Clinical Endocrinology (1979) 11,141-150.
PLASMA HORMONE LEVELS A N D OESTROGEN PRODUCTION IN A POSTMENOPAUSAL WOMAN WITH E N D O M E T R I A L CARCINOMA A N D AN O V A R I A N THECOMA M . J . REED, J. D. HUTTON, R. W. B E A R D , H. S . J A C O B S AND V . H. T. J A M E S Departments of Chemical Pathology and Obstetrics and Gynaecology, St Mary’s Hospital Medical School, London, W.2 (Received 28 October 19 78; revised 16 January 1979; accepted 30 January 1979)
SUMMARY
Plasma hormone concentrations, the production rate of oestrone from androstenedione, and the total production rate of oestrone have been measured in a 57year-old woman with an ovarian thecoma and endometrial carcinoma before and 2 months after hysterectomy and bilateral ovariectomy. The plasma concentration of oestradiol (184 pmol/l), but not oestrone (144 pmol/l), was elevated preoperatively and FSH secretion was suppressed (1 15.0 pg/l). Post-operatively the plasma concentration of oestradiol was significantly reduced (80 pmol/l) and the concentration of FSH (571 pg/l) had increased to a postmenopausal level. The concentration of oestradiol in samples of plasma obtained simultaneously at operation from the ovarian vein of the ovary containing the thecoma (3.38 nmol/l) was fifteen times greater than in the periphery (228.0 pmol/l), and this confirmed that oestradiol was secreted by the thecoma. The total production rates of oestrone preand post-operatively were 101.3 nmo1/24 h and 29.2 nmo1/24 h respectively and the production rates of oestrone from androstenedione were 41.4 nmo1/24 h and 32.9 nmo1/24 h. Thus, postaperatively, all the oestrone was produced from circulating androstenedione, whereas pre-operatively only 40% of the oestrone was produced by the extraglandular conversion of androstenedione; the remaining 60% presumably originated from the peripheral conversion of oestradiol secreted by the thecoma. It is concluded that the oestradiol secreted by the thecoma was probably the major factor which caused the development of endometrial carcinoma in this subject. The association of endometrial cancer with an oestrogen producing ovarian tumour, first reported by Schroeder in 1922, has since been confirmed by several studies (Diddle, 1951; Larson, 1954; McDonald et al., 1976). The incidence of feminizing tumours of the ovary have been estimated to be between 3-6% of all ovarian tumours (Larmont & Ashton, 1975; Correspondence: Dr M. J . Reed, Department of Chemical Pathology, St Mary’s Hospital Medical School, London, W.2. 03304664/79/08004 141$02.00
0 1979 Blackwell Scientific Publications
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Norris & Chorlton, 1974) but may be less than 1% (Anikwue et al., 1978). A recent survey of the literature (Anikwue et al. 1978) showed that as many as 17% of the subjects with an ovarian thecoma also had endometrial carcinoma. Because of the clinical association of endometrial carcinoma with ovarian thecomas it has been suggested that unopposed secretion of oestrogens by the tumour may cause endometrial carcinoma (Gusberg & Kardon, 1971). Measurements of steroid excretion before and after surgery have shown that a large proportion of urinary oestrogens may originate from ovarian tumours (Brown et af., 195Y; Brown & Beischer, 1972; Targett, 1974). In vitro studies have demonstrated that tissue obtained from oestrogen producing ovarian tumours has the ability t o synthesize both oestrone and oestradiol (Griffiths et al., 1964; Besch et al., 1966) although in v i secretion ~ ~ o f these steroids by such tumours has not yet been demonstrated. It has recently been shown b y the use o f isotope techniques that the increased oestrone production in a subject with a mucinous cystadenocarcinoma of the ovary resulted from excessive secretion of androstenedione, although ext raglandular conversion of androstenedione to oestrone was normal (MacDonald ef al., 1976). The present study was carried out to determine the hormonal status o f a postmenopausal woman with an ovarian thecoma and endometrial carcinoma and to assess the contributionof the thecoma to oestrogen production. CASE REPORT
A 57-year-old woman (height 151 cm, weight 4 7 kg) was admitted to hospital for investigation of continuous vaginal bleeding o f 1 year’s duration. When aged 4 5 years, her regular periods had become heavier and, 5 years later, she developed irregular vaginal bleeding. A swelling, about 8 cm diameter, was palpable posterior to a normal-sized anteverted uterus. The vaginal epithelium was not atrophic (Hammond, 1976) and no other abnormality was detected. Her blood haemoglobin concentration was 9.7 g/dl; liver, renal and thyroid function tests were normal. The karyopyknotic index of a lateral vaginal wall smear was 21%. The cytological diagnosis of an aspirate smear from the endometrial cavity was of a well differentiated adenocarcinoma (Hutton et al., 1978) and this was confirmed at curettage. The patient was readmitted one month later having meanwhile taken 200 mg of iron daily; her blood haemoglobin was then 11.9 g/dl. Before undergoing laparotomy the patient gave her informed consent to hormone studies using radioactive isotopes. Approval for these studies was obtained from the hospital ethical committee and Department of Health and Social Security Committee for the use of isotopes. Four days after the completion of t h e first endocrine study the patient underwent laparotomy; the left ovary was enlarged and posterior t o the normal sized uterus. The fallopian tubes and atrophied right ovary appeared normal. A total abdominal hysterectomy and bilateral salpingoophorectomy was then performed. The second endocrine study was carried out 2 months after the operation at which time the karyopyknotic index was 0%. Pathology Pathological examination showed that the left ovary, which had a smooth surface, was replaced by a tumour measuring 7 X 4 X 4 cm, which was soft and when cut was yellow. This soft yellow area showed the classical histological features of a benign thecoma (McGoldrick & Lapp, 1944) whereas the residue was a fibroma. No granulosa cell elements, follicles or germ cells were seen in either ovary. l-bstological examination of the thick poly-
Oestrogen production b y a thecoma
I43
poid endometrium showed a well differentiated adenocarcinoma; there were also a few areas of cystic hyperplasia, adenomatous hyperplasia and atypical adenomatous hyperplasia (Dallenb ach-He llweg , 19 7 5).
Biochemical studies Hormone concentrations. Plasma hormones were measured by specific radioimmunoassays: oestrone and oestradiol by a modification of the procedure described by Hotchkiss et al. (1971); androstenedione and testosterone as described by Goodall et a f . (1979). Cortisol was measured after extraction from plasma with dichloromethane and evaporation of an aliquot of the extract. Progesterone was measured after extraction from plasma with hexane using ammonium sulphate precipitation to separate free from anti-body bound hormone. Plasma FSH (MRC 68/39) and LH (MRC 68/40) were measured using antisera supplied by Dr W. D. Ode11 (Jacobs & Lawton, 1974) and reference preparations by the Division of Biological Standards of the MRC of the U.K. So that accurate estimates of the plasma hormone concentrations throughout the day could be determined, heparinized blood samples (8 ml) were obtained through an indwelling cannula in a forearm vein every 2 h for 24 h. The plasma was separated immediately after collection and stored at -20°C until processed. The significance of differences between plasma hormone concentrations measured pre- and post-operatively was analysed by Student's t test. Urine was collected during this 24 h sampling period and the urinary excretion of oestrogens measured (Brown, 1955). Blood samples (10 ml) were also obtained at operation simultaneously from a peripheral vein and a vein close to the left ovary. Dynamic studies. Oestrone production and the conversion of androstenedione to oestrone was determined by the double isotope technique described by Grodin et al. (1973), whose symbols and abbreviations are used in the present study. At the end of the 24 h period during which sequential blood samples were obtained, approximately 12 pCi [6, 7-3H] oestrone (50 Ci/mmol) and 24 pCi [4-14C] androstenedione (50 mCi/mmol) in 4% human serum albumin were infused into an arm vein over a 4.5 h period. Blood samples (50 ml) were obtained 3.5, 4.0 and 4.5 h after tracer infusion was started. Urine was collected for 72 ti. Radioisotopes were obtained from the Kadiochemical Centre, Amersham, U.K., and purified before use by paper chromatography, using light petroleum (100"-120" fraction): to1uene:me;hanol:water (33:17:40:10 by volume) as solvents. Determination of the plasma production rate of androstenedione (PRp-A) The metabolic clearance rate for androstenedione (MCR-A) was calculated from the equilibrium concentration of [4-14C] androstenedione in plasma (Baird et al., 1969). This latter was measured after ether extraction and Sephadex LH 20 column chromatography (Olivo et af., 1973). The coefficient of variation for the determination of the metabolic clearance rate for androstenedione by this method in plasma samples obtained from nine subjects undergoing similar investigations was 7.4%. Determination of the Blood Transfer Constant [ p ] [3H]- and [I4C]-Oestrone extracted from plasma (Olivo et al., 1973) were further purified by thin-layer chromatography (TLC) using dichloromethane:ethylacetate (4:l V/V) as solvents. Determination of the isotopic ratios in replicate plasma samples obtained from eight subjects gave a coefficient of variation of 15.9% from this method.
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Determination of the extent of the conversion of plasma androstenedione to oestrone as measured in urine / p ] '2; Steroid conjugates were recovered from urine by passing an aliquot of the pooled 3 day urine collection through an Amberlite XAD-2 ion exchange resin and subsequently eluting with methanol (Bradlow, 1968). The steroid conjugates were hydrolyzed after evaporation of the methanol and reconstitution in buffer with a P-glucuronidase enzyme preparation (Ketodase, W. R. Warner & Co. Ltd., Eastleigh, Hampshire, U.K.) for 48 h at 37°C and the liberated steroids were extracted with chloroform. After washing the chloroform with water and drying with anhydrous sodium sulphate, the chloroform was evaporated under vacuum. The residue was used to prepare a phenolic fraction (Brown, 1955) and oestrone, oestradiol and oestriol were subsequently separated using a Sephadex LH20 (20 g) column with toluene:methanol(85: I5 v/v) as solvents. Each oestrogen was subjected t o three further purification steps b y TLC. Tritiated steroid standards were also chromatographed and were located using a radiochromatogram imaging system (Panax Equipment Ltd., Surrey, U.K.). The oestrogens were eluted with ether and the ether evaporated under nitrogen. The residue was dissolved in ethanol and an aliquot transferred to 10 ml o f a toluene-based scintillator t o determine the 3H/'4C ratio o f the urinary oestrogens. A Beckman (Model No. LS-200B) liquid scintillation spectrometer was used for radioactivity determinations. The counting efficiency was 45% for tritium and 67% for carbon-14. Discriminator and gain settings were such that 21% of the carbon-I4 counts appeared in t h e tritium channel. The coefficients of variation in 3H/'4C ratios, estimated from the duplicate analysis of urines obtained from seven subjects were 8.4%, 5.4% and 1.7% for oestrone, oestradiol and oestriol respectively. Determination of the total production rate of oestrone (PREI) The total production rate of oestrone was estimated by an isotope dilution method (Gurpide, 1975). In order to calculate the specific activity of urinary oestrone, the concentration of oestrone, isolated and purified by t h e procedure previously described, was determined by radioimmunoassay. The total production rate of oestrone was calculated by dividing the amount o f tritiated oestrone infused by the cumulative specific activity of urinary oestrone isolated from the 3 day pooled urine collection. The coefficient of variation for determination of the specific activity o f oestrone, estimated from the duplicate analysis o f urines from seven subjects, was 7.0%. RESULTS
Plasma hormone levels The mean (k S.D.) hormone concentrations o n the blood obtained during the pre- and post-operative 24 h sampling periods, and the statistical significance o f differences between the means are shown in Table 1 . The concentrations of the hormones determined in plasma samples obtained simultaneously from the unclarnped left ovarian vein and a peripheral vein are also shown in Table 1 : the ratios for the concentrations o f the steroids in the left ovarian vein t o that in a peripheral vein (also included in Table 1 ) showed that oestradiol was the predominant unconjugated steroid secreted by t h e ovarian tumour. Dynamic studies The results are shown in Tables 2, 3 and 4. Removal o f the ovarian thecoma did not alter
115 5.3 t
f
59.0 0.9
< 3.2
144 f 55 184 t 40 534 t 87 1.30 f 0.4 1 9 3 t 130
(n = 12)
* 0.7
< 3.2 571 2 63.0** 10.6 t 2.0**
1 6 6 + 121
1.43
< 36** 80 t 4 * * 4 2 3 + 101*
After operation
* P < 0.01; * * P < 0.001. Gonadotrophins : Normal premenopausal range LH 0.5-2.0 &l, FSH < 16-50 pg/L Normal postmenopausal range LH > 4.5 #g/l, FSH > 170 #g/l.
Oestrone pmol/l Oestradiol pmol/l Testosterone pmol/l Androstenedione nmol/l Cortisol nmol/l Progesterone nmoyl FSH ccdl LH r g / l
Hormone
Before operation
499 3,380 4,5 10 5.59 618 19.1
Left ovarian vein
At operation
Table 1. Plasma hormone concentrations (mean f S.D.)
3.8 1.3 0.9 3.0 1,I 80 4.43 69 8 6.4
I .a
1 5 .o
281 228
Peripheral vein
Ratio of concentrations in left ovarian vein and peripheral vein
P
wl
M.J. Reed et al,
146
P
Table 2. Plasma production rates of androstenedione (PR -A), transfer constants determined by the AE1 urinary method ([PIS" ) and production rates of oestrone from androstenedione (PREI-A)
Before operation After operation
MCR-A L/24 h
Plasma concentration of androstenedione (A) (n mo I/])
PRP-A* (fimo1/24 h )
1328.0 1149.0
1.3 1.4
1.7 1.6
tU"'t '?o
PR131-A** (nrno1/24 h)
2.4 1.9
41.4 33.9
[PI
* PR'-A = MCR-A x A. * * PREI-A = PRP-A X [ p ] ABEu1 . AE1
t l P ' ~ u=
311/'4C ratio of administered dose, ratio of urinary oestrone
Table 3. Tritium to carbon-14 ratios of urinary oestrogen metabolites, specific activity of urinary oestrone and total production rates of oestrone (PRE-I)
3H/14Cratios of urinary metabolites
Beloreoperation After operation
El
E2
E3
Specific activity of urinary oestrone d.p.rn. X I03/ng
18.6 26.8
11.5 23.6
17.0 27.2
276.0 1227.0
* PREl
=
%-El infused (fiCi)
PRE-I * (nmo1/24 h)
10.1 12.9
101.0 29.0
'11 oestrone administered
Specific activity of urinary oestrone X time of urine collection
Table 4. Blood transfer constants
31H/'4Cratio o f administered dose
311/1qCratio of plasma oestrone
lpltBE1*
0.45 0.52
30.4 40.2
1.5
Re fo re operation After operation AE1
%
I .3
3H/14Cratio of administered dose of plasma oestrone '
* ' P l ~= ~3 k i / l ~ratio
the plasma production rate of androstenedione, nor the extent to which androstenedione was converted t o oestrone, as measured by the urinary method (Table 2). Thus the production o f oestrone from androstenedione before operation of 41.4 nmo1/24 h was similar t o the 32.9 nmo1/24 h production rate after operation. The total production o f oestrone from aU sources (Table 3) was 101 nmo1/24 11 pre-operatively, and 2 9 nmo1/24 h post-operatively. Thus, after operation nearly all the oestrone was derived by the peripheral aromatiiz~tiono f androstenedione, whereas pre-operatively only 40% was derived from plasma androstenedione.
Oestrogen production b y a thecoma
147
The blood transfer constants ( [ p ] ;,"I) for the conversion of androstenedione t o plasma oestrone are shown in Table 4. Although these pre- and post-operative constants of 1.5% and 1.3% are similar to each other (Table 4) they are approximately 40% lower than the values for the urinary transfer constants ( [ p ] of 2.4% and 1.9% respectively, calculated from the 3H/'4C ratio of urinary oestrone (Table 2).
Total urinary oestrogens Before operation excretion of total urinary oestrogens was 33.9 p o 1 / 2 4 h whereas after operation it had decreased to 16.1 pmo1/24 h.
DISCUSSION The results of the present study have shown that oestradiol was the major unconjugated hormone secreted by the ovarian thecoma and that pre-operatively peripheral conversion of androstenedione was not the major source of oestrone. Based on a single blood level there was evidence that some oestrone was secreted by the thecoma. The secretion of oestradiol by the thecoma produced a mean peripheral plasma oestradiol level w h c h was at the upper limit of o u r normal range of 55-187 pmol/l for postmenopausal women of similar age. This mean concentration was therefore not as high as the 220-440 pmol/l that occurs in the midfollicular phase of the cycle in younger women, but was higher than the mean plasma oestrone level. The normal oestradio1:oestrone ratio of 0.60:l that occurs in postmenopausal women was therefore reversed. Although the oestradiol :oestrone ratio in the ovarian vein was 6.8:1, as a result of peripheral metabolism of the oestrogens secreted by the thecoma the ratio in the periphery was 1.3:l. Due to the limitsof sensitivity of the oestrogen assays and the imprecision a t levels < 40 pmol/l, the ratio could not be determined after the operation with accuracy. Post-operatively the low level of oestrone and slightly higher level of oestradiol was unusual for a postmenopausal woman. Clinically there appeared to be no evidence of residual tumour which would have been a source of oestradiol. The pre-operative unconjugated plasma oestrogen levels in peripheral blood in this patient showed fluctuations during the 24 h sampling period. Although the mean plasma oestradiol and oestrone levels were w i t h our normal range for postmenopausal women, the plasma oestrogen levels that normally result solely from the peripheral conversion of adrenal androgens (i.e. the post-operative values in Table 1) were significantly augmented pre-operatively by oestrogens secreted by the thecoma. The development of abnormal hyperplasia and adenocarcinoma of the endometrium in this woman may, therefore, have been due to these peripheral levels being maintained for some considerable time. Although there are theoretical objections to the determination of the conversion rates from urinary metabolites (Tait & Horton, 1966; Rizkallah ef al., 1975) the method does allow comparisons to be made. Dissimilar isotopic ratios of the three urinary oestrogen metabolites such as were observed in this study have previously been reported in patients with endometrial carcinoma (Kelly & Rizkallah, 1973), and in a patient with a granulosa cell tumour (Vaughn er al., 1976). The conversion of androstenedione to oestrone as measured in urine resulted in higher values than obtained by the plasma method: this finding is in agreement with the results of Grodin et al. (1973) who have recently discussed possible reasons for the discrepancy (Edman et al., 1978). The values obtained for the conversion of androstenedione to oestrone, and for the production rates for oestrone were both pre- and
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post-operatively , within the range normally found for women after the menopause (Grodin et al., 1973) or with endometrial carcinoma (hzkallah et ul., 1975). An increase in the rate of conversion of androstenedione to oestrone and the production of oestrone soon after surgery has been reported by Rizkallah et ul. (1975) and MacDonald el al. (1976). In order t o reduce any possible effect of surgery on steroid metabolism (Strong et al., 1956) in this investigation the repeat study was postponed until 2 months after the operation. The similar conversion rates that were obtained pre- and post-operatively in this study suggest that neither the ovaries nor the uterus are substantially involved in the extraglandular aroniatization o f androstenedione. Adipose tissue has usually been regarded as the tissue where extraglandular aromatization occurs (Schindler et al., 1972; Nimrod & Ryan, 1975) but recently the adult liver has also been shown t o be able t o convert androstenedione t o oestrone (Smuk & Schwers, 1977). The results of this study also show that pre-operatively the peripheral conversion o f androstensdione was not the major source of plasma oestrone as is usual after the menopause (Crodin et al., 1973). The 60% o f oestrone that was not produced from plasma androstenedione presumably originated from direct secretion or by peripheral conversion of other oestrogens secreted b y the thecoma, or both. Although the most likely plasma oestrogen t o be converted to oestrone is unconjugated oestradiol, iri vitro studies have shown that ovarian tuniours may produce oestrone sulphate (Flickinger et al., 1965) which may be an alternative source of plasma oestrone (Longcope, 1971). However, total urinary oestrogen levels were within the normal ranges for postmenopausal women (Brown et al., 1968) so it is unlikely that the amount of oestrone sulphate secreted by the thecoma exceeded that o f unconjugated oestradiol. Oestradiol is biologically more potent than oestrone, and, despite an hypothesis implicating oestrone in endometrial carcinogenesis (Siiteri & MacDonald, 1973; Siiteri et al., 1974) we consider it likely that the oestradiol secreted into the circulation by the thecoma was probably the major factor which caused this woman’s abnormal endometrial hyperplasia and carcinoma. ACKNOWLEDGMENTS
We would like t o thank Dr M. C. Anderson for his expert histological opinion. This work was supported by the Cancer Research Campaign and Abbott Laboratories Ltd.
REFERENCES ANIKWUE, C., DAWOOD, Y. & KRAMER, E. (1978) Granulosa and theca cell tumors. Obstetrics arid Gynecology, 52,214-220. BAIRD, D.T., HORTON, R . , LONCCOPE, C. & TAIT, J.F. (1969) Steroid dynamics under steady-state conditions. Recent Progress in Hormone Research, 25, 61 1-656. BESCH, P.K., WATSON, D.J., VORYS, N., HAMWI, G.J., BARRY, R.D. & BARNETT, E.B. (1966) I n v i t n , biosynthetic studies of endocrine tumors. American Journal of Obstetrics and GynecoloKy, 46, 466-471. BRADLOW, H.L. (1968) Extraction of steroid conjugates with a neutral resin. Steroids, 11, 265-272. BROWN, J.B. (1955) A chemical method for the determination of oestriol, oestrone and oestradiol in human urine. Biochemical Journal, 60, 185-193. BROWN, J.B. (1955) A chemical method for the determination of oestriol, oestrone and oestradiol in I: Estrogen assays in gynecology and early pregnancy. Obstetric and Gynecological Survey, 27, 205235.
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BROWN, J.B., KELLAR, R. & MATTHEW, G.D. (1959) Preliminary observations o n urinary oestrogen excretion in certain gynaecological disorders. Journal of Obstetrics and Gynaecology of the British Empire, 66, 177-21 1. BROWN, J . B . , MACLEOD, S.C., MACNAUGHTON, C., SMITH, M.A. & SMYTH, B. (1968) A rapid method for estimating oestrogens in urine using a semi-automatic extractor. Journal of Endocrinology, 42,5-15. DALLENBACH-HELLWEG, G. (1975) Histopathology of the endometrium. Springer Verlag, Berlin. DIDDLE, A.W. (1951) Granulosa and theca cell ovarian tumors. Cancer, 5,215-228. EDMAN, C.D., AIMAN, E.J., PORTER, J.C. & MACDONALD, P.C. (1978) Identification of the oestrogen product of extraglandular aromatization of plasma androstenedione. American Journal of Obstetrics and Gynecology, 130,439-447. FLICKINGER, G.L., MURAWEC, T. & TOUCHSTONE, J.C. (1965) Free and conjugated oestrogens of an ovarian cystadenoma and granulosa cell tumor. Journal of Clinical Endocrinology and Metabolism, 25, 1231-1236. GOODALL, A.B., RIPPON, A.E. & JAMES, V.H.T. (1979) A simple nonchromatographic radioimmunoassay for Androstenedione. Journal ofSteroid Biochemistry (in press). GRIFFITHS, K., GRANT, J.K. & SYMINGTON, T. (1964) Steroid biosynthesis in vitro by granulosa-theca cell tumour tissue. Journal of Endocrinology, 30, 247-254. GRODIN, J.M., SIITERI, P.K. & MACDONALD, P.C. (1973) Source of estrogen production in postmenopausal women. Journal of Clinical Endocrinology and Metabolism, 36,207-21 4. GURPIDE, E. (1975) Tracer methods in hormone research, p. 161. Springer-Verlag, Berlin, Heidelberg & New York. GUSBERG, S.B. & KARDON, P. (1971) Proliferative endometrial response to theca-granulosa cell tumors. American Journal of Obstetrics and Gynecology, 1 1 1, 633-64 1. HAMMOND, C.B. (1976) T h e menopause-an American view. In TheManagement of'the Menopause and Postmenopausal Years (ed. S . Campbell). LMTP Press, Lancaster. I-IOTCHKISS, J., ATKINSON, L.E. & KRIOBIL, E. (1971) Time course of serum estrogen and luteinizing hormone concentrations during the menstrual cycle of the Rhesus monkey. Endocrinology, 89, 177183. HUTTON, J.D., MORSE, A.R., BEARD, R.W. & ANDERSON, M.C. (1978) Endometrical assessment with lsaacs cell sampler. British Medical Journal, i, 947-949. JACOBS, H.S. & LAWTON, N.F. (1974) Pituitary and placental glycopeptide hormones. British Medical Bulletin, 30,55-6 1. KELLY, W.G. & RIZKALLAH, T.W. (1973) Dissimilar isotopic ratios in urinary estrogens following simultaneous injection of [ I4C] androstenedione and ['HI estrone. Journal of Clinical Endocrinology and Metabolism, 36, 196-199. LARMONT, C.A.R. & ASHTON, R.W. (1975) Observations o n granulosa cell tunours: A clinical review of 2 3 patients. Excerpta Medica, 364,241-248. LARSON, J.A. (1954) Estrogens and endometrial carcinoma. Obstetrics and Gynecology, 3,551-572. LONGCOPE, C. (1971) Metabolic clearance and blood production rates of oestrogens in postmenopausal women. American Journal of Obstetricsand Gynecology, I l l , 778-781. MACDONALD, P.C., GRODIN, J.M., EDMAN, C.D., VELLIOS, F. & SIITERI, P.K. (1976) Origin of estrogen in a postmenopausal woman with a nonendocrine tumour of the ovary and endometrial hyperplasia. Obstetrics and Gynecology, 47, 644-650. MCDONALD, T.W., MALKASIAN, G.D. & GAFFEY, T.A. (1976) Endometrial cancer associated with feminizing ovarian tumor and polycystic ovarian disease. Obstetrics and Gynecology, 49,654-658. MCGOLDRICK, J.L. & LAPP, W.A. (1944) Theca cell tumors of the ovary. American JournalofObstetrics & Gynecology. 48,409-41 5 . NIMROD, A. & RYAN, K.H. (1975) Aromatization of androgens by human abdominal and breast fat tissue. Journal of Clinical Endocrinology and Metabolism, 40, 367-312. NORRIS, H.J. & CHORLTON, I. (1974) Functioning tumors of the ovary. Clinical Obstetrics and Gynecology, 17, 184-228. OLIVO, J., VITTEK, J., SOUTHREN, A.L., GORDON, G.G. & RAFII, F. (1973) Rapid method for the measurement of androgen kinetics and conversion to estrogens using Sephadex LH-20 column chromatography. Journal of Clinical Endocrinology and Metabolism, 36, 153-1 59. RIZKALLAH, T.H., TOVEL, H.M.M. & KELLY, W.G. (1975) Production of estrone and fractional con-
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version of circulating androstenedione to estrone in women with endometrial carcinoma. Journal of Clinical Endocrinology and Metabolism. 40, 1045-1056. SCHINDLER, A.E., EBERT, A. & FRIEDRICH, E.F. (1972) Conversion of androstenedione to estrone by human fat tissue. Journal of Clinical Endocrinology and Metabolism, 35, 627-630. SCHROEDEK, R. (1 922) Granulosazelltumor des Ovars mit glandulircystischer Hyperplasie des Endometriums und beginnenedem Karzinom auf diesem Boden.Zentralblattfir Gynukologie, 46, 195-1 96 SIITERI, P.K. & MACDONALD, P.C. (1973) Handbook of Physiology-section 7, Endocrinology, p. 615 (FAs. Astwood, G.B. & Creep, R.O., American Physiological Society: Washington, D.C.). SIITERI, P.K., SCHWARZ, B.E. & MACDONALD, P.C. (1974) Estrogen receptors and the estrone hypothesis in relation to endometrial and breast cancer. Gynecologic Oncology, 2 , 228-238. SMUK, M . & SCHWERS, J . (1977) Aromatization of androstenedione by human adult liver in vitro. Journal of Clinical Endocrinology and Metabolism, 45,1009- 10 12. STRONG, J.A., BROWN, J.B., BRUCE, J., DOUGLAS, M., KLOPPER, A.1. & LORAINE, J.A. (1956) Sex-hormone excretion after bilateral adrenalectomy and oophorectomy in patients with mammary carcinoma. Lancet, ii, 955-959. TAIT, J . & HORTON, R. (1966) Steroid Dynamics. Academic Press, New York. TARGETT, C.S. (1974) Estrogen excretion in a case of theca-granulosa cell tumor. American Journal of Obstetrics and Gynecology, 119,859-861. VAUGHN, C.B., KOLAKOWSKI, D., ZYLKA, U. & BROOKS, S.C. (1976) The fate of 17p estradiol in the plasma of premenopausal and postmenopausal patients with cancer. Journal of Clinical Endocrinology and Metabolism, 43, 381-39 3.
PLASMA HORMONE LEVELS A N D OESTROGEN PRODUCTION IN A POSTMENOPAUSAL WOMAN WITH E N D O M E T R I A L CARCINOMA A N D AN O V A R I A N THECOMA M . J . REED, J. D. HUTTON, R. W. B E A R D , H. S . J A C O B S AND V . H. T. J A M E S Departments of Chemical Pathology and Obstetrics and Gynaecology, St Mary’s Hospital Medical School, London, W.2 (Received 28 October 19 78; revised 16 January 1979; accepted 30 January 1979)
SUMMARY
Plasma hormone concentrations, the production rate of oestrone from androstenedione, and the total production rate of oestrone have been measured in a 57year-old woman with an ovarian thecoma and endometrial carcinoma before and 2 months after hysterectomy and bilateral ovariectomy. The plasma concentration of oestradiol (184 pmol/l), but not oestrone (144 pmol/l), was elevated preoperatively and FSH secretion was suppressed (1 15.0 pg/l). Post-operatively the plasma concentration of oestradiol was significantly reduced (80 pmol/l) and the concentration of FSH (571 pg/l) had increased to a postmenopausal level. The concentration of oestradiol in samples of plasma obtained simultaneously at operation from the ovarian vein of the ovary containing the thecoma (3.38 nmol/l) was fifteen times greater than in the periphery (228.0 pmol/l), and this confirmed that oestradiol was secreted by the thecoma. The total production rates of oestrone preand post-operatively were 101.3 nmo1/24 h and 29.2 nmo1/24 h respectively and the production rates of oestrone from androstenedione were 41.4 nmo1/24 h and 32.9 nmo1/24 h. Thus, postaperatively, all the oestrone was produced from circulating androstenedione, whereas pre-operatively only 40% of the oestrone was produced by the extraglandular conversion of androstenedione; the remaining 60% presumably originated from the peripheral conversion of oestradiol secreted by the thecoma. It is concluded that the oestradiol secreted by the thecoma was probably the major factor which caused the development of endometrial carcinoma in this subject. The association of endometrial cancer with an oestrogen producing ovarian tumour, first reported by Schroeder in 1922, has since been confirmed by several studies (Diddle, 1951; Larson, 1954; McDonald et al., 1976). The incidence of feminizing tumours of the ovary have been estimated to be between 3-6% of all ovarian tumours (Larmont & Ashton, 1975; Correspondence: Dr M. J . Reed, Department of Chemical Pathology, St Mary’s Hospital Medical School, London, W.2. 03304664/79/08004 141$02.00
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Norris & Chorlton, 1974) but may be less than 1% (Anikwue et al., 1978). A recent survey of the literature (Anikwue et al. 1978) showed that as many as 17% of the subjects with an ovarian thecoma also had endometrial carcinoma. Because of the clinical association of endometrial carcinoma with ovarian thecomas it has been suggested that unopposed secretion of oestrogens by the tumour may cause endometrial carcinoma (Gusberg & Kardon, 1971). Measurements of steroid excretion before and after surgery have shown that a large proportion of urinary oestrogens may originate from ovarian tumours (Brown et af., 195Y; Brown & Beischer, 1972; Targett, 1974). In vitro studies have demonstrated that tissue obtained from oestrogen producing ovarian tumours has the ability t o synthesize both oestrone and oestradiol (Griffiths et al., 1964; Besch et al., 1966) although in v i secretion ~ ~ o f these steroids by such tumours has not yet been demonstrated. It has recently been shown b y the use o f isotope techniques that the increased oestrone production in a subject with a mucinous cystadenocarcinoma of the ovary resulted from excessive secretion of androstenedione, although ext raglandular conversion of androstenedione to oestrone was normal (MacDonald ef al., 1976). The present study was carried out to determine the hormonal status o f a postmenopausal woman with an ovarian thecoma and endometrial carcinoma and to assess the contributionof the thecoma to oestrogen production. CASE REPORT
A 57-year-old woman (height 151 cm, weight 4 7 kg) was admitted to hospital for investigation of continuous vaginal bleeding o f 1 year’s duration. When aged 4 5 years, her regular periods had become heavier and, 5 years later, she developed irregular vaginal bleeding. A swelling, about 8 cm diameter, was palpable posterior to a normal-sized anteverted uterus. The vaginal epithelium was not atrophic (Hammond, 1976) and no other abnormality was detected. Her blood haemoglobin concentration was 9.7 g/dl; liver, renal and thyroid function tests were normal. The karyopyknotic index of a lateral vaginal wall smear was 21%. The cytological diagnosis of an aspirate smear from the endometrial cavity was of a well differentiated adenocarcinoma (Hutton et al., 1978) and this was confirmed at curettage. The patient was readmitted one month later having meanwhile taken 200 mg of iron daily; her blood haemoglobin was then 11.9 g/dl. Before undergoing laparotomy the patient gave her informed consent to hormone studies using radioactive isotopes. Approval for these studies was obtained from the hospital ethical committee and Department of Health and Social Security Committee for the use of isotopes. Four days after the completion of t h e first endocrine study the patient underwent laparotomy; the left ovary was enlarged and posterior t o the normal sized uterus. The fallopian tubes and atrophied right ovary appeared normal. A total abdominal hysterectomy and bilateral salpingoophorectomy was then performed. The second endocrine study was carried out 2 months after the operation at which time the karyopyknotic index was 0%. Pathology Pathological examination showed that the left ovary, which had a smooth surface, was replaced by a tumour measuring 7 X 4 X 4 cm, which was soft and when cut was yellow. This soft yellow area showed the classical histological features of a benign thecoma (McGoldrick & Lapp, 1944) whereas the residue was a fibroma. No granulosa cell elements, follicles or germ cells were seen in either ovary. l-bstological examination of the thick poly-
Oestrogen production b y a thecoma
I43
poid endometrium showed a well differentiated adenocarcinoma; there were also a few areas of cystic hyperplasia, adenomatous hyperplasia and atypical adenomatous hyperplasia (Dallenb ach-He llweg , 19 7 5).
Biochemical studies Hormone concentrations. Plasma hormones were measured by specific radioimmunoassays: oestrone and oestradiol by a modification of the procedure described by Hotchkiss et al. (1971); androstenedione and testosterone as described by Goodall et a f . (1979). Cortisol was measured after extraction from plasma with dichloromethane and evaporation of an aliquot of the extract. Progesterone was measured after extraction from plasma with hexane using ammonium sulphate precipitation to separate free from anti-body bound hormone. Plasma FSH (MRC 68/39) and LH (MRC 68/40) were measured using antisera supplied by Dr W. D. Ode11 (Jacobs & Lawton, 1974) and reference preparations by the Division of Biological Standards of the MRC of the U.K. So that accurate estimates of the plasma hormone concentrations throughout the day could be determined, heparinized blood samples (8 ml) were obtained through an indwelling cannula in a forearm vein every 2 h for 24 h. The plasma was separated immediately after collection and stored at -20°C until processed. The significance of differences between plasma hormone concentrations measured pre- and post-operatively was analysed by Student's t test. Urine was collected during this 24 h sampling period and the urinary excretion of oestrogens measured (Brown, 1955). Blood samples (10 ml) were also obtained at operation simultaneously from a peripheral vein and a vein close to the left ovary. Dynamic studies. Oestrone production and the conversion of androstenedione to oestrone was determined by the double isotope technique described by Grodin et al. (1973), whose symbols and abbreviations are used in the present study. At the end of the 24 h period during which sequential blood samples were obtained, approximately 12 pCi [6, 7-3H] oestrone (50 Ci/mmol) and 24 pCi [4-14C] androstenedione (50 mCi/mmol) in 4% human serum albumin were infused into an arm vein over a 4.5 h period. Blood samples (50 ml) were obtained 3.5, 4.0 and 4.5 h after tracer infusion was started. Urine was collected for 72 ti. Radioisotopes were obtained from the Kadiochemical Centre, Amersham, U.K., and purified before use by paper chromatography, using light petroleum (100"-120" fraction): to1uene:me;hanol:water (33:17:40:10 by volume) as solvents. Determination of the plasma production rate of androstenedione (PRp-A) The metabolic clearance rate for androstenedione (MCR-A) was calculated from the equilibrium concentration of [4-14C] androstenedione in plasma (Baird et al., 1969). This latter was measured after ether extraction and Sephadex LH 20 column chromatography (Olivo et af., 1973). The coefficient of variation for the determination of the metabolic clearance rate for androstenedione by this method in plasma samples obtained from nine subjects undergoing similar investigations was 7.4%. Determination of the Blood Transfer Constant [ p ] [3H]- and [I4C]-Oestrone extracted from plasma (Olivo et al., 1973) were further purified by thin-layer chromatography (TLC) using dichloromethane:ethylacetate (4:l V/V) as solvents. Determination of the isotopic ratios in replicate plasma samples obtained from eight subjects gave a coefficient of variation of 15.9% from this method.
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Determination of the extent of the conversion of plasma androstenedione to oestrone as measured in urine / p ] '2; Steroid conjugates were recovered from urine by passing an aliquot of the pooled 3 day urine collection through an Amberlite XAD-2 ion exchange resin and subsequently eluting with methanol (Bradlow, 1968). The steroid conjugates were hydrolyzed after evaporation of the methanol and reconstitution in buffer with a P-glucuronidase enzyme preparation (Ketodase, W. R. Warner & Co. Ltd., Eastleigh, Hampshire, U.K.) for 48 h at 37°C and the liberated steroids were extracted with chloroform. After washing the chloroform with water and drying with anhydrous sodium sulphate, the chloroform was evaporated under vacuum. The residue was used to prepare a phenolic fraction (Brown, 1955) and oestrone, oestradiol and oestriol were subsequently separated using a Sephadex LH20 (20 g) column with toluene:methanol(85: I5 v/v) as solvents. Each oestrogen was subjected t o three further purification steps b y TLC. Tritiated steroid standards were also chromatographed and were located using a radiochromatogram imaging system (Panax Equipment Ltd., Surrey, U.K.). The oestrogens were eluted with ether and the ether evaporated under nitrogen. The residue was dissolved in ethanol and an aliquot transferred to 10 ml o f a toluene-based scintillator t o determine the 3H/'4C ratio o f the urinary oestrogens. A Beckman (Model No. LS-200B) liquid scintillation spectrometer was used for radioactivity determinations. The counting efficiency was 45% for tritium and 67% for carbon-14. Discriminator and gain settings were such that 21% of the carbon-I4 counts appeared in t h e tritium channel. The coefficients of variation in 3H/'4C ratios, estimated from the duplicate analysis of urines obtained from seven subjects were 8.4%, 5.4% and 1.7% for oestrone, oestradiol and oestriol respectively. Determination of the total production rate of oestrone (PREI) The total production rate of oestrone was estimated by an isotope dilution method (Gurpide, 1975). In order to calculate the specific activity of urinary oestrone, the concentration of oestrone, isolated and purified by t h e procedure previously described, was determined by radioimmunoassay. The total production rate of oestrone was calculated by dividing the amount o f tritiated oestrone infused by the cumulative specific activity of urinary oestrone isolated from the 3 day pooled urine collection. The coefficient of variation for determination of the specific activity o f oestrone, estimated from the duplicate analysis o f urines from seven subjects, was 7.0%. RESULTS
Plasma hormone levels The mean (k S.D.) hormone concentrations o n the blood obtained during the pre- and post-operative 24 h sampling periods, and the statistical significance o f differences between the means are shown in Table 1 . The concentrations of the hormones determined in plasma samples obtained simultaneously from the unclarnped left ovarian vein and a peripheral vein are also shown in Table 1 : the ratios for the concentrations o f the steroids in the left ovarian vein t o that in a peripheral vein (also included in Table 1 ) showed that oestradiol was the predominant unconjugated steroid secreted by t h e ovarian tumour. Dynamic studies The results are shown in Tables 2, 3 and 4. Removal o f the ovarian thecoma did not alter
115 5.3 t
f
59.0 0.9
< 3.2
144 f 55 184 t 40 534 t 87 1.30 f 0.4 1 9 3 t 130
(n = 12)
* 0.7
< 3.2 571 2 63.0** 10.6 t 2.0**
1 6 6 + 121
1.43
< 36** 80 t 4 * * 4 2 3 + 101*
After operation
* P < 0.01; * * P < 0.001. Gonadotrophins : Normal premenopausal range LH 0.5-2.0 &l, FSH < 16-50 pg/L Normal postmenopausal range LH > 4.5 #g/l, FSH > 170 #g/l.
Oestrone pmol/l Oestradiol pmol/l Testosterone pmol/l Androstenedione nmol/l Cortisol nmol/l Progesterone nmoyl FSH ccdl LH r g / l
Hormone
Before operation
499 3,380 4,5 10 5.59 618 19.1
Left ovarian vein
At operation
Table 1. Plasma hormone concentrations (mean f S.D.)
3.8 1.3 0.9 3.0 1,I 80 4.43 69 8 6.4
I .a
1 5 .o
281 228
Peripheral vein
Ratio of concentrations in left ovarian vein and peripheral vein
P
wl
M.J. Reed et al,
146
P
Table 2. Plasma production rates of androstenedione (PR -A), transfer constants determined by the AE1 urinary method ([PIS" ) and production rates of oestrone from androstenedione (PREI-A)
Before operation After operation
MCR-A L/24 h
Plasma concentration of androstenedione (A) (n mo I/])
PRP-A* (fimo1/24 h )
1328.0 1149.0
1.3 1.4
1.7 1.6
tU"'t '?o
PR131-A** (nrno1/24 h)
2.4 1.9
41.4 33.9
[PI
* PR'-A = MCR-A x A. * * PREI-A = PRP-A X [ p ] ABEu1 . AE1
t l P ' ~ u=
311/'4C ratio of administered dose, ratio of urinary oestrone
Table 3. Tritium to carbon-14 ratios of urinary oestrogen metabolites, specific activity of urinary oestrone and total production rates of oestrone (PRE-I)
3H/14Cratios of urinary metabolites
Beloreoperation After operation
El
E2
E3
Specific activity of urinary oestrone d.p.rn. X I03/ng
18.6 26.8
11.5 23.6
17.0 27.2
276.0 1227.0
* PREl
=
%-El infused (fiCi)
PRE-I * (nmo1/24 h)
10.1 12.9
101.0 29.0
'11 oestrone administered
Specific activity of urinary oestrone X time of urine collection
Table 4. Blood transfer constants
31H/'4Cratio o f administered dose
311/1qCratio of plasma oestrone
lpltBE1*
0.45 0.52
30.4 40.2
1.5
Re fo re operation After operation AE1
%
I .3
3H/14Cratio of administered dose of plasma oestrone '
* ' P l ~= ~3 k i / l ~ratio
the plasma production rate of androstenedione, nor the extent to which androstenedione was converted t o oestrone, as measured by the urinary method (Table 2). Thus the production o f oestrone from androstenedione before operation of 41.4 nmo1/24 h was similar t o the 32.9 nmo1/24 h production rate after operation. The total production o f oestrone from aU sources (Table 3) was 101 nmo1/24 11 pre-operatively, and 2 9 nmo1/24 h post-operatively. Thus, after operation nearly all the oestrone was derived by the peripheral aromatiiz~tiono f androstenedione, whereas pre-operatively only 40% was derived from plasma androstenedione.
Oestrogen production b y a thecoma
147
The blood transfer constants ( [ p ] ;,"I) for the conversion of androstenedione t o plasma oestrone are shown in Table 4. Although these pre- and post-operative constants of 1.5% and 1.3% are similar to each other (Table 4) they are approximately 40% lower than the values for the urinary transfer constants ( [ p ] of 2.4% and 1.9% respectively, calculated from the 3H/'4C ratio of urinary oestrone (Table 2).
Total urinary oestrogens Before operation excretion of total urinary oestrogens was 33.9 p o 1 / 2 4 h whereas after operation it had decreased to 16.1 pmo1/24 h.
DISCUSSION The results of the present study have shown that oestradiol was the major unconjugated hormone secreted by the ovarian thecoma and that pre-operatively peripheral conversion of androstenedione was not the major source of oestrone. Based on a single blood level there was evidence that some oestrone was secreted by the thecoma. The secretion of oestradiol by the thecoma produced a mean peripheral plasma oestradiol level w h c h was at the upper limit of o u r normal range of 55-187 pmol/l for postmenopausal women of similar age. This mean concentration was therefore not as high as the 220-440 pmol/l that occurs in the midfollicular phase of the cycle in younger women, but was higher than the mean plasma oestrone level. The normal oestradio1:oestrone ratio of 0.60:l that occurs in postmenopausal women was therefore reversed. Although the oestradiol :oestrone ratio in the ovarian vein was 6.8:1, as a result of peripheral metabolism of the oestrogens secreted by the thecoma the ratio in the periphery was 1.3:l. Due to the limitsof sensitivity of the oestrogen assays and the imprecision a t levels < 40 pmol/l, the ratio could not be determined after the operation with accuracy. Post-operatively the low level of oestrone and slightly higher level of oestradiol was unusual for a postmenopausal woman. Clinically there appeared to be no evidence of residual tumour which would have been a source of oestradiol. The pre-operative unconjugated plasma oestrogen levels in peripheral blood in this patient showed fluctuations during the 24 h sampling period. Although the mean plasma oestradiol and oestrone levels were w i t h our normal range for postmenopausal women, the plasma oestrogen levels that normally result solely from the peripheral conversion of adrenal androgens (i.e. the post-operative values in Table 1) were significantly augmented pre-operatively by oestrogens secreted by the thecoma. The development of abnormal hyperplasia and adenocarcinoma of the endometrium in this woman may, therefore, have been due to these peripheral levels being maintained for some considerable time. Although there are theoretical objections to the determination of the conversion rates from urinary metabolites (Tait & Horton, 1966; Rizkallah ef al., 1975) the method does allow comparisons to be made. Dissimilar isotopic ratios of the three urinary oestrogen metabolites such as were observed in this study have previously been reported in patients with endometrial carcinoma (Kelly & Rizkallah, 1973), and in a patient with a granulosa cell tumour (Vaughn er al., 1976). The conversion of androstenedione to oestrone as measured in urine resulted in higher values than obtained by the plasma method: this finding is in agreement with the results of Grodin et al. (1973) who have recently discussed possible reasons for the discrepancy (Edman et al., 1978). The values obtained for the conversion of androstenedione to oestrone, and for the production rates for oestrone were both pre- and
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M. J. Reed et al.
post-operatively , within the range normally found for women after the menopause (Grodin et al., 1973) or with endometrial carcinoma (hzkallah et ul., 1975). An increase in the rate of conversion of androstenedione to oestrone and the production of oestrone soon after surgery has been reported by Rizkallah et ul. (1975) and MacDonald el al. (1976). In order t o reduce any possible effect of surgery on steroid metabolism (Strong et al., 1956) in this investigation the repeat study was postponed until 2 months after the operation. The similar conversion rates that were obtained pre- and post-operatively in this study suggest that neither the ovaries nor the uterus are substantially involved in the extraglandular aroniatization o f androstenedione. Adipose tissue has usually been regarded as the tissue where extraglandular aromatization occurs (Schindler et al., 1972; Nimrod & Ryan, 1975) but recently the adult liver has also been shown t o be able t o convert androstenedione t o oestrone (Smuk & Schwers, 1977). The results of this study also show that pre-operatively the peripheral conversion o f androstensdione was not the major source of plasma oestrone as is usual after the menopause (Crodin et al., 1973). The 60% o f oestrone that was not produced from plasma androstenedione presumably originated from direct secretion or by peripheral conversion of other oestrogens secreted b y the thecoma, or both. Although the most likely plasma oestrogen t o be converted to oestrone is unconjugated oestradiol, iri vitro studies have shown that ovarian tuniours may produce oestrone sulphate (Flickinger et al., 1965) which may be an alternative source of plasma oestrone (Longcope, 1971). However, total urinary oestrogen levels were within the normal ranges for postmenopausal women (Brown et al., 1968) so it is unlikely that the amount of oestrone sulphate secreted by the thecoma exceeded that o f unconjugated oestradiol. Oestradiol is biologically more potent than oestrone, and, despite an hypothesis implicating oestrone in endometrial carcinogenesis (Siiteri & MacDonald, 1973; Siiteri et al., 1974) we consider it likely that the oestradiol secreted into the circulation by the thecoma was probably the major factor which caused this woman’s abnormal endometrial hyperplasia and carcinoma. ACKNOWLEDGMENTS
We would like t o thank Dr M. C. Anderson for his expert histological opinion. This work was supported by the Cancer Research Campaign and Abbott Laboratories Ltd.
REFERENCES ANIKWUE, C., DAWOOD, Y. & KRAMER, E. (1978) Granulosa and theca cell tumors. Obstetrics arid Gynecology, 52,214-220. BAIRD, D.T., HORTON, R . , LONCCOPE, C. & TAIT, J.F. (1969) Steroid dynamics under steady-state conditions. Recent Progress in Hormone Research, 25, 61 1-656. BESCH, P.K., WATSON, D.J., VORYS, N., HAMWI, G.J., BARRY, R.D. & BARNETT, E.B. (1966) I n v i t n , biosynthetic studies of endocrine tumors. American Journal of Obstetrics and GynecoloKy, 46, 466-471. BRADLOW, H.L. (1968) Extraction of steroid conjugates with a neutral resin. Steroids, 11, 265-272. BROWN, J.B. (1955) A chemical method for the determination of oestriol, oestrone and oestradiol in human urine. Biochemical Journal, 60, 185-193. BROWN, J.B. (1955) A chemical method for the determination of oestriol, oestrone and oestradiol in I: Estrogen assays in gynecology and early pregnancy. Obstetric and Gynecological Survey, 27, 205235.
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BROWN, J.B., KELLAR, R. & MATTHEW, G.D. (1959) Preliminary observations o n urinary oestrogen excretion in certain gynaecological disorders. Journal of Obstetrics and Gynaecology of the British Empire, 66, 177-21 1. BROWN, J . B . , MACLEOD, S.C., MACNAUGHTON, C., SMITH, M.A. & SMYTH, B. (1968) A rapid method for estimating oestrogens in urine using a semi-automatic extractor. Journal of Endocrinology, 42,5-15. DALLENBACH-HELLWEG, G. (1975) Histopathology of the endometrium. Springer Verlag, Berlin. DIDDLE, A.W. (1951) Granulosa and theca cell ovarian tumors. Cancer, 5,215-228. EDMAN, C.D., AIMAN, E.J., PORTER, J.C. & MACDONALD, P.C. (1978) Identification of the oestrogen product of extraglandular aromatization of plasma androstenedione. American Journal of Obstetrics and Gynecology, 130,439-447. FLICKINGER, G.L., MURAWEC, T. & TOUCHSTONE, J.C. (1965) Free and conjugated oestrogens of an ovarian cystadenoma and granulosa cell tumor. Journal of Clinical Endocrinology and Metabolism, 25, 1231-1236. GOODALL, A.B., RIPPON, A.E. & JAMES, V.H.T. (1979) A simple nonchromatographic radioimmunoassay for Androstenedione. Journal ofSteroid Biochemistry (in press). GRIFFITHS, K., GRANT, J.K. & SYMINGTON, T. (1964) Steroid biosynthesis in vitro by granulosa-theca cell tumour tissue. Journal of Endocrinology, 30, 247-254. GRODIN, J.M., SIITERI, P.K. & MACDONALD, P.C. (1973) Source of estrogen production in postmenopausal women. Journal of Clinical Endocrinology and Metabolism, 36,207-21 4. GURPIDE, E. (1975) Tracer methods in hormone research, p. 161. Springer-Verlag, Berlin, Heidelberg & New York. GUSBERG, S.B. & KARDON, P. (1971) Proliferative endometrial response to theca-granulosa cell tumors. American Journal of Obstetrics and Gynecology, 1 1 1, 633-64 1. HAMMOND, C.B. (1976) T h e menopause-an American view. In TheManagement of'the Menopause and Postmenopausal Years (ed. S . Campbell). LMTP Press, Lancaster. I-IOTCHKISS, J., ATKINSON, L.E. & KRIOBIL, E. (1971) Time course of serum estrogen and luteinizing hormone concentrations during the menstrual cycle of the Rhesus monkey. Endocrinology, 89, 177183. HUTTON, J.D., MORSE, A.R., BEARD, R.W. & ANDERSON, M.C. (1978) Endometrical assessment with lsaacs cell sampler. British Medical Journal, i, 947-949. JACOBS, H.S. & LAWTON, N.F. (1974) Pituitary and placental glycopeptide hormones. British Medical Bulletin, 30,55-6 1. KELLY, W.G. & RIZKALLAH, T.W. (1973) Dissimilar isotopic ratios in urinary estrogens following simultaneous injection of [ I4C] androstenedione and ['HI estrone. Journal of Clinical Endocrinology and Metabolism, 36, 196-199. LARMONT, C.A.R. & ASHTON, R.W. (1975) Observations o n granulosa cell tunours: A clinical review of 2 3 patients. Excerpta Medica, 364,241-248. LARSON, J.A. (1954) Estrogens and endometrial carcinoma. Obstetrics and Gynecology, 3,551-572. LONGCOPE, C. (1971) Metabolic clearance and blood production rates of oestrogens in postmenopausal women. American Journal of Obstetricsand Gynecology, I l l , 778-781. MACDONALD, P.C., GRODIN, J.M., EDMAN, C.D., VELLIOS, F. & SIITERI, P.K. (1976) Origin of estrogen in a postmenopausal woman with a nonendocrine tumour of the ovary and endometrial hyperplasia. Obstetrics and Gynecology, 47, 644-650. MCDONALD, T.W., MALKASIAN, G.D. & GAFFEY, T.A. (1976) Endometrial cancer associated with feminizing ovarian tumor and polycystic ovarian disease. Obstetrics and Gynecology, 49,654-658. MCGOLDRICK, J.L. & LAPP, W.A. (1944) Theca cell tumors of the ovary. American JournalofObstetrics & Gynecology. 48,409-41 5 . NIMROD, A. & RYAN, K.H. (1975) Aromatization of androgens by human abdominal and breast fat tissue. Journal of Clinical Endocrinology and Metabolism, 40, 367-312. NORRIS, H.J. & CHORLTON, I. (1974) Functioning tumors of the ovary. Clinical Obstetrics and Gynecology, 17, 184-228. OLIVO, J., VITTEK, J., SOUTHREN, A.L., GORDON, G.G. & RAFII, F. (1973) Rapid method for the measurement of androgen kinetics and conversion to estrogens using Sephadex LH-20 column chromatography. Journal of Clinical Endocrinology and Metabolism, 36, 153-1 59. RIZKALLAH, T.H., TOVEL, H.M.M. & KELLY, W.G. (1975) Production of estrone and fractional con-
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version of circulating androstenedione to estrone in women with endometrial carcinoma. Journal of Clinical Endocrinology and Metabolism. 40, 1045-1056. SCHINDLER, A.E., EBERT, A. & FRIEDRICH, E.F. (1972) Conversion of androstenedione to estrone by human fat tissue. Journal of Clinical Endocrinology and Metabolism, 35, 627-630. SCHROEDEK, R. (1 922) Granulosazelltumor des Ovars mit glandulircystischer Hyperplasie des Endometriums und beginnenedem Karzinom auf diesem Boden.Zentralblattfir Gynukologie, 46, 195-1 96 SIITERI, P.K. & MACDONALD, P.C. (1973) Handbook of Physiology-section 7, Endocrinology, p. 615 (FAs. Astwood, G.B. & Creep, R.O., American Physiological Society: Washington, D.C.). SIITERI, P.K., SCHWARZ, B.E. & MACDONALD, P.C. (1974) Estrogen receptors and the estrone hypothesis in relation to endometrial and breast cancer. Gynecologic Oncology, 2 , 228-238. SMUK, M . & SCHWERS, J . (1977) Aromatization of androstenedione by human adult liver in vitro. Journal of Clinical Endocrinology and Metabolism, 45,1009- 10 12. STRONG, J.A., BROWN, J.B., BRUCE, J., DOUGLAS, M., KLOPPER, A.1. & LORAINE, J.A. (1956) Sex-hormone excretion after bilateral adrenalectomy and oophorectomy in patients with mammary carcinoma. Lancet, ii, 955-959. TAIT, J . & HORTON, R. (1966) Steroid Dynamics. Academic Press, New York. TARGETT, C.S. (1974) Estrogen excretion in a case of theca-granulosa cell tumor. American Journal of Obstetrics and Gynecology, 119,859-861. VAUGHN, C.B., KOLAKOWSKI, D., ZYLKA, U. & BROOKS, S.C. (1976) The fate of 17p estradiol in the plasma of premenopausal and postmenopausal patients with cancer. Journal of Clinical Endocrinology and Metabolism, 43, 381-39 3.
