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Virilization of two siblings by maternal androgen-secreting adrenal adenoma
840
Clinical and laboratory observations
3. Jacobs PA, Hunt P'A, Matsuvra JS, Wilson CC. Complete and partial hydatidiform mole in Hawaii: cytogenetics, morphology and epidemiology. Br J Obstet Gynecol 1982;89:258266. 4. Boue J. Phenotypic expression of lethal chromosomal anomalies in human abortuses. Teratology 1976;14:3. 5. Kajii T, Chama K. Androgenic origin of hydatidiform mole. Nature 1977;268:633. 6. Wallace DC, Suiti U, Adams CW. Complete moles have paternal chromosomes but maternal mitochondrial DNA. Human Genet 1982;61:145-7. 7. Szulman AE, Surti V. The syndromes of hydatidiform mole. Am J Obstet Gynecol 1978;131:665-71. 8. Santos-Ramos R, Forney JP, Schwarz BE. Sonographic findings and clinical correlation in molar pregnancy. Obstet Gynecol 1980;56:186-92. 9. Heath JM, Bu TH, Breseton WF. Hydatidiform moles. Ann Fam Physician 1985;31(5):123-30. 10. Matalon M, Paz B, Modan M, et al. Malignant trophoblastic
The Journal of Pediatrics November 1986
11.
12.
13.
14.
15.
disorders: epidemiologic aspects and relationship to hydatidiform mole. Am J Obstet Gyneeol 1972;112:101-6. Lurain JR, Brewer JI, Torok Ee. Gestational trophoblastic disease treatment results at the Brewer Trophoblastic Center. Obstet Gynecol 1982;60:354-60. Stone M, Dent J, Kardana A, et al. Relationship of oral contraception to development of trophoblastic tumor after evacuation of a hydatidiform mole. Br J Gynecol 1976; 83:913. Berkowitz RS, Marea AR, Goldstein DP, et al. Oral contraceptives and post molar trophoblastic tumors [Letter]. Lancet 1980;2:752. Berkowitz RS, Goldstein DP, Marean AR, Bernstein M. Oral contraceptives and post molar trophoblastic disease. Obstet Gyneeol 1981;58:474-7. Felici O, Trojerouni A, Robyn C. The reproductive function following a hydatidiform mole. J Gynecol Obstet Biol Reprod (Paris) 1984;13(1):67-76.
Virilization of two siblings by maternal androgen-secreting adrenal adenoma T. J. O'Leary, M.D., T. C. Ooi, M.D., J. D. Miller, M.D., C. L. Branchaud, Ph.D., and J. Kalra, M.D. From the Division of Endocrinology and Metabolism, Ottawa Civic Hospital, University of Ottawa, and the Department of Pediatrics, Children's Hospital of Eastern Ontario, University of Ottawa, Ontario, Canada; and Montreal Children's Hospital Research Institute, McGill University, Montreal, Quebec, Canada
Virilization of the female fetus may occur as a result of exposure to excessive androgen activity during the critical period of genital development in utero. T h e c o m m o n causes are congenital adrenal hyperplasia in the fetus and administration of anabolic or progestational agents to the mother during pregnancy. 1 Rarely, m a t e r n a l ovarian tumors have led to masculinization of female offspring? Only four previous cases caused by a m a t e r n a l adrenal t u m o r have been reported? -6 W e describe the virilization of two female siblings by a m a t e r n a l adrenal adenoma.
Submitted for publication June 16, 1986; accepted July 16, 1986. Reprint rexlUCStS: T. C. Ooi, M.D., Division of Endocrinology & Metabolism, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, Ontario, Canada K1Y 4E9.
CASE REPORT After a normal pregnancy, a 3550 g infant was born at term to a healthy 28-year-old primigravida. Physical examination of the infant was unremarkable except for the genitalia, which revealed mild clitoromegaly, posterior labial fusion, and a single urogenital orifice. Chromosomal analysis confirmed a female karyotype. Serum 17-hydroxyprogesterone, testosterone, and dehydroepiandrosterone sulfate concentrations were normal. A cloacogram demonstrated the vagina and urethra entering a 2 cm cloaca. DHEAS hCG 17-OHP
Dehydroepiandrosterone sulfate Human chorionic gonadotropin 17- Hydroxyprogesterone
Intravenous pyelogram and cystoscopy showed normal development of the remainder of the urinary tract. The mother had no symptoms of virilization at that time, and was not tested. Twenty months after the birth of the first infant, a second child was born to the same parents after a normal term pregnancy. This 3375 g infant was designated as male at birth but was referred for further examination because of mieropenis (stretched length 1.5
Volume 109 Number 5
Clinical and laboratory observations
841
Figure. Computed tomography of abdomen with intravenous contrast enhancement showing large right-sided suprarenal mass.
Table. H o r m o n e studies of m o t h e r with adrenal a d e n o m a before and during d e x a m e t h a s o n e suppression
Serum (8:00 AM) Cortisol (nmol/L) Testosterone (nmole/L) Dehydroepiandrosterone sulfate (~.mol/L) Sex hormone binding globulin (nmol/L) 17-OH-progesterone (nmol/L) Aldosterone (pmol/L) Plasma renin activity supine (ng/L/se~j Adrenocorticotrophic hormone (pmol/L) Follicle-stimulating hormone (IU/L) Luteinizing hormone (IU/L) Urine (24 hr) 17-Ketosteroids (/~mol/d) Androsterone (/~moi/d) Etiocholanolone (#mol/d) Dehydroepiandrosterone 0zmol/d) 17-Ketogenic steroids (#mot/d) Free cortisol (nmol/d)
Normal range
Baseline levels
200-600 0.3-2.2 2.2-9.2 35-93 1.5-7.0 260-940 0.14-0.44 3.3-22.0 2-15 2-20
281 4.9 39.5 29 1.2 341 0.27 11.0 11 19
20-50 2.1-20.7 2.1-13.8 0-4.9 10-50 40-260
627.6 94.2 54 7.3 40.7 44
Dexamethasone suppression 61 9.8 99.5 29 4.6 481 0.38 7.0
1043.2 140.5 111.5 36.1 17.3 6
*Dexamethasone phosphate 2 mg orally every 6 hours for 48 hours. Serum samples taken 5 hours after last dose. Urine collection done during second day of dexamethasone suppression.
cm) and hypospadias. The apparent urethral orifice was at the phallic-perineal junction. The scrotum was well formed, but testes were not palpable. Further investigation revealed a 46,XX karyotype. Serum DHEAS, 17-OHP, and cortisol levels were normal, and serum testosterone was 1.6 nmol/L (normal range 0.1 to 1.0 nmol/L). Pelvic ultrasound study showed a 2.9 cm uterus. A retrograde cystogram demonstrated the vagina opening into then
membranous urethra. Other urinary tract abnormalities were excluded by intravenous pyelogram. The mother had noticed mild hirsutism of the face and lower abdomen prior to the birth of the second child. She denied menstrual irregularity, acne, or voice change. Physical examination revealed mild hirsutism, borderline clitoral hypertrophy (3.0 • 0.7 cm), and mild temporal recession of scalp hair. No
842
Clinical and laboratory observations
abnormal masses were detected on abdominal or pelvic examination. Serum testosterone and DHEAS values were elevated, with a paradoxical rise after dexamethasone 2.0 mg orally every 6 hours for 48 hours (Table). There was no further elevation of testosterone, DHEAS, and 17-OHP in response to a single, 5000 U intramuscularly administered dose of human chorionic gonadotropin. Computed tomography demonstrated a large right,sided suprarenal mass (Figure). At laparotomy, a well-encapsulated, right adrenal tumor, measuring 11.8 • 6.7 • 4.9 cmand weighing 239 g, was resected. Histologic examination showed that it was a benign adrenocortical adenoma. Explant cultures were established from the tumor tissue by the method described previously. 7 The cultures were challenged with ACTH (10 and 100 ng/ml), hCG (0.5 and 5.0 ug/mi), or 50% conditioned placental medium.* The media were coUocted every 24 hours and assayedT: DHEAS production was 47 _+ 5.1 ng/grid/24 hr (mean + SE) on day 2, and fell to 29 --- 3.6 on day 5 and 10 _+ 1.1 on day 7 in culture. Testosterone production was 8.3 _+ 2.0 on day 3, and fell to 4.3 _+ 2.4 on day 7. ACTH, hCG, and conditioned placental medium failed to stimulate DHEAS or testosterone production or prevent the fall in steroid production over time. DISCUSSION Four previous reports 3-6 of single infants virilized by a maternal adrenal adenoma indicated the same pattern of marked virilization of the infant and minimal maternal signs. Two of the previous studies demonstrated an increase in androgen levels in response to administered hCG. In our study, there was no increase in androgen production by the tumor, in vivo or in vitro. Errors in the management of female pseudohcrmaphroditism can occur when a maternal adrenal adenoma is not adequately excluded. A normal maternal serum testosterone concentration is not adequate for exclusion, inasmuch as this occurred in two of the four cases in the literature. Maternal serum D H E A S concentration appears to be more consistently elevated. Severe fetal virilization despite minimal maternal signs is a consistent and intriguing finding in these cases. In two of the previous reports 3,5 maternal androgens increased in response to hCG administration (5000 U IM). This led Fuller et al. 5 to suggest that endogenous hCG of pregnancy enhanced androgen production by the adrenal adenoma, resulting in fetal virilization, but the lower androgen levels between pregnancies accounted for the minimal maternal signs. Our patients also showed this discordance between the degree of maternal and fetal virilization but failed to show stimulation of testosterone or D H E A S production by hCG, in rive and in vitro. Therefore, this explanation for the disproportionate fetal virilization must be questioned. Excess androgenic activity during a critical period between the seventh and twelfth weeks of gestation is
The Journal of Pediatrics November 1986
necessary to cause the labial fusion and persistent urogenital sinus present in our patients? In the normal male fetus, testosterone from the fetal testes is converted by 5areductase to 5a-dihydrotestosterone in the external anlage. 9 The availability of testosterone determines the degree of maSculinization, because 5a,reduetase levels are similar in both sexes at the time of sexual differentiation. 9 Transplaeentai passage of testosterone would provide the simplest mechanism for fetal virilization in utero. In normal pregnancy, maternal serum total testosterone increases because of increased sex hormone binding globulin, whereas free testosterone remains normal. In late pregnancy the placenta provides a barrier to transfer of maternal testosterone by aromatization to estradiol. ~However, it is not known whether this harrier is effective in early pregnancy, when sexual differentiation occurs. Two of the previously reported infants 3,6 had normal maternal tc~tostexone levels, and both authors queried whether D H E A S may be the transplaeental virilizing agent. When congenital virilizing adrenal hyperplasia has been excluded as a cause of fetal virilization, the differential diagnosis must include the presence of an adrenal adenoma in the mother. REFERENCES
1. Lauritzen C, Klopper A. Estrogens and androgens. In: Fuchs F, Kloppers A, eds. Endocrinology of pregnancy. Philadelphia: Harper & Row, 1983:73-91. 2. Verkauf BS, Reiter EO, Hernandez L, Burns SA. Virilization of mother and fetus associated with luteomas of pregnancy: a case report with endocrinologie studies. Am J Obstet Gynecol 1977;129:274-80. 3. Murset G, Zachmann M, Prader A, Fischer J, Labhart A. Male external genitalia of a girl caused by a virilizing adrenal tumonr in the mother. Acta Endocrinol 1970;65:627-38. 4. Elterman J J, Hagen GA. Aldosteronism in pregnancy: association with virilization of female offspring. South Med J 1983;76:514-6. 5. Fuller P J, Pettigrew IG, Peke JW, Stockigt JR. An adrenal adenoma causing virilization of mother and infant. Clin Endocrinol 1983;18:143-53. 6. Van dc Kamp JJP, van Seters AP, Moolenaar A J, van Geldcren HH. Female pseudohermaphroditism due to an adrenal tumour in the mother. Eur J Pediatr 1984;142:1402. 7. Branchaud CL, Goodyer CG, Shore P, Lipowski LS, Lefebvre Y. Functional zonation of the midgestation human fetal adrenal cortex: fetal versus definitive zone use of progesterone for eortisol synthesis. Am J Obstet Gynecol 1985;151:2717. 8. Branchaud CL, Goodyer CG, Lipowski LS. Progesterone and estrogen production by placental monolayer cultures: effect of dehydroepiandrosterone and luteinizing hormone releasing hormone. J Clin Endocrinol Metab 1983;56:761.6. 9. Siiteri PK, Wilson JD. Testosterone formation and metabolism during male sexual differentiation in the human embryo. J Clin Endocrinol Metab 1974;38:113-25.
Clinical and laboratory observations
3. Jacobs PA, Hunt P'A, Matsuvra JS, Wilson CC. Complete and partial hydatidiform mole in Hawaii: cytogenetics, morphology and epidemiology. Br J Obstet Gynecol 1982;89:258266. 4. Boue J. Phenotypic expression of lethal chromosomal anomalies in human abortuses. Teratology 1976;14:3. 5. Kajii T, Chama K. Androgenic origin of hydatidiform mole. Nature 1977;268:633. 6. Wallace DC, Suiti U, Adams CW. Complete moles have paternal chromosomes but maternal mitochondrial DNA. Human Genet 1982;61:145-7. 7. Szulman AE, Surti V. The syndromes of hydatidiform mole. Am J Obstet Gynecol 1978;131:665-71. 8. Santos-Ramos R, Forney JP, Schwarz BE. Sonographic findings and clinical correlation in molar pregnancy. Obstet Gynecol 1980;56:186-92. 9. Heath JM, Bu TH, Breseton WF. Hydatidiform moles. Ann Fam Physician 1985;31(5):123-30. 10. Matalon M, Paz B, Modan M, et al. Malignant trophoblastic
The Journal of Pediatrics November 1986
11.
12.
13.
14.
15.
disorders: epidemiologic aspects and relationship to hydatidiform mole. Am J Obstet Gyneeol 1972;112:101-6. Lurain JR, Brewer JI, Torok Ee. Gestational trophoblastic disease treatment results at the Brewer Trophoblastic Center. Obstet Gynecol 1982;60:354-60. Stone M, Dent J, Kardana A, et al. Relationship of oral contraception to development of trophoblastic tumor after evacuation of a hydatidiform mole. Br J Gynecol 1976; 83:913. Berkowitz RS, Marea AR, Goldstein DP, et al. Oral contraceptives and post molar trophoblastic tumors [Letter]. Lancet 1980;2:752. Berkowitz RS, Goldstein DP, Marean AR, Bernstein M. Oral contraceptives and post molar trophoblastic disease. Obstet Gyneeol 1981;58:474-7. Felici O, Trojerouni A, Robyn C. The reproductive function following a hydatidiform mole. J Gynecol Obstet Biol Reprod (Paris) 1984;13(1):67-76.
Virilization of two siblings by maternal androgen-secreting adrenal adenoma T. J. O'Leary, M.D., T. C. Ooi, M.D., J. D. Miller, M.D., C. L. Branchaud, Ph.D., and J. Kalra, M.D. From the Division of Endocrinology and Metabolism, Ottawa Civic Hospital, University of Ottawa, and the Department of Pediatrics, Children's Hospital of Eastern Ontario, University of Ottawa, Ontario, Canada; and Montreal Children's Hospital Research Institute, McGill University, Montreal, Quebec, Canada
Virilization of the female fetus may occur as a result of exposure to excessive androgen activity during the critical period of genital development in utero. T h e c o m m o n causes are congenital adrenal hyperplasia in the fetus and administration of anabolic or progestational agents to the mother during pregnancy. 1 Rarely, m a t e r n a l ovarian tumors have led to masculinization of female offspring? Only four previous cases caused by a m a t e r n a l adrenal t u m o r have been reported? -6 W e describe the virilization of two female siblings by a m a t e r n a l adrenal adenoma.
Submitted for publication June 16, 1986; accepted July 16, 1986. Reprint rexlUCStS: T. C. Ooi, M.D., Division of Endocrinology & Metabolism, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, Ontario, Canada K1Y 4E9.
CASE REPORT After a normal pregnancy, a 3550 g infant was born at term to a healthy 28-year-old primigravida. Physical examination of the infant was unremarkable except for the genitalia, which revealed mild clitoromegaly, posterior labial fusion, and a single urogenital orifice. Chromosomal analysis confirmed a female karyotype. Serum 17-hydroxyprogesterone, testosterone, and dehydroepiandrosterone sulfate concentrations were normal. A cloacogram demonstrated the vagina and urethra entering a 2 cm cloaca. DHEAS hCG 17-OHP
Dehydroepiandrosterone sulfate Human chorionic gonadotropin 17- Hydroxyprogesterone
Intravenous pyelogram and cystoscopy showed normal development of the remainder of the urinary tract. The mother had no symptoms of virilization at that time, and was not tested. Twenty months after the birth of the first infant, a second child was born to the same parents after a normal term pregnancy. This 3375 g infant was designated as male at birth but was referred for further examination because of mieropenis (stretched length 1.5
Volume 109 Number 5
Clinical and laboratory observations
841
Figure. Computed tomography of abdomen with intravenous contrast enhancement showing large right-sided suprarenal mass.
Table. H o r m o n e studies of m o t h e r with adrenal a d e n o m a before and during d e x a m e t h a s o n e suppression
Serum (8:00 AM) Cortisol (nmol/L) Testosterone (nmole/L) Dehydroepiandrosterone sulfate (~.mol/L) Sex hormone binding globulin (nmol/L) 17-OH-progesterone (nmol/L) Aldosterone (pmol/L) Plasma renin activity supine (ng/L/se~j Adrenocorticotrophic hormone (pmol/L) Follicle-stimulating hormone (IU/L) Luteinizing hormone (IU/L) Urine (24 hr) 17-Ketosteroids (/~mol/d) Androsterone (/~moi/d) Etiocholanolone (#mol/d) Dehydroepiandrosterone 0zmol/d) 17-Ketogenic steroids (#mot/d) Free cortisol (nmol/d)
Normal range
Baseline levels
200-600 0.3-2.2 2.2-9.2 35-93 1.5-7.0 260-940 0.14-0.44 3.3-22.0 2-15 2-20
281 4.9 39.5 29 1.2 341 0.27 11.0 11 19
20-50 2.1-20.7 2.1-13.8 0-4.9 10-50 40-260
627.6 94.2 54 7.3 40.7 44
Dexamethasone suppression 61 9.8 99.5 29 4.6 481 0.38 7.0
1043.2 140.5 111.5 36.1 17.3 6
*Dexamethasone phosphate 2 mg orally every 6 hours for 48 hours. Serum samples taken 5 hours after last dose. Urine collection done during second day of dexamethasone suppression.
cm) and hypospadias. The apparent urethral orifice was at the phallic-perineal junction. The scrotum was well formed, but testes were not palpable. Further investigation revealed a 46,XX karyotype. Serum DHEAS, 17-OHP, and cortisol levels were normal, and serum testosterone was 1.6 nmol/L (normal range 0.1 to 1.0 nmol/L). Pelvic ultrasound study showed a 2.9 cm uterus. A retrograde cystogram demonstrated the vagina opening into then
membranous urethra. Other urinary tract abnormalities were excluded by intravenous pyelogram. The mother had noticed mild hirsutism of the face and lower abdomen prior to the birth of the second child. She denied menstrual irregularity, acne, or voice change. Physical examination revealed mild hirsutism, borderline clitoral hypertrophy (3.0 • 0.7 cm), and mild temporal recession of scalp hair. No
842
Clinical and laboratory observations
abnormal masses were detected on abdominal or pelvic examination. Serum testosterone and DHEAS values were elevated, with a paradoxical rise after dexamethasone 2.0 mg orally every 6 hours for 48 hours (Table). There was no further elevation of testosterone, DHEAS, and 17-OHP in response to a single, 5000 U intramuscularly administered dose of human chorionic gonadotropin. Computed tomography demonstrated a large right,sided suprarenal mass (Figure). At laparotomy, a well-encapsulated, right adrenal tumor, measuring 11.8 • 6.7 • 4.9 cmand weighing 239 g, was resected. Histologic examination showed that it was a benign adrenocortical adenoma. Explant cultures were established from the tumor tissue by the method described previously. 7 The cultures were challenged with ACTH (10 and 100 ng/ml), hCG (0.5 and 5.0 ug/mi), or 50% conditioned placental medium.* The media were coUocted every 24 hours and assayedT: DHEAS production was 47 _+ 5.1 ng/grid/24 hr (mean + SE) on day 2, and fell to 29 --- 3.6 on day 5 and 10 _+ 1.1 on day 7 in culture. Testosterone production was 8.3 _+ 2.0 on day 3, and fell to 4.3 _+ 2.4 on day 7. ACTH, hCG, and conditioned placental medium failed to stimulate DHEAS or testosterone production or prevent the fall in steroid production over time. DISCUSSION Four previous reports 3-6 of single infants virilized by a maternal adrenal adenoma indicated the same pattern of marked virilization of the infant and minimal maternal signs. Two of the previous studies demonstrated an increase in androgen levels in response to administered hCG. In our study, there was no increase in androgen production by the tumor, in vivo or in vitro. Errors in the management of female pseudohcrmaphroditism can occur when a maternal adrenal adenoma is not adequately excluded. A normal maternal serum testosterone concentration is not adequate for exclusion, inasmuch as this occurred in two of the four cases in the literature. Maternal serum D H E A S concentration appears to be more consistently elevated. Severe fetal virilization despite minimal maternal signs is a consistent and intriguing finding in these cases. In two of the previous reports 3,5 maternal androgens increased in response to hCG administration (5000 U IM). This led Fuller et al. 5 to suggest that endogenous hCG of pregnancy enhanced androgen production by the adrenal adenoma, resulting in fetal virilization, but the lower androgen levels between pregnancies accounted for the minimal maternal signs. Our patients also showed this discordance between the degree of maternal and fetal virilization but failed to show stimulation of testosterone or D H E A S production by hCG, in rive and in vitro. Therefore, this explanation for the disproportionate fetal virilization must be questioned. Excess androgenic activity during a critical period between the seventh and twelfth weeks of gestation is
The Journal of Pediatrics November 1986
necessary to cause the labial fusion and persistent urogenital sinus present in our patients? In the normal male fetus, testosterone from the fetal testes is converted by 5areductase to 5a-dihydrotestosterone in the external anlage. 9 The availability of testosterone determines the degree of maSculinization, because 5a,reduetase levels are similar in both sexes at the time of sexual differentiation. 9 Transplaeentai passage of testosterone would provide the simplest mechanism for fetal virilization in utero. In normal pregnancy, maternal serum total testosterone increases because of increased sex hormone binding globulin, whereas free testosterone remains normal. In late pregnancy the placenta provides a barrier to transfer of maternal testosterone by aromatization to estradiol. ~However, it is not known whether this harrier is effective in early pregnancy, when sexual differentiation occurs. Two of the previously reported infants 3,6 had normal maternal tc~tostexone levels, and both authors queried whether D H E A S may be the transplaeental virilizing agent. When congenital virilizing adrenal hyperplasia has been excluded as a cause of fetal virilization, the differential diagnosis must include the presence of an adrenal adenoma in the mother. REFERENCES
1. Lauritzen C, Klopper A. Estrogens and androgens. In: Fuchs F, Kloppers A, eds. Endocrinology of pregnancy. Philadelphia: Harper & Row, 1983:73-91. 2. Verkauf BS, Reiter EO, Hernandez L, Burns SA. Virilization of mother and fetus associated with luteomas of pregnancy: a case report with endocrinologie studies. Am J Obstet Gynecol 1977;129:274-80. 3. Murset G, Zachmann M, Prader A, Fischer J, Labhart A. Male external genitalia of a girl caused by a virilizing adrenal tumonr in the mother. Acta Endocrinol 1970;65:627-38. 4. Elterman J J, Hagen GA. Aldosteronism in pregnancy: association with virilization of female offspring. South Med J 1983;76:514-6. 5. Fuller P J, Pettigrew IG, Peke JW, Stockigt JR. An adrenal adenoma causing virilization of mother and infant. Clin Endocrinol 1983;18:143-53. 6. Van dc Kamp JJP, van Seters AP, Moolenaar A J, van Geldcren HH. Female pseudohermaphroditism due to an adrenal tumour in the mother. Eur J Pediatr 1984;142:1402. 7. Branchaud CL, Goodyer CG, Shore P, Lipowski LS, Lefebvre Y. Functional zonation of the midgestation human fetal adrenal cortex: fetal versus definitive zone use of progesterone for eortisol synthesis. Am J Obstet Gynecol 1985;151:2717. 8. Branchaud CL, Goodyer CG, Lipowski LS. Progesterone and estrogen production by placental monolayer cultures: effect of dehydroepiandrosterone and luteinizing hormone releasing hormone. J Clin Endocrinol Metab 1983;56:761.6. 9. Siiteri PK, Wilson JD. Testosterone formation and metabolism during male sexual differentiation in the human embryo. J Clin Endocrinol Metab 1974;38:113-25.