Estrogen-Androgen Balance in Hirsutism

FERTIUTY AND STERILITY Copyright © 1979 The American Fertility Society

Vol. 32, No.3, September 1979 Printed in U.SA.

ESTROGEN-ANDROGEN· BALANCE IN·HIRSUTISM

CHUNG H. WU; M.D.*

Department of Obstetrics and Gynecology, Uniuersity of Pennsyluania School of Medicine, Philadelphia, Pennsyluania 19104

Random blood samples were obtained from a group of patients having oligomenorrhea or secondary amenorrhea, who responded to progesterone with uterine withdrawal bleeding, associated with hirsutism (N = 35) or not associated with hirsutism (N = 9). A few blood samples were obtained during the midfollicular phase of normal cycles in age-matched normal females (N = 10) as controls for comparison. Plasma gonadotropins (follicle-stimulating hormone and luteinizing hormone [LHVand steroid hormones including testosterone (T), androstenedione (A), estradiol (E 2), estrone (E I ), and progesterone (P) were quantitated by radioimmunoassay. The percentage of free fraction of E 2 and T was determined by equilibrium dialysis at 37° C, and the percentage of T-E "binding globulin (TeBG}-bound fraction of E 2 and T was estimated by the specific steroid displacement-charcoal adsorption technique. The binding capacity of plasma TeBG was measured by the modified Scatchard plot technique. The E 2:T ratios of concentrations and the binding parameters were also evaluated. The weight (but not height) of hirsute females was significantly higher than that of normal females. Plasma LH, T, A, and E I levels were significantly elevated in hirsute females. Plasma T concentrations, including free, index, and total, correlated positively with degree of hirsutism. E 2:T ratios of concentration also suggested a similar, but negative, correlation. The percentage of free E2 increased more than the percentage offree T when the percentage of TeBG-bound E2 decreased more than the percentage of TeBG-bound T in hirsute females. The E2:T ratios of the percentage of free and TeBG-bound fractions did not show any correlation with degree of hirsutism. The T-binding capacity of plasma TeBG decreased in hirsute females and was also correlated significantly with degree of hirsutism. The elevated plasma T concentrations (including free, index, and total concentrations) in hirsute females overlapped significantly with that of normal females, thus they failed to discriminate each other. Fertil Steril 32:269, 1979

The testosterone (T) production rate in hirsute women is increased, 1 yet the T concentration in the peripheral blood of hirsute women is not always elevated. 1, 2 This difference is explained by the increased T metabolic clearance rate, 1, 3 perhaps secondary to the decreased testosterone-estradiolbinding globulin (TeBG) in hirsute women. 3 Plasma TeBG concentration is influenced by estradiol (E 2) and T. Elevated E2 stimulates TeBG

synthesis in the liver, thus peripheral TeBG concentration increases in the hyperestrogenic state; when T suppresses TeBG synthesis, the TeBG concentration in plasma decreases in the hyperandrogenic state. TeBG, because of its high affinity for E2 andT, plays a significant role in controllhig the biologic activity of these two sex hormones. TeBG seems to amplify the biologic activity of E2 and T by a self-serving phenomenon. 4 Follicular hair growth is stimulated by androgen and suppressed by estrogen, 5 and a delicate estrogenandrogen balance may playa role in hair growth in hirsute women. A study was thus carried out to elucidate the estrogen-androgen balance in a group of hirsute women with menstrual disorders.

Received February 16, 1979; accepted April 9, 1979. *Reprint requests: Dr. Chung H. Wu, Department ofObstetrics and Gynecology, Hospital of the University ofPennsylvania, Philadelphia, Pa. 19104.

269

wu

270 MATERIALS AND METHODS

Hirsute women with menstrual disorders (oligomenorrhea or secondary amenorrhea) who responded to progesterone injection by uterine withdrawal bleeding were recruited from the fertilitygynecologic endocrine service at the Hospital of the University of Pennsylvania. Patients between the ages of 18 and 27 were included to narrow the age range. The degree of hirsutism was described as mild (mild facial and lower abdominal hirsutism), moderate (moderate facial and lower abdominal hirsutism with periareolar hirsutism), or severe (marked facial, lower abdominal and periareolar hirsutism with or without clitor~meg­ aly. Age-matched women with menstrual disorders without hirsutism (N = 9) and age-matched normal females with normal ovulatory cycles (N = 10) were included as control groups. All patients had normal thyroid function and normal skull xrays of the sella turcica area. Random blood samples were obtained from these patients between 8 and 11 A.M., and the plasma was separated and frozen at -20 C until assayed. Concentrations of plasma gonadotropins (follicle-stimulating hormone [FSH] and luteinizing hormone [LH] and steroid hormones (including T, androstenedione [A], E 2, estrone [E I], and progesterone [P]) were measured by radioimmunoassay.6.10 The percentage of free fraction of E2 and T was determined by equilibrium dialysis at 37 C, and the precentage ofTeBG-bound fraction ofE2 and T was estimated by the specific steroid displacement charcoal adsorption technique. II The binding capacity of plasma TeBG was measured by the modified Scatchard plot technique. l l Statistical analysis was carried out by Student's t-test for differences between means, and linear regression was applied for the correlation analysis. 0

0

RESULTS

Height and Weight. Means ± standard error for

September 1979 age, height, and weight of these patients are summarized in Table 1. There was no statistical difference in age, as they were age-matched groups. The heights of these groups of patients also showed no significant difference. However, the weights of hirsute women tended to be higher than those of patients without hirsutism. There was a trend to suggest that the more severe the hirsutism, the higher the body weight. Plasma Hormones. E2 levels in patients with menstrual disorders, either with or without hirsutism, were comparable to those in the midfollicular phase of normal cycles and were not significantly different from each other. Therefore, normal female control values were obtained from the midfollicular phase of normal cycles for comparison. The plasma concentrations of the hormones, including LH, FSH, T, A, E 2, Eh and P, of these patients are summarized in Table 2. As compared with normal female values, there was a significant increase in LH levels in moderately and severely hirsute females. Plasma T, A, and EI levels were also elevated in hirsute females, with some correlation with degree of hirsutism. Estradiol and Testosterone Concentration. E2 and T concentrations, including total, index, and free concentrations, and the E2:T ratio of these three concentration parameters, are summarized in Figure 1. A parallelism was observed among the three concentration parameters in relation to E 2, T concentration, and even the E2:T ratio. Markedly elevated T levels yet relatively unchanged E2 levels were noted in the hirsute women. The E2:T ratios of these concentration parameters showed significant decreases in hirsute females as compared with normal females. Slightly elevated free and total T concentrations were also observed in women with menstrual disorders without hirsutism. Estradiol arid Testosterone-Binding Parameters. The percentage of free and percentage of TeBGbound fractions plus binding capacity of TeBG for

TABLE 1. Age, Height, and Weight of Patients Having Menstrual Disorders with and without Hirsutism a Menstrual disorders Normal females (N

~

10)

With hirsutism Without hirsutism (N

Age (yr) Height (em) Weight (kg)

23 + 1.1 159 ± 1.3 55.7 ± 1.3

"Values are means ± standard error. bp < 0.05 as compared with the normal value. cp < 0.01 as compared with the normal value.

~

9)

23 ± 1.4 161 ± 1.5 63.0 ± 5.9

Mild (N

~

10)

22 ± 1.0 159 ± 1.5 65.7 + 3.2b

Moderate (N

13)

23 ± 1.2 164 ± 2.5 66.6 + 3.4b

Severe (]V

12)

21 ± 1.2 161 ± 1.8 81.1 + 6.8c

Vol. 32, No.3

ESTROGEN-ANDROGEN BALANCE IN IDRSUTISM 116

E ..... a. '" ....I <[

b I-

0.1

o 0.3

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NORMAL WITHOUT FEMALE \ HIRSUTISM

WITH HIRSUTISM

271

definition of the degree of hirsutism was arbitrary and subject to error, an attempt was made in this group of patients to correlate the endocrine parameters with the clinical subjective observation of the degree of hirsutism. Only those patients with menstrual disorders with or without hirsutism were included in this correlation analysis W = 44). The correlation coefficients and statistical analysis are summarized in Table 3. A significant correlation was noted in free T, T index, and total T concentrations, but only a fair and negative correlation was observed between degree of hirsutism and percentage of TeBG-bound T and T-binding capacity. In E2 parameters only the percentage of free E2 showed a positive correlation; the percentage of TeBG-bound E2 showed a significantly negative correlation. Although there was a significant decrease in E2:T ratios of concentration parameters in hirsute females, all of the E2:T ratio parameters failed to show any significant correlation with degree of hirsutism. In addition, there were some positive correlations observed between degree of hirsutism and plasma A, Eh and P concentrations as well as body weight of the patients.

MENSTRUAL DISORDER

FIG. 1. Plasma E. and T concentrations and their E.:T ratios in normal females and women with menstrual disorders associated with or without hirsutism.

E2 and T, and their ratios, are summarized in Figure 2. A significant increase in the percentage of free E2 was observed in patients with menstrual disorders, either with or without hirsutism, and a slightly increased percentage of free T was noted only in the severely hirsute females. The E2:T ratio of the percentage offree fraction did not show any trend. The percentage ofTeBG-bound E2 decreased significantly in all hirsute women, and even in women with menstrual disorders without hirsutism. The decrease in percentage ofTeBG-bound T was observed only in severely hirsute females. The E2 T ratio of percentage of TeBG-bound fraction decreased in those moderately and severely hirsute females as well as in patients without hirsutism. A significant decrease in T-binding capacity of TeBG and a nonsignificant decrease in E 2-binding capacity of TeBG were observed in the moderately and severely hirsute females. Thus the E2:T ratio of this parameter did not show a significant difference in hirsute women; however, a slight increase in this E2:T ratio was noted in patients without hirsutism. Correlation Analysis. Although the clinical

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MENSTRUAL DISORDER

FIG. 2. The percentage of free and TeBG-bound fractions of plasma E. and T plus the binding capacity of TeBG and their . E.:T ratios in normal females and women with menstrual disorders associated with or without hirsutism.

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272

September 1979

TABLE 2. Plasma Hormone Concentrations in Patients Having Menstrual Disorders with and without Hirsutism a Plasma honnone

LH FSH T A E2 E, P

(mIU/ml) (mIU/ml) (pg/mD (ng/ml) (pg/ml) (pg/ml) (ng/ml)

Menstrual disorders

Normal females (midfollicular phase), days -5 to - 9 IN = 25)

6.0 ± 5.8 ± 308 ± 0.94 ± 85 ± 54 ± 0.61 ±

Without hirsutism IN = 9)

0.7 0.8 34 0.08 6.5 7.8 0.06

12.4 ± 5.2 ± 449 ± 0.94 ± 69± 87 ± 1.10 ±

4.0 1.2 45 C 0.24 19 16 0.32

With hirsutism Mild IN - 10)

16.5 ± 5.5 ± 762 ± 1.57 ± 67 ± 91 ± 0.96 ±

6.9 1.3 132b 0.26c 14 16" 0.10

Moderate IN -

14.0 ± 4.2 ± 705 ± 1.88 ± 82 ± 89 ± 1.23 ±

13)

2.8b 0.83 56 b 0.20b 13 11 c 0.37

Severe IN -

21.0 ± 6.4 ± 966 ± 2.43 ± 81 ± 129 ± 1.22 ±

12)

6.4c 1.0 116b 0.54b 12 17 b 0.26

aValues are means ± standard error. bp < 0.01 as compared with the normal value. cp < 0.05 as compared with the normal value.

Correlation analyses were also obtained between the plasma hormones in those patients with hirsutism (N = 35). The correlation coefficients and their statistical probability are summarized in Table 4. A positive correlation was noted between E1 and FSH, T, A, and E 2 • There were also significantly positive correlations between E2 and P, between A and FSH, T and LH, and between FSH and LH. Testosterone Concentration in Hirsutism. Since TABLE 3. Correlation Analysis between Endocrine Parameters and Degree a of Hirsutism in Women with Menstrual Disorders (N = 44) Endocrine parameter

coefficient

Statistical analysis

Free T T index Total T % Free T % TeBG-bound T T binding capacity

+0.464 +0.638 +0.446 +0.153 -0.229 -0.348

P < 0.01 P < 0.01 P < 0.01 NS P < 0.05 P < 0.01

Free E2 E2 index Total E2 % Free E2 %TeBG-bound E2 E2 binding capacity

+0.192 +0.185 +0.197 +0.254 -0.266 -0.221

NS NS NS P < 0.01 P < 0.01 NS

E2:T ratios Free Index Total % Free % TeBG-bound Binding capacity

-0.199 -0.150 -0.190 +0.123 -0.145 -0.047

NS NS NS NS NS NS

Androstenedione Estrone Progesterone LH FSH Weight Height

+0.414 +0.292 +0.244 +0.001 +0.112 +0.217 -0.203

P < 0.01 P < 0.01 P < 0.01 NS NS P < 0.05 NS

Correlation

aDegrees of hirsutism are numerically assigned as 0, 1,2, and 3 for none, mild, moderate, and severe hirsutism, respectively.

the plasma T concentration showed a good correlation with degree of hirsutism, the individual data ofT concentrations, including free T, T index, and total T, are plotted in Figure 3 for comparison. Although there was a trend toward increased concentrations in these parameters as degree of hirsutism advanced, there were approximately 57% of free T, 40% ofthe T index, and 51% ofthe total T concentration in hirsute females overlapping into the range of normal females. Therefore, despite the evidence of elevated T and an increasing trend ofT concentration as hirsutism advanced, the concentration measurement, regardless oftotal T, the T index, or even free T as the parameter, was unable to distinguish normal females from hirsute females. DISCUSSION

Increased body weight in hirsute females could be explained by the anabolic effect of T; however, obesity with oligomenorrhea itself can also increase androgen production. 12 Therefore it is not clear whether androgen causes weight gain or weight gain increases androgen production. However, a definite correlation was found. The slightly elevated LH levels in these hirsute females were probably due to the frequent clinical diagnosis of polycystic ovarian disease (PCO). Approximately two-thirds of PCO patients have elevated LH levels. 13,14 The elevated T, A, and El levels observed in these hirsute females may also be related to the characteristic findings of these hormones in PCO.13,14 Progesterone levels in the hirsute females, although suggesting anovulation (less than 3 ng/mI) , were slightly higher than during the preovulatory follicular phase levels of normal cycles. This phenomenon can be explained by possible luteinization of some follicular component due to elevated LH levels. The plasma E2 levels in

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273

ESTROGEN-ANDROGEN BALANCE IN IDRSUTISM

TABLE 4. Correlation Analysis of Plasma Hormones in Hirsute Females (N LH

LH

FSH

1.00

T

+0.247 a P < 0.05 b 1.00

FSH T

E,

A

+0.249 P < 0.05 +0.138 NS 1.00

+0.002 NS +0.309 P < 0.01 +0.076 NS 1.00

A

=

35) E,

+0.083 NS -0.199 NS -0.141 NS -0.107 NS 1.00

P P P P

+0.086 NS +0.252 < 0.05 +0.238 < 0.05 +0.355 < 0.01 +0.390 < 0.01 1.00

p

p

+0.040 NS -0.066 NS +0.085 NS +0.127 NS +0.363 P < 0.01 -0.048 NS 1.00

aCoITelation coefficient (y). bProbability; NS, not significant.

normal females. Free T concentrations in the hirsute females were reported 16 to be elevated, and not overlapping those of normal females; however, this phenomenon was not confirmed in this study. Perhaps the differences in methodology for determining the percentage offree T may playa role in this discrepancy. A decreased E2:T ratio of total and free steroid concentration in hirsute females was previously reported. 15 These results were confirmed in this study and, in addition, the E2:T ratio of index concentration was also found to be decreased. The correlation analysis failed to show significance between degree of hirsutism and decrease in E2:T ratio of concentration parameters, although there

hirsute females showed a decrease in a previous report,15 yet this was not confirmed in this study. Instead, the E2 levels tended to stay in the midfollicular phase range. This discrepancy cannot be explained, or possibly is due to different patient groups. Because of the E 2 level in these hirsute females, normal control data were obtained from the midfollicular phase for comparison. Plasma T concentrations, including free T, T index, and total T, were definitely elevated in hirsute females, and even correlated with degree of hirsutism. However, the significant overlapping of these values with that in normal females makes this parameter unfavorable to distinguish hirsute females from

TOTAL T

T INDEX

FREE T P9/ml

P9/ml

P9/ml

80

800

1600

60

600

1200

~1850

-I-

40

400

800

20

200

400

o

N

9

S

I

t

"It fit,

H. c HIRSUTISM, MENSTRUAL DISORDER

I

OL-~~--~~~-

N

S

~

IH.

It it fit,

c HIRSUTISM,

MENSTRUAL DISOROER

O~A-~~~~~-

N

<;j?

S

It it itt,

c HIRSUTISM, MENSTRUAL DISORDER IH.

FIG. 3. Individual plasma T concentrations, including free T, T index, and total T, in normal females and women with menstrual disorders associated with or without hirsutism. Horizontal lines indicate mean levels; uerticallines include the ranges.

274

wu

was a slight trend toward a decrease in these E2:T ratios. Thus the E2:T ratio of concentration (total, index, or even free) can be utilized to discriminate between hirsute and normal females, yet they cannot be used to differentiate degrees of hirsutism. The increased percentage of free T fraction in hirsute females 15 was confirmed in this study; furthermore, a significantly increased percentage of free E2 was also observed in hirsute females. The magnitude of the increase in the percentage offree E2 was greater than that in the percentage offree T. This can be explained by the different affinity of TeBG to T and E 2, i.e., higher affinity to T than to E2. 4 Because of parallel shift in percentage offree T and percentage of free E 2, the E2:T ratio of this parameter seems useless for evaluating hirsute females. The percentage ofTeBG-bound E2 drastically decreased in hirsutism, whereas that ofT was only slightly decreased. Therefore, the E2:T ratio of this parameter did reveal a significant decrease in hirsute females. The drastic decrease in percentage of TeBG-bound E2 in hirsutism as compared with the only slight dect:ease in the T fraction can also be explained by the differences in affinity ofE 2 and T to TeBG. The elevated T level suppressed TeBG synthesis, thus the concentration ofTeBG in the plasma decreases, which subsequently decreases the percentage ofTeBG-bound T and E 2. In addition, elevated T, because of its higher affinity to TeBG, may displace competitively the TeBGbound E 2, thus the fraction ofE 2bound to the TeBG is further reduced. Therefore measurement of percentage ofTeBG-bound E2 may reflect or discriminate better between the normal and hirsute females than measurement of percentage of TeBG-bound T. The TeBG concentration in a plasma sample should be a constant, thus the specific binding sites for sex steroids, either E2 or T, should also be a constant. Therefore, if the binding capacity of TeBG is expressed as the TeBG "concentration" itself, it should be the same regardless for E2 or T. However, if the "functional" binding capacity of existing TeBG in the plasma is considered, perhaps the different concentrations of E2 and T present in plasma may affect the binding capacity of the existing TeBG to the additional exogenous E2 or T. The Scatchard plot technique, as used in this study to evaluate the binding capacity, did yield different values for E2 and T. This difference was approximately 80% (i.e., E2~binding capacity = 80% of T-binding capacity) in normal female plasma. 17 If we accept the measurement of binding

September 1979 capacity of TeBG as a functional phenomenon rather than concentration ofTeBG, then there was a definite decrease in T-binding capacity of TeBG in hirsute females as compared with normal females, yet only an insignificant decrease was observed in the E 2-binding capacity ofTeBG in the same samples. Therefore, despite the fact that the concentration of TeBG in a plasma sample is a constant, its functional binding capacity to E2 or T can be varied or influenced by the existing T and E2 in the plasma sample. In a hyperandrogenic state such as hirsutism, the decrease in T-binding capacity may reflect the decreased TeBG concentration, while the E 2-binding capacity of TeBG may not reflect the TeBG concentration well. If the displacement technique is used to quantitate the binding capacity of TeBG, perhaps the T-binding capacity is more meaningful than the E 2-binding capacity. A good correlation was observed in T concentration parameters and degree of hirsutism, and the T index gave the best correlation (r = +0.63) as compared with free T (r = +0.464) or total T (r = +0.446). The difficulty involved in grading the degree of hirsutism by subjective clinical observation might explain this magnitude of correlation despite the knowledge that T stimulates hair growth in the hair follicles. The significant fluctuation of T concentration observed in daily and/or hourly samples may also reveal a significant variation in random blood samples obtained in this study. A better correlation between the degree of hirsutism and the precentage ofTeBG-bound E2 as compared with that versus the percentage of TeBG-bound T observed in this study also suggests that in hirsute females the percentage of TeBGbound E2 fraction will reflect the degree of hirsutism (although negatively) better than the T fraction. Whereas the percentage of free E2 showed some positive correlation with hirsutism, the percentage of free T failed to show any correlation. This may also be explained by differences in the affinity of these steroids to TeBG. The significant correlations observed in A, Eb and P concentrations with the degree of hirsutism may possibly be due to the clinical diagnosis of pea in some of these hirsute females. The slight increase in LH levels observed in moderately or severely hirsute females can be explained by the pulsatile release of LH18 and its short biologic half-life causing variation in random blood samples. Thus when degree of hirsutism was compared with LH level, a correlation could not be shown. Gonadotropins and steroids evaluated in this

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ESTROGEN-ANDROGEN BALANCE IN IDRSUTISM

study may mutually be related in the feedback system or in the metabolic pathway; therefore, an attempt was made to analyze the mutual correlation ofthese hormones only in those patients with hirsutism. A slightly significant correlation observed between LH and FSH could be explained by their common hypothalamic releasing hormone (GnRH) or it could be coincidental. The correlation observed between LH andT is perhaps due to LHdependent T secretion in the thecal component of ovarian follicles, and both LH and T are known to be elevated in patients with PCOP' 14 Significant correlations between FSH and A, and between FSH and E 1, observed in this group of hirsute females are difficult to explain. However, the correlations between A and E1 and between E2 and E1 are explainable by their related metabolic pathway, and the correlation between T and E1 can also be indirectly explained by their metabolic relation. The correlation between E2 and P may suggest that perhaps a large portion of progesterone in hirsute females may derive from the ovaries rather than the adrenals, as the primary source ofE 2is known to be the ovary. The failure to see any correlation between FSH and E 2, although it is known to be a sensitive negative feedback system, is attributed to the fact that the midfollicular phase range of E2 in these females may have sufficiently suppressed the FSH level to the low-normal range, and only a minimal amount of FSH is released from the pituitary. The relatively low correlation observed with all of these hormones is perhaps due to a biologic variation in the plasma concentration of these steroids plus the narrow range of hormone concentrations we were dealing with. Thus a high degree of correlation is unlikely even if some correlation exists. Therefore, it cannot be overemphasized that interpretation of these correlations between hormones in hirsute females should be cautiously exercised. REFERENCES 1. 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

275

2. 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 Endocrinol Metab 26:314, 1966 3. Vermeulen V, Verdonck L, Van der Straeten M, Orie N: Capacity of the testosterone-binding globulin in human plasma and influence of specific binding of testosterone on its metabolic clearance rate. J Clin Endocrinol Metab 29:1470, 1969 4. Burke CW, Anderson DC: Sex-hormone-binding globulin is an oestrogen amplifier. Nature 240:38, 1972 5. Casey JH, Moxham A, Nabarro JDN: Idiopathic hirsutism: seasonal variation of hair growth and response to oestrogen administration. Lancet 1:587, 1964 6. Midgley AR: Radioimmunoassay for human folliclestimulating hormone. J Clin Endocrinol Metab 27:295, 1967 7. Midgley AR: Radioimmunoassay: a method for human chorionic gonadotropin and human luteinizing hormone. Endocrinology 79:10, 1966 8. Wu CH, Blasco L, Flickinger GL, Mikhail G: Ovarian function in the preovulatory rabbit. BioI Reprod 17:304, 1977 9. Wu CH, Lundy LE: Radioimmunoassay of plasma estrogens. Steroids 18:91, 1971 10. DeVilla GO Jr, Roberts K, Wiest WG, Mikhail G, Flickinger GL: A specific radioimmunoassay of plasma progesterone. J Clin Endocrinol Metab 35:458, 1972 11. Wu CH, Motohashi T, Abdel-Rahman HA, Flickinger GL, Mikhail G: Free and protein-bound plasma estradiol-17.B during the menstrual cycle. J Clin Endocrinol Metab 43:436, 1976 12. Hosseinian AH, Kim MH, Rosenfield RL: Obesity and oligomenorrhea are associated with hyperandrogenism independent of hirsutism. J Clin Endocrinol Metab 42:765, 1976 13. De Vane GW, Czekala NM, Judd HL, Yen SSC: Circulating gonadotropins, estrogens, and androgens in polycystic ovarian disease. Am J Obstet Gynecol 121:496, 1975 14. Duignan NM: Polycystic ovarian disease. Br J Obstet Gynecol 83:593, 1976 15. Tulchinsky D, Chopra IJ: Estrogen-androgen imbalance in patients with hirsutism and amenorrhea. J Clin Endocrinol Metab 39:164, 1974 16. Paulson JD, Keller DW, Wiest WG, Warren JC: Free testosterone concentration in serum: elevation is the hallmark of hirsutism. Am J Obstet GynecoI128:851, 1977 17. Motohashi T, Wu CH, Abdel-Rahman HA, Marymor N, Mikhail G: Estrogen/androgen balance in health and disease. Am J Obstet Gynecol. In press, 1979 18. Rebar R, Judd HL, Yen SSC, Rakoff J, VandenBerg G, Naftolin F: Characterization of the inappropriate gonadotropin secretion in polycystic ovary syndrome. J Clin Invest 57:1320, 1976