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Department of Endocrinology (C.R., A.B., D.M., T.G., J.-P.L., P.C.), Centre Hospitalier Universitaire (CHU) Rangueil; Departments of Clinical Biochemistry (M.P.) and Gynecology (X.M.), CHU La Grave; and Department of Histopathology (P.R.), Institut Claudius Régaud, Toulouse, France
Address all correspondence and requests for reprints to: P. Caron, M.D., Service d’Endocrinologie, Hôpital Rangueil, avenue Jean Poulhès, 31403 Toulouse Cedex, France. E-mail: . caron.p{at}chu-toulouse.fr
Abstract
Ovarian virilizing tumors are rare and can lead to assessment difficulties because of their small size. A 41-yr-old female was referred for evaluation of hirsutism that had increased within the previous 3 yr. Menstrual cycle length was normal. Plasma testosterone was 3.9 ng/ml (normal range, 0.2–0.8 ng/ml), was not suppressible by 2 mg dexamethasone (4.3 ng/ml), and was increased (6.3 ng/ml) after three daily injections of hCG (5000 IU). Abdominal computed tomography scan showed an adrenal nodule (13 x 6 mm) that remained unchanged after 3 months. Ultrasound examination of the pelvis was normal. Ovarian and adrenal venous catheterization did not yield additional information. Topographic assessment was made by intraoperative measurement of testosterone in the samples taken from each ovarian vein (competitive chemiluminescent immunoassay ADVIA Centaur; right ovarian vein, 105 ng/ml; left ovarian vein, 5 ng/ml; peripheral blood, 7 ng/ml). Right annexectomy resulted in normalization of testosterone levels (0.22 ng/ml). Histopathological examination found a Leydig cell tumor of hilar type (1.5 cm). This observation illustrates the usefulness of intraoperative measurement of testosterone by a rapid automated technique for topographic assessment of ovarian virilizing tumor in premenopausal women.
VIRILIZING TUMORS are rare; they develop from the ovary or adrenal cortex and are observed more frequently in postmenopausal women. The diagnosis of ovarian virilizing tumors can be difficult in women of reproductive age, because the size of such tumors is often too small to allow detection by ultrasound or radiological examination (1).
We report the case of a virilizing ovarian tumor in a premenopausal woman. The diagnosis of the tumor location was made using an intraoperative measurement of testosterone levels in blood samples taken directly from the ovarian veins, thereby leading to conservative surgery.
Case Report
A 41-yr-old female, without significant past medical history, was referred to our Department of Endocrinology for evaluation of hirsutism. Her hirsutism had developed within the last 3 yr and had become more severe within the last 6 months. The patient did not report any alteration regarding her libido or length of her menstrual cycles. The patient was taking no medication.
On physical examination the patient was found to have hirsutism on the chin, breasts, and lower abdomen. Mild clitoral enlargement was observed. No other clinical abnormality was found: the body mass index was within the normal range (22 kg/m2), and blood pressure was 110/80 mm Hg. The patient had no specific symptoms of Cushing’s syndrome. The abdominal palpation and gynecological examination did not provide additional information.
The patient had high serum testosterone (3.9 ng/ml), which implied the diagnosis of a virilizing tumor (1). Serum dehydroepiandrosterone sulfate (DHEAS) was normal (92 µg/dl), which argued against an adrenal source of testosterone. The increase in testosterone levels during hCG administration (5000 IU/d for 3 d) (2) was consistent with the hypothesis of an ovarian virilizing tumor. Other hormonal data comprised normal levels of 
4-androstenedione, slightly elevated LH and FSH levels (LH, 10 IU/liter; FSH, 9 IU/liter), SHBG levels at the lower limit of the normal range (3.0 mg/liter; normal range in premenopausal women, 3–6 mg/liter), normal urinary 17-cetocorticosteroids (7.5 mg/d) and 17-hydroxycorticosteroids (4.8 mg/d), normal urinary free cortisol (38.5 µg/d), and normal suppression of urinary free cortisol by dexamethasone (2 mg/d for 2 d), ruling out Cushing’s syndrome (Table 1
).
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Selective catheterization of the ovarian and adrenal veins by an experienced radiologist did not reveal any gradient of testosterone concentration in the samples taken from ovarian and adrenal veins. Catheterization was performed under basal (unstimulated) conditions and after hCG administration (3) (Table 2). However, no correct catheterization of the right ovarian vein was possible during any of the two examinations. Because no technical difficulty occurred during catheterization of the adrenal veins, it was concluded that the location of the virilizing tumor was probably in one of the ovaries.
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Materials and Methods
Blood samples were taken by the surgeon from the right and left ovarian veins and from an antecubital vein. Serum testosterone levels were measured intraoperatively using an automated direct chemiluminescent immunoassay (Advia Centaur, provided by Bayer Corp., Puteaux, France). The intraassay coefficient of variation is less than 6.2%. The percentage of cross-reaction is 5.4% with 5
-dihydrotestosterone, 0.94% with 4-androstenedione, and less than 0.1% with other steroids. Serum testosterone concentrations can be measured approximately 18 min after centrifugation of the sample. No manual aliquoting of the centrifuged sample is required, and primary centrifuged samples can be loaded directly onto the instrument. The ADVIA Centaur method enables the measurement of testosterone concentrations up to 15 ng/ml and preprograming of dilutions before running the assay; measurement of testosterone in the primary samples and in 1:10 diluted samples was made for each sample taken from the ovarian veins. The sample volume is 15 µl, but because of the dead volume, a minimum 50-µl sample is necessary. As with the more recent automated methods, it is possible to load an additional sample once the automaton has been started, which allows an emergency test to be performed. Thus, the testosterone concentration result is available less than 30 min after the sample has been received in the laboratory, including all of the steps required (centrifugation, loading of the tube, and measurement by the automated method).
Results
An unequivocal gradient of testosterone concentrations was found, localizing the source of testosterone secretion in the right ovary (Table 2
). The patient underwent right annexectomy. The ovary contained several cysts at various maturation steps; the biggest cysts were 9 mm large. Macroscopic examination detected a 1.5-cm nodule in the ovarian hilus. Microscopic examination showed a hilar, well circumscribed tumor composed of steroid-type cells with eosinophilic cytoplasm sometimes containing crystals of Reinke. The cells were diffusely arranged, but frequently separated in nests or cords by anuclear eosinophilic areas. Fibrinoid replacement of the walls of moderate-sized vessels was frequent. Thus, the histopathological diagnosis was a Leydig cell tumor of hilar type (Fig. 1).
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Discussion
Virilizing tumors of the ovaries constitute less than 5% of all ovarian neoplasms (4). Many histological tumor types have been described, and different classifications have been proposed. The most frequent type is the Sertoli-Leydig cell tumor (arrhenoblastoma), which is observed in premenopausal women. In our patient the virilizing tumor is a preferentially hilar cell tumor, which is one of the steroid cell tumors, representing 0.1% of all ovarian tumors (5). Eighty percent of such tumors result in hyperandrogenism (5). They are small tumors (<4 cm), unilateral in more than 95% of cases, with excellent prognosis because almost all of them are benign. Hilar Leydig cell tumors are more often observed in postmenopausal women and are gonadotropin dependent. The rare hilar non-Leydig cell tumors that are diagnosed in younger women belong to another histological type classified as nonspecific tumors. Only 50% of them are associated with hyperandrogenism, and 20–40% are malignant (6). However, when located in the hilus, they are more likely to be benign, and a small size is a criterion for better prognosis. In our premenopausal patient, the histopathological findings are those of a Leydig cell tumor, because the tumor contains large or medium-size cells with an abundant eosinophilic cytoplasm, crystals of Reinke, small centered nuclei and unique nucleolus, and rare mitoses.
In our patient localization of the virilizing tumor was provided only by intraoperative measurement of testosterone, whereas hormonal data, radiological investigations, scintigraphy imaging, selective venous catheterization, and even inspection of the ovaries during laparotomy failed to localize the tumor. First we had to determine that the virilizing tumor was not the left adrenal nodule; such a nodule is actually a silent adrenal adenoma, as detected by CT scan in 2% of subjects (incidentaloma) (7). Typical virilizing adrenal tumors are large malignant masses with DHEAS levels and are unresponsive to hCG. However, small virilizing adrenal tumors may be observed, and it is known that DHEAS levels are normal in as many as 20% of adrenal virilizing tumors, and that several virilizing adrenal tumors express hCG receptors (7). Finally, the lack of gradient between the two adrenal veins, which had been catheterized without technical problems, provided evidence against an adrenal source of testosterone.
The next step was to show that the virilizing tumor was actually in the right ovary, because correct catheterization of the right ovarian vein had not been possible. None of the radiological investigations showed the tumor. Ovarian virilizing tumors of the adult are often too small to allow detection by ultrasound examination, and adult premenopausal patients with virilizing ovarian tumors may have micropolycystic ovaries (8). Tumor size should be at least 0.5 or 2 cm to allow correct detection by transvaginal (9) or pelvic (10) sonography. Gray scale transvaginal sonography combined with color Doppler studies can more easily allow the diagnosis of small steroid cell tumors (5). MRI scan could give more contrasted imaging and better detection of ovarian tumors. Recently, an MRI technique comprising transvaginal examination has appeared promising (11). Iodomethylnorcholesterol scanning can also be used (12). Selective catheterization of ovarian veins can be very informative (13, 14, 15), but is associated with technical problems, especially for catheterization of the right ovarian vein for anatomical reasons (12, 16). Finally, exploration by laparoscopy or laparotomy does not always lead to correct determination of the location of small ovarian tumors (1). Thus, intraoperative measurement of testosterone in the ovarian veins is valuable, because it can localize the source of testosterone secretion, probably better than intraoperative direct sonography of the ovaries, which provides only morphological information.
In our patient intraoperative measurement of testosterone levels in samples taken by the surgeon from each ovarian vein revealed a gradient of testosterone levels and led to the correct localization and conservative surgery of the ovarian virilizing tumor. To date, several direct immunoassays for testosterone are available. Eight nonradioactive direct testosterone immunoassays [ACS 180 (Ciba Corning Diagnostics, Norwood, MA), Architect i2000 (Abbot Diagnostics, Maidenhead, UK), AutoDelfia (Pharmacia Wallac, Milton Keynes, UK), Elecsys 2010 (Roche Diagnostics, East Sussex, UK), Immulite 2000 (Diagnostic Products Ltd., Gwynedd, Wales, UK), Immuno 1 (Bayer, Tarrytown, NY), Vidas (BioMerieux, Marcy L’Etoile, France), Vitros Eci (Ortho-Clinical Diagnostics, Cardiff, Wales, UK)] and two RIAs [Immunotech (Beckman Coulter, Westbrook, ME) and DPC (Diagnostic Products, Los Angeles, CA)] have been recently evaluated by Taieb et al. (17), whereas Wheeler (18) reviewed the performances of several automated immunoassay analyzers, including a few methods for measurement of testosterone levels (ACS:180 and Advia-Centaur, provided by Bayer Corp., and Immulite and Immulite 2000, provided by Diagnostic Products). The study by Taieb et al. (17) showed that all direct assays had good interassay reproducibility and provided accurate evaluation of plasma testosterone for concentrations within the male normal range (
5 ng/ml), i.e. the same range of values as that observed in most female patients with virilizing tumors. A strong correlation was also observed by Fitzgerald et al. (19) between the results given by ACS 180 and those of isotopic dilution gas chromatography-mass spectrometry for this range of testosterone concentrations. ACS 180 is the first fully automated nonradioactive testosterone immunoassay approved by the U.S. FDA (20). The ADVIA-Centaur method, which we used for the present study, employs the same reagents as those of ACS 180; the latter method requires 15 min for testosterone evaluation, according to Wheeler et al. (20). In the ADVA-Centaur method, only the automation employed is different. More recently, a new modular chemiluminescence immunoassay analyzer, compared with gas chromatography-mass spectrometry, showed better reproducibility than ACS 180 (21). All of the more recent automated direct nonradioactive testosterone immunoassays allow the insertion of additional emergency samples once the instrument has been started, the loading of centrifuged primary samples without manual aliquoting, and the preprograming of the preparation of diluted samples. All such direct testosterone assays make possible rapid and accurate evaluation of testosterone levels within the range observed in patients with virilizing tumors. Thus, the short time required for the assay makes such direct assays especially suitable for intraoperative localization of testosterone-secreting tumors.
Conclusion
In women with recent-onset or rapidly progressive hirsutism, with or without normal menstrual cycles, measurement of testosterone levels is mandatory to screen for virilizing tumors. The finding of an adrenal nodule on CT scan does not rule out an ovarian source of the tumoral secretion of androgens.
Intraoperative measurement of testosterone in samples taken directly from the ovarian veins can be useful to localize the source of testosterone secretion when selective catheterization of adrenal and ovarian veins fails to provide full diagnostic evidence for the localization of the tumor. This probably represents one major advantage of the recent automated methods of testosterone measurements.
Footnotes
Abbreviations: CT, Computed tomography; DHEAS, dehydroepiandrosterone sulfate; MRI, magnetic resonance imaging.
Received November 15, 2001.
Accepted April 11, 2002.
References
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  G. Mengozzi, D. Rossato, C. Bertello, C. Garrone, A. Milan, R. Pagni, F. Veglio, and P. Mulatero Rapid Cortisol Assay during Adrenal Vein Sampling in Patients with Primary Aldosteronism Clin. Chem., November 1, 2007; 53(11): 1968 - 1971. [Abstract] [Full Text] [PDF]
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L. J. Sokoll, F. H. Wians Jr, and A. T. Remaley Rapid Intraoperative Immunoassay of Parathyroid Hormone and Other Hormones: A New Paradigm for Point-of-Care Testing Clin. Chem., July 1, 2004; 50(7): 1126 - 1135. [Abstract] [Full Text] [PDF]
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