patient B showed an increment from 20 to 35 ng/ml for T, and from 55 to 180 pg/ml for E 2 • The daily administration of hCG was followed by an increment in both T and E2 levels, which was of lesser degree for estrone concentration. These results suggest that endogenous estrogens, particularly E 2 , modulate LH secretion in patients with partial androgen insensitivity; however, it appears that estrogens had no physiologic effect on FSH secretion. Fertil Steril52:239, 1989

The Reifenstein's syndrome represents a form of hereditary male pseudohermaphroditism characterized by incomplete virilization with perineoscrotal hypospadia, small penis, and gynecomastia at the time of puberty. 1 Recent studies have demonstrated that male pseudohermaphroditism is caused by a partial or complete androgen resistance at target tissues. 2- 4 The hormonal profile in the complete and incomplete androgen insensitivity syndrome type I has been well established. 5- s Elevated luteinizing hormone (LH) levels in the presence of augmented circulating testosterone (T)

Received November 15, 1988; revised and accepted April 21, 1989. * Supported in part by grant PCSACNA-050126 from the Consejo Nacional de Ciencia y Tecnologia. t Reprint requests: Arturo Zarate, M.D., Endocrine Research Unit, Apdo. Postal 107-115, 06760 Mexico, D.F., Mexico. VoL 52, No.2, August 1989

results from the absence of the feedback mechanism, which in turn is due to the androgen resistance present in these affected individuals. In order to explain why follicle-stimulating hormone (FSH) levels remain normal or even lower than in normal patients, it has been proposed that the elevated levels of endogenous estrogens have a negative feedback at pituitary level and are responsible for this finding. 4 ,7,8 However, recent studies on patients with testicular feminization syndrome, a complete form of androgen insensitivity, demonstrated the absence of a significant effect of estrogens on FSH secretion. 9 To explore further the mechanism of gonadotropin secretion in patients with the syndrome of Reifenstein, we studied the effect of long-term administration of clomiphene citrate (CC), a nonsteroidal antiestrogen compound, on LH, FSH, T, and estradiol (E 2 ) serum levels in two siblings with the

Medina et al.

LH and FSH secretion in Reifenstein's syndrome

239

Each time represents five replicates. Then CC was given at a daily dose of 200 mg for 28 consecutive days. Serum LH, FSH, T, and E2 were measured by duplicate before and at weekly intervals during the CC administration. Two weeks later, hCG 2,500 U was administered intramuscularly daily during 4 days, and serum levels of T, E 2, estrone (E 1 ), and estriol (E3) were measured. Assays

Figure 1 Two siblings with incomplete virilization syndrome type 1 showing normal breast development and a female phenotype.

syndrome. We also studied the effect of luteinizing hormone-releasing hormone (LH-RH) and human chorionic gonadotropin (hCG) stimulation on the pituitary and testes, respectively. MATERIALS AND METHODS Patients

Two postpubertal siblings ("A," a 20-year-old and "B," a 15-year-old) with male pseudohermaphroditism type I were studied (Fig. 1A). Diagnosis was established on the basis of (1) ambiguous external genitalia as clitoral enlargement, partial scrotal fusion, perineoscrotal hypospadia, and bilateral partially descended testes; (2) breast development, Tanner IV to V; (3) scanty pubic and axillary hair; (4) absence of miillerian structures; and (5) 46, XY karyotype in peripheral leukocytes. A 12-year-old prepubertal sibling was studied as a control for the hCG stimulation test. Informed consent was obtained from all participants, and the protocol of study was approved by the ethical committee. Gonads from the two patients were removed after completing their study, and they were raised as women.

Serum LH and FSH levels were determined by specific radioimmunoassay as previously described1o; results are expressed as milli-International Unit per milliliter. Samples were analyzed by duplicate in two different dilutions. Steroid assays included measurements of T, Elo E 2, and E3 by the use of specific antiseraY Steroid serum extraction and thin-layer chromatography (TLC) were used to increase the specificity and accuracy of the assays. Steroid serum extraction was performed with ethyl ether. Thin-layer chromatography separation of estrogens was performed by using the following solvent systems: (1) benzene, (2) benzene/ethyl acetate 65/35, and (3) benzene/ methanol 85/15. The recovery was 75%. The interassay and intra-assay coefficients of variation were for E 3, 18% and 8%; E 2, 10% and 5%; E 1 , 14% and 9%, respectively; the results were expressed as picogram per milliliter. Serum T extraction was performed with ethylic ether, and the recovery was 95%. Thin-layer chromatography isolation of T was performed using the solvent systems: (1) benzene 100%, (2) benzene-ethyl acetate 70/30, and (3) benzene/methanol 95/5. The recovery obtained in this system was 81.3 ± 4.3%; the results were expressed as nanograms per milliliter. Thin-layer chromatography plates of silica gel (60F 254 20 X 20 cm and layer thickness of 0.25 mm) were purchased PATIENT B

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LH and FSH secretion in Reifenstein's syndrome

Fertility and Sterility

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from E. Merck (Darmstadt, West Germany). The solvents were analytic reagent (Merck, Mexico City, Mexico) distilled to obtain the highest purity. All other reagents were analytic grade. The 3H-Iabeled hormones were purchased from New England Nuclear Corporation (Boston, MA). The radiochemicals were assessed with the use of TLC. Nonradioactive steroids were purchased from Steraloids Inc. (Pauling, NY) and recrystallized before use. Specific antibodies were obtained as previously described with dilutions of 1:10,000 or higher. Dextran T-70 was purchased from Pharmacia Chemicals (Stockholm, Sweden) and charcoal from Nort-A (New York, NY)

nificative changes for E3 levels were found. The hCG stimulation in the normal sibling was not followed by any change in T and E2 levels (Fig. 3). A significant increase (P < 0.005) in LH levels was observed during the CC administration in both patients; however, no significant changes were noted in FSH levels (Fig. 4). Both patients with the Reifenstein's syndrome showed an increment in T and E2 under CC administration, from 20 to 48 ng/ml and 78 to 220 pg/ml, respectively, for patient A. In patient B, T increased from 20 to 35 ng/ml and E2 from 55 to 180 pg/ml. PATIENT

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Basal LH and FSH serum levels for patient A were 12.5 ± 1.1 mIU/ml and 8.0 ± 0.40 mIU/ml, respectively. The normal values for adult males are for LH 10.0 ± 1.2, and for FSH 8.5 ± 0.9 mIU / ml. The administration of synthetic LH -RH elicited a hypersecretion of LH from 12.5 to 78.0 mIU/ml in patient A and from 19.8 to 98.0 mIU/ ml in patient B. On the contrary, a hyporesponse was observed for FSH in both cases (Fig. 2). Mean basal serum T and E 2 levels in five determinations in patient A were 20 ng/ml and 115 pg/ ml, respectively, whereas patient B had a mean value of 19 ng/ml for T and 105 pg/ml for E2 levels. The hCG stimulation produced a rise in T and E2 levels of approximately 75% and over 200%, respectively, in patient A. In patient B, the T augmented 100% and E2 over 200%. No sigVol. 52, No.2, August 1989

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Medina et al.

LH and FSH secretion in Reifenstein's syndrome

241

DISCUSSION

The defective androgen receptors at pituitary level in patients with Reifenstein's syndrome results from the absence of feedback mechanism by T on gonadotropin secretion. 2 It is generally recognized that pituitary LH secretion in normal men is primarily modulated by T 5 ; therefore, in Reifenstein's syndrome and in other androgen-insensitive syndromes, the LH levels are found increased despite the elevated basal T levels. Conversely, FSH regulation seems to be modulated either by endogenous estrogens or some testicular nonsteroidal factors.12 Patients with androgen resistancesuch as Reifenstein's syndrome-present azoospermia with arrest of spermatogenesis at the spermatogonial level; therefore, elevated FSH levels also might be expected secondary to the testicular abnormality. The peripheral aromatization of T to estrogens and the elevated testicular estrogen production 2,13 produce elevated circulating levels of estrogens in Reifenstein's syndrome that could be an explanation for the normal or even low FSH levels found in these patients. The present study conducted in two siblings with partial androgen insensitivity confirms that these patients have augmented basal levels of LH with a hyperresponse to LH-RH; on the contrary, the FSH concentration was normal and LH -RH administration induced an abnormal response. Long-term administration of CC, whose mechanism of action is to compete for estrogen receptors at hypothalamic and pituitary levels, produced a significative rise in serum LH, suggesting that endogenous estrogens play a role in LH regulation. The administration of CC was unable to modify FSH levels, demonstrating that neither endogenous T nor endogenous estrogens participate in FSH regulation. Similar findings have been reported in testicular feminization syndrome9 ; however, Lacroix et alY reported an increase in serum FSH during 6 days of CC administration in patients with testicular feminization syndrome. Since both LH and FSH increased after castration, it may suggest that in Reifenstein's syndrome as well as in testicular feminization syndrome, estrogens and another nonesteroidal testicular factor, probably inhibin, are involved in gonadotropin regulation. The overall data demonstrate a different regulatory mechanism for LH as compared with normal men. However, the mechanism of gonadotropin regulation seems to be similar in the complete and the incomplete forms of androgen resistance despite the clinical and ge242

Medina et al.

netic heterogeneity reported m male pseudohermaphroditism "type 1." Acknowledgment. We are greatly indebted to Salvador Raiti, M.D., National Hormone and Pituitary Program (University of Maryland School of Medicine, Baltimore, Maryland), for supplying the reagents for the radioimmunoassays. REFERENCES 1. Reifenstein EC, Jr: Hereditary familiar hypogonadism.

Clin Res 3:86,1947 2. Wilson JD, Harrod MJ, Goldstein NL, Hamsell LC, MacDonald PC: Familiar incomplete male pseudohermaphroditism, type 1. N Engl J Med 290:1,097,1974 3. Griffin JE, Wilson JD: Studies on the pathogenesis of the incomplete forms of androgen resistance in man. J Clin Endocrinol Metab 45:1,137,1977 4. Medina M, Chavez B, Perez-Palacios G: Defective androgen action at the cellular level in the androgen resistance syndromes. 1. Differences between the complete and incomplete testicular feminization syndromes. J Clin Endocrinol Metab 53:1,243, 1981 5. Judd HL, Hamilton CR, Barlow JJ, Yen SSC, Kliman B: Androgen and gonadotropin dynamics in testicular femini~ zation syndrome. J Clin Endocrinol Metab 34:229, 1972 6. Tremblay RR, Foley TP, Corvol P, In-Joo Park, Kowarski A, Blizzard RM, Jones HW, Migeon CJ: Plasma concentration oftestorerone, dihydrostestosterone, testosterone-oestradiol binding globulin, and pituitary gonadotrophins in the syndrome of male pseudo- hermaphoroditism with testicular feminization. Acta Endocrinol (Copenh) 70:301, 1972 7. Boyar RM, Moore RJ, Rosner W, Aiman J, Chipman J, Madden JD, Marks JF, Griffin JE: Studies of gonadotropin-gonadal dynamics in patients with androgen insensitivity. J Clin Endocrinol Metab 47:115,1978 8. Faiman C, Winter J: The control of gonadotropin secretion in complete testicular feminization. J Clin Endocrinol Metab 39:531, 1978 9. Medina M, Ulloa AA, Fernandez MA, Perez-Palacios G: The role of oestrogens on gonadotrophin function in the testicular feminization syndrome. Acta Endocrinol (Copenh) 95:314, 1980 10. Medina M, Herrera J, Flores M, Martin 0, Bermudez JA, Zarate A: Normal ovarian function in a mild form of lateonset 3(3 hydroxysteroid dehydrogenase deficiency. Fertil Steril46:1,021, 1986 11. Bermudez J A, Coronado VM, Mijares A, Leon C, Velazquez A, Noble P, Mateos JL: Stereochemical approach to increase the specificity of steroid antibodies. J Steroid Biochern 6:283, 1975 12. Steinberger A, Steinberger E: Secretion of an FSH-inhibiting factor by cultured Sertoli cells. Endocrinology 99:918, 1976 13. Schweikart HU, Milawich L, Wilson JD: Aromatization of androstandione by cultured human fibroblast. J Clin Endocrinol Metab 43:781, 1975 14. Vaitukaitis JL, Bermudez JA, Cargille CM, Lippsett MB, Ross GT: New evidence for an anti-estrogenic action of climiphen citrate (CC) in women. J Clin Endocrinol Metab 32:503, 1971 15. Lacroix A, McKanna T J, Rabinowitz D: Sex steroid modulation of gonadotropins in normal men and in androgen insensitivity syndrome. J Clin Endocrinol Metab 48:235, 1979

LH and FSH secretion in Reifenstein's syndrome

Fertility and Sterility