A SCHEMATIC APPROACH TO THE WORK-UP OF AMENORRHEA

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1 FERTLTY AND STERLTY Copyright 977 The American Fertility Society Vol. 28, No.3, March 977 Printed in U.SA. A SCHEMATC APPROACH TO THE ORK-UP OF AMENORRHEA RYOSUKE NAKANO, M.D. NOBUYUK HASHBA, M.D. FUMKAZU KOTSUJ, M.D. SHMPE TOJO, M.D. Department of Obstetrics and Gynecology, Kobe University, School of Medicine, Kobe, Hyogo 650, Japan Amenorrhea is a symptom having many possible causes. Since amenorrhea can result from disturbed function anywhere in the hypothalamic-pituitary-ovarian-uterine axis, a specific etiologic diagnosis must be made if treatment is to be effective. For this purpose, a diagnostic scheme for the differential diagnosis of the etiology of primary and secondary amenorrhea is proposed. This scheme includes a progestin test, a cyclic estrogen and progestin test, a luteiniing hormone-releasing hormone () loading test, and a gonadotropin (human menopausal gonadotropin and human chorionic gonadotropin) loading test. A specific pattern of responses to and gonadotropins exists in patients with hypothalamic, pituitary, and ovarian amenorrheas, respectively, and the character of the response may facilitate the etiologic diagnosis of amenorrhea. The clinical usefulness and/or value of the scheme in the diagnosis and treatment of amenorrheas is discussed. Amenorrhea is the complete absence of menstruation in a woman of reproductive age. A menorrhea is a symptom having many possible causes. t is important to realie that there are times when absence of menstruation is to be regarded as normal: prior to puberty, during pregnancy and lactation, after the menopause, and during administration of certain drugs. However, pathologic amenorrhea due to endocrine disorder can result from disturbed function anywhere in the hypothalamic-hypophyseal-ovarian-uterine axis, with or without an associated organic lesion. Thus, a specific etiologic diagnosis must be made in the treatment of amenorrheic patients. Fortunately, there is a rational strategy to be followed in the investigation of amenorrhea which can limit both the time and the number of tests required to prove the diagnosis. The essential features of this strategy have been admirably described by Schrank and Kupperman and Lefkovics. 2 Recently, diagnostic methods in the field of clinical endocrinology have made remarkable Accepted November 8, progress. The purpose of the present communication is to report the clinical diagnostic scheme, including the luteiniing hormone-releasing hormone () loading test and the gonadotropin loading test, in the differential diagnosis and treatment of patients amenorrheic as a result of an endocrine disturbance in the hypothalamicpituitary-ovarian-uterine axis. MATERALS AND METHODS Seven normal female volunteers whose ages ranged from 25 to 29 years served as controls. All of the women had regular ovulatory menstrual cycles with intervals between periods varying from 28 to 32 days. The subjects were studied during the follicular phase of the menstrual cycle (cycle day 7). Determination of the follicular phase of the cycle was based on () knowledge of the length of five or more of the most recent, consecutive menstrual cycles; (2) daily basal body temperature readings; and (3) the timing of the subsequent menstrual cycle. Thirty-three amenorrheic patients were studied. Eight patients had primary amenorrhea and

2 230 NAKANO ET AL. twenty-five patients had secondary amenorrhea. No patient had postpill amenorrhea and/or galactorrhea-amenorrhea in the secondary amenorrheic group. An injection of progesterone (25 mg) was first given intramuscularly to each amenorrheic patient. Patients who did not respond to this test dose of progesterone were given an increased dose of progesterone (50 mg) intramuscularly. Patients with persistence of amenorrhea after the increased dose of progesterone were given a trial of cyclic estrogen and progestin therapy. Mestranol, 0.06 mg daily, was given for days and, days after the initiation of estrogen medication, chlormadinone acetate, 2 mg daily, was given for days. n order to make a specific etiologic diagnosis, seven normal control subjects and thirty-three amenorrheic patients were each subjected to an loading test and a gonadotropin loading test. The used in this study was synthesied by a classic method and generously donated by Daiichi Seiyaku Co. Ltd., Tokyo. Synthetic was administered intravenously in a dose of 0 Lg to each subject. Blood was sampled at time ero and at 5, 30, 45, 60, and minutes for determination of serum follicle-stimulating hormone (FSH) and luteiniing hormone (LH) concentrations. Serum FSH and LH were measured in duplicate by a double-antibody radioimmunoassay utiliing reagents provided by the Human Pituitary Program of the National nstitute of Arthritis, Metabolism and Digestive Diseases, according to the methods of Midgley3 and Odell et al.,4 with modifications. Both Second nternational Reference Preparation of Human Menopausal Gonadotropin (2nd RP-HMG) and LER-907 were used to obtain standard curves. The average relative potencies of these preparations were 44 U of 2nd RP-HMG/mg of LER- 907 for FSH and 304 U of 2nd RP-HMG/mg of LER-907 for LH. The results of this study were expressed as mu of 2nd RP-HMG/ml of serum. Exogenous gonadotropins, human menopausal gonadotropin (HMG) and human chorionic gonadotropin (HCG), were administered to normal subjects and amenorrheic patients according to the following schedule, and the ovarian response was evaluated by daily estimation of the 24-hour urinary excretion of total estrogens. The HMG used was Humegon (Organon; Amsterdam, The Netherlands). Each ampule contained 75 U (2nd RP-HMG) of FSH activity and 44 U (2nd RP HMG) of LH activity. The HCG was generously supplied by Teikoku Hormone, Tokyo, and its activity was calibrated against Second nter- March 977 national Standard of Human Chorionic Gonadotropin (2nd S-HCG). HMG (50 U) and HCG (0 U) were administered by intramuscular injection on the st and the 3rd days of the gonadotropin loading test. Total urinary estrogens were measured in 24- hour samples according to the method of Brown et aj.5 for 5 consecutive days. The area of gonadotropin or estrogen responses above the base line before and after or HMG and HCG administration was integrated by a computer and used for comparing responses for each group by Student's t-test. nformed consent was obtained from each subject after the- purpose and nature of the studies had been fully explained. RESULTS Fifteen amenorrheic patients responded to a test dose (25 mg) of progesterone with uterine bleeding, and eighteen patients did not. These 8 patients were given an increased dose (50 mg) of progesterone intramuscularly, but none responded with bleeding. The 8 patients with persistence of amenorrhea after an increased dose of progesterone were given a trial of cyclic estrogen and progestin therapy. All were responsive to cyclic estrogen and progestin therapy, and experienced uterine bleeding. Eight patients had amenorrhea due to ovarian hypogonadism; the diagnosis of ovarian hypogonadism was made on the basis of ovarian biopsy, chromosomal analysis, and hormone assay. Three of the eight had Turner's syndrome and five had primary ovarian failure such as atrophic or hypoplastic ovary. Five patients had amenorrhea due to hypophyseal hypogonadism. Three of the five had Sheehan's syndrome with a past history of massive hemorrhage at delivery and succeeding complaints of lack of lactation and amenorrhea, and two patients had chromophobe adenomas. Five patients had amenorrhea due to hypothalamic hypogonadism. The diagnosis of hypothalamic hypogonadism was based upon the result of the loading test. The intravenous administration of synthetic stimulated a concomitant release of FSH and LH in normal volunteers in the follicular phase of the menstrual cycle. The serum FSH levels rose at 5 minutes, reached a peak 45 minutes after injection, and fell subsequently (Fig. ). The serum LH levels, also elevated at 5 minutes, reached a peak of 45 minutes after injection and fell gradually (Fig. ). The magni-

3 Vol. 28, No.3 SCHEMATC APPROACH TO THE ORK-UP OF AMENORRHEA. 23 HMG& 70 HCG >- 60 Z -.t 0 c0 50 (!) :J ]. :J 0 f- :: :: 40 l. 50.J 50 > :. :. :J :J «it :J ::, o a min. min. day FG.. The effect of synthetic on serum FSH and LH levels and the effect of gonadotropins (HMG and HCG) on the 24-hour urinary excretion of total estrogens in normal, menstruating women. Brackets indicate ± standard error of the mean :-0 0 E... :J ]. :: :: 50.J 50 :. :. :J :J :J 0 0 t.. HMG& 70. HCG - 60 > w (!) 0 f- 40 > «it r o o min. min. day FG.2. The effect of synthetic on serum FSH and LH levels and the effect of gonadotropins (HMG and HCG) on the 24-hour urinary excretion of total estrogens in patients with amenorrhea responding to progesterone with uterine bleeding. Brackets indicate ± standard error of the mean.

4 232 NAKANO ET AL. tude of response to synthetic was greater in LH than in FSH (Fig. ). The combined intramuscular administration of HMG and HCG evoked a secretion of estrogen. The urinary excretion of total estrogens increased gradually after an injection of HMG and HCG (Fig. ). Synthetic also stimulated a concomitant release of FSH and LH in the 5 patients with amenorrhea responding to progesterone with uterine bleeding (Fig. 2). The pattern and magnitude of response to were similar to those seen in normal subjects in the follicular phase. HMG and HCG enhanced the urinary excretion of total estrogens in amenorrheic patients responding to progesterone, and the response pattern was approximately analogous to that in normal subjects (Fig. 2). The base line levels of serum FSH in eight patients with amenorrhea due to ovarian hypogonadism were extraordinarily high, and the response to was great (Fig. 3). The FSH response to was greater in patients with ovarian amenorrhea than in normal subjects in the follicular phase of the menstrual cycle (p < 0.0). The serum LH levels in patients with ovarian amenorrhea were markedly high, and the response to 0 f.j,g of synthetic was apparently greater than that in normal volunteers March 977 (P < 0.0) (Fig. 3). Conversely, the urinary excretion of total estrogens after HMG and HCG stimulation did not increase at all in patients with ovarian amenorrhea, and the estrogen response to HMG and HCG was lower than that seen in normal control subjects (P < 0.0) (Fig. 3). The base line levels of serum FSH and LH in five patients with amenorrhea due to hypophyseal hypogonadism were low, and synthetic failed to stimulate FSH or LH release from the anterior pituitary (Fig. 4). The FSH and LH levels after administration in patients with hypophyseal amenorrhea were significantly lower than those in normal subjects in the follicular phase (p < 0.05 and P < 0.0, respectively). n contrast, the urinary excretion of total estrogens after intramuscular injection of HMG and HCG in patients with hypophyseal amenorrhea rose day by day (Fig. 4). The pattern of estrogen response was similar to that seen in the follicular phase of the normal menstrual cycle (Fig. 4). Although the response magnitude in hypophyseal amenorrhea was slightly smaller than that in the follicular phase, no statistical difference could be demonstrated. The serum FSH and LH levels in five patients with amenorrhea due to hypothalamic hypogonadism were similar to those seen in normal vol E <=... E E l. 50 J 50 :< :< r r 0 0 "" > , 50 w (!l 0 Cl: f- 40 >- r..: cr HMG.\ 70 HCG o o min. FG. 3. The effect of synthetic on serum FSH and LH levels and the effect of gonadotropins (HMG and HeG) on the 24-hour urinary excretion of total estrogens in patients with ovarian amenorrhea due to primary hypogonadism. Brackets indicate ± standard error of the mean. day

5 Vol. 28, No.3 SCHEMATC APPROACH TO THE ORK-UP OF AMENORRHEA i ! J: 50 J 50 :E :E /)! /) 0 0,., Z 50 Cl 0 t- /) 40 > c( it 30 HMGo HCG T l' T T T o o min. min. day FG.4. The effect of synthetic on serum FSH and LH levels and the effect of gonadotropins (HMG and HCG) on the 24-hour urinary excretion of total estrogens in patients with hypophyseal amenorrhea due to secondary hypogonadism. Brackets indicate ± standard error of the mean J: 50 /) LL :E /) !. J: 50.J /) ,3, 50 w 8 40 /) > 30 ii' HMGo HCG o min. o min FG. 5. The effect of synthetic on serum FSH and LH levels and the effect of gonadotropins (HMG and HCG) on the 24-hour urinary excretion of total estrogens in patients with hypothalamic amenorrhea due to tertiary hypogonadism. Brackets indicate ± standard error of the mean. day

6 234 NAKANO ET AL. unteers in the follicular phase (Fig. 5). The pattern and magnitude of the FSH and LH responses to synthetic were also analogous to those seen in the normal follicular phase, and no statistical difference was demonstrated. The estrogen response to HMG and HCG in hypothalamic amenorrhea was also similar to that in the follicular phase of the normal cycle (Fig. 5). No serious side effects were noted in any patients given synthetic or HMG and HCG. DSCUSSON Diagnosing the amenorrheas and their potentially complex causes requires an organied and rational approach to the problem as well as adequate understanding of normal and pathologic endocrine physiology. The essential features of the differential diagnosis have been described by Schrankl and Kupperman and Lefkovics.2 According to their diagnostic scheme, an injection of progesterone should first be given intramuscularly to amenorrheic patients. f uterine bleeding occurs within 2 weeks, the following conclusions are warranted: () The endometrium is capable offunctioning. (2) Endogenous estrogenic activity is present, as the progesterone can act only on an endometrium adequately primed with estrogen. (3) The anterior pituitary must be providing FSH and LH. These conclusions imply that the patient's hypothalamic-pituitary-ovarian-uterine axis is at least relatively intact and the endocrine disorder might be due to hypothalamic dysfunction. n contrast, persistence of amenorrhea after a test dose of progesterone does not delimit the site of the relevant lesion. Such amenorrheic patients are given a trial of cyclic estrogen and progestin treatment. f bleeding does not occur, the diagnostic interest centers upon uterine and endometrial causes. f bleeding occurs after a course of cyclic estrogen and progestin therapy, it is clear that the endometrium is relatively intact. n such patients, it is desirable to distinguish among ovarian, pituitary, and hypothalamic causes of amenorrhea. For this purpose, a gonadotropin loading test should be performed. Several investigators have reported the ovarian response to exogenous gonadotropins. 6-9 HMG and/or HCG are used, and a positive response of the ovary to exogenous gonadotropin is recognied by an increase in the urinary excretion of total estrogens, increased production of cervical mucus, and, eventually, uterine bleeding. Ovarian refractoriness to gonadotropins suggests ovarian failure, and ovarian March 977 responsiveness indicates hypothalamic-pituitary failure. The recent elucidation of the structure and synthesis of LH_RHlO-l2 made it possible to investigate the pituitary responsiveness to this material. t is now apparent that synthetic LH RH is effective in stimulating a concomitant release offsh and LH from the pituitary in normal men and women. 3-6 Consequently, the pituitary responsiveness to has been studied in certain disorders of the hypothalamic-pituitarygonadal axis, and the diagnostic value of the synthetic decapeptide for evaluation of the pituitary gonadotropin reserve has been discussed Thus, it is now agreed that is an effective means of testing the pituitary secretory reserve for FSH and LH, and it is possible to distinguish hypothalamic from hypophyseal dysfunction. n this series, the loading test and the HMG and HCG loading test were performed in seven normal, menstruating subjects and thirtythree amenorrheic patients. Seven normal subjects in the follicular phase of the menstrual cycle responded to with a concomitant increase in serum FSH and LH levels. They were also responsive to HMG and HCG, with an increase in the urinary excretion of total estrogens. Fifteen amenorrheic patients responding to progesterone with uterine bleeding exhibited a response to and gonadotropins analogous to that seen in normal control subjects. The pituitary and ovarian reserve function in these patients was at least relatively intact. Eight patients with ovarian amenorrhea due to Turner's syndrome or primary ovarian failure showed an extraordinary over-response to. The base line levels of FSH and LH in these patients were markedly high, and administration e voked a remarkable release of FSH and LH. Conversely, administration of HMG and HCG failed to stimulate ovarian estrogen production, and no response was observed. Five patients with hypophyseal amenorrhea due to Sheehan's syndrome or pituitary tumors showed little or no response to. n contrast, an ovarian response to gonadotropins with an increase in urinary estrogens was observed. Five patients with hypothalamic amenorrhea due to tertiary hypogonadism showed relatively good responses to and gonadotropins. The patterns of pituitary and ovarian responses in these patients were similar to those in normal control subjects. The results suggest that a specific pattern of response to and HMG and HCG exists in hypothalamic, hypophyseal, and ovarian a-

7 Vol. 28, No.3 SCHEMATC APPROACH TO THE ORK-UP OF AMENORRHEA 235 HYPOTHALAMUS Bleeding (CENTER) HYPOTHALAMUS - (CENTER) Bleeding Amenorrhea CD PROGESTN TEST Amenorrhea ESTROGEN-PROGESTN GONADOTROPN TEST Estrogen response LH. FSH TEST Amenorrhea llut!eru!.:s No response- OV ARY No response... PTUTARY FG. 6. Scheme for diagnosing the amenorrheas. menorrhea, respectively, and the response pattern may facilitate the differential diagnosis of idiopathic amenorrheas. Thus, a diagnostic scheme in the differential diagnosis of idiopathic amenorrheas might be established (Fig. 6). First, a test dose of progestin should be given to amenorrheic patients. f uterine bleeding occurs, the lesion in the hypothalamic-pituitary-ovarian-uterine axis lies in the hypothalamus. Persistence of amenorrhea after progestin administration does not delimit the site of the relevant lesion. Cyclic therapy of estrogen and progestin should be tried. f bleeding does not occur, uterine refractoriness would be suspected. The gonadotropin (HMG and HCG) loading test should be tried in amenorrheic patients responding to cyclic estrogen and progestin therapy with bleeding. f no ovarian response to gonadotropins is observed, the cause of amenorrhea might be of ovarian origin. The LH RH loading test should be performed in amenorrheic patients responding to exogenously administered gonadotropins with an increase in the urinary excretion of total estrogens. Pituitary responsiveness to suggests a hypothalamic lesion, and little or no responsiveness suggests a hypophyseal lesion. However, one should bear in mind that responses to gonadotropin (HMG and HCG) and loading tests are not always clear-cut, and differentiating and exceptional cases may exist. Acknowledgments. e wish to thank the National Pituitary Agency and the Endocrinology Study Section of the National nstitute of Arthritis, Metabolism and Digestive Diseases for the generous supply of materials for radioimmunoassay of human pituitary gonadotropins. The Second nternational Reference Preparation of Human Menopausal Gonadotropin was obtained from the National nstitute for Medical Research, London. REFERENCES. Schrank P: Uber einen in der Allgemeinpraxis anwendbaren Amenorrhoetest. Zentralbl Gynaekol 74:569, Kupperman HS, Lefkovics SC: Progesterone in problem of sterility; diagnostic and therapeutic use. Fertil Steril 8:3, Midgley AR: Radioimmunoassay of human follicle-stimulating hormone. J Clin Endocrinol Metab 27:295, Odell D, ayford PL, Ross GT: Simplified, partially automated method for radioimmunoassay of human thyroid-stimulating, growth, luteiniing, and folliclestimulating hormones. J Lab Clin Med 70:973, Brown JB, MacLeod SC, Manaughtan C, Smith MA, Smyth B: A rapid method for estimating oestrogens in urine using a semi-automatic extractor. J Endocrinol 42:5, Crooke AC, Butt R, Bertrand PV: Clinical trial of human gonadotropins. Tl. Variation in sensitivity between patients and standardisation of treatment. Acta Endocrinol [Supp] (Kbh) 53:3, Crooke AC, Butt R, Bertrand PV: Treatment of idiopathic secondary amenorrhoea with single injections of follicle-stimulating hormone and chorionic gonadotropin. Lancet 2:54, Cox R, Cox L, Black TL: Test for ovarian function and responsiveness leading to ovulation induction. Lancet 2: 888, 966

8 236 NAKANO ET AL. 9. ashio M: Fundamental and clinicopathological studies on human gonadotropin actions on anovulatory diseases.. Studies on the clinical results of artificial induction of ovulation with human gonadotropins and a new trial classification of anovulatory ovaries. Kobe J Med Sci 8: 4, 972. Matsuo H, Baba Y, Nair RMG, Arimura A, Schally A V: Structure of the porcine LH- and FSH-releasing hormone.. The proposed amino acid sequence. Biochem Biophys Res Commun 43:334, 97. Baba Y, Matsuo H, Schally AV: Structure of porcine LHand FSH-releasing hormone.. Confirmation of the proposed structure by conventional sequential analyses. Biochem Biophys Res Commun 44:459, Matsuo H, Arimura A, Nair RMG, Schally AV: Synthesis of porcine LH- and FSH-releasing hormone by the solidphase method. Biochem Biophys Res Commun 45:822, Yen SSC, Rebar R, VandenBerg G, Naftolin F, Ehara Y, Engblom S, Ryan KJ, Benirschke K, Rivier J, Amoss M, Guillemin R: Synthetic luteiniing hormone-releasing factor: a potent stimulator of gonadotropin release in man. J Clin Endocrinol Metab 34:8, Rebar R, Yen SSC, VandenBerg G, Naftolin F, Ehara Y, Engblom S, Ryan KJ, Rivier J, Amoss M, Guillemin R: Gonadotropin responses to synthetic LRF: dose-response relationship in men. J Clin Endocrinol Metab 36:, Roth JC, Grumbach MM, Kaplan SL: Effect of synthetic luteiniing hormone-releasing factor on serum March 977 testosterone and gonadotropins in prepubertal, pubertal and adult males. J Clin Endocrinol Metab 37:680, Nakano R, Kotsuji F, Miuno T, Hashiba N, ashio M, Tojo S: Response to luteiniing hormone releasing factor (LRF) in normal subjects and anovulatory patients. Acta Obstet Gynecol Scand 52:7, Siler TM, Yen SSC: Augmented gonadotropin response to synthetic LRF in hypo gonadal state. J Clin Endocrinol Metab 37:49, Nillius SJ, ide L: The LH-releasing hormone test in 3 women with secondary amenorrhoea. J Obstet Gynaecol Br Commonw 79:874, Yen SSC, Rebar R, VandenBerg G, Judd H: Hypothalamic amenorrhea and hypogonadotropinism: response to synthetic LRF. J Clin Endocrinol Metab 36:8, 973. Zarate A, Canales ES, De La Crus A, Soria J, Schally AV: Pituitary response to synthetic in Stein-Leventhal syndrome and functional amenorrhea. Obstet Gynecol4: 803, AonoT, MinagawaJ,Kinugasa T, TaniawaO, KurachiK: Response of pituitary LH and FSH to synthetic LH-releasing hormone in normal subjects and patients with Sheehan's syndrome. Am J Obstet Gynecol 7:46, Nakano R, Kotsuji F, Tojo S: Pituitary responsiveness to synthetic luteiniing hormone releasing hormone () during the menstrual cycle and in female hypogonadism. Br J Obstet Gynaecol 82:805, 975