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Plasma testosterone stimulation-suppression dynamics in hirsute women
Plasma Testosterone Stimulation-Suppression Dynamics in Hirsute Women
BRUCE ETTINGER, M.D. KLAUS von WERDER, M.D.* GUY C. THENAERS, M.D.? PETER H. FORSHAM. M.D. San Francisco, California
From the Metabolic Research Unit, General Clinical Research Center, and Department of Medicine, University of California, San Francisco, California 94122. This paper was supported in part by the Levi J. and Mary Skaggs Foundation. Studies were carried out in the General Clinical Research Center of the University of California, San Francisco, supported by a grant from the National Institutes of Health (RR-79). Requests for reprints should be addressed to Dr. P. H. Forsham, Metabolic Research Unit, 1143-HSW, University of California, San Francisco, Calif. 94122. Manuscript received August 17, 1970. * Present address: Second Medical
Clinic, University of Munich, Munich, Germany. f Present address: 67, Genebos, 3920 Lummen,
170
Belgium.
Of forty-seven hirsute women referred to a metabolic clinic, 60 per cent were found to have excessive levels of testosterone in the plasma, yet only 20 per cent of this group had increased urinary excretion of testosterone. The plasma testosterone levels served as a useful screening test for hirsutism. Nineteen patients with hirsutism were further evaluated using a six day protocol for adrenal suppression-stimulation and ovarian stimulation (human chorionic gonadotropin), as well as ovarian visualization procedures. These tests gave inconsistent results in individual cases and no general patterns of response: they were of no use in predicting the pathologic source of testosterone. Hirsutism in women, frequently associated with menstrual abnormalities, is a common diagnostic problem. The relevance of excessive testosterone production in this syndrome has been stressed by many investigators [l-3]. It has been well documented that the adrenals [4] and ovaries [5] are capable of secreting considerable amounts of testosterone. Since there is no evidence that excessive peripheral conversion of androgenie precursors (e.g., ,dehydroepiandrosterone and Ad-androstenedione) accounts for elevation of testosterone levels [6-lo], investigation of the anatomic source of excessive androgen production might focus treatment on the secretory organ(s) responsible. We report our experience in screening hirsute women for excessively high levels of testosterone and attempt to define the pathologic source by the patterns of testosterone response. SUBJECTS AND METHODS Subjects.
Hirsute patients referred to the Metabolic Clinic were screened by twenty-four hour urinary and plasma1 testostero,ne determinations, while receiving no treatment. Significant hirsutism was defined as (1) facial hair growth with “side burns,” beard or anterior neck hair, (2) intermammalry hair growth, and (3) male hair distribution on arms, legs and trunk. In all patients hair growth was of typical male distribution and texture. Patients showing periareolar hair growth or a male escutcheon only were exclu,ded since such a finding
The American
Journal
of Medicine
PLASMA TESTOSTERONE
is common in normal women. Menstrual abnormalities were present in all the patients selected, usually oligomenorrhea. Obesity (greater than 50 per cent above ideal weight) was noted in 75 per cent of our patients. Base line urinary 17-hydroxycorticosteroids (17. OHCS [ll], 17-ketosteroids (17.KS) [12] and pregnanetriols [13] were measured, and appropriate tests were used to rule out Cushing’s syndrome, 21p- and lip-hydroxylase deficiencies, and other endocrinopathies. Levels of urinary testosterone were measured by gas-liquid chromatography of an acetate derivative of testosterone glucuronide [14]. Plasma testosterone was determined by a radioligand method [15], as modified by Maeda et al. [16]. Twenty-five units of ACTH was infused intravenously for eight hours, and plasma testosterone was measured at the end of the infusion. Plasma ll-OHCS [17] levels were determined to document adequate adrenal stimulation. The adrenals were suppressed by the oral administration of dexamethasone, 1.0 mg every six hours for two days. At the end of the suppression period, blood was drawn for testosterone and llOHCS determinations; the latter assured adequacy of adrenal suppression. The ovaries were stimulated for two days (3,000 units human chorionic gonadotropin [HCG], intramuscularly-Follutein@, Squibb, luteinizing hormone activity: 0.924 IU/unit HCG), while adrenal suppression was maintained. Four hours after the second administration of HCG, plasma testosterone was measured. This sampling time was determined after analyzing the plasma testosterone hourly, following intramuscular administration of HCG in several women with idiopathic hirsutism and Stein-Leventhal ovaries, and in three normal women. If clinically indicated, tomography of the adrenals was performed. When ovarian pathology was suspected and adequate pelvic examination was precluded by obesity, a pelvic pneumogram was obtained using the technic of Schulz and Rosen [18]. When indicated, further study by laparoscopy, via a transperitoneal fibroscope insertion, allowed direct visualization of the ovaries. In six of eight patients with a roentgenologic diagnosis of SteinLeventhal ovaries, wedge resection was performed and the diagnosis confirmed.
RESULTS Screening Tests. In 60 per cent of patients referred to us for hirsutism and menstrual irregularity, the basal plasma testosterone level was clearly elevated (Figure 1). However, urinary testosterone excretion was borderline or increased in only a third of the patients with elevated plasma levels. Twenty per cent of patients had borderline abnormal plasma testosterone levels, and usually had normal or slightly increased urinary testosterone excretion. Twenty per cent of patients had
Volume
51, August
1971
PLASMA mfig/lOOml
IN HIRSUTE
WOMEN -
URINE fig/24
hr
ETTINGER
PLASMA
ET AL.
URINE
m~g/lOOml
Agl24
hr
200
100
Patients STEIN
OVARIES
Figure 1. performed Leventhal hirsutism.
with
- LEVENTHAL n=12
Patients
with
IDIOPATHIC HIRSUTISM
n=35
Plasma and urinary
base line measurements on the same day in twelve patients with Steinovaries and thirty-five patients with idiopathic Shaded areas represent normal levels.
normal plasma testosterone levels, and with rare exception had normal urinary testosterone excretion. There was no quantitative relation between the basal urinary 17-OHCS, 17-KS and pregnanetrio1 levels, and the testosterone excretion (Table I) [l]. Base line laboratory tests did not differentiate idiopathic hirsutism from Stein-Leventhal ovaries. Plasma and urinary testosterone levels did not correlate with obesity. Stimulation-Suppression Tests. To compare the patterns of stimulation and suppression tests we arbitrarily established the following criteria for response: (1) doubling of base line testosterone levels after ACTH infusion, (2) halving of base line testosterone level after dexamethasone suppression, and (3) doubling of the post dexamethasonesuppressed base line testosterone level by HCG stimulation. Table II demonstrates the dynamics of plasma testosterone in three normal women, eight patients with Stein-Leventhal ovaries and eleven patients with idiopathic hirsutism. A summary of test responses in eight patients with Stein-Leventhal ovaries revealed (1) 50 per cent adrenal suppression in one patient and 40 per cent suppression in three other patients, (2) ACTH response in two patients, and (3) response to HCG in four of seven tested. Categorizing responses as “adrenal” if ACTH and dexamethasone
171
PLASMA TESTOSTERONE
TABLE I
IN HIRSUTE
WOMEN-ETTINGER
Basal Urinary Steroids and Plasma Patients with Idiopathic Hirsutism
ET AL
Testosterone
in Eight Patients
with Stein-Leventhal
Ovaries
Urinary
Patient
17-OHCS (mg/24 hr)
Patients S.B. M.M. V.Y. D.Ro D.Re M.H. P.H. J.D.
5.2 9.7 14.7 13.9 9.2 10.4 18.2 7.3
N.P. N.G. J.F. J.De L.A. J.G. E.B. L.B. P.J. G.H. AX. Normal range
6.6 10.0 13.6 20.5 14.9 13.9 7.3 6.6 16.0 8.9 10.3 5-12
Pregnanetriol (mg/24 hr)
17-KS (mg/24 hr)
with Stein-Leventhal
5.0 8.5 8.5 7.5 11.4 11.5 16.6 10.3 Patients
with Idiopathic
responses were noted and as “ovarian” if HCG response was noted, the individual patterns of response in these patients with Stein-Leventhal ovaries were: ovarian, three patients; adrenal, one; mixed ovarian/adrenal, one; and no significant response to any test, three. Ten of eleven patients with idiopathic hirsutism showed adrenal or ovarian responses, or both (Table II). Adrenal suppression response was definite in two patients and borderline in three. Six of eleven responded to ACTH, but four of the six showed no adrenal suppression. Stimulation with HCG occurred in seven of eleven patients tested. In the patients responding, the individual patterns were as follows: ovarian, two patients; adrenal, none; mixed adrenal/ovarian, two; inconsistent adrenal (stimulation with ACTH without suppression by dexamethasone, or vice versa), three: and inconsistent adrenal/ovarian, three. In twelve of the hirsute patients and in three normal subjects, studies of urinary testosterone excretion were made at the same time that the plasma testosterone was being measured. Only four patients showed stimulation with ACTH (doubling of basal excretion, and at least 5’Pg/ twenty-four hours), but none showed suppression
172
(424
hr)
Plasma Testosterone (m&100
ml)
Ovaries
0.4 1.6 2.6 0.9 2.7 2.2 0.7 1.8
9.1 8.6 18.9 11.6 19.7 13.4 7.7 6.7 7.8 9.8 12.0 5-15
Testosterone
and in Eleven
16.1 11.4
118 &l 126 127 225 130 172 215
1.2
378 267 82 65 99 276 185 52 99 93 288 40-100
Hirsutism 3.2 2.1 3.3 2.4 2.3 2.6 2.6 1.5 0.4 3.4 1.9
to 50 per cent of base line levels with dexamethasone. In four patients urinary testosterone excretion was suppressed with dexamethasone but in none was there stimulation with ACTH. Two patients with Stein-Leventhal ovaries showed stimulation with HCG and ACTH but not suppression with dexamethasone. The remaining patients showed no significant changes in urinary testosterone excretion with any of the test maneuvers. Thus, in all twelve patients the urinary testosterone responses yielded no clinically useful or consistent data. COMMENTS Base Line Studies. The usefulness of the plasma testosterone determination as a screening procedure in hirsute women is shown. Two-thirds of the hirsute patients studied were found to have normal basal urinary testosterone excretion, yet more than half this group had elevations in plasma testosterone levels. Normal levels of plasma testosterone have been accompanied by normal urinary testosterone excretion, with rare exceptions. Hence, plasma testosterone is a more sensitive indicator of hypertestosteronism and rarely yields falsely negative results. In our series,
The American
Journal
of Modiclne
PLASMA TESTOSTERONE
Testosterone Dynamics in Three Normal Women, Eight Patients with Stein-Leventhal with Idiopathic Hirsutism Base Line
Patient
Plasma (mFg/lDO ml)
ACTH Stimulation
UrinZ (jig/24 hr)
Plasma (m&CO
Dexamethasone Suppression Plasma
Urine b&!/24 hr)
ml)
(m&100
Normal J.S. M.M. Y.V.
56 91 46
1.0 0.5 2.0
292 229 239
S.B. M.M. V.Y. D.Ro D.Re M.H. P.H. J.D.
118 94
16.1 11.4
138 74 131 414 151
1.5 0.3 1.8
Urine @g/24 hr)
ml)
126 127 225 130 172 215
... 260 739
... 2.4 ... ...
...
1.0 0.3 1.1
31 86 91
331 116 154 79 129 79 214 105
Patients with Idiopathic N.P. N.G. J.F. J.D.E. L.A. J.G. E.B. L.B. P.J. G.H. A.C.
* h = inconsistent
Volume
1.2
378 267 82 65 99 276 185 52 99 93 288
adrenal
51, August 1971
749 260 267 172 113 762 53 260 326 238 194
stimulation-suppression
... ... ... 22.2 10.0
ETTINGER
ET AL.
Ovaries and Eleven Women
HCG Stimulation Urine
Plasma (m&/100
ml)
r&z/24 hr)
Plasma Testosterone Pattern*
Subjects
Patients with Stein-Leventhal 41.4
WOMEN -
version of dehydroepiandrosterone may also account for 15 per cent of the testosterone produced [8,9]. Excluding the peripheral conversion of other precursors, as yet unknown, glandular secretion of testosterone is likely to be the important cause of hypertestosteronism. Dexamethasone, 0.5 mg orally every six hours for two days, is sufficient to suppress ACTH-dependent adrenal steroid production in normal subjects [22]. In all our patients urinary 17-OHCS and 17-KS excretion was adequately suppressed. The plasma testosterone levels, after adrenal stimulation and suppression in the normal and hirsute women studied, were quite similar to those reported by Casey and Nabarro [23] who showed that despite inconstant dexamethasone suppression, significant ACTH stimulation of plasma testosterone occurred in normal women. These results are in conflict with the majority of reports [24-271, however, which indicate minimal response of the plasma testosterone to adrenal stimulation in normal women. In one of five patients with idiopathic hirsutism and high basal
obesity did not affect the plasma testosterone level. These findings are in agreement with other investigators using either the radioligand method or the double-isotope technic [16,19,20]. All base line determinations were performed in patients who were not taking estrogenic or progestational agents, since these are well known to influence plasma testosterone levels. For similar reasons, plasma testosterone, after estrogen suppression, could not be determined. All patients studied were shown to be euthyroid by appropriate tests. This obviates the problem of false elevation or depression of plasma testosterone noted in hyperthyroidism and hypothyroidism, respectively [21]. The source of testosterone in normal and hirsute women has been partially elucidated [5-lo]. In the normal woman, a,-androstenedione is peripherally converted to testosterone, accounting for 50 to 60 per cent of the total testosterone production. In the hirsute woman, A,-androstenedione production may be twice normal, yet its conversion to testosterone accounts for only 20 to 25 per cent of testosterone production [5,7]. ConTABLE II
IN HIRSUTE
234 172 70 100 60 284 64 41 119 240 193
191 261 251
Adrenal-ovarian Adrenal-ovarian Adrenal-ovarian
310 230 110 276 764 640 85 151
48.5 8.8
None Ovarian None Adrenal-ovarian Ovarian Ovarian None Adrenal
303 538 165 578 423 1,323 188 394 63 243 133
1.2 2.0 5.2 5.2 8.5 7.5 ...
Ovaries 29.4 14.2 1.2 ...
...
...
... ... 3.5 43.2
Hirsutism
... ...
... 14.6 14.1
... 25.5 3.0
+ Adrenal Ovarian Adrenal-ovarian Z!Z Adrenal-ovarian Ovarian rfr Adrenal-ovarian It Adrenal-ovarian Adrenal-ovarian z!= Adrenal =t Adrenal None
responses.
173
PLASMA TESTOSTERONE
IN HIRSUTE
WOMEN-ETTINGER
ET AL
plasma testosterone levels, excessive testosterone levels were suppressed 50 per cent or more with dexamethasone, whereas three others showed only slight suppression. In our patients with idiopathic hirsutism (unlike those reported by Casey and Nabarro), dexamethasone suppressiveness of testosterone did not correlate with ACTH-responsiveness of testosterone. The converse also held, since of the seven patients with idiopathic hirsutism who responded to ACTH, dexamethasone suppression was noted in none. In similar testing of patients with Stein-Leventhal ovaries, plasma testosterone levels were suppressed with dexamethasone in one of eight and stimulated by ACTH in two of seven. The lack of suppression and the inconsistency of adrenal stimulation and suppression responses agree with the findings of Lloyd et al. [27]. We agree with Bardin et al. [26] that acute changes in the plasma testosterone level are largely independent of endogenous ACTH in both normal women and those with hypertestosteronism. Thus the testosterone contribution of the adrenal glands cannot be assessed by short-term ACTH suppression. In all three normal subjects plasma testosterone was stimulated with HCG. This was also true in four of eight patients with proved ovarian pathology and seven of eleven with idiopathic hirsutism. The plasma testosterone response to HCG shows no correlation with the anatomic lesion. HCG stimulation has been variously reported to elevate or to have no effect on plasma testosterone levels in normal subjects and in patients with idiopathic hirsutism or Stein-Leventhal ovaries [23-25,271. The results of our studies in patients with SteinLeventhal ovaries and idiopathic hirsutism concur with previous reports showing a marked hetero-
geneity of responses of plasma testosterone. Furthermore, the day to day variation in plasma testosterone averaged 50 mpg/100 ml in our patients and 60 mpg/100 ml in normal subjects reported by others [23,24,26]. A mid-cycle increase in plasma testosterone has been inconstantly reported [23,24,26]. Although the response of plasma testosterone to suppression/stimulation tests appears to correlate with testosterone production rates [28], there are marked inconsistencies of responses to both adrenal and ovarian test procedures in hirsute women. Similarly, measurements of changes in urinary testosterone excretion during various suppression and stimulation maneuvers have not yielded clinically useful data. The determination of plasma testosterone appears to be a sensitive screening test for hypertestosteronism in women. It could prove valuable in providing a rapid chemical means of assessing the need for, and the adequacy of, treatment of hirsutism [29], even though there is good evidence for the contrary [27]. We doubt its clinical value in the performance of any short-term stimulation/ suppression tests of testosterone dynamics in the hirsute woman. We agree with other investigators [28-301 that the patterns of response elicited in such testing have no value in predicting the pathologic source of testosterone and do not appear to offer a rational guide to the choice of therapy. Although studies of urine and plasma testosterone response can show patterns in the means of groups of patients [2,27,30], there is marked inconsistency in each individual case. ACKNOWLEDGMENT We wish to thank Miss Jeanette Shinsako and Mr. Shiro Horita for their technical assistance.
REFERENCES 1.
2.
3.
4.
5.
174
Migeon CJ, Kirschner MA, Bardin CW: Physiologic basis of disorders of androgen metabolism. Ann Intern Med 68: 1327, 1968. Deller JJ, Wegienka LC, Conte NF, Rosner JM, Forsham PH: Testosterone metabolism in idiopathic hirsutism. Ann Intern Med 63: 369, 1965. Conrad SH, Lindberg MC, Herrmann WL: Significance of plasma dehydroisoandrosterone and androsterone sulfates in the diagnosis of virilizing disorders. Amer J Obstet Gynec 91: 449, 1965. Burger HG, Kent JR, Kellie AE: Determination of testosterone in human peripheral and adrenal venous plasma. J Clin Endocr 24: 432, 1964. Migeon CJ, Saez JM, Rivarola MA: Comparison of
6.
7.
8.
the results obtained for the production rate of testosterone by “blood” and “urinary” methods. Testosterone. Proceedings of the Workshop Conference, April 20-22, 1967, Tremsbijttel (Tamm J, ed), Stuttgart, Georg Thieme Verlag, 1968, p 42. Horton R, Neisler J: Plasma androgens in patients with the polycystic ovary syndrome. J Clin Endocr 28: 479,1968. Bardin CW, Lipsett MB: Testosterone and androstenedione blood production rates in normal women and women with idiopathic hirsutism or polycystic ovaries. J Clin Invest 46: 891, 1967. Horton R, Tait JF: In vivo conversion of dehydroisoandrosterone to plasma androstenedione and testosterone in man. J Clin Endocr 27: 79, 1967.
The American
Journal
of Mediche
PLASMA
9.
10.
11.
12.
13.
14.
15.
16.
17.
18. 19.
20.
Volume
Horton R, Tait JF: Androstenedione production and interconversion rates measured in peripheral blood and studies on the possible site of its conversion to testosterone. J Clin Invest 45: 301, 1966. Tait JF, Horton R: The in vivo estimation of blood production and ‘interconversion rates of androstenedione and testosterone and the calculations of their secretion rates, Steroid Dynamics. Proceedings, Symposium on Dynamics of Steroid Hormones, Mav 1965. Tokvo (Pincus G. Nakao T, Tatit JF, eds): New York, Academic Press, 1966, p 393. Reddy WJ: Modification of the Reddy-Jenkins-Thorn method for the estimation of 17-hydroxycorticoids in urine. Metabolism 3: 489, 1954. Drekter IJ, Heisler A, Scism GR, Stern S, Pearson S, McGavack TH: The determinati’on of urinary steroids. I. The preparation of pigment-free extracts and a simplified procedure for the estimation of total 17-ketosteroids. J Clin Endocr 12: 55. 1952. Bongiovanni AM, Eberlein WR: Critical analysis of methods for measurement of pregnane-3-alpha, 17-alpha, 20.alpha-trio1 in human urine. Anal Chem 30: 388, 1958. Wegienka LC, Bower BF, Shinsako J, Elattar TM, Hane S, Mimica N, Demertze E, Stutheit JE, Forsham PH: Gas chromatographic method for the determination of testosterone in urine. Anal Biochem 18: 203, 1967. Murphy BEP: Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J Clin Endocr 27: 973, 1967. Maeda R, Okamoto M, Wegienka LC, Forsham PH: A clinically useful method for plasma testosterone determination. Steroids 13: 83, 1969. Mattingly D: A simple fluorimetric method for the estimation of free 11-hydroxycorticoids in human plasma. J Clin Path 15: 374, 1962. Schulz E, Rosen SW: Gynecography: technique and interpretation. Amer J Roentgen 86: 866, 1961. Mayes D, Nugent CA: Determination of plasma testosterone by the ulse ‘of competitive protein binding. J Clin Endocr 28: 1169, 1968. Gandy HM, Peterson RE: Measurement of testoster-
51,
August
1971
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
TESTOSTERONE
IN
HIRSUTE
WOMEN
-
ETTINGER
ET
AL.
one and 17.ketosteroids in plasma by the double isotope dilution derivative technique. J Clin Endocr 28: 949, 1968. Gordon GG, Southren AL, Tochimoto S, Rand JJ, Olivo J: Effect of hyperthyroidism and hypothyroidism on the metabolism of testosterone and androstenedione in man. J Clin Endocr 29: 164, 1969. Liddle GW: Tests of pituitary-adrenal suppressibility in the diagnosis of Cushing’s syndrome. J Clin Endocr 20: 1539. 1960. Casey JH, Nabarrb JDN: Plasma testosterone in idiopathic hirsutism and the changes produced by adrenal and ovarian stimulation and suppression. J Clin Endocr 27: 1431, 1967. (a) Lamb EJ, Dignam WJ, Pion RJ, Simmer HH: Plasma androgens in women. I. Normal and nonhirsute females, oophorectomized and adrenalectomized patients. Acta Endocr 45: 243, 1964. (b) Dignam WJ, Pion RJ, Lamb EJ, Simmer HH: Plasma androgens in women. II. Patients with polycystic ovaries and hirsutism. Acta Endocr 45: 254, 1964. Korenman SG, Kirschner MA, Lipsett MB: Testosterone production in normal and virilized women and in women with the Stein-Leventhal syndrome or idiopathic hirsutism. J Clin Endocr 25: 798, 1965. Lobotsky J, Wyss HI, Segre EJ, Lloyd CW: Plasma testosterone in the normal woman. J Clin Endocr 24: 1261, 1964. Lloyd CW, Lobotsky J, Segre EJ, Kobayashi T, Taymor ML, Batt RE: Plasma testosterone and urinary 17.ketosteroids in women with hirsutism and polycystic ovaries. J Clin Endocr 26: 314, 1966. Bardin CW, Hembree WC, Lippsett MD: Suppression of testosterone and androstenedione production rates with dexamethasone in women with idiopathic hirsutism and polycystic ovaries. J Clin Endocr 28: 1300, 1968. Casey JH, Burger HG, Kent JR, Kellie AE, Moxham A, Nabarro J, Nabarro JDN: Treatment of hirsutism by adrenal and ovarian suppression. J Clin Endocr 26: 1370, 1966. Wieland RG, Vorys N, Folk RL, Besch PK, Neri A, Hamwi GJ: Studies of female hirsutism. Amer J Med 41: 927, 1966.
175
BRUCE ETTINGER, M.D. KLAUS von WERDER, M.D.* GUY C. THENAERS, M.D.? PETER H. FORSHAM. M.D. San Francisco, California
From the Metabolic Research Unit, General Clinical Research Center, and Department of Medicine, University of California, San Francisco, California 94122. This paper was supported in part by the Levi J. and Mary Skaggs Foundation. Studies were carried out in the General Clinical Research Center of the University of California, San Francisco, supported by a grant from the National Institutes of Health (RR-79). Requests for reprints should be addressed to Dr. P. H. Forsham, Metabolic Research Unit, 1143-HSW, University of California, San Francisco, Calif. 94122. Manuscript received August 17, 1970. * Present address: Second Medical
Clinic, University of Munich, Munich, Germany. f Present address: 67, Genebos, 3920 Lummen,
170
Belgium.
Of forty-seven hirsute women referred to a metabolic clinic, 60 per cent were found to have excessive levels of testosterone in the plasma, yet only 20 per cent of this group had increased urinary excretion of testosterone. The plasma testosterone levels served as a useful screening test for hirsutism. Nineteen patients with hirsutism were further evaluated using a six day protocol for adrenal suppression-stimulation and ovarian stimulation (human chorionic gonadotropin), as well as ovarian visualization procedures. These tests gave inconsistent results in individual cases and no general patterns of response: they were of no use in predicting the pathologic source of testosterone. Hirsutism in women, frequently associated with menstrual abnormalities, is a common diagnostic problem. The relevance of excessive testosterone production in this syndrome has been stressed by many investigators [l-3]. It has been well documented that the adrenals [4] and ovaries [5] are capable of secreting considerable amounts of testosterone. Since there is no evidence that excessive peripheral conversion of androgenie precursors (e.g., ,dehydroepiandrosterone and Ad-androstenedione) accounts for elevation of testosterone levels [6-lo], investigation of the anatomic source of excessive androgen production might focus treatment on the secretory organ(s) responsible. We report our experience in screening hirsute women for excessively high levels of testosterone and attempt to define the pathologic source by the patterns of testosterone response. SUBJECTS AND METHODS Subjects.
Hirsute patients referred to the Metabolic Clinic were screened by twenty-four hour urinary and plasma1 testostero,ne determinations, while receiving no treatment. Significant hirsutism was defined as (1) facial hair growth with “side burns,” beard or anterior neck hair, (2) intermammalry hair growth, and (3) male hair distribution on arms, legs and trunk. In all patients hair growth was of typical male distribution and texture. Patients showing periareolar hair growth or a male escutcheon only were exclu,ded since such a finding
The American
Journal
of Medicine
PLASMA TESTOSTERONE
is common in normal women. Menstrual abnormalities were present in all the patients selected, usually oligomenorrhea. Obesity (greater than 50 per cent above ideal weight) was noted in 75 per cent of our patients. Base line urinary 17-hydroxycorticosteroids (17. OHCS [ll], 17-ketosteroids (17.KS) [12] and pregnanetriols [13] were measured, and appropriate tests were used to rule out Cushing’s syndrome, 21p- and lip-hydroxylase deficiencies, and other endocrinopathies. Levels of urinary testosterone were measured by gas-liquid chromatography of an acetate derivative of testosterone glucuronide [14]. Plasma testosterone was determined by a radioligand method [15], as modified by Maeda et al. [16]. Twenty-five units of ACTH was infused intravenously for eight hours, and plasma testosterone was measured at the end of the infusion. Plasma ll-OHCS [17] levels were determined to document adequate adrenal stimulation. The adrenals were suppressed by the oral administration of dexamethasone, 1.0 mg every six hours for two days. At the end of the suppression period, blood was drawn for testosterone and llOHCS determinations; the latter assured adequacy of adrenal suppression. The ovaries were stimulated for two days (3,000 units human chorionic gonadotropin [HCG], intramuscularly-Follutein@, Squibb, luteinizing hormone activity: 0.924 IU/unit HCG), while adrenal suppression was maintained. Four hours after the second administration of HCG, plasma testosterone was measured. This sampling time was determined after analyzing the plasma testosterone hourly, following intramuscular administration of HCG in several women with idiopathic hirsutism and Stein-Leventhal ovaries, and in three normal women. If clinically indicated, tomography of the adrenals was performed. When ovarian pathology was suspected and adequate pelvic examination was precluded by obesity, a pelvic pneumogram was obtained using the technic of Schulz and Rosen [18]. When indicated, further study by laparoscopy, via a transperitoneal fibroscope insertion, allowed direct visualization of the ovaries. In six of eight patients with a roentgenologic diagnosis of SteinLeventhal ovaries, wedge resection was performed and the diagnosis confirmed.
RESULTS Screening Tests. In 60 per cent of patients referred to us for hirsutism and menstrual irregularity, the basal plasma testosterone level was clearly elevated (Figure 1). However, urinary testosterone excretion was borderline or increased in only a third of the patients with elevated plasma levels. Twenty per cent of patients had borderline abnormal plasma testosterone levels, and usually had normal or slightly increased urinary testosterone excretion. Twenty per cent of patients had
Volume
51, August
1971
PLASMA mfig/lOOml
IN HIRSUTE
WOMEN -
URINE fig/24
hr
ETTINGER
PLASMA
ET AL.
URINE
m~g/lOOml
Agl24
hr
200
100
Patients STEIN
OVARIES
Figure 1. performed Leventhal hirsutism.
with
- LEVENTHAL n=12
Patients
with
IDIOPATHIC HIRSUTISM
n=35
Plasma and urinary
base line measurements on the same day in twelve patients with Steinovaries and thirty-five patients with idiopathic Shaded areas represent normal levels.
normal plasma testosterone levels, and with rare exception had normal urinary testosterone excretion. There was no quantitative relation between the basal urinary 17-OHCS, 17-KS and pregnanetrio1 levels, and the testosterone excretion (Table I) [l]. Base line laboratory tests did not differentiate idiopathic hirsutism from Stein-Leventhal ovaries. Plasma and urinary testosterone levels did not correlate with obesity. Stimulation-Suppression Tests. To compare the patterns of stimulation and suppression tests we arbitrarily established the following criteria for response: (1) doubling of base line testosterone levels after ACTH infusion, (2) halving of base line testosterone level after dexamethasone suppression, and (3) doubling of the post dexamethasonesuppressed base line testosterone level by HCG stimulation. Table II demonstrates the dynamics of plasma testosterone in three normal women, eight patients with Stein-Leventhal ovaries and eleven patients with idiopathic hirsutism. A summary of test responses in eight patients with Stein-Leventhal ovaries revealed (1) 50 per cent adrenal suppression in one patient and 40 per cent suppression in three other patients, (2) ACTH response in two patients, and (3) response to HCG in four of seven tested. Categorizing responses as “adrenal” if ACTH and dexamethasone
171
PLASMA TESTOSTERONE
TABLE I
IN HIRSUTE
WOMEN-ETTINGER
Basal Urinary Steroids and Plasma Patients with Idiopathic Hirsutism
ET AL
Testosterone
in Eight Patients
with Stein-Leventhal
Ovaries
Urinary
Patient
17-OHCS (mg/24 hr)
Patients S.B. M.M. V.Y. D.Ro D.Re M.H. P.H. J.D.
5.2 9.7 14.7 13.9 9.2 10.4 18.2 7.3
N.P. N.G. J.F. J.De L.A. J.G. E.B. L.B. P.J. G.H. AX. Normal range
6.6 10.0 13.6 20.5 14.9 13.9 7.3 6.6 16.0 8.9 10.3 5-12
Pregnanetriol (mg/24 hr)
17-KS (mg/24 hr)
with Stein-Leventhal
5.0 8.5 8.5 7.5 11.4 11.5 16.6 10.3 Patients
with Idiopathic
responses were noted and as “ovarian” if HCG response was noted, the individual patterns of response in these patients with Stein-Leventhal ovaries were: ovarian, three patients; adrenal, one; mixed ovarian/adrenal, one; and no significant response to any test, three. Ten of eleven patients with idiopathic hirsutism showed adrenal or ovarian responses, or both (Table II). Adrenal suppression response was definite in two patients and borderline in three. Six of eleven responded to ACTH, but four of the six showed no adrenal suppression. Stimulation with HCG occurred in seven of eleven patients tested. In the patients responding, the individual patterns were as follows: ovarian, two patients; adrenal, none; mixed adrenal/ovarian, two; inconsistent adrenal (stimulation with ACTH without suppression by dexamethasone, or vice versa), three: and inconsistent adrenal/ovarian, three. In twelve of the hirsute patients and in three normal subjects, studies of urinary testosterone excretion were made at the same time that the plasma testosterone was being measured. Only four patients showed stimulation with ACTH (doubling of basal excretion, and at least 5’Pg/ twenty-four hours), but none showed suppression
172
(424
hr)
Plasma Testosterone (m&100
ml)
Ovaries
0.4 1.6 2.6 0.9 2.7 2.2 0.7 1.8
9.1 8.6 18.9 11.6 19.7 13.4 7.7 6.7 7.8 9.8 12.0 5-15
Testosterone
and in Eleven
16.1 11.4
118 &l 126 127 225 130 172 215
1.2
378 267 82 65 99 276 185 52 99 93 288 40-100
Hirsutism 3.2 2.1 3.3 2.4 2.3 2.6 2.6 1.5 0.4 3.4 1.9
to 50 per cent of base line levels with dexamethasone. In four patients urinary testosterone excretion was suppressed with dexamethasone but in none was there stimulation with ACTH. Two patients with Stein-Leventhal ovaries showed stimulation with HCG and ACTH but not suppression with dexamethasone. The remaining patients showed no significant changes in urinary testosterone excretion with any of the test maneuvers. Thus, in all twelve patients the urinary testosterone responses yielded no clinically useful or consistent data. COMMENTS Base Line Studies. The usefulness of the plasma testosterone determination as a screening procedure in hirsute women is shown. Two-thirds of the hirsute patients studied were found to have normal basal urinary testosterone excretion, yet more than half this group had elevations in plasma testosterone levels. Normal levels of plasma testosterone have been accompanied by normal urinary testosterone excretion, with rare exceptions. Hence, plasma testosterone is a more sensitive indicator of hypertestosteronism and rarely yields falsely negative results. In our series,
The American
Journal
of Modiclne
PLASMA TESTOSTERONE
Testosterone Dynamics in Three Normal Women, Eight Patients with Stein-Leventhal with Idiopathic Hirsutism Base Line
Patient
Plasma (mFg/lDO ml)
ACTH Stimulation
UrinZ (jig/24 hr)
Plasma (m&CO
Dexamethasone Suppression Plasma
Urine b&!/24 hr)
ml)
(m&100
Normal J.S. M.M. Y.V.
56 91 46
1.0 0.5 2.0
292 229 239
S.B. M.M. V.Y. D.Ro D.Re M.H. P.H. J.D.
118 94
16.1 11.4
138 74 131 414 151
1.5 0.3 1.8
Urine @g/24 hr)
ml)
126 127 225 130 172 215
... 260 739
... 2.4 ... ...
...
1.0 0.3 1.1
31 86 91
331 116 154 79 129 79 214 105
Patients with Idiopathic N.P. N.G. J.F. J.D.E. L.A. J.G. E.B. L.B. P.J. G.H. A.C.
* h = inconsistent
Volume
1.2
378 267 82 65 99 276 185 52 99 93 288
adrenal
51, August 1971
749 260 267 172 113 762 53 260 326 238 194
stimulation-suppression
... ... ... 22.2 10.0
ETTINGER
ET AL.
Ovaries and Eleven Women
HCG Stimulation Urine
Plasma (m&/100
ml)
r&z/24 hr)
Plasma Testosterone Pattern*
Subjects
Patients with Stein-Leventhal 41.4
WOMEN -
version of dehydroepiandrosterone may also account for 15 per cent of the testosterone produced [8,9]. Excluding the peripheral conversion of other precursors, as yet unknown, glandular secretion of testosterone is likely to be the important cause of hypertestosteronism. Dexamethasone, 0.5 mg orally every six hours for two days, is sufficient to suppress ACTH-dependent adrenal steroid production in normal subjects [22]. In all our patients urinary 17-OHCS and 17-KS excretion was adequately suppressed. The plasma testosterone levels, after adrenal stimulation and suppression in the normal and hirsute women studied, were quite similar to those reported by Casey and Nabarro [23] who showed that despite inconstant dexamethasone suppression, significant ACTH stimulation of plasma testosterone occurred in normal women. These results are in conflict with the majority of reports [24-271, however, which indicate minimal response of the plasma testosterone to adrenal stimulation in normal women. In one of five patients with idiopathic hirsutism and high basal
obesity did not affect the plasma testosterone level. These findings are in agreement with other investigators using either the radioligand method or the double-isotope technic [16,19,20]. All base line determinations were performed in patients who were not taking estrogenic or progestational agents, since these are well known to influence plasma testosterone levels. For similar reasons, plasma testosterone, after estrogen suppression, could not be determined. All patients studied were shown to be euthyroid by appropriate tests. This obviates the problem of false elevation or depression of plasma testosterone noted in hyperthyroidism and hypothyroidism, respectively [21]. The source of testosterone in normal and hirsute women has been partially elucidated [5-lo]. In the normal woman, a,-androstenedione is peripherally converted to testosterone, accounting for 50 to 60 per cent of the total testosterone production. In the hirsute woman, A,-androstenedione production may be twice normal, yet its conversion to testosterone accounts for only 20 to 25 per cent of testosterone production [5,7]. ConTABLE II
IN HIRSUTE
234 172 70 100 60 284 64 41 119 240 193
191 261 251
Adrenal-ovarian Adrenal-ovarian Adrenal-ovarian
310 230 110 276 764 640 85 151
48.5 8.8
None Ovarian None Adrenal-ovarian Ovarian Ovarian None Adrenal
303 538 165 578 423 1,323 188 394 63 243 133
1.2 2.0 5.2 5.2 8.5 7.5 ...
Ovaries 29.4 14.2 1.2 ...
...
...
... ... 3.5 43.2
Hirsutism
... ...
... 14.6 14.1
... 25.5 3.0
+ Adrenal Ovarian Adrenal-ovarian Z!Z Adrenal-ovarian Ovarian rfr Adrenal-ovarian It Adrenal-ovarian Adrenal-ovarian z!= Adrenal =t Adrenal None
responses.
173
PLASMA TESTOSTERONE
IN HIRSUTE
WOMEN-ETTINGER
ET AL
plasma testosterone levels, excessive testosterone levels were suppressed 50 per cent or more with dexamethasone, whereas three others showed only slight suppression. In our patients with idiopathic hirsutism (unlike those reported by Casey and Nabarro), dexamethasone suppressiveness of testosterone did not correlate with ACTH-responsiveness of testosterone. The converse also held, since of the seven patients with idiopathic hirsutism who responded to ACTH, dexamethasone suppression was noted in none. In similar testing of patients with Stein-Leventhal ovaries, plasma testosterone levels were suppressed with dexamethasone in one of eight and stimulated by ACTH in two of seven. The lack of suppression and the inconsistency of adrenal stimulation and suppression responses agree with the findings of Lloyd et al. [27]. We agree with Bardin et al. [26] that acute changes in the plasma testosterone level are largely independent of endogenous ACTH in both normal women and those with hypertestosteronism. Thus the testosterone contribution of the adrenal glands cannot be assessed by short-term ACTH suppression. In all three normal subjects plasma testosterone was stimulated with HCG. This was also true in four of eight patients with proved ovarian pathology and seven of eleven with idiopathic hirsutism. The plasma testosterone response to HCG shows no correlation with the anatomic lesion. HCG stimulation has been variously reported to elevate or to have no effect on plasma testosterone levels in normal subjects and in patients with idiopathic hirsutism or Stein-Leventhal ovaries [23-25,271. The results of our studies in patients with SteinLeventhal ovaries and idiopathic hirsutism concur with previous reports showing a marked hetero-
geneity of responses of plasma testosterone. Furthermore, the day to day variation in plasma testosterone averaged 50 mpg/100 ml in our patients and 60 mpg/100 ml in normal subjects reported by others [23,24,26]. A mid-cycle increase in plasma testosterone has been inconstantly reported [23,24,26]. Although the response of plasma testosterone to suppression/stimulation tests appears to correlate with testosterone production rates [28], there are marked inconsistencies of responses to both adrenal and ovarian test procedures in hirsute women. Similarly, measurements of changes in urinary testosterone excretion during various suppression and stimulation maneuvers have not yielded clinically useful data. The determination of plasma testosterone appears to be a sensitive screening test for hypertestosteronism in women. It could prove valuable in providing a rapid chemical means of assessing the need for, and the adequacy of, treatment of hirsutism [29], even though there is good evidence for the contrary [27]. We doubt its clinical value in the performance of any short-term stimulation/ suppression tests of testosterone dynamics in the hirsute woman. We agree with other investigators [28-301 that the patterns of response elicited in such testing have no value in predicting the pathologic source of testosterone and do not appear to offer a rational guide to the choice of therapy. Although studies of urine and plasma testosterone response can show patterns in the means of groups of patients [2,27,30], there is marked inconsistency in each individual case. ACKNOWLEDGMENT We wish to thank Miss Jeanette Shinsako and Mr. Shiro Horita for their technical assistance.
REFERENCES 1.
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174
Migeon CJ, Kirschner MA, Bardin CW: Physiologic basis of disorders of androgen metabolism. Ann Intern Med 68: 1327, 1968. Deller JJ, Wegienka LC, Conte NF, Rosner JM, Forsham PH: Testosterone metabolism in idiopathic hirsutism. Ann Intern Med 63: 369, 1965. Conrad SH, Lindberg MC, Herrmann WL: Significance of plasma dehydroisoandrosterone and androsterone sulfates in the diagnosis of virilizing disorders. Amer J Obstet Gynec 91: 449, 1965. Burger HG, Kent JR, Kellie AE: Determination of testosterone in human peripheral and adrenal venous plasma. J Clin Endocr 24: 432, 1964. Migeon CJ, Saez JM, Rivarola MA: Comparison of
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