Aust. N.Z. J. Med. (1978), 8, pp. 171-175 CASE REPORT
A Testosterone-Secreting Adrenal Cortical Adenoma H. C. Smith", S. Posent, P. Clifton-BlighS and J. Casey""
From the Department of Medicine, Sydney Hospital, and Garvan Institute, St. Vincent's Hospital, Sydney
Summary: A testosterone-secreting adrenal cortical adenoma. H. C. Smith, S. Posen, P. CliftonBligh and J. Casey, Aust. N.Z. J. Med., 1978, 8, pp.171175.
A
virilizing, gonadotrophin -responsive adrenal cortical adenoma was removed from a 54- year-old woman. Following removal of the tumour, which contained and secreted androgens and estradiol, serum gonadotrophins rose to menopausal levels and the patient experienced menopausal symptoms. Pre-operative attempts to localize the source of androgens were unsuccessful.
loss of scalp hair (Fig. 1) and a male type of facial hair distribution. Dark, coarse hair was found on the upper chest, breasts, abdomen, arms and legs. The muscle bulk was increased. The clitoris was minimally enlarged, while the uterus was normal in size and position. Both ovaries were palpable and considered to be of normal size. Serum electrolytes, uric acid and creatinine were normal. The haemoglobin was 17.1 g/lOO ml, the haematocrit 48'39/d. The serum cholesterol was 330 mg/100 ml. Chromosome analysis of peripheral leucocytes revealed a normal female karyotype (46, XX). After a series of investigations (see "Results"), the patient declined to have further tests or treatment and was not seen until four years later. When she reappeared in 1975 complaining of increasing libido, the clinical appearances were
Adrenal cortical adenomas rarely cause virilization.' Testosterone-secreting, gonadotrophin-responsive adrenal cortical adenomas in adult women are particularly rare and we found only three such cases in the English 33 A fourth patient is described in this paper. Case History
N.B. (SH. 353132), a 50-year-old married woman was referred in 1971 because of virilization. Shortly after the cessation of menstruation at 45 years of age she had noticed the growth of excess facial and body hair and, by the time of presentation. she shaved each day. When aged 47 years she noticed loss of scalp hair which progressed over the next three years to a pattern of advanced male baldness. She had also noted deepening of the voice, clitoral enlargement, acne, gain in body weight and increased libido. After her menarche at the age of 13 years, she had menstruated regularly but she had never succeeded in becoming pregnant. Vaginal examination and tuba1 insufflation had been performed at the age of 25 years and had revealed no abnormality. There was no other relevant past medical history. Physical examination showed a blood pressure of 130/80, a height of 163 cm and a weight of 61 kg. There was marked 'Endocrine Registrar, Sydney Hospital. tAssoc. Professor of Medicine, Sydney Hospital fEndocrinologist, Sydney Hospital. "Endocrinologist, St. Vincent's Hospital. Correspondence: Professor S. Posen, Sydney Hospital, Sydney, NSW 2000 Accepted for publication: 20 December, 1977
FIGURE 1.
Patient's appearance on presentation in 1971. The face and upper chest were shaved regularly.
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SMITH ET AL.
unchanged. The uterus and ovaries were removed in March 1976 (see “Results”) but their removal did not lead to any significant clinical or biochemical improvement. After further investigations (see “Results”), the right adrenal gland was removed by Dr. Ross Campbell in August 1976. It contained a bilobed partially encapsulated adenoma (Fig. 2). The left adrenal gland was palpated during the operation and was considered normal. Left adrenal exploration was therefore not performed. After her unilateral adrenalectomy the patient did not require glucocorticoid medication and signs of virilization gradually diminished.
FIGURE 2. Cut surface of the right adrenal gland containing the tumor which measured 4 . 5 ~ 3 . 0 ~ 3c m .0 and weighed 19 g. Assay Methods
The urinary 17 ketosteroids (17-KS) and 17 ketogenic steroids (17-KGS) were determined by the method of Appleby er aL5 The urinary total estrogens were assayed by the method of Outch et aL6 Until 1975, plasma testosterone was measured by competitive protein binding.’ After 1975 testosterone measurements were made by radioimmunoassay with an antiserum to a testosterone-3-oxime HSA complex (Radioassay Systems Laboratories Inc., Carson). Cross-reaction with other 17 Bhydroxysteroids limits the specificity of these assays to a group determination of “total androgens”. Serum LH and FSH were determined by radioimmunoassay, and the results were expressed in microgramsilitre of the LER 907 standard.* Plasma estradiol, 17 hydroxyprogesterone, dehydroepiandrosterone, dehydroepiandrosterone sulphate and androstenedione were measured by radioimmunoassay. Five grams of tumor tissue were homogenized in 20 ml of ether. The volume was reduced to approximately 2 ml by evaporation under nitrogen. mixed with an equal volume of chloroform : n-heptanol :ethanol (100: 100 : 2) applied to a Sephadex LH 20 column (30 x I cm) and eluted with chloroform : hexane : ether. The fractions were evaporated to dryness and the solid residue was dissolved in 0.05 mol/l phosphate buffer pH 7 . 5 prior to testosterone and estradiol assay. Results
During the initial investigations in 1971, the plasma androgens were markedly elevated to *Pituitary Standards, Washington.
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6.1 and 8.0 ng/ml (normal range for adult females 0.8- 1 . 7 ng/ml). Plasma cortisol at 9 am was 14.9 pg/100 ml (normal 8-25 pg/ 100 ml). The 24 hour urinary 17 ketosteroids (17-KS) and 17 ketogenic steroids (17-KGS) were within the normal range (11.2 mg and 11-0 mg respectively). The total urinary estrogens were 17.0 pg per 24 hours (normal post-menopausal value less than 10 pg/24 hours). The urinary pregnanetriol was 0 . 13 mg/24 hours (normal range 0.46-0.91 mg/24 hours). After one week’s administration of dexamethasone (2 mg/day) the plasma cortisol was 7 . 6 pg/100 ml at 9 am, the urinary 17-KS were 4 . 5 mg/24 hours and the urinary 17-KGS were 3.0 mg/24 hours. The plasma androgens remained elevated (6.1 ng/ml) and the total urinary estrogen excretion was unaltered (21.4 pg/24 hours). Dexamethasone in a dose of 8 mg/day for one week resulted in a further suppression of urinary 17-KS and t7-KGS while the plasma androgens fell to 4.4 ngiml. The patient was given human chorionic gonadotrophin (HCG) by intramuscular injection in a dose of 3000 international units per day for four days. Plasma androgens rose to exceed 10 ng/ml, urinary 17-KS increased to 28 mg per 24 hours, but the 17-KGS remained at 4 . 5 mg per 24 hours. Ethinyl estradiol in a dose of 1 mg orally for five days did not suppress the elevated plasma androgens. Adrenal tomography suggested a right-sided adrenal mass. When the patient returned for investigation in September 1975, plasma androgens were 7 . 4 ng/ml, and plasma estradiol was 156 pgjml (normal post-menopausal range 0 to 20 pg/ml). Serum 17-hydroxyprogesterone was 1.9 ng/ml (normal range less than 2 ng/ml); plasma cortisol at 9 am was 21.5 pg/100 ml. The serum LH was 53 pg/l (normal postmenopausal values are in excess of 200 p g / l ) while the serum FSH was 260 pg/l (normal post-menopausal values are greater than 300 pg/l). Twenty-four hour urinary 17-KS and 17-KGS were normal. Plasma androgen concentrations were 8 . 7 ng/ ml before and 11.4 ng/ml after the administration of ethinyl estradiol (0.1 mg daily for three weeks). Serum FSH fell from 260 to
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110 pg/l and the serum LH rose slightly (53 to 73 pg/l) during the period of ethinyl estradiol administration. Adrenal tomography on this occasion disclosed no abnormality. At laparoscopy, in January 1976, a small nodule was seen attached to the right ovary. Because of this finding and the belief that the ovaries were the origin of excess circulating testosterone, the patient underwent a bilateral oophorectomy and hysterectomy in March 1976 (Dr. J. Furber). The left ovary measured 2 . 7 x 1 - 2 cm and appeared normal. The right ovary was 2.0 cm in its greatest diameter and had attached to its free surface a hard, smooth nodule 1.5 x 1.0 cm. The cut surface of this nodule was whorled, pale grey and partly calcified. Microscopic examination of both ovaries showed adenofibromas but no other abnormality. The endometrium was atrophic with patchy cystic changes. Blood samples taken during operation from a peripheral vein and the right and left ovarian veins showed androgen concentrations ranging from 11.1 to 13.1 ng/ml. The peripheral plasma androgen level decreased to 2 . 3 ng/ml on the second post-operative day, but three weeks later the level had risen to its pre-operative value. Three months later plasma androgens ranged from 4 . 3 to 9 . 7 ng/ml on separate occasions. Serum dehydroepiandrosterone (DHEA) was 11.4 and 9 . 6 ng/ml on separate occasions (normal range 1 .3- 11 ' 5 ng/ml); serum dehydroepiandrosterone sulphate (DHEA-S) was 2 . 3 and 1 . 9 pg/ml (normal range 1-1 to 3.8 pg/ml) and serum androstenedione was 2.7 and 2 . 6 ng/ml (normal range 0.9-3.0 ng/ml). Serum LH was unchanged (42 vg/l) and serum FSH was somewhat higher (310 pg/l) than before oophorectomy. Since an adrenal lesion was now suspected, 250 pg Synacthen were infused over eight hours. While plasma cortisol increased five-fold during this period, plasma androgens remained unchanged ; serum estradiol increased to double the basal level (Table 1). An aortic angiogram demonstrated no abnormal vascularity in the region of the adrenal glands.
TABLE 1 Synacthen stimulation (250 pg over eight hours) after oophorectomy and before removal of right adrenal
Time (hours)
0 6 8
Plasma cortisol (,ug/lOO ml)
Plasma androgen (ng/ml)
19.0 61.5 . 62.5
4.3 5.4 4.6
Estradiol (pg/ml)
I22 188 224
In August 1976, the right adrenal gland was explored. A soft, yellowish, spherical tumor was found occupying the upper part of the right adrenal gland which was removed. The left adrenal gland was normal to palpation and was left intact. The right adrenal tumor measured 4 . 5 cm x 3.0 cm x 3 - 0 cm and weighed 19 g (Fig. 2). It protruded through the rim of the adrenal cortex and was surrounded by a thin layer of fibrous tissue or adipose tissue alone. Its cut surface showed poorly defined fibrous areas. On microscopical examination the tumour was composed of compact and loose sheets of cells with the cytological features of testicular interstitial cells (Figs 3a, b, c). They had abundant finely granular eosinophilic cytoplasm and occasional double nuclei. No Reinke crystalloids were seen. Rare mitoses were found. There was no vascular invasion, haemorrhage or necrosis.
FIGURE 3. A. The cells of the adenoma closely resembled interstitial cells of the testis with eosinophilic granular cytoplasm and rounded nuclei. The pale vacuolated cells are adrenal cortex, ~2000. B. The adenoma here merges with periadrenal adipose tissue, x 1030. C. The adenoma in most areas was covered by a band of clear adrenal cortical cells, ~ 3 4 0 .
The tumour contained 109 ng androgens per gram of wet weight and 0.85 ng estradiol per gram of wet weight.
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SMITH ET AL.
Plasma androgens concentration on the day after operation was 0.6 ng/ml. Four weeks after removal of the right adrenal the plasma androgens were 0.5 ng/ml, serum estradiol was 14 pg/ml, urinary 17-KS were 12.6 and urinary 17-KGS were 15.3 mg/24 hours. Serum FSH was greater than 1400 pg/l and serum LH was 370 p g / l . Serum DHEA was 3 . 6 ng/ml, DHEA-S was less than 130 ng/ml, androstenedione was 1 . 0 ng/ml and 17-OHP was 0.5 ng/ml. An ACTH infusion (250 pg of Synacthen over eight hours) produced a marked rise in plasma cortisol and a very slight rise in plasma androgens and estradiol (Table 2). TABLE 3 Synacthen stimulation (250 pg over eight hours) after removal of the right adrenal
Time (hours)
Plasma cortisol ( d l 0 0 mU
Plasma androgen (ng/ml)
0 6
16.5
0.3
Not measured
8
51.5
1.2 0.8
Estradiol (pg/ml)
< 16.2 < 16.2 < 16.2 ~~
The patient began to experience frequent severe hot flushes. She also noticed decreased beard growth, change in voice quality and a little growth of scalp hair. Discussion
This patient demonstrates the rare phenomenon of virilization due to androgen production from an adrenal adenoma without elevation of urinary 17 ketosteroids. We found reports of only four similar cases, three of them in adult
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females2, 3 , and one in a pre-pubertal female.' A testosterone producing adrenal adenoma with minimally elevated urinary 17 ketosteroids has been reported in a young male." Comparison of our patient with the previously reported adult females reveals some interesting similarities and differences (Table 3). All patients had basal plasma androgen values I1 to 18 times the mean level expected for an adult female and, in each case, considerable fluctuations in these values were noted. The adenoma was found in the right adrenal gland in all cases. Three of the four patients underwent initial oophorectomy prior to adrenalectomy. In all cases the adrenal tumor was benign. The onset of virilization occurred in all patients one to four years after cessation of regular menses. As three of these tumours were shown to be responsive to exogenous HCG (Table 3), it seems reasonable to postulate that rising levels of gonadotrophins at the time of the menopause may have facilitated androgen secretion from the adrenal adenoma. Plasma estrogen levels were not measured in the three previously reported patients. There is good evidence that in our patient the adenoma produced significant quantities of estrogen. The estradiol content of the tumour was high and the high plasma level of estrogen fell after removal of the adenoma. The concentrations of steroid metabolites normally associated with production of adrenal androgens and estrogens (DHEA, DHEA-S, 17-hydroxyprogesterone, and androstenedione) were normal in the serum of this patient. This finding plus the low level of urinary 17-KS, suggests that testosterone and
TABLE 3
'
Clinical and biochemical data of four published cases of adult women with testosterone-secreting adrenal cortical adenomas Age at :
Ref.
2 3 4
This case
Menopause
Onset of virilization
Removal of adenoma
49 44 39t 45
50 45 43 47
53 49 76
*Urinary measurements.
Serum gonadotrophins
Plasma testosterone after :
55
tSurgically induced.
HCG
t*
t* Notrecorded t
Estrogen
ACTH
Dexamethasone
I* No change
1 1
No change No change
No change
No change
1
1
1
Slight
1
High Low High Low
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estradiol were produced in the adenoma tissue via a biosynthetic pathway similar to that found in normal gonadal tissue.' ', Exogenous estrogen given orally (see Results") failed to suppress excess androgen production in our patient though two other patients Estrogen also failed to suppress the serum LH level. However, there appeared to be an inverse relationship between the patient's endogenous gonadal steroids and serum gonadotrophins. At the time of presentation serum LH and FSH were lower than the expected post-menopausal values. Following oophorectomy there was no change in the gonadotrophin, testosterone or estradiol levels, while after removal of the source of excess testosterone and estradiol, both FSH and LH rapidly rose to post-menopausal levels (Table 4).
''
'
TABLE 4 Biochemical changes following oophorectomy and adrenalectomy in this patient Serum Serum Plasma Plasma FSH LH androgen estradiol (!Jg/U (PLgll) (ng/ml) (pg/ml) Pre-oophorectom y Post-oophorectomy Post-adrenalectomy
260 310 z 1400
53 42 370
8.6 11.7 0.5
156 122 14
The patient described by Givens et aL3, also showed low gonadotrophin levels prior to removal of the tumor and these authors suggested that LH secretion was suppressed by high plasma testosterone levels. Estrogen levels were not reported. On the other hand, the patients described by Werk et d 2 and Larson et d4,had elevated gonadotrophin levels at the time of presentation. There is no evidence that endogenous plasma testosterone levels regulate gonadotrophin secretion in normal females13, in patients with virilization or in patients with dysgenetic gonad^.'^ We therefore postulate that plasma estrogens inhibited gonadotrophin secretion in this patient. Suppression and stimulation tests did not clearly indicate the source of androgen production in our patient. The normal basal urinary 17-KS were thought to be in favour of an ovarian source', and, in view of the studies
175
reported by Kirschner and Jacobs16, we persisted with this mistaken hypothesis even after 8 mg dexamethasone/day had partially suppressed plasma androgen levels. Various techniques have been described for the localization of the source of excess androgens in virilized women. These include tomography, and arteriography, adrenal venography' 7* hormone assays .after selective catheterization. None of them were diagnostically useful in this patient and a so-called selective catheterization was in fact misleading in a previous case.' Acknowledgements
All plasma cortisol estimations were performed by Mr. Angelo Negrin. Plasma androgens were measured in the Garvan Institute. Serum DHEA, DHEA-S and androstenedione assays were performed in the laboratory of Prof. J. R. Turtle. Dr. S. McCarthy interpreted the histological findings. The expert secretarial assistance of Debbie Reynolds and Christine Child is gratefully acknowledged. This work was supported by the National Health and Medical Research Council of Australia and the NSW State Cancer Council. References 1. KENNY, F. M., HASHIDAY. A~KARI H. A. Smam W. H. and FETTERMAN G. H. (1968): Virilizing ;umours of ihe adrkal co;tex, Amer. J. Dis. Child: 115, 445.
2. WERK,E. E., SHOLITON, L. J. and KALEJS, L. (1973): Testosterone-secreting adrenal adenoma under gonadotrophin control, New Engl. J. Med. 289,767. 3. GIVENSJ. R. ANDERSON R. N. WISERW. L. COLEMAN, S. A. a n d F m S. A. (19j4): A' gonadotrophin-r&ponsi;e adr&ocortical adenoma, J. din. Endocr. 38, 126. 4. LAPSON, B. A., VANDERLAAN, W. P., JUDO,H. L. and MCCULLWCH, D. L. (1976): A testosterone-producing adrenal cortical adenoma in an elderly woman, J. din. Endocr. 42, 882. 5 . APPLEBY, J. I., GIESON,G., NORYMBERSKI, J. K. and SNBES,R.D. (1955): Indirect analysis of corticostesoids, Biochemistry 60, 453. 6. OUTCH, K. H., DENNIS, P. M. and LARSEN, A. (1972): A rapid method forthe estimation of estrogens in non-pregnancy urine, Clin. chim. Acra 40, 377. 7. HORTON,R.,KAM, T. and SHERINS,9 . J. (1967): A rapid method for the estimation of testosterone in male plasma, Steroids 10, 245. 8. Case records ofThe Massachusetts General Hospital (Case 8-1969) (1969): New Engl. J. Med. 280, 438. 9. Bum, I. M., SULLIVAN, J., GRAHAM, T., HARTMAN, W. H. and ONEILL,J. (1973):A testosterone-secreting tumour of the adrenal producing virilization in a female infant, Lancet 2, 643. 10. DAVID,R., AUGUST,G. and CANDY,H. M. (1968): A virilizing adrenal tumour with borderline elevation of urinary 17-ketosteroids, Pediatrics 42, 139. 11. HUDSON,B.,COGHLAN,J. P. a n d D u ~ h i A ~ i ~(1967):Testicularfunctionin ,A. man, Ciba Foundation, Colloquia on Endocrinology 16, 140, Churchill, London. 12. SAVARD.K. (1966): Biogenesis of sterolds in the human ovary. In: Endocrrnologic and Morphologic Correlation of the Ovary, The Florentine Conference. Inguilla, W. and Greenblatt, R. B. (eds.), Thomas, Springfield, p. 264. 13. KIRSCHNER, M. A. and BARDIN,C. W. (1972): Androgen production and 1 metabolism in normal and virilized women, Metabolism 21, 667. 14. BARDIN,C. W., ROSEN,S., LE MAIRE W. J. Ti10 J. H. GALLUPJ. MARSHALL, 1. and SAVARD, K. (1969): In Live and in v i r h studids of andrdged metabolism in a patient with pure gonadal dysgenesis and Leydig cell hyperplasia, J. clin. Endocr. 29, 1429. 15. BEITINS. I. Z., BAYARD, F., KOWARSKI, A. and MICEON,C. I. (1973): The effect of ACTH administration on plasma testosterone dihydrotestosterone and serum LH concentrations in normal man, Steroih 21, 553. M. A. and JACOBS, 16. KIRSCHNU(, 1. B. (1971): Combined ovarian and adrenal vein catheterization to determine the site(s) of androgen overproduction in hirsute women, J. clin. Endocr. 33, 199. 17. BLAIR,A. J. and REIITER, S . R. (1970): Adrenal venography in virilized women, J. Amer. med. Ass. 213. 1623. 18. GABRILOVE, J. L., NICHOLIS,G. L. and MITTY,H. A. (1976): Virilizing adrenocortical adenoma studied by selective adrenal venography, Amer. J. Obstet. Gynec. 125, 180.
A Testosterone-Secreting Adrenal Cortical Adenoma H. C. Smith", S. Posent, P. Clifton-BlighS and J. Casey""
From the Department of Medicine, Sydney Hospital, and Garvan Institute, St. Vincent's Hospital, Sydney
Summary: A testosterone-secreting adrenal cortical adenoma. H. C. Smith, S. Posen, P. CliftonBligh and J. Casey, Aust. N.Z. J. Med., 1978, 8, pp.171175.
A
virilizing, gonadotrophin -responsive adrenal cortical adenoma was removed from a 54- year-old woman. Following removal of the tumour, which contained and secreted androgens and estradiol, serum gonadotrophins rose to menopausal levels and the patient experienced menopausal symptoms. Pre-operative attempts to localize the source of androgens were unsuccessful.
loss of scalp hair (Fig. 1) and a male type of facial hair distribution. Dark, coarse hair was found on the upper chest, breasts, abdomen, arms and legs. The muscle bulk was increased. The clitoris was minimally enlarged, while the uterus was normal in size and position. Both ovaries were palpable and considered to be of normal size. Serum electrolytes, uric acid and creatinine were normal. The haemoglobin was 17.1 g/lOO ml, the haematocrit 48'39/d. The serum cholesterol was 330 mg/100 ml. Chromosome analysis of peripheral leucocytes revealed a normal female karyotype (46, XX). After a series of investigations (see "Results"), the patient declined to have further tests or treatment and was not seen until four years later. When she reappeared in 1975 complaining of increasing libido, the clinical appearances were
Adrenal cortical adenomas rarely cause virilization.' Testosterone-secreting, gonadotrophin-responsive adrenal cortical adenomas in adult women are particularly rare and we found only three such cases in the English 33 A fourth patient is described in this paper. Case History
N.B. (SH. 353132), a 50-year-old married woman was referred in 1971 because of virilization. Shortly after the cessation of menstruation at 45 years of age she had noticed the growth of excess facial and body hair and, by the time of presentation. she shaved each day. When aged 47 years she noticed loss of scalp hair which progressed over the next three years to a pattern of advanced male baldness. She had also noted deepening of the voice, clitoral enlargement, acne, gain in body weight and increased libido. After her menarche at the age of 13 years, she had menstruated regularly but she had never succeeded in becoming pregnant. Vaginal examination and tuba1 insufflation had been performed at the age of 25 years and had revealed no abnormality. There was no other relevant past medical history. Physical examination showed a blood pressure of 130/80, a height of 163 cm and a weight of 61 kg. There was marked 'Endocrine Registrar, Sydney Hospital. tAssoc. Professor of Medicine, Sydney Hospital fEndocrinologist, Sydney Hospital. "Endocrinologist, St. Vincent's Hospital. Correspondence: Professor S. Posen, Sydney Hospital, Sydney, NSW 2000 Accepted for publication: 20 December, 1977
FIGURE 1.
Patient's appearance on presentation in 1971. The face and upper chest were shaved regularly.
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SMITH ET AL.
unchanged. The uterus and ovaries were removed in March 1976 (see “Results”) but their removal did not lead to any significant clinical or biochemical improvement. After further investigations (see “Results”), the right adrenal gland was removed by Dr. Ross Campbell in August 1976. It contained a bilobed partially encapsulated adenoma (Fig. 2). The left adrenal gland was palpated during the operation and was considered normal. Left adrenal exploration was therefore not performed. After her unilateral adrenalectomy the patient did not require glucocorticoid medication and signs of virilization gradually diminished.
FIGURE 2. Cut surface of the right adrenal gland containing the tumor which measured 4 . 5 ~ 3 . 0 ~ 3c m .0 and weighed 19 g. Assay Methods
The urinary 17 ketosteroids (17-KS) and 17 ketogenic steroids (17-KGS) were determined by the method of Appleby er aL5 The urinary total estrogens were assayed by the method of Outch et aL6 Until 1975, plasma testosterone was measured by competitive protein binding.’ After 1975 testosterone measurements were made by radioimmunoassay with an antiserum to a testosterone-3-oxime HSA complex (Radioassay Systems Laboratories Inc., Carson). Cross-reaction with other 17 Bhydroxysteroids limits the specificity of these assays to a group determination of “total androgens”. Serum LH and FSH were determined by radioimmunoassay, and the results were expressed in microgramsilitre of the LER 907 standard.* Plasma estradiol, 17 hydroxyprogesterone, dehydroepiandrosterone, dehydroepiandrosterone sulphate and androstenedione were measured by radioimmunoassay. Five grams of tumor tissue were homogenized in 20 ml of ether. The volume was reduced to approximately 2 ml by evaporation under nitrogen. mixed with an equal volume of chloroform : n-heptanol :ethanol (100: 100 : 2) applied to a Sephadex LH 20 column (30 x I cm) and eluted with chloroform : hexane : ether. The fractions were evaporated to dryness and the solid residue was dissolved in 0.05 mol/l phosphate buffer pH 7 . 5 prior to testosterone and estradiol assay. Results
During the initial investigations in 1971, the plasma androgens were markedly elevated to *Pituitary Standards, Washington.
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6.1 and 8.0 ng/ml (normal range for adult females 0.8- 1 . 7 ng/ml). Plasma cortisol at 9 am was 14.9 pg/100 ml (normal 8-25 pg/ 100 ml). The 24 hour urinary 17 ketosteroids (17-KS) and 17 ketogenic steroids (17-KGS) were within the normal range (11.2 mg and 11-0 mg respectively). The total urinary estrogens were 17.0 pg per 24 hours (normal post-menopausal value less than 10 pg/24 hours). The urinary pregnanetriol was 0 . 13 mg/24 hours (normal range 0.46-0.91 mg/24 hours). After one week’s administration of dexamethasone (2 mg/day) the plasma cortisol was 7 . 6 pg/100 ml at 9 am, the urinary 17-KS were 4 . 5 mg/24 hours and the urinary 17-KGS were 3.0 mg/24 hours. The plasma androgens remained elevated (6.1 ng/ml) and the total urinary estrogen excretion was unaltered (21.4 pg/24 hours). Dexamethasone in a dose of 8 mg/day for one week resulted in a further suppression of urinary 17-KS and t7-KGS while the plasma androgens fell to 4.4 ngiml. The patient was given human chorionic gonadotrophin (HCG) by intramuscular injection in a dose of 3000 international units per day for four days. Plasma androgens rose to exceed 10 ng/ml, urinary 17-KS increased to 28 mg per 24 hours, but the 17-KGS remained at 4 . 5 mg per 24 hours. Ethinyl estradiol in a dose of 1 mg orally for five days did not suppress the elevated plasma androgens. Adrenal tomography suggested a right-sided adrenal mass. When the patient returned for investigation in September 1975, plasma androgens were 7 . 4 ng/ml, and plasma estradiol was 156 pgjml (normal post-menopausal range 0 to 20 pg/ml). Serum 17-hydroxyprogesterone was 1.9 ng/ml (normal range less than 2 ng/ml); plasma cortisol at 9 am was 21.5 pg/100 ml. The serum LH was 53 pg/l (normal postmenopausal values are in excess of 200 p g / l ) while the serum FSH was 260 pg/l (normal post-menopausal values are greater than 300 pg/l). Twenty-four hour urinary 17-KS and 17-KGS were normal. Plasma androgen concentrations were 8 . 7 ng/ ml before and 11.4 ng/ml after the administration of ethinyl estradiol (0.1 mg daily for three weeks). Serum FSH fell from 260 to
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110 pg/l and the serum LH rose slightly (53 to 73 pg/l) during the period of ethinyl estradiol administration. Adrenal tomography on this occasion disclosed no abnormality. At laparoscopy, in January 1976, a small nodule was seen attached to the right ovary. Because of this finding and the belief that the ovaries were the origin of excess circulating testosterone, the patient underwent a bilateral oophorectomy and hysterectomy in March 1976 (Dr. J. Furber). The left ovary measured 2 . 7 x 1 - 2 cm and appeared normal. The right ovary was 2.0 cm in its greatest diameter and had attached to its free surface a hard, smooth nodule 1.5 x 1.0 cm. The cut surface of this nodule was whorled, pale grey and partly calcified. Microscopic examination of both ovaries showed adenofibromas but no other abnormality. The endometrium was atrophic with patchy cystic changes. Blood samples taken during operation from a peripheral vein and the right and left ovarian veins showed androgen concentrations ranging from 11.1 to 13.1 ng/ml. The peripheral plasma androgen level decreased to 2 . 3 ng/ml on the second post-operative day, but three weeks later the level had risen to its pre-operative value. Three months later plasma androgens ranged from 4 . 3 to 9 . 7 ng/ml on separate occasions. Serum dehydroepiandrosterone (DHEA) was 11.4 and 9 . 6 ng/ml on separate occasions (normal range 1 .3- 11 ' 5 ng/ml); serum dehydroepiandrosterone sulphate (DHEA-S) was 2 . 3 and 1 . 9 pg/ml (normal range 1-1 to 3.8 pg/ml) and serum androstenedione was 2.7 and 2 . 6 ng/ml (normal range 0.9-3.0 ng/ml). Serum LH was unchanged (42 vg/l) and serum FSH was somewhat higher (310 pg/l) than before oophorectomy. Since an adrenal lesion was now suspected, 250 pg Synacthen were infused over eight hours. While plasma cortisol increased five-fold during this period, plasma androgens remained unchanged ; serum estradiol increased to double the basal level (Table 1). An aortic angiogram demonstrated no abnormal vascularity in the region of the adrenal glands.
TABLE 1 Synacthen stimulation (250 pg over eight hours) after oophorectomy and before removal of right adrenal
Time (hours)
0 6 8
Plasma cortisol (,ug/lOO ml)
Plasma androgen (ng/ml)
19.0 61.5 . 62.5
4.3 5.4 4.6
Estradiol (pg/ml)
I22 188 224
In August 1976, the right adrenal gland was explored. A soft, yellowish, spherical tumor was found occupying the upper part of the right adrenal gland which was removed. The left adrenal gland was normal to palpation and was left intact. The right adrenal tumor measured 4 . 5 cm x 3.0 cm x 3 - 0 cm and weighed 19 g (Fig. 2). It protruded through the rim of the adrenal cortex and was surrounded by a thin layer of fibrous tissue or adipose tissue alone. Its cut surface showed poorly defined fibrous areas. On microscopical examination the tumour was composed of compact and loose sheets of cells with the cytological features of testicular interstitial cells (Figs 3a, b, c). They had abundant finely granular eosinophilic cytoplasm and occasional double nuclei. No Reinke crystalloids were seen. Rare mitoses were found. There was no vascular invasion, haemorrhage or necrosis.
FIGURE 3. A. The cells of the adenoma closely resembled interstitial cells of the testis with eosinophilic granular cytoplasm and rounded nuclei. The pale vacuolated cells are adrenal cortex, ~2000. B. The adenoma here merges with periadrenal adipose tissue, x 1030. C. The adenoma in most areas was covered by a band of clear adrenal cortical cells, ~ 3 4 0 .
The tumour contained 109 ng androgens per gram of wet weight and 0.85 ng estradiol per gram of wet weight.
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SMITH ET AL.
Plasma androgens concentration on the day after operation was 0.6 ng/ml. Four weeks after removal of the right adrenal the plasma androgens were 0.5 ng/ml, serum estradiol was 14 pg/ml, urinary 17-KS were 12.6 and urinary 17-KGS were 15.3 mg/24 hours. Serum FSH was greater than 1400 pg/l and serum LH was 370 p g / l . Serum DHEA was 3 . 6 ng/ml, DHEA-S was less than 130 ng/ml, androstenedione was 1 . 0 ng/ml and 17-OHP was 0.5 ng/ml. An ACTH infusion (250 pg of Synacthen over eight hours) produced a marked rise in plasma cortisol and a very slight rise in plasma androgens and estradiol (Table 2). TABLE 3 Synacthen stimulation (250 pg over eight hours) after removal of the right adrenal
Time (hours)
Plasma cortisol ( d l 0 0 mU
Plasma androgen (ng/ml)
0 6
16.5
0.3
Not measured
8
51.5
1.2 0.8
Estradiol (pg/ml)
< 16.2 < 16.2 < 16.2 ~~
The patient began to experience frequent severe hot flushes. She also noticed decreased beard growth, change in voice quality and a little growth of scalp hair. Discussion
This patient demonstrates the rare phenomenon of virilization due to androgen production from an adrenal adenoma without elevation of urinary 17 ketosteroids. We found reports of only four similar cases, three of them in adult
VOL.
8, NO. 2
females2, 3 , and one in a pre-pubertal female.' A testosterone producing adrenal adenoma with minimally elevated urinary 17 ketosteroids has been reported in a young male." Comparison of our patient with the previously reported adult females reveals some interesting similarities and differences (Table 3). All patients had basal plasma androgen values I1 to 18 times the mean level expected for an adult female and, in each case, considerable fluctuations in these values were noted. The adenoma was found in the right adrenal gland in all cases. Three of the four patients underwent initial oophorectomy prior to adrenalectomy. In all cases the adrenal tumor was benign. The onset of virilization occurred in all patients one to four years after cessation of regular menses. As three of these tumours were shown to be responsive to exogenous HCG (Table 3), it seems reasonable to postulate that rising levels of gonadotrophins at the time of the menopause may have facilitated androgen secretion from the adrenal adenoma. Plasma estrogen levels were not measured in the three previously reported patients. There is good evidence that in our patient the adenoma produced significant quantities of estrogen. The estradiol content of the tumour was high and the high plasma level of estrogen fell after removal of the adenoma. The concentrations of steroid metabolites normally associated with production of adrenal androgens and estrogens (DHEA, DHEA-S, 17-hydroxyprogesterone, and androstenedione) were normal in the serum of this patient. This finding plus the low level of urinary 17-KS, suggests that testosterone and
TABLE 3
'
Clinical and biochemical data of four published cases of adult women with testosterone-secreting adrenal cortical adenomas Age at :
Ref.
2 3 4
This case
Menopause
Onset of virilization
Removal of adenoma
49 44 39t 45
50 45 43 47
53 49 76
*Urinary measurements.
Serum gonadotrophins
Plasma testosterone after :
55
tSurgically induced.
HCG
t*
t* Notrecorded t
Estrogen
ACTH
Dexamethasone
I* No change
1 1
No change No change
No change
No change
1
1
1
Slight
1
High Low High Low
APRIL
1978
A TESTOSTERONE-SECRETING ADRENAL ADENOMA -
estradiol were produced in the adenoma tissue via a biosynthetic pathway similar to that found in normal gonadal tissue.' ', Exogenous estrogen given orally (see Results") failed to suppress excess androgen production in our patient though two other patients Estrogen also failed to suppress the serum LH level. However, there appeared to be an inverse relationship between the patient's endogenous gonadal steroids and serum gonadotrophins. At the time of presentation serum LH and FSH were lower than the expected post-menopausal values. Following oophorectomy there was no change in the gonadotrophin, testosterone or estradiol levels, while after removal of the source of excess testosterone and estradiol, both FSH and LH rapidly rose to post-menopausal levels (Table 4).
''
'
TABLE 4 Biochemical changes following oophorectomy and adrenalectomy in this patient Serum Serum Plasma Plasma FSH LH androgen estradiol (!Jg/U (PLgll) (ng/ml) (pg/ml) Pre-oophorectom y Post-oophorectomy Post-adrenalectomy
260 310 z 1400
53 42 370
8.6 11.7 0.5
156 122 14
The patient described by Givens et aL3, also showed low gonadotrophin levels prior to removal of the tumor and these authors suggested that LH secretion was suppressed by high plasma testosterone levels. Estrogen levels were not reported. On the other hand, the patients described by Werk et d 2 and Larson et d4,had elevated gonadotrophin levels at the time of presentation. There is no evidence that endogenous plasma testosterone levels regulate gonadotrophin secretion in normal females13, in patients with virilization or in patients with dysgenetic gonad^.'^ We therefore postulate that plasma estrogens inhibited gonadotrophin secretion in this patient. Suppression and stimulation tests did not clearly indicate the source of androgen production in our patient. The normal basal urinary 17-KS were thought to be in favour of an ovarian source', and, in view of the studies
175
reported by Kirschner and Jacobs16, we persisted with this mistaken hypothesis even after 8 mg dexamethasone/day had partially suppressed plasma androgen levels. Various techniques have been described for the localization of the source of excess androgens in virilized women. These include tomography, and arteriography, adrenal venography' 7* hormone assays .after selective catheterization. None of them were diagnostically useful in this patient and a so-called selective catheterization was in fact misleading in a previous case.' Acknowledgements
All plasma cortisol estimations were performed by Mr. Angelo Negrin. Plasma androgens were measured in the Garvan Institute. Serum DHEA, DHEA-S and androstenedione assays were performed in the laboratory of Prof. J. R. Turtle. Dr. S. McCarthy interpreted the histological findings. The expert secretarial assistance of Debbie Reynolds and Christine Child is gratefully acknowledged. This work was supported by the National Health and Medical Research Council of Australia and the NSW State Cancer Council. References 1. KENNY, F. M., HASHIDAY. A~KARI H. A. Smam W. H. and FETTERMAN G. H. (1968): Virilizing ;umours of ihe adrkal co;tex, Amer. J. Dis. Child: 115, 445.
2. WERK,E. E., SHOLITON, L. J. and KALEJS, L. (1973): Testosterone-secreting adrenal adenoma under gonadotrophin control, New Engl. J. Med. 289,767. 3. GIVENSJ. R. ANDERSON R. N. WISERW. L. COLEMAN, S. A. a n d F m S. A. (19j4): A' gonadotrophin-r&ponsi;e adr&ocortical adenoma, J. din. Endocr. 38, 126. 4. LAPSON, B. A., VANDERLAAN, W. P., JUDO,H. L. and MCCULLWCH, D. L. (1976): A testosterone-producing adrenal cortical adenoma in an elderly woman, J. din. Endocr. 42, 882. 5 . APPLEBY, J. I., GIESON,G., NORYMBERSKI, J. K. and SNBES,R.D. (1955): Indirect analysis of corticostesoids, Biochemistry 60, 453. 6. OUTCH, K. H., DENNIS, P. M. and LARSEN, A. (1972): A rapid method forthe estimation of estrogens in non-pregnancy urine, Clin. chim. Acra 40, 377. 7. HORTON,R.,KAM, T. and SHERINS,9 . J. (1967): A rapid method for the estimation of testosterone in male plasma, Steroids 10, 245. 8. Case records ofThe Massachusetts General Hospital (Case 8-1969) (1969): New Engl. J. Med. 280, 438. 9. Bum, I. M., SULLIVAN, J., GRAHAM, T., HARTMAN, W. H. and ONEILL,J. (1973):A testosterone-secreting tumour of the adrenal producing virilization in a female infant, Lancet 2, 643. 10. DAVID,R., AUGUST,G. and CANDY,H. M. (1968): A virilizing adrenal tumour with borderline elevation of urinary 17-ketosteroids, Pediatrics 42, 139. 11. HUDSON,B.,COGHLAN,J. P. a n d D u ~ h i A ~ i ~(1967):Testicularfunctionin ,A. man, Ciba Foundation, Colloquia on Endocrinology 16, 140, Churchill, London. 12. SAVARD.K. (1966): Biogenesis of sterolds in the human ovary. In: Endocrrnologic and Morphologic Correlation of the Ovary, The Florentine Conference. Inguilla, W. and Greenblatt, R. B. (eds.), Thomas, Springfield, p. 264. 13. KIRSCHNER, M. A. and BARDIN,C. W. (1972): Androgen production and 1 metabolism in normal and virilized women, Metabolism 21, 667. 14. BARDIN,C. W., ROSEN,S., LE MAIRE W. J. Ti10 J. H. GALLUPJ. MARSHALL, 1. and SAVARD, K. (1969): In Live and in v i r h studids of andrdged metabolism in a patient with pure gonadal dysgenesis and Leydig cell hyperplasia, J. clin. Endocr. 29, 1429. 15. BEITINS. I. Z., BAYARD, F., KOWARSKI, A. and MICEON,C. I. (1973): The effect of ACTH administration on plasma testosterone dihydrotestosterone and serum LH concentrations in normal man, Steroih 21, 553. M. A. and JACOBS, 16. KIRSCHNU(, 1. B. (1971): Combined ovarian and adrenal vein catheterization to determine the site(s) of androgen overproduction in hirsute women, J. clin. Endocr. 33, 199. 17. BLAIR,A. J. and REIITER, S . R. (1970): Adrenal venography in virilized women, J. Amer. med. Ass. 213. 1623. 18. GABRILOVE, J. L., NICHOLIS,G. L. and MITTY,H. A. (1976): Virilizing adrenocortical adenoma studied by selective adrenal venography, Amer. J. Obstet. Gynec. 125, 180.
