THE DIAGNOSTIC USE OF GONADOTROPINS*

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FERTILITY AND STERILITY Copyright 1970 by The Williams & Wilkins Co. Vol. 21, No.2, February 1970 Printed in U.S.A. THE DIAGNOSTIC USE OF GONADOTROPINS* PAUL G. McDONOUGH, M.D., V. B. MAHESH, PH.D., D.

1 FERTILITY AND STERILITY Copyright 1970 by The Williams & Wilkins Co. Vol. 21, No.2, February 1970 Printed in U.S.A. THE DIAGNOSTIC USE OF GONADOTROPINS* PAUL G. McDONOUGH, M.D., V. B. MAHESH, PH.D., D. PHIL. AND J. ROGERS BYRD, PH.D. Department of Obstetrics and Gynecology and Department of Endocrinology, Medical College of Georgia, A ugusta, Georgia The therapeutic utility of homologous gonadotropins in ovulation induction is well established.a, la A few investigators have used human gonadotropins as a diagnostic aid in determining ovarian competence, and thereby corroborated a pituitary deficit of endogenous gonadotropins. I, 4 Other investigators have used clomiphene citrate to test the integrity of the hypothalamic-pituitary axis, and its potential for releasing gonadotropins. l1 They no doubt saw the parallel between the metyrapone test for adrenocorticotropin (ACTH) reserve, and the possible potential of clomiphene citrate as a test for gonadotropin reserve. Clinically it is sometimes difficult to ascertain whether gonadal failure is at a pituitary or ovarian level. Although significant advances have been made in the measurement of follicle-stimulating hormones and luteinizing hormones in serum and urine by radioimmunoassays, the availability of these technics is limited at the present time.9, 10 The clinical tool which has been of assistance in distinguishing the defect level has been total urinary gonadotropins. In most clinical laboratories this method is based on an 100~ enlargement of the mouse uterus on a test dose without the use of an appropriate standard. The variability of the response of individual experimental animals and the toxicity of urine extracts makes "Supported by research grant AM from the National Institute of Arthritis and Metabolic Diseases; hospitalization of the patient supported by Grant MOI-FR from the National Institutes of Health, United States Public Health Seryice. the method somewhat unpredictable, and leaves the clinician many times with contradictory values. 14 It would seem, therefore, that a stimulation test can establish the diagnosis of ovarian failure as well as of pituitary hypogonadotropism. Another way of stating the same theorem is to say that a provocative challenge with human gonadotropins can assist in differentiating between hypergonadotropic hypogonadism and hypogonadotropic hypogonadism. However, the results obtained from a diagnostic gonadotropin challenge must be interpreted with caution regarding the dormancy and reserve of the end organ in question. One must also take into consideration the endocrine milieu in 126 which one is attempting to evoke an ovarian response. Thyroid function, adrenal function, the prior presence of endogenous estrogen, and also systemic disease may alter the response to gonadotropin provocation. This paper points out the diagnostic use of human menopausal gonadotropins in two different clinical situations, and discusses some of the problems in interpreting the results of gonadotropin stimulation. The patients discussed here are examples of gonadotropin failure and primary ovarian failure respectively. CASE 1 S. T. wa" an 18-year-old Negro female who presented with primary amenorrhea and sexual infantilism. The patient was of tall eunuchoid habitus with a height of 167 cm. and a shoulder span of 180 cm. Breast development and axillary hair were absent. Pelvic examination revealed infantile ex-

$February 1970 DIAGNOSTIC USE OF GONADOTROPIl\'S 127 ternal genitalia and cervix, and a small uterus. Sparse strands of pubic hair were present on the labia majora (Fig. 1).$ ; p3l uptake was 15.5% in 24 hr. (normal values 15-45%/24 hr.). A vaginal smear revealed predominately parabasal FIG. 2. Small ovaries with infantile uterus evident on pelvic pneumoperitoneum. FIG. 1. Patient S.

; p3l uptake was 15.5% in 24 hr. (normal values 15-45%/24 hr.). A vaginal smear revealed predominately parabasal FIG. 2. Small ovaries with infantile uterus evident on pelvic pneumoperitoneum. FIG. 1. Patient S.

Skull, films, visual fields, intravenous pyelograms, and hand films were all normal except for the findings of moderate osteoporosis and retarded bone age (13 years).

2 February 1970 DIAGNOSTIC USE OF GONADOTROPIl\'S 127 ternal genitalia and cervix, and a small uterus. Sparse strands of pubic hair were present on the labia majora (Fig. 1). Family history revealed 1 brother (173 cm and 1 sister (165 cm.). Her sister is 25 and has 5 children. Laboratory studies revealed the following: protein-bound iodine was 7.5 ",g./loo ml.; p3l uptake was 15.5% in 24 hr. (normal values 15-45%/24 hr.). A vaginal smear revealed predominately parabasal FIG. 2. Small ovaries with infantile uterus evident on pelvic pneumoperitoneum. FIG. 1. Patient S. T. cells (maturation index 53/44/3). Skull, films, visual fields, intravenous pyelograms, and hand films were all normal except for the findings of moderate osteoporosis and retarded bone age (13 years). Polyuria and polydysia were absent. Mean 24-hr. urine volumes were 1800 cc. and maximum urinary concentration after an overnight water deprivation was Transabdominal pelvic pneumoperitoneum demonstrated the presence of a small uterus and two bilaterally symmetrical gonadal structures (Fig. 2). Culdoscopic visualization confirmed the presence of a small uterus and two small, but normal, quiescent appearing ovaries (Fig. 3). Cytogenetic Findings. Buccal smear demonstrated that over 40% of the cells had a single sex chromatin mass. Chromosomal analysis performed on peripheral leukocytes revealed a normal female 46/XX karyotype. Routine Endocrine Assays. Routine assays of 17-ketosteroids and 17 -ketogenic steroids were performed and the results were within the limits of the normal range. Multiple determinations of total urinary gonadotropins (5 x) were all less than 6.6 M.D./24 hr. (normal values 6.6 M.D.D. to 52 M.D.D./24 hr.). ACTH

These results were interpreted as indicating an intact and functional ACTH-adrenal mechanism.

3 128 Vol. 21 MCDONOUGH ET AL. stimulation over a 3-day period resulted in a 4-fold augmentation of the 17-ketogenic steroids to 25.5 mg./24 hr. A metyrapone test for pituitary reserve for ACTH also resulted in an elevation of the 17ketogenic steroids to 6 times the control values (Table 1). These results were interpreted as indicating an intact and functional ACTH-adrenal mechanism. Fractionation and individual estimation of various urinary neutral steroids were carried out in control urine samples after adrenal suppression and gonadal stimulation with human menopausal gonadotropin using the method described by Mahesh et au (Table 2). In the control urine samples the urinary ll-deoxy -17 -ketosteroids (dehydroepiandrosterone, etiocholanolone, and androsterone), ll-oxygenated17-ketosteroids (ll-hydroxyetiocholanolone, ll-keto etiocholanolone and ll-hydroxy androsterone), and tetrahydrocorticords were somewhat below the normal values for our laboratory. They all decreased to undetectable levels when the adrenals were FIG. 3. Close-up view of ovary through culdoscope. TABLE 1. Urinary 17 -Ketogenic Steroids in a Patient with Hypogonadotropic Hypogonadism after ACTH* Stimulation and after Metyraponet Administration ACTH r-----l ~ ::r: '" E [ '" '"0 METYRAPONE, , r- es>-'" u <.:> 15 rr- 0 t;:; "" :::; 10 5 March..- IT II S. T. * ACTH (adrenocorticotropic hormone), 25 U. intravenously infused over G hr. followed by 25 U. intramuscularly. t Metyrapone, 750 mg. orally every 6 hr. suppressed with dexamethasone (2 mg. every 6 hr.) for 4 days (Table 2). The neutral steroids remained below detectable levels during gonadal stimulation with human menopausal gonadotropins (HMGt) (75 LU. of follicle-stimulating hormones and 25 LU. of luteinizing hormones twice daily for 5 days followed by human chorionic gonadotropin (HCG) 5000 LU.) for 3 days. Continuous adrenal suppression was maintained throughout this period of gonadal stimulation. Table 3 depicts the clinical re~ponse to the first course of HMG and HCG in terms of total urinary estrogens and changes in vaginal cytology. Urinary estrogens were measured by the method of Mahesh. 6 Although there was a marked increase in the number of superficial cells the total urinary estrogen values neyer rose aboye 6.2,..,g./24 hr. of the urine. It was after this negligible response to the first provocation with gonadotropins t The HMO used in this study was supplied by Ortho Research Foundation, Raritan, N. J., as HMO-Ortho (Pregova), Lot 2105, 2316.

4 February 1970 DIAGNOSTIC USE OF GONADOTROPIXS 129 that culdoscopy was performed to supplement roentgen visualization of the gonads. Table 4 illustrates the response to the second course of therapy with human menopausal gonadotropin. This time the course of therapy was extended over a period of 8 days and the patient received 2 ampules/day for 4 days followed by 3 ampules/day for 4 days. On the 10th day following the initiation of the HMG there was marked augmentation in the number of superficial cells on the vaginal smear TABLE 2. Urinary Steroids Excretion Patterns before and after Dexamethasone Suppression in a Patient with Hypogonadotropic Hypogonadism- t, t MEDICATION CONTROL DEXAMETHASONE ROUTINE 17-KS ROUT I NE 17-KGS 14 GONADOTROPINS < 6.6 < 6.6 P'TRIOL 0.8 < P'DIOL DHA < < ANDRO < HlO 0.5 < ll-oh [TIO 0.5 < ll-oh ANDRO 0.4 < ll-keto ET I < Tn F 0.7 < ALLO Tn F < < Tn E 1.0 < - All values are the average of two determinations or two 24-hr. mine specimens. With dexamethasone suppression, day 3 and -t of suppressioll were used for the study. t All results are expressed as mg./24 hr except for the urinary gonadotropins which are expressed M.U./24 hr. all gonadotropin determinations were < 6.6 M.U. t The following abbreviations have been used: routine 17 -KS, routine 17 -ketosteroids; routine 17-KGS, routine 17-ketogenic steroids; P'triol, pregnanetriol; P'diol, pregnanediol; DHA, dehyoepiandrosterone; Andro, androsterone; Etio, etiocholanolone; ll-oh ETIO, 11-hydroxy etiocholano-lone; 11-0H ANDRO, ll-hydroxy androsterone; ll-keto ETIO, ll-keto etiocholanolone; TET F, tetrahydrocortisol; ALLO TET F, allotetrahydro-cortisol; TET E, tetrahydrocortisone. TABLE 3. Response to First Course of Stimulation with Human Menopausal Gonadotropin Followed by Human Chorionic Gonadotropin while under Dexamethasone Suppression as Measul'ed by the Maturation Index, Cervical MuclIs, and the Urinary Estrogens , 60 ' \- DEXAMETHASONE ~on~ No ne None 19317/0 I /16/01119/65/191 L~'~~-LJ_~_L~-LJ_LL_L~~ JUNE JULY - The following abbreviations have been used: DEX, dexamethasone 2 mg. q 6 hr. for 12 days; HMG, human menopausal gonadotropin, 2 ampules/day for 5 days, Pregova, lot 2316; licg, human chorionic gonadotropin 5000 I.U./da~ for 3 days, lot TABLE 4. Response to Second Course of Stinwlalion with Human Menopausal Gonadotropin as Measured by Ihe Matul'ation Index and the Urinary Estrogens ~ loo " ~ August HMG September - The following abbreviations have been used:. HMG, human menopausal gonadotropin, 2 ampules/day for 4 days followed by 3 ampules/day for 4 days, Pregova, lot (maturation index 4/21/75) and the total urinary estrogens rose to p.g./ 24 hr. of urine. The patient experienced an episode of withdrawal bleeding lasting 3 days and occurring 19 days after the first dose of human menopausal gonadotropins. Diminished gonadotropin activity of the 5.1.

$130 :\ICDONOUGH ET AL. Vol. 21 pituitary may occasionally be a temporal and functional disturbance.$

5 130 :\ICDONOUGH ET AL. Vol. 21 pituitary may occasionally be a temporal and functional disturbance. Therefore, in an effort to distinguish functional failure of gonadotropins from an organic etiology, this patient was subsequently given a course of 1000 mg. of clomiphene citrate over a 5-day period.h However, there was no evidence of gonadotropin release as assessed by basal body temperature, vaginal smear, cervical mucus, and withdrawal bleeding. CASE 2 A. H. was an 18-year-old white female with secondary amenorrhea of 3- year duration.s The patient noted some breast development and pubic hair at age 13. Menarche occurred at 14 years and she had normal, cyclic, painless menses at 28- to 30-day intervals, lasting 4-5 days. After 7 months, menses ceased abruptly and she has had none since. Development of secondary sex characteristics also ceased at 14 years of age. The patient was 147 cm. in height and weighed 41.8 kg. She had a small amount of breast development and scanty pubic hair. Axillary hair was absent. She had no significant somatic anomalies other than her slightly decreased stature. Pelvic examination revealed a cervix and a small uterus. Adnexal structures were not palpable. Laboratory and roentgen studies were unremarkable except for moderate osteoporosis and a bone age of 12 years. Vaginal smear revealed predominately intermediate cells (maturation index 0/81/19). Transabdominal pelvic pneumoperitoneum demonstrated two very small rudimentary gonadal structures and uterus. Cytogenetic Findings. Buccal smear demonstrated that 18% of the cells had 1 Barr body and 2% of the cells had 2 sex chromatin masses. Chromosomal analysis of peripheral blood leukocytes revealed a sex chromosome mosaic of the type 45/ XO/47/XXX. Endocrine Responses to Gonadal Stimulation. Routine assay of 17-ketosteroids and 17-ketogenic steroids were within the limits of normal. Urinary gonadotropin levels were also within normal limits. Fractionation and individual estimation of various urinary neutral steroids was carried out in control urinary samples, after adrenal suppression and during gonadal stimulation with human menopausal gonadotropin (Table 5). Urinary estorgens were measured by the method of Mahesh7 and the results are also shown in Table 5. In the control urine samples all the fractionated elements of the 17- ketosteroids and 17-ketogenic steroids were within the limits of the normal range. They decreased to undetectable levels when the adrenals were suppressed with dexamethasone. The neutral steroids remained below detectable levels during gonadal stimulation with human gonadotropins (75 I.U. of follicle-stimulating hormones and 5 I.U. of luteinizing hormones twice daily for 5 days followed by human chorionic gonadotropin 5000 I.U.jday for.1 days. Continued adrenal suppression was maintained throughout this period of gonadal stimulation. Urinary estrogen levels were low during the controlled period and showed an appreciable depression when the adrenals were suppressed. When the gonads were stimulated with human menopausal gonadotropin while the adrenals were suppressed the urinary estrogen level showed a 4-fold increase although the total level of urinary estrogens still remained low. No further increase was noted when the ovaries were stimulated with human chorionic gonadotropin. Clinically there was no alteration in the patient's cervical mucous, vaginal smear, or basal body temperature, nor did she experience withdrawal bleeding after gonadal stimulation. Apparently her estrogen augmentation was not sufficient to stimulate dormant target organs.

6 February 1970 DIAGNOSTIC USE OF GONADOTROPIXS 131 TABLE 5. Steroid Excretion Pal tern before and after Adrenal Suppression and Stimulation of Gonadal Function with HMG and HCG (Case 2) II-de0xy-17-ketosteroids (mg./24 hr.) Dehydr0epiandrosterone Androsterone Etiocholanolone Total II-oxygenated-17-ket0steroids (mg./24 hr.) ('pnt r"l Urinary excretion Dexamethasone AI"ne + IIMG + HCG II-hydroxy etiocholanolone II-hydroxy androsterone ll-ketoetischolanolone Total Tetrahydrocorticoids (mg./24 hr.) Tetrahydrocortisol Allotetrahydrocortisol Tetrahydrocortisone Total Estrogens (ug./24 hr.) Estrone Estradiol Estriol Total COMMENT We believe that the first patient is an example of monotropic hypogonadotropic hypogonadism. This patient did not exhibit a significant response to the first course of therapy with human menopausal gonadotropin. Although there was a significant change in her vaginal smear her total urinary estrogens never rose above 6.2 {tg./24 hr. It may be that this patient indeed had a peak level of estrogens which was missed since we did not do daily urinary estrogens. When she did not respond significantly to this first course of therapy it was then that the culdoscopic procedure was performed. When culdoscopy further corroborated the presence of small ovaries we elected to stimulate her a second time but with a longer course of therapy and with 3 ampules/day rather than 2. The second course of therapy demonstrates significant changes in the vaginal smear and total urinary estrogens. No at- tempt was made to induce ovulation by adding HCG to the program. No doubt the excellent response to the second course of therapy is a reflection of the larger dose of HMG and also the overcoming of ovarian dormancy. Adrenal suppression with dexamethasone was not maintained during the responsive course to exogenous gonadotropins. Crooke et al,2 in a patient with Simmon's disease have found no alteration in the response to gonadotropins regardless of the presence or absence of replacement therapy with cortisone. But, as has been pointed out, the endocrine milieu in which the provocating agent is used certainly should be considered as a possible modifying factor. This case also points out that the lack of a response may only reflect a long period of ovarian inactivity and refractoriness to stimulation. Thus a lack of response to one course of gonadotropins does not necessarily indicate failure at an ovarian level. In situa-

7 132 MCDONOUGH ET AL. Vol. 21 tions where the ovary has not been subject to stimulation for long periods of time it is difficult to know the amount of exogenous gonadotropins and duration of therapy necessary to elicit a response. Abrams et au have been able to induce ovulation with the first course of therapy in a 15- year-old female who had a surgical hypophysectomy at 7 years of age for a craniopharyngioma. Polishuk et al. 12 have successfully induced ovulation, which was followed by pregnancy, in a 24-year-old female with Sheehan's syndrome of 4- year duration. However, Crooke et al.2 failed to induce ovulation in a 27-year-old female with an 8-year history of Simmond's disease. Again due consideration must be given to the total endocrine environment in which the ovary is being stimulated. Factors such as the amount of pre-existing endogenous gonadotropin, the metabolism of the administered drug, concomitantly administered hormones, and gonadotropin-inhibiting material must be considered. 5 The relationship has yet to be established between the duration of end organ latency and the degree of responsiveness after stimulation with human gonadotropins. The second patient is an example of gonadal dysgenesis with menses who apparently had a limited endowment of primordial follicles and therefore some residual ovarian function.s Even in patients with primary ovarian failure one can not be certain whether there may be some residual ovarian reserve left in the ovary. Therefore, the second point of caution with regard to the diagnostic use of human menopausal gonadotropin has to do with a positive response. Before concluding that such a patient indeed has a deficiency in pituitary or hypothalamicpituitary function one must weigh the possibility that some abnormality in ovarian responsiveness or a limited ovarian reserve has been overcome by massive nonphysiologic stimulation with HMO. In instances where a marked response is obtained, as exemplified by the first patient, this explanation is not very likely. In our second patient with primary ovarian failure the response to gonadotropin stimulation was only minimal. The slight augmentation which we obtained in the second case with regard to total urinary estrogens may represent some functional ovarian reserve capable of responding to a gonadotropin challenge. In general, one would not anticipate that patients with primary ovarian failure would respond to gonadotropin provocation. Vande Wiele and Turksoy14 have given Pergonal to 5 patients with primary ovarian failure and have failed to elicit a response in any of the subjects. SUMMARY AND CONCLUSIONS The diagnosis of gonadotropin failure or hypogonadotropic hypogonadism is a tenuous one when made on the basis of clinical gonadotropins alone. As has been proposed by Oemzell, Diczfalusy, and Tillinger,3 in the evaluation of ovarian competence HMO may be a more useful diagnotic tool than urinary gonadotropins. However, as had been pointed out and demonstrated, such a test of ovarian presence, competence, function, or reserve must be used with caution. In spite of these shortcomings the diagnostic value inherent in HMO therapy is indeed worthwhile. This paper illustrates the diagnostic use of human menopausal gonadotropin in determining pituitary gonadotropin failure, and also in assessing ovarian reserve in a patient with primary ovarian failure. Further studies along these lines will be very helpful to ascertain the extent to which immature unprimed ovaries are capable of responding to stimulation with exogenous gonadotropins. REFERENCES 1. ABRA)IS, C. A. L.. GRUMBACH, M. M., DYREN FURTH, 1.. AND VAXDE WIELE, R. 1,. Ovarian stimulation with human menopausal and chorionic gonadotropins in a prepubertal

8 February 1970 DIAGNOSTIC USE OF GOXADOTROPL,\S 133 hypophysectomized female. J Clin Endocl' 27 : CROOKE. A. C. Bt:TT, W. R.. PALMER. R. F.. MORRIS. R.. AXD MORGAN, D. B. The effect of human gonadotropins on a patient with Simmon's disease. Acta Endocr 46 :292, GEMZELL, C. A.. DICZFALUSY, E.. AND TILLINGER. K. G. Clinical effect of human pituitary follicle-stimulation hormone (FSH). J Clin Endocr 18 : Jmms, G. S., AND DEMoRAES-RuEHSEX, M. Induction of ovulation with human gonadotropins and with rlomiphen. Fertil Steril 16 :461, LANDAU. B., SCHWARTZ, H. S.. AXD SOFFER, L. J. Presence of a gonadotropin-inhibiting factor in urine of ~'oung children. Af etabolism 9 : MAHESH. V. B. A paper chromatographic method for estrogen determination. Steroids 3 :647, MAHESH. V. B.. GREENBLATT. R. B.. AYDAR, C. K.. Roy, S., PUEBLA. R. A.. AND ELLEGOOD, J. O. Urinary steroid excretion patterns in hirsutism. Use of adrenal and O\'arian suppression tests in the stud" of hirsutism. J Clin Endocr 24 : McDONOUGH, P. G., BYRD, J. R.. MAHESH, V. B. Gonadal dysgenesis with spontaneous menses: Report of a patient stimulated with HMG and HCG. Fertil Steril. In press. 9. MIDGLEY, A. R.. JR. Radioimmunoassay-A method for human chorionic gonadotropin and human luteinizing hormones. Endocrinology 79: 10, MIDGLEY, A. R., JR. Radioimmunoassay for human follicle-stimulating hormone. J Clin Endocr 27 :295, PLUXKETT, E. R., AXD YUZPE, A. Effect of clomiphene citrate upon pituitary gonadotropins. Amer J Obstet Gynec 100:506, POLISHUK, W. Z., PALTI. Z.. RABAU, E., LUNEN FELD. B., AXD DAVID, A. Pregnancy in a case of Sheehan's syndrome following treatment with human gonadotropins. J Obstet Gynaec Brit Comm 72: ROSE~IBERG, E.. MAHER. R. E.. STERX, A.. AND DEMANY, M. Clinical effect of gonadotropins of human origin: Case report with two year follow-up. J Clin Endocr 24 :105, VANDE WIELE, R. L., AND TURKSOY, R. N. Treatment of amenorrhea and of anovulation with human menopausal and chorionic gonadotropins. J Clin Endocr 25 :369, 1965.

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