Vol. 30, No. 6, December 1978 Printed in U.S.A.
FERTILITY AND STERILITY Copyright ~ 1978 The American Fertility Society
EFFECT OF EPIMESTROL ON GONADOTROPIN AND PROLACTIN PLASMA LEVELS AND RESPONSE TO LUTEINIZING HORMONE-RELEASING HORMONE/THYROTROPINRELEASING HORMONE IN SECONDARY AMENORRHEA AND OLIGOMENORRHEA
ANDREA R. GENAZZANI, M.D.* FABIO FACCHINETTI VICENZO DE LEO, M.D. ENRICO PICCIOLINI, M.D. FRANCO FRANCHI, M.D. DONATELLA PARRINI, PH.D. PETAR M. KICOVIC, M.D.
Department of Obstetrics and Gynecology, University of Siena, Siena, Italy, and Scientific Development Group, Medical Unit, Organon, Oss, Holland
The effects of epimestrol (5 mg every 6 hours for 5 days) on basal levels of luteinizing hormone (UI), follicle-stimulating hormone (FSH), prolactin (Prl), estradiol,progesterone, and dehydroepiandrosterone sulfate, and on the response to Ul-releasing hormone (UI-RH) and thyrotropin-releasing hormone (TRH) stimulation, were studied in 18 cases of secondary amenorrhea and oligomenorrhea of hypothalamic-pituitary origin, in three cases of anorexia nervosa, in two cases of long-lasting progestin-induced amenorrhea, and in one case of precocious menopause. The results in the first 18 patients indicate that epimestrol treatment induces a significant increase in LH and Prllevels after 24 hours, while the FSH increase becomes significant only after4 days of therapy. Twelve hours after discontinuation of treatment, all three hormone levels decreased signifwantly to values similar to the basal levels, while the pituitary response to UI-RH indicated a much more marked LH secretion than before treatment. A second test, performed 36 hours after the last drug administration, again showed a significantly higher Ul response than that found under basal conditions. No significant variations were observed in the FSH response to UI-RH, nor in the Prl response to TRH. These data suggest that epimestrol interferes at the level of the centers responsible for Prl and gonadotropin secretion in the manner of a weak estrogen. Fertil Steril30:654, 1978
Recently Melis et aL, 5 in a preliminary paper, observed that in a few cases of secondary amenorrhea, epimestrol treatment induced a greater luteinizing hormone (LH) and follicle-stimulating hormone (FSH) response to LH-releasing hormone (LH-RH) than those observed prior to treatment. Furthermore, Luisi et aL 6 reported that in males 15 mg of epimestrol given for 10 days causes a significant increase in plasma gonadotropin levels, while a rise is also noted in testosterone and estradiol plasma levels after 7 days of treatment. The present results indicate that in cases of oligomenorrhea and secondary amenorrhea of
Some data have recently been published on the effects of epimestrol (17-epiestriol-3-methy1ether) in the induction of ovulation in anovulatory cycles. 1"4 On the other hand, no data are available in literature which indicate the manner in which epimestrol acts on the centers responsible for gonadotropin secretion in humans. Received April 26, 1978; revised July 6, 1978; accepted Au-
gust 8, 1978. *Reprint requests: ProfeBBOr A. R. Genazzani, Department of Obstetrics and Gynecology, University of Siena, 53100 Siena, Italy.
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EFFECT OF EPIMESTROL IN SECONDARY AMENORRHEA AND OLIGOMENORRHEA
hypothalamic-pituitary origin, epimestrol treatment at a dose of 20 mg/day for 5 days induces a rise in basal LH, FSH, and prolactin (Prl) levels, while a greater LH response to LH-RH stimulation is found after therapy. These data suggest that epimestrol interferes positively, in a manner similar to that of a weak estrogen, with the hypothalamic-pituitary mechanism controlling gonadotropin secretion. MATERIALS AND METHODS
Sixteen patients with secondary amenorrhea, three of whom also had anorexia nervosa, and eight patients with oligomenorrhea were studied; their ages ranged from 17 to 33 years. Oral treatment with epimestrol (5 mg every 6 hours) was given for 5 days. Blood samples were obtained daily between 8 and 9 A.M. starting before treatment and continuing throughout, the last samples being obtained 12 and 36 hours after the last tablet was given. Intravenous stimulation with 50 1-Lg of LH-RH (Relisorm L; Serono, Rome, Italy) and 200 1-Lg of thyrotropin-releasing hormone (TRH) (Biodata, Rome, Italy) was performed both before treatment and 12 and 36 hours afterward. Plasma LH, FSH, and Prllevels were measured, using the Biodata-Serono kits, in the basal samples and in those collected during the LH-RH and TRH tests. LH and FSH values were expressed as milliunits of Second International Reference Prep-
.
655
aration of human menopausal gonadotropin per milliliter. Estradiol (E 2 ), progesterone (P), and dehydroepiandrosterone sulfate (DHA-S) were also determined in the basal samples and in those obtained 12 and 36 hours afterward. E 2 and P were extracted from the plasma with ethyl ether (10:1) and measured using CEA-IRE-SORIN kits, while DHA-S was measured directly in the plasma using an antiserum kindly supplied by Dr. Guy Abraham, Torrance, Calif. The radioimmunoassays were effected by using an overnight incubation at 4 o C and dextran-coated charcoal separation. 7 The statistical analysis of the results was performed using the paired t-test. RESULTS
The patients were separated into two groups according to the clinical pathogenesis of their menstrual disorders. The first group included 10 cases of secondary amenorrhea and 8 cases of oligomenorrhea, presumably of hypothalamicpituitary origin (Table 1). The basal LH, FSH, Prl, and E 2 levels and the pituitary response to LH-RH and TRH (described later) allowed all 18 cases to be included in a single group. The second group included three cases of anorexia nervosa, two cases of long-lasting progestin-induced amenorrhea, and one case of precocious menopause. The results of epimestrol treatment in these two groups are discussed separately.
TABLE 1. Group 1 Patients: Age, Menstrual Disorders, and Plasma LH, FSH, Prl, and Estradiol Levels Basal levels Patient
Age
1 2 3
19 22 17
4 5
23 17
6 7 8 9 10 11
29 22 35 22 28 21
12 13 14 15 16 17 18
32 21 25 22 26 29 24
Diagnosis
Secondary amenorrhea, obesity Oligomenorrhea Secondary amenorrhea, delayed puberty Oligomenorrhea Secondary amenorrhea, hirsutism, obesity Secondary amenorrhea Secondary amenorrhea Secondary amenorrhea, obesity Oligomenorrhea Secondary amenorrhea Secondary amenorrhea, hirsutism Oligomenorrhea, obesity Secondary amenorrhea Oligomenorrhea Oligomenorrhea, obesity Oligomenorrhea, obesity Oligomenorrhea, obesity Secondary amenorrhea
LH
FSH
Prl
Estradiol
mU/ml
mU/ml
nglml
pg/ml
11.37 4.84
2.58 9.50
20.21 13.81
280 80
13.37 1.17
8.27 5.35
2.74 5.05
10 256
18.10 13.00 2.35 21.57 5.37 2.47
2.73 9.67 4.76 8.90 3.88 6.84
18.00 5.27 8.54 4.91 19.00 3.50
118 216 60 54 356 168
8.75 5.80 2.10 18.00 6.60 10.10 4.00 4.80
13.22 7.90 6.85 11.25 10.35 12.25 7.40 8.70
12.64 8.05 5.95 21.00 5.20 5.85 7.70 21.00
20 92 172 164 252 164 204 127
656
GENAZZANI ET AL.
December 1978 ~ before I reatment e-e12hrs. After
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90
120
FIG. 1. Effect of epimestrol treatment in 18 patients with secondary amenorrhea and oligomenorrhea on basal levels of plasma LH (left) and the LH response to 50 p.g of LH-RH intravenously (right) determined before and 12 and 36 hours after the end of therapy.
Plasma Prllevels (Fig. 3) increased from 8;9 ± 1.6 ng/ml to 15.5 ± 3.4 ng/ml after 24 hours (P < 0.05); on the 5th day of therapy they had risen to 23.8 ± 8.5 ng/ml, with a subsequent decrease to 11.6± 3.9 after 12 hours and then to 6.0± 0.9 ng/ml 36 hours after the end of treatment. Epimestrol did not induce significant changes in E 2 , P, or DHA-S plasma levels (Table 2). The single values of plasma P show that in two cases the rise in gonadotropins led to formation of the corpus luteum. Lh-RH Stimulation Test. Intravenous stimulation with 50 JLg ofLH-RH, given before the start of the 5-day therapy with epimestrol and 12 and 36 hours afterward, induced a prompt, significant, and sustained increase in the levels of both hor-
Group 1 A significant and early increase in basal LH levels (Fig. 1) was observed after 24 hours of treatment (9.66 ± 1.79 to 20.3 ± 3.64 mU:ml; P < 0.02). These levels then remained constant until the end of treatment, when a significant decrease was found 12 hours later (25.5 ± 4.6 to 16.6 ± 4.2 mU/ml; P < 0.01). No further decrease was observed 24 hours later. FSH plasma levels (Fig. 2) rose significantly 72 hours after the start of treatment (from 8.1 ± 0.9 to 10.3 ± 1.0 mU/ml; P < 0.02); they then remained stable until a significant decrease was observed (7.3 ± 0.7 mU/ml;P < 0.01) 12 hours after the last tablet. Plasma concentrations subsequently remained constant at these levels. Epimeslrol 5 mp x4)iay
• : 50 }19 LH·RH i. v.
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-
12 hrs. after
~-oil.
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0
2
3
4
5
&days 7
0
15
30
45
&0
90 min:
120·
FIG. 2. Effect of epimestrol treatment in 18 patients with secondary amenorrhea and oligomenorrhea on basal levels of plasma FSH (left) and the FSH response to 50 p.g ofintravenously. LH-RH (right) determined before and 12 and .36 hours after the end of therapy.
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657
EFFECT OF EPIMESTROL IN SECONDARY AMENORRHEA AND OLIGOMENORRHEA
Vol. 30, No.6
Epomestrol Smgxlo/day
'• 200,.g TRH o.v /~
120
before treatment
•-• 12 hrs. after 11--11 36 •
•
( I . n. of cases 80
0
~days
0
S
o...---1.,..S-..,30--,T"os-s""o-m-,n-.-9..-o-----,do 1
FIG. 3. Effect of epimestrol treatment of 18 patients with secondary amenorrhea and oligomenorrhea on basal levels of plasma Prl (left) and the Prl response to 200 p.g of TRH intrave· nously (right) determined before and 12 and 36 hours after the end of therapy.
mones in all patients. However, the LH response was significantly higher than that found under basal conditions, both at 12 and 36 hours (Fig. 1), while the FSHpituitary response was similarboth before and after treatment (Fig. 2). However, determinations made 30 and 45 minutes after stimulation showed significantly higher levels when the test was performed 12 hours after the end of treatment than those found after 36 hours (P < 0.05).
TRH Stimulation Test. The Prl response to TRH did not show any significant difference in values found before treatment and 12 or 36 hours afterward (Fig. 3).
Group 2 The basal levels of LH, FSH, and Prl (Table 3) were not affected significantly by treatment with epimestrol in the three patients with anorexia nervosa. In the patients with long-lasting progestin-induced amenorrhea, LH basal levels decreased after treatment, while FSH and Prl levels remained unaltered. The one patient with precocious menopause showed no variations in FSH levels throughout the treatment (4 days), while an LH peak was observed, with values rising from 9.6 to 82 mU/ml. Twelve hours after therapy, the LH levels decreased to 8.9 mU/mL
TABLE 2. Plasma Levels of E 2 , P, and DHA-S before and after Treatment of Secondary Amenorrhea and Oligomenorrhea p
E, Patient Before
After 12 hr
Alter 36 hr
Before
pglml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean SE
280 80 10 256 118 216·
DHA.S After 36 hr
Before
54 356 168 20 92 172 164 252 164· 204 127
244 40 10 228 84 236 84 104 208 149
155.16 22.33
135.50 18.31
138.7 27.27
3,600 450 750 870 890 1,400 680 1,150 600 990 390 980 220 620
750 850 380 770 430 760 900 420 5,500 780 280 390 390 320
710 730 650
800 480 490
922.35 180.90
821.23 299.72
Alter 12 hr
After 36 hr
nglml
pg!ml
244 64 10 228 60 216 40 48 216 176 44 95 164 132 222 204 164 112
60•
After 12 hr
650 1,400 340 1,500 320 480 12,000 5,100 1,080 650
2,352.0. 1,16L2
968 1,460 1,000 500 540 1,160 1,020 1,040 1,400 1,440 1,140 1,040 1,000 610
960 1,060 1,080 620 860 880 1,440 688 980 1,250 1,640 1,076 1,080 880
900 1,100 880
740 1,400 910
1,011.64 68.08
1,032.0 66.37 .
960 1,120 900 600 540 1,130 1,320 696 1,220 1,300
978.60 90.87
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GENAZZANI ET AL.
December 1978
TABLE 3. Basal Levels of LH, FSH, and Prl in Patients with Anorexia Neroosa, Long-Lasting Progestin-Induced Amenorrhea, and Precocious Menopause Day 2
LH (mU!ml) Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorrhea Patient 1 Patient 2 Precocious menopause FSH (mU!ml) Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorrhea Patient 1 Patient 2 Precocious menopause Prl (ng/ml) Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorrhea Patient 1 Patient 2 Precocious menopause
0.84 1.27 1.68 18.94 7.18 9.65 1.02 2.20 6.34 1.67 5.27 33.5 7.20 5.60 6.90
1.20 1.40
0.39 2.00
0.86 2.30
0.62 2.40
8.20 76.0
9.30 82.0
9.40 71.0
9.60 8.88
4.30 2.30
3.00 2.50
2.70 2.00
2.08 1.60
10.20 29
8.10 60
65
8.30 32
12.0 10.0
12.5 17.5
11.6 21.0
9.10 17.0
2.40 2.60 5.55
As shown in Table 4, LH-RH stimulation, evaluated as the a maximal response for each test and for each hormone, caused a smaller LH and FSH response in two of the anorectic patients after treatment, in comparison with the results found before epimestrol treatment. The third patient showed no increase in plasma LH levels and only a very slight FSH response. Prl responses were in the same range as found in group 1, all subjects showing the highest levels after treatment.
7
6
4
3
1.10 1.47 1.14
1.52 1.55
6.35 4.40
7.94 8.13
3.6 1.52 1.97
2.68 4.48
1.37 7.30
1.26 6.64
7.50 4.10 6.70
6.40 6.20
10.90 11.50
8.90 13.90
In the first patient with long-lasting progestininduced amenorrhea, epimestrol caused a reduction in the LH maximum after stimulation, while in the other patient this response was consistently poor (Table 4). In both of these patients FSH was only very slightly responsive. In the first patient the Prl response to TRH decreased after epimestrol administration, while progressively rising levels were found in the second patient. The patient with precocious menopause showed a greater LH and FSH response to stimulation
TABLE 4. Maximal Release of LH and FSH after LH-RH Stimulation, and of Prl after TRH Stimulation Before and 12 and 36 Hours after Epimestrol Treatment LH plasma levels after LH-RH stimulation (4 max.) After After Before 12 hr 36 hr
FSH plasma levels after LH·RH stimulation (4 max.) After After Before 12 hr 36 hr
mU/ml
Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorrhea Patient 1 Patient 2 Precocious menopause
Prl plasma levels after TRH stimulation (4 max.) Before
After 12 hr
After 36hr
nglml
mU/ml
33.2 1.7 54.1
26.2 1.6 35.1
16.8 0.9 21.9
26.3 5.3 35.2
15.3 10.5 9.3
9.8 6.5 6.5
69.9 22.2 31.6
78.9 38.3 59.5
43.9 51.5 58.7
113.8 7.8 19.4
62.7 7.2 34.3
62.1 5.0
3.0 4.4 10.5
3.4 5.2 30.0
1.2 2.8
71.0 18.7 49.5
41.5 34.5
64.7 80.7
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659
EFFECT OF EPIMESTROL IN SECONDARY AMENORRHEA AND OLIGOMENORRHEA
TABLE 5. Plasma Levels of E 2 , P, and DHA-S before and 12 and 36 Hours after Epimestrol Treatment in Patients with Anorexia Nervosa, Long-Lasting Progestin-lnudced Amenorrhea, and Precocious Menopause p
E, Before
After 12 hr
After 36 hr
Before
pglml
•
Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorhea Patient 1 Patient 2 Precocious menopause
After 36 hr
Before
pglml
After 12 hr
After 36 hr
nglml
296 104 217
276 64 267
220 126 270
384 350 1220
960 260 1060
340 780 1080
370 700 740
4 440 860
12 616 940
228 54 204
152 84 140
172 76
700 350 440
350 600 810
1040 1040
1480 1080 1360
1340 940 1160
1640 940
after therapy (Table 4). Plasma steroid levels did not show significant variations when measured before and after treatment (Table 5). It is interesting to note that all three patients with anorexia nervosa showed low DHA-S levels (Table 5); there was, in fact, a significant decrease in comparison with those seen in normal females. 7
..
DHA-S
After 12 hr
DISCUSSION
The present results confirm the ability of epimestrol to cause an increase in basal levels of gonadotropins in cases of secondary amenorrhea and oligomenorrhea, as was shown in anovulatory cycles by Melis et al. 5 and in normal males by Luisi et al. 6 In particular, the effect on LH appears to be prompter and more marked than that on FSH. The present data also show that the stimulatory effect of epimestrol on basal gonadotropin levels is no longer apparent, or is markedly reduced, 12 hours after the last 5-mg dose of the agent. As an ether of naturally occurring 17-epiestriol, epimestrol is quickly metabolized. This drug also seems able to increase not only the basal plasma gonadotropin levels but also the intrapituitary concentrations, chiefly ofLH, as shown by the results of the LH-RH stimulation test. This effect is the opposite of that generally described for the antiestrogenic compound clomiphene citrate. In fact, in normal females, 8 in secondary amenorrhea, and in oligomenorrhea, 9 as well as in normal males, 10 clomiphene citrate causes an increase in basal gonadotropins but, with the exception of delayed puberty, 11 a decrease is found in the pituitary response to releasing hormones. This later phenomenon has been explained on the basis of clomiphene's antiestrogenic activity, stimulating the continuous secretion of LH-RH from the hypothalamus and thus inducing a reduction in the intrapituitary gonadotropin storage. 9 In this context, epimestrol seems to act as a weak estro-
gen and not as an antiestrogenic compound. The increase in basal gonadotropin levels and in the intrapituitary storage of LH supports this hypothesis, in accordance with the findings of Yen's group 12 - 17 on the effect of estrogens on gonadotropin regulation. The augmented Prl plasma levels caused by epimestrol in hypogonadal women indicate a greater sensitivity of the hypothalamic response of this hormone in women than in men, in whom epimestrol at a dose of 15 mg/day for 10 days had no effect on plasma Prl levels. 6 The Prl response to this agent was in fact the same as that observed after estrogen therapy in hypogonadal 18 and postmenopausal women. 19 The failure of this drug to increase either basal gonadotropin levels or the gonadotropic response to releasing hormone in cases of anorexia nervosa is in accordance with the well-known impairment of hypothalamic centers in this clinical syndrome. 9 • 20 Similarly, the effects of therapy in the two cases of long-lasting progestin-induced amenorrhea indicate the existence of a hyothalamic disorder in this situation, chiefly affecting gonadotropin secretion. The reported effects of epimestrol in secondary amenorrhea and oligomenorrhea, together with the LH peak induced in the patient with precocious menopause and the increase in the maximal response to LH-RH of both gonadotropins in this patient, are in agreement with Yen's concept regarding the effect of estrogenic compounds in regulating the pituitary gonadotropins and hypothalamic LH-RH synthesis, storage, and secretion. 12- 17 The observation that the 5-day treatment with epimestrol does not provoke a rise in E 2 plasma levels supports the findings of Luisi et al. 6 that in males treated with this agent, plasma testosterone and E2 show a significant rise only after 7 to 10 days of therapy. In conclusion, the present data demonstrate the
660
GENAZZANI ET AL.
stimulatory effect of epimestrol on gonadotropin and Prl plasma levels and on the response to Lh-Rh in patients with secondary amenorrhea. They also suggest that, in these patients, the effect of this agent differs from that described for clomiphene citrate 8• 11 in that it acts as an estrogenic compound and not as an antiestrogenic agent. REFERENCES 1. Demol R, Vanderkerckove D, Vermijlen P, Lepoutre L: Induction de I'ovulation par l'epimestrol. Brux Med 12:653,
1975 2. Meckies J, Leo-Rossberg I, Felshart R, Moltz L, Hammerstein J: Behandlung Steriler Frauen mit Epimestrol. Erfahrungsbericht ueber 143 Patientinnen mit 30 Graviditaeten. Dtsch Med Wochenschr 47:1711, 1976 3. Schmidt-Elmendorff H, Kaemmerling R: Vergleichende Klinische Untersuchungen von Clomiphen, Cyclophenil und Epimestrol. Geburtshilfe Frauenheikd 37:531, 1977 4. Portuondo JA, Busturia MA, Esteban J, Roman MD, Riego AG: Pituitary, ovarian, and peripheral changes after epimestrol (Stimovul) therapy (abstr). Fertil Steril28:390, 1977 5. Melis GB, Paoletti A, Genazzani AR, Fioretti P: Effect of epimestrol on pituitary response to exogenous Gn/RH and its validity in the treatment of anovulatory cycles. Acta Endocrinol [Suppl) (Kbh) 212:137, 1977 6. Luisi M, Kicovic PM, Franchi F: Placebo-controlled study of effects of epimestrol on the pituitary-testicular axis in normal men. Reproduccion. In press, 1978 7. Genazzani AR, Magrini G, Facchinetti F, Romagnino S, Pintor C, Felber JP, Fioretti P: Behavior and origin of plasma androgens throughout the menstural cycle. In Androgens and Antiandrogens, Edited by L Martini, M Motta. New York, Raven Press, 1977, p 247 8. Wang CF, Yen SSC: Direct evidence of estrogen modulation of pituitary sensitivity to luteinizing hormonereleasing factor during the menstrual cycle. J Clin Invest 55:201, 1975
December 1978 9. Wentz AC, Jones GS, Sapp KC: Effect of clomiphene citrate on gonadotropin responses to LH-RH administration in secondary amenorrhea and oligomenorrhea. Obstet Gynecol 47:677, 1976 10. Dhont M, De Gezelle H, Vanderkerckove D: Modulation of pituitary responsiveness to exogenous LH-RH by an oestrogenic and an anti-oestrogenic compound in the normal male. Clin Endocrinol (Oxf) 5:175, 1976 11. Snoep MC, de Lange WE, Sluited WJ, Doorenbos H: Differential response of serum LH in hypogonadotropic hypogonadism and delayed puberty to LH-RH stimulation before and after clomiphene citrate administration. J Clin Endocrinol Metab 44:603, 1977 12. Yen SSC, Vandenberg G, SilerTM: Modulation of pituitary responsiveness to LRF by estrogen. J Clin Endocrinol Metab 39:170, 1974 13. Lasley BL, Wang CF, Yen SSC: The effect of estrogen and progesterone on the functional capacity of the gonadotrops. J Clin Endocrinol Metab 41:820, 1975 14. Yen SSC, Lasley BL, Wang CF, Leblanc H, Siler TM: The operating characteristic of the hypothalamic-pituitary system during the menstrual cycle and observation of biological action of somatostatin. Recent Prog Horm Res 31:321, 1975 15. Wang CF, Lasley BL, Lein A, Yen SSC: The functional changes of the pituitary gonadotrops during the menstrual cycle. J Clin Endocrinol Metab 42:718, 1976 16. Yen SSC, Lein A: The apparent paradox of the negative and positive feedback control system on gonadotropin secretion. Am J Obstet Gynecol 126:942, 1976 17. Hoff JD, Lasley BL, Wang CF, Yen SSC: The two pools of pituitary gonadotropin: regulation during the menstrual cycle. J Clin Endocrinol Metab 44:302, 1977 18. Yen SSC, Ebara Y, Siler TM: Augmentation of prolactin secretion by estrogen in hypogonadal women. J Clin Invest 53:652, 1974 19. Robyn C, Vekemans M: Influence oflow dose oestrogen on circulating prolactin, LH and FSH in postmenopausal women. Acta Endocrinol (Kbh) 83:9, 1976 20. Aro A, Lamberg BA, Pelkonen R: Hypothalamic endocrine dysfunction in anorexia nervosa. Acta Endocrinol (Kbh) 85:673, 1977
FERTILITY AND STERILITY Copyright ~ 1978 The American Fertility Society
EFFECT OF EPIMESTROL ON GONADOTROPIN AND PROLACTIN PLASMA LEVELS AND RESPONSE TO LUTEINIZING HORMONE-RELEASING HORMONE/THYROTROPINRELEASING HORMONE IN SECONDARY AMENORRHEA AND OLIGOMENORRHEA
ANDREA R. GENAZZANI, M.D.* FABIO FACCHINETTI VICENZO DE LEO, M.D. ENRICO PICCIOLINI, M.D. FRANCO FRANCHI, M.D. DONATELLA PARRINI, PH.D. PETAR M. KICOVIC, M.D.
Department of Obstetrics and Gynecology, University of Siena, Siena, Italy, and Scientific Development Group, Medical Unit, Organon, Oss, Holland
The effects of epimestrol (5 mg every 6 hours for 5 days) on basal levels of luteinizing hormone (UI), follicle-stimulating hormone (FSH), prolactin (Prl), estradiol,progesterone, and dehydroepiandrosterone sulfate, and on the response to Ul-releasing hormone (UI-RH) and thyrotropin-releasing hormone (TRH) stimulation, were studied in 18 cases of secondary amenorrhea and oligomenorrhea of hypothalamic-pituitary origin, in three cases of anorexia nervosa, in two cases of long-lasting progestin-induced amenorrhea, and in one case of precocious menopause. The results in the first 18 patients indicate that epimestrol treatment induces a significant increase in LH and Prllevels after 24 hours, while the FSH increase becomes significant only after4 days of therapy. Twelve hours after discontinuation of treatment, all three hormone levels decreased signifwantly to values similar to the basal levels, while the pituitary response to UI-RH indicated a much more marked LH secretion than before treatment. A second test, performed 36 hours after the last drug administration, again showed a significantly higher Ul response than that found under basal conditions. No significant variations were observed in the FSH response to UI-RH, nor in the Prl response to TRH. These data suggest that epimestrol interferes at the level of the centers responsible for Prl and gonadotropin secretion in the manner of a weak estrogen. Fertil Steril30:654, 1978
Recently Melis et aL, 5 in a preliminary paper, observed that in a few cases of secondary amenorrhea, epimestrol treatment induced a greater luteinizing hormone (LH) and follicle-stimulating hormone (FSH) response to LH-releasing hormone (LH-RH) than those observed prior to treatment. Furthermore, Luisi et aL 6 reported that in males 15 mg of epimestrol given for 10 days causes a significant increase in plasma gonadotropin levels, while a rise is also noted in testosterone and estradiol plasma levels after 7 days of treatment. The present results indicate that in cases of oligomenorrhea and secondary amenorrhea of
Some data have recently been published on the effects of epimestrol (17-epiestriol-3-methy1ether) in the induction of ovulation in anovulatory cycles. 1"4 On the other hand, no data are available in literature which indicate the manner in which epimestrol acts on the centers responsible for gonadotropin secretion in humans. Received April 26, 1978; revised July 6, 1978; accepted Au-
gust 8, 1978. *Reprint requests: ProfeBBOr A. R. Genazzani, Department of Obstetrics and Gynecology, University of Siena, 53100 Siena, Italy.
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.. ..
EFFECT OF EPIMESTROL IN SECONDARY AMENORRHEA AND OLIGOMENORRHEA
hypothalamic-pituitary origin, epimestrol treatment at a dose of 20 mg/day for 5 days induces a rise in basal LH, FSH, and prolactin (Prl) levels, while a greater LH response to LH-RH stimulation is found after therapy. These data suggest that epimestrol interferes positively, in a manner similar to that of a weak estrogen, with the hypothalamic-pituitary mechanism controlling gonadotropin secretion. MATERIALS AND METHODS
Sixteen patients with secondary amenorrhea, three of whom also had anorexia nervosa, and eight patients with oligomenorrhea were studied; their ages ranged from 17 to 33 years. Oral treatment with epimestrol (5 mg every 6 hours) was given for 5 days. Blood samples were obtained daily between 8 and 9 A.M. starting before treatment and continuing throughout, the last samples being obtained 12 and 36 hours after the last tablet was given. Intravenous stimulation with 50 1-Lg of LH-RH (Relisorm L; Serono, Rome, Italy) and 200 1-Lg of thyrotropin-releasing hormone (TRH) (Biodata, Rome, Italy) was performed both before treatment and 12 and 36 hours afterward. Plasma LH, FSH, and Prllevels were measured, using the Biodata-Serono kits, in the basal samples and in those collected during the LH-RH and TRH tests. LH and FSH values were expressed as milliunits of Second International Reference Prep-
.
655
aration of human menopausal gonadotropin per milliliter. Estradiol (E 2 ), progesterone (P), and dehydroepiandrosterone sulfate (DHA-S) were also determined in the basal samples and in those obtained 12 and 36 hours afterward. E 2 and P were extracted from the plasma with ethyl ether (10:1) and measured using CEA-IRE-SORIN kits, while DHA-S was measured directly in the plasma using an antiserum kindly supplied by Dr. Guy Abraham, Torrance, Calif. The radioimmunoassays were effected by using an overnight incubation at 4 o C and dextran-coated charcoal separation. 7 The statistical analysis of the results was performed using the paired t-test. RESULTS
The patients were separated into two groups according to the clinical pathogenesis of their menstrual disorders. The first group included 10 cases of secondary amenorrhea and 8 cases of oligomenorrhea, presumably of hypothalamicpituitary origin (Table 1). The basal LH, FSH, Prl, and E 2 levels and the pituitary response to LH-RH and TRH (described later) allowed all 18 cases to be included in a single group. The second group included three cases of anorexia nervosa, two cases of long-lasting progestin-induced amenorrhea, and one case of precocious menopause. The results of epimestrol treatment in these two groups are discussed separately.
TABLE 1. Group 1 Patients: Age, Menstrual Disorders, and Plasma LH, FSH, Prl, and Estradiol Levels Basal levels Patient
Age
1 2 3
19 22 17
4 5
23 17
6 7 8 9 10 11
29 22 35 22 28 21
12 13 14 15 16 17 18
32 21 25 22 26 29 24
Diagnosis
Secondary amenorrhea, obesity Oligomenorrhea Secondary amenorrhea, delayed puberty Oligomenorrhea Secondary amenorrhea, hirsutism, obesity Secondary amenorrhea Secondary amenorrhea Secondary amenorrhea, obesity Oligomenorrhea Secondary amenorrhea Secondary amenorrhea, hirsutism Oligomenorrhea, obesity Secondary amenorrhea Oligomenorrhea Oligomenorrhea, obesity Oligomenorrhea, obesity Oligomenorrhea, obesity Secondary amenorrhea
LH
FSH
Prl
Estradiol
mU/ml
mU/ml
nglml
pg/ml
11.37 4.84
2.58 9.50
20.21 13.81
280 80
13.37 1.17
8.27 5.35
2.74 5.05
10 256
18.10 13.00 2.35 21.57 5.37 2.47
2.73 9.67 4.76 8.90 3.88 6.84
18.00 5.27 8.54 4.91 19.00 3.50
118 216 60 54 356 168
8.75 5.80 2.10 18.00 6.60 10.10 4.00 4.80
13.22 7.90 6.85 11.25 10.35 12.25 7.40 8.70
12.64 8.05 5.95 21.00 5.20 5.85 7.70 21.00
20 92 172 164 252 164 204 127
656
GENAZZANI ET AL.
December 1978 ~ before I reatment e-e12hrs. After
Epimestrol Smg" 4/day
I
lO
1
.
T·M:tsem
120
1
1.
20
·,~J 1 •
1
•1 • : p c .02
0
0
2
3
'
5
0
15
lO
45
&o min:
90
120
FIG. 1. Effect of epimestrol treatment in 18 patients with secondary amenorrhea and oligomenorrhea on basal levels of plasma LH (left) and the LH response to 50 p.g of LH-RH intravenously (right) determined before and 12 and 36 hours after the end of therapy.
Plasma Prllevels (Fig. 3) increased from 8;9 ± 1.6 ng/ml to 15.5 ± 3.4 ng/ml after 24 hours (P < 0.05); on the 5th day of therapy they had risen to 23.8 ± 8.5 ng/ml, with a subsequent decrease to 11.6± 3.9 after 12 hours and then to 6.0± 0.9 ng/ml 36 hours after the end of treatment. Epimestrol did not induce significant changes in E 2 , P, or DHA-S plasma levels (Table 2). The single values of plasma P show that in two cases the rise in gonadotropins led to formation of the corpus luteum. Lh-RH Stimulation Test. Intravenous stimulation with 50 JLg ofLH-RH, given before the start of the 5-day therapy with epimestrol and 12 and 36 hours afterward, induced a prompt, significant, and sustained increase in the levels of both hor-
Group 1 A significant and early increase in basal LH levels (Fig. 1) was observed after 24 hours of treatment (9.66 ± 1.79 to 20.3 ± 3.64 mU:ml; P < 0.02). These levels then remained constant until the end of treatment, when a significant decrease was found 12 hours later (25.5 ± 4.6 to 16.6 ± 4.2 mU/ml; P < 0.01). No further decrease was observed 24 hours later. FSH plasma levels (Fig. 2) rose significantly 72 hours after the start of treatment (from 8.1 ± 0.9 to 10.3 ± 1.0 mU/ml; P < 0.02); they then remained stable until a significant decrease was observed (7.3 ± 0.7 mU/ml;P < 0.01) 12 hours after the last tablet. Plasma concentrations subsequently remained constant at these levels. Epimeslrol 5 mp x4)iay
• : 50 }19 LH·RH i. v.
f l (I: n.
20
of cases
f.......-·--.
1en:z: ...
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10
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I "~--!
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1
10
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1
e
I
•: poe .02
M:Uam·
0
l
,Y~~:: Jl/;::1· 1 1 1
P" 1
::::~5
T
treatment
-
12 hrs. after
~-oil.
3& •
0
2
3
4
5
&days 7
0
15
30
45
&0
90 min:
120·
FIG. 2. Effect of epimestrol treatment in 18 patients with secondary amenorrhea and oligomenorrhea on basal levels of plasma FSH (left) and the FSH response to 50 p.g ofintravenously. LH-RH (right) determined before and 12 and .36 hours after the end of therapy.
..
657
EFFECT OF EPIMESTROL IN SECONDARY AMENORRHEA AND OLIGOMENORRHEA
Vol. 30, No.6
Epomestrol Smgxlo/day
'• 200,.g TRH o.v /~
120
before treatment
•-• 12 hrs. after 11--11 36 •
•
( I . n. of cases 80
0
~days
0
S
o...---1.,..S-..,30--,T"os-s""o-m-,n-.-9..-o-----,do 1
FIG. 3. Effect of epimestrol treatment of 18 patients with secondary amenorrhea and oligomenorrhea on basal levels of plasma Prl (left) and the Prl response to 200 p.g of TRH intrave· nously (right) determined before and 12 and 36 hours after the end of therapy.
mones in all patients. However, the LH response was significantly higher than that found under basal conditions, both at 12 and 36 hours (Fig. 1), while the FSHpituitary response was similarboth before and after treatment (Fig. 2). However, determinations made 30 and 45 minutes after stimulation showed significantly higher levels when the test was performed 12 hours after the end of treatment than those found after 36 hours (P < 0.05).
TRH Stimulation Test. The Prl response to TRH did not show any significant difference in values found before treatment and 12 or 36 hours afterward (Fig. 3).
Group 2 The basal levels of LH, FSH, and Prl (Table 3) were not affected significantly by treatment with epimestrol in the three patients with anorexia nervosa. In the patients with long-lasting progestin-induced amenorrhea, LH basal levels decreased after treatment, while FSH and Prl levels remained unaltered. The one patient with precocious menopause showed no variations in FSH levels throughout the treatment (4 days), while an LH peak was observed, with values rising from 9.6 to 82 mU/ml. Twelve hours after therapy, the LH levels decreased to 8.9 mU/mL
TABLE 2. Plasma Levels of E 2 , P, and DHA-S before and after Treatment of Secondary Amenorrhea and Oligomenorrhea p
E, Patient Before
After 12 hr
Alter 36 hr
Before
pglml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean SE
280 80 10 256 118 216·
DHA.S After 36 hr
Before
54 356 168 20 92 172 164 252 164· 204 127
244 40 10 228 84 236 84 104 208 149
155.16 22.33
135.50 18.31
138.7 27.27
3,600 450 750 870 890 1,400 680 1,150 600 990 390 980 220 620
750 850 380 770 430 760 900 420 5,500 780 280 390 390 320
710 730 650
800 480 490
922.35 180.90
821.23 299.72
Alter 12 hr
After 36 hr
nglml
pg!ml
244 64 10 228 60 216 40 48 216 176 44 95 164 132 222 204 164 112
60•
After 12 hr
650 1,400 340 1,500 320 480 12,000 5,100 1,080 650
2,352.0. 1,16L2
968 1,460 1,000 500 540 1,160 1,020 1,040 1,400 1,440 1,140 1,040 1,000 610
960 1,060 1,080 620 860 880 1,440 688 980 1,250 1,640 1,076 1,080 880
900 1,100 880
740 1,400 910
1,011.64 68.08
1,032.0 66.37 .
960 1,120 900 600 540 1,130 1,320 696 1,220 1,300
978.60 90.87
658
GENAZZANI ET AL.
December 1978
TABLE 3. Basal Levels of LH, FSH, and Prl in Patients with Anorexia Neroosa, Long-Lasting Progestin-Induced Amenorrhea, and Precocious Menopause Day 2
LH (mU!ml) Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorrhea Patient 1 Patient 2 Precocious menopause FSH (mU!ml) Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorrhea Patient 1 Patient 2 Precocious menopause Prl (ng/ml) Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorrhea Patient 1 Patient 2 Precocious menopause
0.84 1.27 1.68 18.94 7.18 9.65 1.02 2.20 6.34 1.67 5.27 33.5 7.20 5.60 6.90
1.20 1.40
0.39 2.00
0.86 2.30
0.62 2.40
8.20 76.0
9.30 82.0
9.40 71.0
9.60 8.88
4.30 2.30
3.00 2.50
2.70 2.00
2.08 1.60
10.20 29
8.10 60
65
8.30 32
12.0 10.0
12.5 17.5
11.6 21.0
9.10 17.0
2.40 2.60 5.55
As shown in Table 4, LH-RH stimulation, evaluated as the a maximal response for each test and for each hormone, caused a smaller LH and FSH response in two of the anorectic patients after treatment, in comparison with the results found before epimestrol treatment. The third patient showed no increase in plasma LH levels and only a very slight FSH response. Prl responses were in the same range as found in group 1, all subjects showing the highest levels after treatment.
7
6
4
3
1.10 1.47 1.14
1.52 1.55
6.35 4.40
7.94 8.13
3.6 1.52 1.97
2.68 4.48
1.37 7.30
1.26 6.64
7.50 4.10 6.70
6.40 6.20
10.90 11.50
8.90 13.90
In the first patient with long-lasting progestininduced amenorrhea, epimestrol caused a reduction in the LH maximum after stimulation, while in the other patient this response was consistently poor (Table 4). In both of these patients FSH was only very slightly responsive. In the first patient the Prl response to TRH decreased after epimestrol administration, while progressively rising levels were found in the second patient. The patient with precocious menopause showed a greater LH and FSH response to stimulation
TABLE 4. Maximal Release of LH and FSH after LH-RH Stimulation, and of Prl after TRH Stimulation Before and 12 and 36 Hours after Epimestrol Treatment LH plasma levels after LH-RH stimulation (4 max.) After After Before 12 hr 36 hr
FSH plasma levels after LH·RH stimulation (4 max.) After After Before 12 hr 36 hr
mU/ml
Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorrhea Patient 1 Patient 2 Precocious menopause
Prl plasma levels after TRH stimulation (4 max.) Before
After 12 hr
After 36hr
nglml
mU/ml
33.2 1.7 54.1
26.2 1.6 35.1
16.8 0.9 21.9
26.3 5.3 35.2
15.3 10.5 9.3
9.8 6.5 6.5
69.9 22.2 31.6
78.9 38.3 59.5
43.9 51.5 58.7
113.8 7.8 19.4
62.7 7.2 34.3
62.1 5.0
3.0 4.4 10.5
3.4 5.2 30.0
1.2 2.8
71.0 18.7 49.5
41.5 34.5
64.7 80.7
..
•
Vol. 30, No.6
659
EFFECT OF EPIMESTROL IN SECONDARY AMENORRHEA AND OLIGOMENORRHEA
TABLE 5. Plasma Levels of E 2 , P, and DHA-S before and 12 and 36 Hours after Epimestrol Treatment in Patients with Anorexia Nervosa, Long-Lasting Progestin-lnudced Amenorrhea, and Precocious Menopause p
E, Before
After 12 hr
After 36 hr
Before
pglml
•
Anorexia nervosa Patient 1 Patient 2 Patient 3 Long-lasting progestininduced amenorhea Patient 1 Patient 2 Precocious menopause
After 36 hr
Before
pglml
After 12 hr
After 36 hr
nglml
296 104 217
276 64 267
220 126 270
384 350 1220
960 260 1060
340 780 1080
370 700 740
4 440 860
12 616 940
228 54 204
152 84 140
172 76
700 350 440
350 600 810
1040 1040
1480 1080 1360
1340 940 1160
1640 940
after therapy (Table 4). Plasma steroid levels did not show significant variations when measured before and after treatment (Table 5). It is interesting to note that all three patients with anorexia nervosa showed low DHA-S levels (Table 5); there was, in fact, a significant decrease in comparison with those seen in normal females. 7
..
DHA-S
After 12 hr
DISCUSSION
The present results confirm the ability of epimestrol to cause an increase in basal levels of gonadotropins in cases of secondary amenorrhea and oligomenorrhea, as was shown in anovulatory cycles by Melis et al. 5 and in normal males by Luisi et al. 6 In particular, the effect on LH appears to be prompter and more marked than that on FSH. The present data also show that the stimulatory effect of epimestrol on basal gonadotropin levels is no longer apparent, or is markedly reduced, 12 hours after the last 5-mg dose of the agent. As an ether of naturally occurring 17-epiestriol, epimestrol is quickly metabolized. This drug also seems able to increase not only the basal plasma gonadotropin levels but also the intrapituitary concentrations, chiefly ofLH, as shown by the results of the LH-RH stimulation test. This effect is the opposite of that generally described for the antiestrogenic compound clomiphene citrate. In fact, in normal females, 8 in secondary amenorrhea, and in oligomenorrhea, 9 as well as in normal males, 10 clomiphene citrate causes an increase in basal gonadotropins but, with the exception of delayed puberty, 11 a decrease is found in the pituitary response to releasing hormones. This later phenomenon has been explained on the basis of clomiphene's antiestrogenic activity, stimulating the continuous secretion of LH-RH from the hypothalamus and thus inducing a reduction in the intrapituitary gonadotropin storage. 9 In this context, epimestrol seems to act as a weak estro-
gen and not as an antiestrogenic compound. The increase in basal gonadotropin levels and in the intrapituitary storage of LH supports this hypothesis, in accordance with the findings of Yen's group 12 - 17 on the effect of estrogens on gonadotropin regulation. The augmented Prl plasma levels caused by epimestrol in hypogonadal women indicate a greater sensitivity of the hypothalamic response of this hormone in women than in men, in whom epimestrol at a dose of 15 mg/day for 10 days had no effect on plasma Prl levels. 6 The Prl response to this agent was in fact the same as that observed after estrogen therapy in hypogonadal 18 and postmenopausal women. 19 The failure of this drug to increase either basal gonadotropin levels or the gonadotropic response to releasing hormone in cases of anorexia nervosa is in accordance with the well-known impairment of hypothalamic centers in this clinical syndrome. 9 • 20 Similarly, the effects of therapy in the two cases of long-lasting progestin-induced amenorrhea indicate the existence of a hyothalamic disorder in this situation, chiefly affecting gonadotropin secretion. The reported effects of epimestrol in secondary amenorrhea and oligomenorrhea, together with the LH peak induced in the patient with precocious menopause and the increase in the maximal response to LH-RH of both gonadotropins in this patient, are in agreement with Yen's concept regarding the effect of estrogenic compounds in regulating the pituitary gonadotropins and hypothalamic LH-RH synthesis, storage, and secretion. 12- 17 The observation that the 5-day treatment with epimestrol does not provoke a rise in E 2 plasma levels supports the findings of Luisi et al. 6 that in males treated with this agent, plasma testosterone and E2 show a significant rise only after 7 to 10 days of therapy. In conclusion, the present data demonstrate the
660
GENAZZANI ET AL.
stimulatory effect of epimestrol on gonadotropin and Prl plasma levels and on the response to Lh-Rh in patients with secondary amenorrhea. They also suggest that, in these patients, the effect of this agent differs from that described for clomiphene citrate 8• 11 in that it acts as an estrogenic compound and not as an antiestrogenic agent. REFERENCES 1. Demol R, Vanderkerckove D, Vermijlen P, Lepoutre L: Induction de I'ovulation par l'epimestrol. Brux Med 12:653,
1975 2. Meckies J, Leo-Rossberg I, Felshart R, Moltz L, Hammerstein J: Behandlung Steriler Frauen mit Epimestrol. Erfahrungsbericht ueber 143 Patientinnen mit 30 Graviditaeten. Dtsch Med Wochenschr 47:1711, 1976 3. Schmidt-Elmendorff H, Kaemmerling R: Vergleichende Klinische Untersuchungen von Clomiphen, Cyclophenil und Epimestrol. Geburtshilfe Frauenheikd 37:531, 1977 4. Portuondo JA, Busturia MA, Esteban J, Roman MD, Riego AG: Pituitary, ovarian, and peripheral changes after epimestrol (Stimovul) therapy (abstr). Fertil Steril28:390, 1977 5. Melis GB, Paoletti A, Genazzani AR, Fioretti P: Effect of epimestrol on pituitary response to exogenous Gn/RH and its validity in the treatment of anovulatory cycles. Acta Endocrinol [Suppl) (Kbh) 212:137, 1977 6. Luisi M, Kicovic PM, Franchi F: Placebo-controlled study of effects of epimestrol on the pituitary-testicular axis in normal men. Reproduccion. In press, 1978 7. Genazzani AR, Magrini G, Facchinetti F, Romagnino S, Pintor C, Felber JP, Fioretti P: Behavior and origin of plasma androgens throughout the menstural cycle. In Androgens and Antiandrogens, Edited by L Martini, M Motta. New York, Raven Press, 1977, p 247 8. Wang CF, Yen SSC: Direct evidence of estrogen modulation of pituitary sensitivity to luteinizing hormonereleasing factor during the menstrual cycle. J Clin Invest 55:201, 1975
December 1978 9. Wentz AC, Jones GS, Sapp KC: Effect of clomiphene citrate on gonadotropin responses to LH-RH administration in secondary amenorrhea and oligomenorrhea. Obstet Gynecol 47:677, 1976 10. Dhont M, De Gezelle H, Vanderkerckove D: Modulation of pituitary responsiveness to exogenous LH-RH by an oestrogenic and an anti-oestrogenic compound in the normal male. Clin Endocrinol (Oxf) 5:175, 1976 11. Snoep MC, de Lange WE, Sluited WJ, Doorenbos H: Differential response of serum LH in hypogonadotropic hypogonadism and delayed puberty to LH-RH stimulation before and after clomiphene citrate administration. J Clin Endocrinol Metab 44:603, 1977 12. Yen SSC, Vandenberg G, SilerTM: Modulation of pituitary responsiveness to LRF by estrogen. J Clin Endocrinol Metab 39:170, 1974 13. Lasley BL, Wang CF, Yen SSC: The effect of estrogen and progesterone on the functional capacity of the gonadotrops. J Clin Endocrinol Metab 41:820, 1975 14. Yen SSC, Lasley BL, Wang CF, Leblanc H, Siler TM: The operating characteristic of the hypothalamic-pituitary system during the menstrual cycle and observation of biological action of somatostatin. Recent Prog Horm Res 31:321, 1975 15. Wang CF, Lasley BL, Lein A, Yen SSC: The functional changes of the pituitary gonadotrops during the menstrual cycle. J Clin Endocrinol Metab 42:718, 1976 16. Yen SSC, Lein A: The apparent paradox of the negative and positive feedback control system on gonadotropin secretion. Am J Obstet Gynecol 126:942, 1976 17. Hoff JD, Lasley BL, Wang CF, Yen SSC: The two pools of pituitary gonadotropin: regulation during the menstrual cycle. J Clin Endocrinol Metab 44:302, 1977 18. Yen SSC, Ebara Y, Siler TM: Augmentation of prolactin secretion by estrogen in hypogonadal women. J Clin Invest 53:652, 1974 19. Robyn C, Vekemans M: Influence oflow dose oestrogen on circulating prolactin, LH and FSH in postmenopausal women. Acta Endocrinol (Kbh) 83:9, 1976 20. Aro A, Lamberg BA, Pelkonen R: Hypothalamic endocrine dysfunction in anorexia nervosa. Acta Endocrinol (Kbh) 85:673, 1977
