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Studies on the mechanism of the production of the testicular feminization syndrome
Studies
on the Mechanism
the Testicular
of the Production
Feminization
of
Syndrome*
GRANT GWINUP, M.D., t RALPH G. WIELAND, M.D., PAIGE K. BESCH, PH.D.$ and GEORGE
J. HAMWI, M.D.
Columbus,
T
testicular feminization syndrome (TFS) is an uncommon form of intersex which is perhaps the most bizarre of this group of anomalies. Affected subjects generally are wholly feminine in appearance with fully developed breasts, and usually normal female external genitalia and very sparse pubic and axillary hair. However, Morris [7] has recently emphasized that an enlarged clitoris may be found in the “incomplete” form of the syndrome. Their psychosexual orientation is typically feminine, yet these subjects are genetic males and their gonads are testes. Amenorrhea and infertility are the complaints which most often bring them to a physician; they rarely suspect that they are afflicted with a more fundamental abnormality. Two principal theories have been proposed to explain the striking discrepancy between gonada1 morphology and secondary sexual development [7]. The first suggests that the gonads, although morphologically testes, function biochemically as ovaries and produce estrogens instead of androgens. The second theory is based upon the initial observations of Wilkins [2] that the administration of large doses of androgen to these subjects fails completely to produce any androgenic effect. This could be due to insensitivity of the tissues to androgens or an excessive conversion of androgens either to estrogens or inactive metabolites. We have studied a patient with classic testicular feminization syndrome in an attempt to define further the mechanisms through which
Ohio this interesting syndrome is produced and have concluded that the testes produce both testosterone and estrogens, but that the usual end organ effects of testosterone are minimal or absent.
HE
CASE REPORT The patient was a nineteen year old phenotypic female (Fig. 1) with primary amenorrhea. Breast development began at age thirteen. The height was 5 feet, 10.5 inches. There was complete absence of axillary hair and pubic hair was sparse. The breasts were well developed and contained palpable.glandular tissue. The external genitalia were entirely normal. The vagina was 5 cm. in length and ended in a blind pouch. The gonads could not be palpated. No history of a similar occurrence of amenorrhea in the family was obtained. METHODS
Chromosome analysis was performed by the Moorhead technic 131. Twenty-four hour urinary collections were analyzed for follicle-stimulating hormone (FSH) , luteinizing hormone (LH) , 17-hydroxycorticoids, 17-ketosteroids, estrone, estradiol, estriol and testosterone during a basal state, after the administration of Pergonal,! following gonadectomy and after the administration of testosterone. At the time of gonadectomy, blood was obtained from the left spermatic vein by direct catheterization and, simultaneously, from an antecubital vein. This was analyzed for testosterone, dehydroepiandrosterone (3B$ Pergonal, an extract of human menopausal urine containing primarily FSH with some LH, was supplied by Cutter Laboratories, Berkeley, California.
* From the Department of Medicine, Division of Endocrinology and Metabolism, the Clinical Research Center, the Department of -Obstetrics and Gynecology and the Department of Pharmacology, of the Ohio State University HosDitals. Columbus. Ohio. This studv was SuoDorted in Dart bv U. S. Public Health Service Research Grants FR-34. HDb075k and CA 07954 and U. S. Public Health Servile Training Grants TI AM 5118 and TI AM 5403 (National Institute of Arthritis and Metabolic Diseases). t Present address: Division of Endocrinology and Metabolism, University of California, California College of Medicine, Los Angeles, California. 1 Research Career Development Award Investigator, 6-K3-HD-22,534, of the National Institute of Child Health and Human Development. 448
AMERICAN
JOURNAL
OF
MEDICINE
Testicular Feminization Syndrome-Gwinup hydroxyandrost-5-en-17-one) and androstenedione (androst-4-ene-3,17-dione). Immediately after removal of the gonads, portions were submitted for routine pathologic examination, but the bulk of each specimen was quick-frozen in liquid nitrogen until approximately thirty days later when in vitro biosynthetic studies were performed. The patient was given one vial of Pergonal intramuscularly every 12 hours for the three days immediately preceding operation. Testosterone propionate, 50 mg., was administered intramuscularly every 12 hours beginning on the sixth postoperative day and continuing for one week. This was followed by the intramuscular administration of testosterone phenylacetate, 200 mg. every week for three months. Ethinyl estradiol, 0.2 mg., was then given daily. Skin sebum specimens were obtained five days after operation, and again eight weeks after the initiation of testosterone administration, and quantitated by the method of Strauss and Pochi [4]. Urinary 17-ketosteroids were measured by the method of Klendshoj et al. [5], 17-hydroxycorticoids by the method of Peterson et al. [6]. FSH was determined by the assay of Steelman and Pohley [7] and LH by the technic of Schmidt-Elmendorff and Loraine (81. Urinary estrogens were determined by the method of Brown et al. [9]. Urinary testosterone was assayed by gas-liquid chromatography and isotope dilution [Xl]. The plasma samples were seeded with testosterone1,2-H3,dehydroepiandrosterone-7-H3 and androstenedione-4~CY4.Steroids were fractionated by the method of Wieland et al. [ 771. Dehydroepiandrosterone and androstenedione were quantitated by the Zimmermann reaction, with corrections for radioactive losses. Testosterone was quantitated by acetylation with acetic anhydride-CY4. Recrystallization after the addition of cold carrier resulted in a constant Cl4 : H3 ratio for the gonadal vein sample. Unfortunately the low levels of peripheral testosterone could not be accurately assayed because a constant 0 : H3 ratio could not be obtained. Plasma dehydroepiandrosterone sulfate was assayed as previously described [ 771. For the in titro biosynthetic studies, the tissue was thawed, minced and incubated with progesterone-4Cl4 (pregn-4-ene-3, 20-dione) and pregnenolone-7-H3 (3B - hydroxypregn - 5 - en - 20 - one). Separation was achieved with paper chromatography and identification of the incubation products was confirmed by oxidation, reduction and acetylation as previously described by Besch et al. 1721. RESULTS
The buccal smear was chromatin-negative and the chromosome analysis revealed a normal male karyotype. Microscopically the testicular tissue showed some thickening of the membranes of the seminiferous tubules. The germinal VOL.
41,
SEPTEMBER
1966
et al.
449
FIG. 1. Patient with testicular feminization syndrome. Note adequate breast development and lack of axillary and pubic hair.
epithelium consisted almost entirely of Sertoli cells. Large clumps of interstitial cells were scattered between the tubules. The preoperative 24 hour urinary excretion of FSH was undetectable and of LH was equivalent to 6.4 Fg. LH-NIH-Sl (normal male range 2 to 6 pg.). The changes in urinary steroids which followed Pergonal stimulation, gonadectomy and testosterone administration are presented in Table I. Urinary estrogens did not change significantly throughout the entire period of study and did not increase when testosterone was administered. The urinary
Testicular
450
Feminization
Syndrome-Gwinup TABLE
URINARY
Condition
STEROID
EXCRETION
TABLE
Steroid Testosterone Dehydroepiandrosterone Androstenedione
SYNDROME
E&O1 (fig./24 hr.)
Testosterone (pg./24 hr.)
17-Hydroxycorticoids (mg./24 hr.)
2-25 4 6 3 3
O-10 1 2 1 3
2-30 7 10 12 12
115 13 138 6 204
2-9 6.5 10.0 7.4 5.5
COMMENTS
TABLE METABOLISM IN VITRO
OF TWO
5* 0 1.4
* This represents a maximal peripheral constant specific activity was not obtained.
76 21.0 ‘1.1 value since
_,’
BY GONADAL TESTICULAR
III
RADIOLABELED TISSUE
19-hydrdxyandrostenedione 17-hydroxyprogesterone Testosterone Dehydroepiandrosterone Androstenedione Pregnenolone sulfate Estradiol sulfate Dehydroepiandrosteronk ‘sulfate Unidentified phenolic conju; gates
AMERICAN
FROM
FEMINIZATION
Steroids Identified
Values in Peripheral Gonadal Vein Vein (pg./100 ml.) (&./lo0 ml.)
5-15 13.2 15.7 5.0 7.9
Our patient represents the classic “complete” form of testicular feminization syndrome with none of the “mixed” features that have been present in some subjects studied ,in a similar manner [7,73]. Differences in the pattern of steroidogenesis between the “complete’! and “mixed” form of the syndrome have recently been demonstrated [ 71. Gonadotropins. In the majority of cases of testicular feminization syndrome in which assays for human pituitary gonadotropins have been performed the values have been either within
IN TESTICULAR
SYNDROME
17-Ketosteroids (mg./24 hr.)
or pubic hair, deepening of the voice, clitoral enlargement or acne. Sebum production was 0.71 mg. per 6.25 cm. ‘[Z] of skin surface in 3 hours before testosterone administration and 1 .O mg. per 6.25 cm. [Z] in 3 hours after testosterone administration. Both of these values are low and are not significantly different. Ethinyl estradiol administration resulted in a prompt increase in breast size.
II
VALUES
FEMINIZATION
FEMINIZATION
E&radio1 (pg./24 hr.)
testosterone level, which initially was in the high female range, rose markedly with Pergonal stimulation, fell after gonadectomy, and showed the expected rise during the period of testosterone administration. Urinary 17-hydroxycorticoids did not change appreciably throughout the study. The level of 17-ketosteroids was markedly decreased after gonadectomy but increased slightly when testosterone was adminis* tered. The values for plasma testosterone, dehydroepiandrosterone and androstenedione are presented in Table II. Both testosterone and dehydroepiandrosterone were present in testicular venous effluent in concentrations many times as great as in peripheral blood. No evidence for gonadal secretion of dehydroepiandrosterone sulfate was obtained. The results of the in vitro incubation studies are shown in Table III. Both testosterone and estrogens were produced from progesterone and pregnenolone. Furthermore, estrogens were recovered only as sulfate conjugates. Immediately after gonadectomy the subject experienced hot flashes and the breasts became progressively smaller. The large doses of testosterone which were administered over more than three months following surgery did not produce any changes in the breasts, nor did testosterone produce growth of facial, axillary STEROID
I
IN TESTICULAR
EStr0ll.Z kg./24 hr.)
Normal female Baseline After pergonal administration After gonadectomy After testostuone administration
PLASMA
et al.
JOURNAL
SUBSTRATES A PATIENT
WITH
SYNDROME
y0 of Total Radioactivity after Incubation of PregnenoProgesterlone-7a-H* one-CC’”
2.58 0.61 6.14 14.17 17.07 4.41 0.19 4.02
2.05 4.82 6.76
0.12
0.66
OF
;:15 d.‘li ...
MEDI,CINE
Testicular Feminization Syndrome-Gwinup the normal range or somewhat higher than normal. In the present case a specific assay for LH gave normal results. However, FSH was undectable, as in another case [ 731 in which specific assays were performed. Androgen Production, Metabolism and Biological Activity. Baseline urinary testosterone excretion was in the high normal female range and increased markedly following Pergonal administration. Slight to marked increases in plasma testosterone levels (which were in the range for male subjects initially) have been reported previously following the administration of human chorionic gonadotropin (HCG) [73,74] to patients with testicular feminization syndrome. That the gonad contributes significantly to the biological testosterone pool is shown by direct gonadal vein assay of testosterone (76 pg. per 100 ml. plasma) and by in vitro incubation studies. It should be noted that in vivo no androstenedione was found in the gonadal vein blood following Pergonal administration whereas a recent report of in vitro studies indicated that Pergonal may favor the synthesis of androstenedione rather than of testosterone, at least in human ovarian tissue [75]. Secretion of free dehydroepiandrosterone but not of its conjugate, dehydroepiandrosterone sulfate, was demonstrated. It is not known whether Pergonal affects the interconversion of the free and conjugated compounds or the intragonadal utilization of dehydroepiandrosterone sulfate as a precursor for other steroids. Following gonadectomy our patient had no discernible clinical response to the administration of massive doses of testosterone, as in other patients [ 7,2]. Furthermore, there was no significant increase in sebum production in response to testosterone administration. Such a change has been demonstrated to be a most sensitive in vivo assay of androgenic activity [4]. During the period of testosterone administration there was essentially no increase in urinary estrogens. These findings suggest that testosterone is not significantly metabolized peripherally to an estrogen. This would confirm earlier findings that radioisotopically-labeled injected testosterone could not be recovered in urinary estrogen fractions [ 731. urinary estroEstrogen Production. Control gen levels were in the normal range for female subjects, in agreement with a recent report [73]. There was a slight but probably insignificant increase in urinary estrogens followVOL.
41,
SEPTEMBER
1966
et al.
451
ing the administration of Pergonal, although definite increases have been reported following more prolonged use of FSH and FSH with HCG [ 731. That the testes in testicular feminization syndrome produce estrogens has been demonstrated by finding the concentration of estradiol in gonadal venous blood to be 25 times as great as in systemic venous blood [ 731. The reason for our failure to observe a decrease in urinary estrogens following gonadectomy is not clear but it should be noted that the control values were quite low. Incubation Studies. Our in vitro biosynthetic studies indicate that the gonad of the patient with testicular feminization syndrome possesses the enzymatic capability for degrading ($1 steroids to androgens and estrogens, and are therefore in agreement with other reports [ 13,14,76, 171. Such degradation could be demonstrated with both b4(progesterone) and A-5(pregnenolone) compounds as the steroid precursors. The in vitro studies indicate that the biosynthetic potential for androgen production by the testes of the subject with testicular feminization syndrome is similar to that reported for testes of normal human subjects [ 781. There is thus direct evidence for gonadal secretion of testosterone in patients with testicular feminization syndrome. Also, the peripheral metabolism of this potent androgen seems to proceed in a normal fashion. However, the tissues do not appear to be responsive to the usual biological actions of this steroid. The ultimate basis for this insensitivity must remain conjectural. There would appear to be strong evidence to suggest that the totally feminine secondary sexual characteristics result from the unopposed effect of estrogens also produced by the testes of these patients. SUMMARY
Studies in a patient with testicular feminization are reported. Evidence is presented that the gonads produce testosterone as measured in the gonadal vein blood and in the urine. Urinary testosterone levels increased in response to gonadal stimulation with Pergonal and decreased following gonadectomy. Low normal urinary levels of estrogen persisted following gonadectomy despite the administration of large amounts of testosterone. In vitro studies of the gonad demonstrated their biosynthetic capacity to produce testosterone. Administration of large doses of exogenous
452
Testicular Feminization Syndrome-Gurinup
testosterone for a period of three months produced no manifestations of virilization. It is concluded that the syndrome represents end organ unresponsiveness to testosterone. This unresponsiveness was demonstrated after gonadectomy, and it is therefore unlikely that it is due to gonadal secretion of an antiandrogenic substance [ 791.
9.
10.
11.
Acknowledgment: We wish to express our gratitude to Dr. John C. Ullery, Chairman of the Department of Obstetrics and Gynecology, who performed the surgery and to Dr. V. C. Stevens, Department of Obstetrics and Gynecology, who performed the gonadotropin assays.
12.
13. REFERENCES
1. MORRIS, J. McL. and MAHESH, V. B. Further observations on the syndrome, “testicular feminization.” Am. J. Obst. & Gynec., 07: 731, 1963. 2. WILKINS, L. The Diagnosis and Treatment of Endocrine Disorders in Childhood and Adolescence, p. 276. Springfield, Ill., 1957. Charles C Thomas. 3. MOORHEAD, P. S., NOWELL, P. C., MELLMAN, W. J., BATTIPS,D. M. and HUNGERFORD,D. A. Chromosome preparations of leukocytes cultured from human peripheral blood. Exper. Cell Res., 20: 613, 1960. 4. STRAUSS, J. S. and POCHI, P. E. The human sebaceous gland: its regulation by steroidal hormones and its use as an end organ for assaying androgenicity in viva. Rec. Progr. Hormone Res., 19: 385, . 1963. 5. KLENDSHOJ,N. C., FELDSTEIN,M. and SPRAGUE,A. Determination of 17-ketosteroids in urine. J. Clin. Endocrinol., 13: 922, 1953. 6. PETERSON,R. E., KARRER, A. and GUERRA, S. L. Evaluation of Silber-Porter procedure for determination of plasma hydrocortisone. ArmI. Chem., 29: 144, 1957. 7. STEELMAN,S. L. and POHLEY, F. M. Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology, 53 : 604, 1953. 8. SCHMIDT-ELMENDORFF, H. and LORAINE,J. A. Some
14.
15.
16.
17.
18.
19.
et al.
observations on the ovarian ascorbic acid depletion method as a test for luteinizing hormone activity. J. Endocrinol., 23: 413, 1962. BROWN, J. B., BULBROOK, R. D. and GREENWOOD, F. C. An additional purification step for a method for estimating oestriol, oestrone, and oestradiol17B in human urine. J. Endocrinol., 16: 49, 1957. WIELAND, R. G., MAYNARD, D. E. and HAM~I, G. J. Urinary testosterone in some endocrine disorders. Am. J. Obst. B Gynec., 94: 806, 1966. WIELAND, R. G., DECOURCY, C., LEVY, R. P., ZALA, A. P. and HIRSCHMANN,H. CIsOe steroids and some of their precursors in blood from normal human adrenals. J. Clin. Znvcst., 44: 159, 1965. BESCH,P. K., WATSON, D. J., BARRY, R. D., HAMWI, G. J., MOSTOW, J. and GWINIJP,G. In vitro cortisol biosynthesis by a testicular tumor. Steroids, 1: 644, 1963. FRENCII, F. S., BAGGETT,B., VAN WYK, J. J., TALBERT, L. M., HUBBARD, W. R., JOHNSTON,F. R., WEAVER, R. P., FORCHIELLI,E., RAO, G. S. and SARDA, I. R. Testicular feminization: clinical morphological and biochemical studies. J. Clin. Endocrinol., 25: 661, 1965. SOUTHERN,A. L., Ross, H., SHARNA,D. C., GORDON, G., WEINGOLD,A. B. and DORFMAN,R. L. Plasma concentration and biosynthesis of testosterone in the syndrome of feminizing testes. J. Clin. Endocrinol., 25: 518, 1965. O’DONNELL, V. J. and MACARTHUR, E. Effect of FSH (Pergonal) on steroidogenesis in a cell-free human ovarian preparation. In: The Endocrine Society Program of the Forty-Seventh Annual Meeting, p. 54, 1965. GRIFFITHS, K., GRANT, J. K. and WHYTE, W. G. Steroid biosynthesis in vitro by cryptorchid testes from a case of testicular feminization. J. Clin. Endocrinol., 23 : 1044, 1963. KASE, N. and MORRIS, J. McL. Steroid synthesis in the cryptorchid testes of three cases of the testicular feminization syndrome. Am. J. Obst. f3 Gyncc., 91: 102, 1965. AXELROD, L. R. Metabolic patterns of steroid biosynthesis in young and aged human testes. B&him. et biophys. acta, 97: 551, 1965. NEHER, R., KAHNT, F. W., ROVERSI, G. D. and BOMPIANI,A. Steroid transformations in vitro by testicular tissue from two cases of testicular feminization. Acta-endocrinol., 49: 177, 1965.
AMERICAN
JOURNAL
OF
YEDICINE
on the Mechanism
the Testicular
of the Production
Feminization
of
Syndrome*
GRANT GWINUP, M.D., t RALPH G. WIELAND, M.D., PAIGE K. BESCH, PH.D.$ and GEORGE
J. HAMWI, M.D.
Columbus,
T
testicular feminization syndrome (TFS) is an uncommon form of intersex which is perhaps the most bizarre of this group of anomalies. Affected subjects generally are wholly feminine in appearance with fully developed breasts, and usually normal female external genitalia and very sparse pubic and axillary hair. However, Morris [7] has recently emphasized that an enlarged clitoris may be found in the “incomplete” form of the syndrome. Their psychosexual orientation is typically feminine, yet these subjects are genetic males and their gonads are testes. Amenorrhea and infertility are the complaints which most often bring them to a physician; they rarely suspect that they are afflicted with a more fundamental abnormality. Two principal theories have been proposed to explain the striking discrepancy between gonada1 morphology and secondary sexual development [7]. The first suggests that the gonads, although morphologically testes, function biochemically as ovaries and produce estrogens instead of androgens. The second theory is based upon the initial observations of Wilkins [2] that the administration of large doses of androgen to these subjects fails completely to produce any androgenic effect. This could be due to insensitivity of the tissues to androgens or an excessive conversion of androgens either to estrogens or inactive metabolites. We have studied a patient with classic testicular feminization syndrome in an attempt to define further the mechanisms through which
Ohio this interesting syndrome is produced and have concluded that the testes produce both testosterone and estrogens, but that the usual end organ effects of testosterone are minimal or absent.
HE
CASE REPORT The patient was a nineteen year old phenotypic female (Fig. 1) with primary amenorrhea. Breast development began at age thirteen. The height was 5 feet, 10.5 inches. There was complete absence of axillary hair and pubic hair was sparse. The breasts were well developed and contained palpable.glandular tissue. The external genitalia were entirely normal. The vagina was 5 cm. in length and ended in a blind pouch. The gonads could not be palpated. No history of a similar occurrence of amenorrhea in the family was obtained. METHODS
Chromosome analysis was performed by the Moorhead technic 131. Twenty-four hour urinary collections were analyzed for follicle-stimulating hormone (FSH) , luteinizing hormone (LH) , 17-hydroxycorticoids, 17-ketosteroids, estrone, estradiol, estriol and testosterone during a basal state, after the administration of Pergonal,! following gonadectomy and after the administration of testosterone. At the time of gonadectomy, blood was obtained from the left spermatic vein by direct catheterization and, simultaneously, from an antecubital vein. This was analyzed for testosterone, dehydroepiandrosterone (3B$ Pergonal, an extract of human menopausal urine containing primarily FSH with some LH, was supplied by Cutter Laboratories, Berkeley, California.
* From the Department of Medicine, Division of Endocrinology and Metabolism, the Clinical Research Center, the Department of -Obstetrics and Gynecology and the Department of Pharmacology, of the Ohio State University HosDitals. Columbus. Ohio. This studv was SuoDorted in Dart bv U. S. Public Health Service Research Grants FR-34. HDb075k and CA 07954 and U. S. Public Health Servile Training Grants TI AM 5118 and TI AM 5403 (National Institute of Arthritis and Metabolic Diseases). t Present address: Division of Endocrinology and Metabolism, University of California, California College of Medicine, Los Angeles, California. 1 Research Career Development Award Investigator, 6-K3-HD-22,534, of the National Institute of Child Health and Human Development. 448
AMERICAN
JOURNAL
OF
MEDICINE
Testicular Feminization Syndrome-Gwinup hydroxyandrost-5-en-17-one) and androstenedione (androst-4-ene-3,17-dione). Immediately after removal of the gonads, portions were submitted for routine pathologic examination, but the bulk of each specimen was quick-frozen in liquid nitrogen until approximately thirty days later when in vitro biosynthetic studies were performed. The patient was given one vial of Pergonal intramuscularly every 12 hours for the three days immediately preceding operation. Testosterone propionate, 50 mg., was administered intramuscularly every 12 hours beginning on the sixth postoperative day and continuing for one week. This was followed by the intramuscular administration of testosterone phenylacetate, 200 mg. every week for three months. Ethinyl estradiol, 0.2 mg., was then given daily. Skin sebum specimens were obtained five days after operation, and again eight weeks after the initiation of testosterone administration, and quantitated by the method of Strauss and Pochi [4]. Urinary 17-ketosteroids were measured by the method of Klendshoj et al. [5], 17-hydroxycorticoids by the method of Peterson et al. [6]. FSH was determined by the assay of Steelman and Pohley [7] and LH by the technic of Schmidt-Elmendorff and Loraine (81. Urinary estrogens were determined by the method of Brown et al. [9]. Urinary testosterone was assayed by gas-liquid chromatography and isotope dilution [Xl]. The plasma samples were seeded with testosterone1,2-H3,dehydroepiandrosterone-7-H3 and androstenedione-4~CY4.Steroids were fractionated by the method of Wieland et al. [ 771. Dehydroepiandrosterone and androstenedione were quantitated by the Zimmermann reaction, with corrections for radioactive losses. Testosterone was quantitated by acetylation with acetic anhydride-CY4. Recrystallization after the addition of cold carrier resulted in a constant Cl4 : H3 ratio for the gonadal vein sample. Unfortunately the low levels of peripheral testosterone could not be accurately assayed because a constant 0 : H3 ratio could not be obtained. Plasma dehydroepiandrosterone sulfate was assayed as previously described [ 771. For the in titro biosynthetic studies, the tissue was thawed, minced and incubated with progesterone-4Cl4 (pregn-4-ene-3, 20-dione) and pregnenolone-7-H3 (3B - hydroxypregn - 5 - en - 20 - one). Separation was achieved with paper chromatography and identification of the incubation products was confirmed by oxidation, reduction and acetylation as previously described by Besch et al. 1721. RESULTS
The buccal smear was chromatin-negative and the chromosome analysis revealed a normal male karyotype. Microscopically the testicular tissue showed some thickening of the membranes of the seminiferous tubules. The germinal VOL.
41,
SEPTEMBER
1966
et al.
449
FIG. 1. Patient with testicular feminization syndrome. Note adequate breast development and lack of axillary and pubic hair.
epithelium consisted almost entirely of Sertoli cells. Large clumps of interstitial cells were scattered between the tubules. The preoperative 24 hour urinary excretion of FSH was undetectable and of LH was equivalent to 6.4 Fg. LH-NIH-Sl (normal male range 2 to 6 pg.). The changes in urinary steroids which followed Pergonal stimulation, gonadectomy and testosterone administration are presented in Table I. Urinary estrogens did not change significantly throughout the entire period of study and did not increase when testosterone was administered. The urinary
Testicular
450
Feminization
Syndrome-Gwinup TABLE
URINARY
Condition
STEROID
EXCRETION
TABLE
Steroid Testosterone Dehydroepiandrosterone Androstenedione
SYNDROME
E&O1 (fig./24 hr.)
Testosterone (pg./24 hr.)
17-Hydroxycorticoids (mg./24 hr.)
2-25 4 6 3 3
O-10 1 2 1 3
2-30 7 10 12 12
115 13 138 6 204
2-9 6.5 10.0 7.4 5.5
COMMENTS
TABLE METABOLISM IN VITRO
OF TWO
5* 0 1.4
* This represents a maximal peripheral constant specific activity was not obtained.
76 21.0 ‘1.1 value since
_,’
BY GONADAL TESTICULAR
III
RADIOLABELED TISSUE
19-hydrdxyandrostenedione 17-hydroxyprogesterone Testosterone Dehydroepiandrosterone Androstenedione Pregnenolone sulfate Estradiol sulfate Dehydroepiandrosteronk ‘sulfate Unidentified phenolic conju; gates
AMERICAN
FROM
FEMINIZATION
Steroids Identified
Values in Peripheral Gonadal Vein Vein (pg./100 ml.) (&./lo0 ml.)
5-15 13.2 15.7 5.0 7.9
Our patient represents the classic “complete” form of testicular feminization syndrome with none of the “mixed” features that have been present in some subjects studied ,in a similar manner [7,73]. Differences in the pattern of steroidogenesis between the “complete’! and “mixed” form of the syndrome have recently been demonstrated [ 71. Gonadotropins. In the majority of cases of testicular feminization syndrome in which assays for human pituitary gonadotropins have been performed the values have been either within
IN TESTICULAR
SYNDROME
17-Ketosteroids (mg./24 hr.)
or pubic hair, deepening of the voice, clitoral enlargement or acne. Sebum production was 0.71 mg. per 6.25 cm. ‘[Z] of skin surface in 3 hours before testosterone administration and 1 .O mg. per 6.25 cm. [Z] in 3 hours after testosterone administration. Both of these values are low and are not significantly different. Ethinyl estradiol administration resulted in a prompt increase in breast size.
II
VALUES
FEMINIZATION
FEMINIZATION
E&radio1 (pg./24 hr.)
testosterone level, which initially was in the high female range, rose markedly with Pergonal stimulation, fell after gonadectomy, and showed the expected rise during the period of testosterone administration. Urinary 17-hydroxycorticoids did not change appreciably throughout the study. The level of 17-ketosteroids was markedly decreased after gonadectomy but increased slightly when testosterone was adminis* tered. The values for plasma testosterone, dehydroepiandrosterone and androstenedione are presented in Table II. Both testosterone and dehydroepiandrosterone were present in testicular venous effluent in concentrations many times as great as in peripheral blood. No evidence for gonadal secretion of dehydroepiandrosterone sulfate was obtained. The results of the in vitro incubation studies are shown in Table III. Both testosterone and estrogens were produced from progesterone and pregnenolone. Furthermore, estrogens were recovered only as sulfate conjugates. Immediately after gonadectomy the subject experienced hot flashes and the breasts became progressively smaller. The large doses of testosterone which were administered over more than three months following surgery did not produce any changes in the breasts, nor did testosterone produce growth of facial, axillary STEROID
I
IN TESTICULAR
EStr0ll.Z kg./24 hr.)
Normal female Baseline After pergonal administration After gonadectomy After testostuone administration
PLASMA
et al.
JOURNAL
SUBSTRATES A PATIENT
WITH
SYNDROME
y0 of Total Radioactivity after Incubation of PregnenoProgesterlone-7a-H* one-CC’”
2.58 0.61 6.14 14.17 17.07 4.41 0.19 4.02
2.05 4.82 6.76
0.12
0.66
OF
;:15 d.‘li ...
MEDI,CINE
Testicular Feminization Syndrome-Gwinup the normal range or somewhat higher than normal. In the present case a specific assay for LH gave normal results. However, FSH was undectable, as in another case [ 731 in which specific assays were performed. Androgen Production, Metabolism and Biological Activity. Baseline urinary testosterone excretion was in the high normal female range and increased markedly following Pergonal administration. Slight to marked increases in plasma testosterone levels (which were in the range for male subjects initially) have been reported previously following the administration of human chorionic gonadotropin (HCG) [73,74] to patients with testicular feminization syndrome. That the gonad contributes significantly to the biological testosterone pool is shown by direct gonadal vein assay of testosterone (76 pg. per 100 ml. plasma) and by in vitro incubation studies. It should be noted that in vivo no androstenedione was found in the gonadal vein blood following Pergonal administration whereas a recent report of in vitro studies indicated that Pergonal may favor the synthesis of androstenedione rather than of testosterone, at least in human ovarian tissue [75]. Secretion of free dehydroepiandrosterone but not of its conjugate, dehydroepiandrosterone sulfate, was demonstrated. It is not known whether Pergonal affects the interconversion of the free and conjugated compounds or the intragonadal utilization of dehydroepiandrosterone sulfate as a precursor for other steroids. Following gonadectomy our patient had no discernible clinical response to the administration of massive doses of testosterone, as in other patients [ 7,2]. Furthermore, there was no significant increase in sebum production in response to testosterone administration. Such a change has been demonstrated to be a most sensitive in vivo assay of androgenic activity [4]. During the period of testosterone administration there was essentially no increase in urinary estrogens. These findings suggest that testosterone is not significantly metabolized peripherally to an estrogen. This would confirm earlier findings that radioisotopically-labeled injected testosterone could not be recovered in urinary estrogen fractions [ 731. urinary estroEstrogen Production. Control gen levels were in the normal range for female subjects, in agreement with a recent report [73]. There was a slight but probably insignificant increase in urinary estrogens followVOL.
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ing the administration of Pergonal, although definite increases have been reported following more prolonged use of FSH and FSH with HCG [ 731. That the testes in testicular feminization syndrome produce estrogens has been demonstrated by finding the concentration of estradiol in gonadal venous blood to be 25 times as great as in systemic venous blood [ 731. The reason for our failure to observe a decrease in urinary estrogens following gonadectomy is not clear but it should be noted that the control values were quite low. Incubation Studies. Our in vitro biosynthetic studies indicate that the gonad of the patient with testicular feminization syndrome possesses the enzymatic capability for degrading ($1 steroids to androgens and estrogens, and are therefore in agreement with other reports [ 13,14,76, 171. Such degradation could be demonstrated with both b4(progesterone) and A-5(pregnenolone) compounds as the steroid precursors. The in vitro studies indicate that the biosynthetic potential for androgen production by the testes of the subject with testicular feminization syndrome is similar to that reported for testes of normal human subjects [ 781. There is thus direct evidence for gonadal secretion of testosterone in patients with testicular feminization syndrome. Also, the peripheral metabolism of this potent androgen seems to proceed in a normal fashion. However, the tissues do not appear to be responsive to the usual biological actions of this steroid. The ultimate basis for this insensitivity must remain conjectural. There would appear to be strong evidence to suggest that the totally feminine secondary sexual characteristics result from the unopposed effect of estrogens also produced by the testes of these patients. SUMMARY
Studies in a patient with testicular feminization are reported. Evidence is presented that the gonads produce testosterone as measured in the gonadal vein blood and in the urine. Urinary testosterone levels increased in response to gonadal stimulation with Pergonal and decreased following gonadectomy. Low normal urinary levels of estrogen persisted following gonadectomy despite the administration of large amounts of testosterone. In vitro studies of the gonad demonstrated their biosynthetic capacity to produce testosterone. Administration of large doses of exogenous
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Testicular Feminization Syndrome-Gurinup
testosterone for a period of three months produced no manifestations of virilization. It is concluded that the syndrome represents end organ unresponsiveness to testosterone. This unresponsiveness was demonstrated after gonadectomy, and it is therefore unlikely that it is due to gonadal secretion of an antiandrogenic substance [ 791.
9.
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Acknowledgment: We wish to express our gratitude to Dr. John C. Ullery, Chairman of the Department of Obstetrics and Gynecology, who performed the surgery and to Dr. V. C. Stevens, Department of Obstetrics and Gynecology, who performed the gonadotropin assays.
12.
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