The Induction of Ovulation in Amenorrheic Patients With Synthetic Luteinizing Hormone-Releasing Hormone: The Significance of Pituitary Responsiveness

FERTILITY AND STERILITY Copyright ~ 1976 The American Fertility Society

Vol. 27, No.1, January 1976 Printed in U.S.A.

THE INDUCTION OF OVULATION IN AMENORRHEIC PATIENTS WITH SYNTHETIC LUTEINIZING HORMONE·RELEASING HORMONE: THE SIGNIFICANCE OF PITUITARY RESPONSIVENESS KO-EN HUANG, M.D.

Department of Obstetrics and Gynecology, College ofMedicine and the Hospital, National Taiwan University, Taipei, Taiwan, Republic of China

Since the structure of porcine luteinizing hormone-releasing hormone (LH-RH) was identified and subsequently synthesized by Matsuo and associates,1, 2 several investigations have been made of pituitary response in normap,4 and amenorrheic5 ,6 women. Synthetic LH-RH had also been used for the treatment of infertility in patients with the anovulatory syndrome. 7-9 The purpose of this study was to evaluate the pituitary response following the administration of synthetic LH-RH and its significance in classifying patients with secondary amenorrhea in whom induction of ovulation is attempted. MATERIALS AND METHODS

Patients. Thirty-two patients who consulted the Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, were studied. They were 22 to 35 years old and had had secondary amenorrhea for 6 to 24 months. All patients were examined thoroughly to exclude pathology of the sella turcica or any endocrine disorders other than anovulation. Hormonal therapy and all other medication were avoided for at least 3 months. Those patients who had markedly high basal level of gonadotropins (more than 45 mIU/ml of serum folliclestimulating hormone (FSH) and/or 70 mIU/ml of serum LH in our laboratory) or those in whom 50 mg of progesterone Received March 26, 1975.

(Proluton; Schering Corporation, Kenilworth, N. J.) could not induce withdrawal bleeding were excluded from this study. All patients were studied for their pituitary response to 25 p.,g of synthetic LH-RH. Previous clomiphene administration to 26 patients (up to 150 mg/day for 5 days) had failed to induce ovulation, yet the pituitary had shown some response to LH-RH; 72 cycles of these women were again treated with clomiphene and subsequent injection of LH-RH in order to induce ovulation. Controls. The responses of five normal, cycling women, ages 23 to 31, to synthetic LH-RH during the late follicular phase (average, 3 days before ovulation) and luteal phase (4 days after ovulation) were studied. The basal body temperatures (BBT) of all of these subjects showed biphasic ovulatory patterns. Five postmenopausal women, ages 51 to 57, who had been admitted to this department because of other conditions and confirmed to be free of endocrine and metabolic diseases, were also studied. ' The duration of amenorrhea in these subjects ranged from 4 to 7 years. LH-RH Test. The LH-RH test was performed on nonfasting ambulant subjects; synthetic LH-RH, generously supplied by Farbwerke Hoechst AG, Frankfurt (Main) as 25-p.,g dry powder ampules, and its solvent were used. One ampule of this preparation was given as a bolus intravenous injection in this study. Blood samples were obtained through an in65

66

January 1976

HUANG

dwelling catheter immediately prior to the test and 5, 15, 30, 60, and 120 minutes after the injection. Serum was collected and stored at - 20° C until assayed. Serum hormone levels of each subject were measured in duplicate in the same assay. Radioimmunoassay. Serum FSH and LH levels were measured by radioimmunoassay with modified double-antibody methods10 for FSHll and LH,12 using Second International Reference Preparation Human Menopausal Gonadotropin (2nd IRP-HMG) as standard reference. The mean immunologic potency of human pituitary standard, LER 907, was 218.0 mIU/J,Lg for LH and 39.0 mIU/J,Lg for FSH in our radioimmunoassay system. The 2nd IRP-HMG and highly purified FSH, LH, antisera, and LER 907 were kindly provided by the National Institute for Medical Research, London, and the N ational Institute of Arthritis, Metabolism and Digestive Diseases, respectively. Induction of Ovulation with LH-RH. Since the results of our previous study9 showed that the route of administration did not significantly affect the number of Ul mJlJ/1II1 300 Ul-RH

~\

1 /l \

F

0----0

1 't,~, ,

,

L

D---.g

P

C5

Mean + S.E.

n' 5

FSH mI1l/ml

~,

I

6----6

>~

200

t

r 100

__ lI'_'

J>--_~

....... ..A-Minutes

HI's f/,

------a

-&- - - - - - -

gh

1;0

Minutes

FIG, 1. Responses (mean ± standard error) ofLH and FSH to intravenous injection of 25 f.Lg of synthetic LH-RH in later follicular phase (ji') and luteal phase CL) of normally cycling women and postmenopausal women <1'),

ovulatory cycles induced, LH-RH was administered either in two intramuscular injections or by intravenous infusion in this study. The initial dose was 100 J,Lg. This dose was doubled whenever 2 months' consecutive treatment failed to induce ovulation. The LH-RH, in a 4-hour 100or 200-J,Lg intravenous infusion or in two 100- or 200-J,Lg intramuscular injections with a 4-hour interval, was given on the 6th day after clomiphene (100 mg/day for 5 days) administration. Ovulation was evaluated with BBT and endometrial biopsies. Ovulation was presumed to have occurred in cycles which showed biphasic BBT, with temperature elevations of more than 0.4° C for 11 days or more, and secretory patterns of endometrium, according to the criteria of Noyes et a1.,13 unless pregnancy occurred. RESULTS

The responses of serum LH and FSH levels following the intravenous infusion of synthetic LH-RH during follicular and luteal phases in five normally ovulating and five postmenopausal women were studied. The results (mean ± standard error) are shown in Figure 1. The basal LH and FSH levels of postmenopausal women were significantly (J> < 0.001) higher than those of normal women. The serum LH levels were elevated at 5 minutes, reached a peak at 30 minutes, then declined but were still elevated 120 minutes after the injection of LH-RH. The serum FSH levels showed a gradual rise at 5 minutes, reached the maximal level at 30 minutes during the normal follicular phase and at 60 minutes during the luteal phase and in postmenopausal women, and remained high until 120 minutes after the injection. The LH response to LH-RH was highest in the normal follicular phase (10.1 ± 1.7), lower in the normal luteal phase (6.3 ± 0.6), and lowest in postmenopausal women (2.3 ± 0.1) when expressed as the ratio

Vol. 27, No.1

LH-RH INDUCTION OF OVULATION: PITUITARY RESPONSIVENESS

67

TABLE 1. Serum LH Response to LH-RH in Normal Women, Postmenopausal Women, and Patients with Secondary A menorrhea a Subjects

No.

Basal level (B)

Stimulated peak value (S)

SoB ratio

to LH(S - B) mIUlml

mIU/ml

Normal Follicular phase Luteal phase Postmenopausal Secondary amenorrhea High response Moderate response Lpw response Nor~nse

5 5 5 14 11 5 2

9.7 ± 1.1 9.0 ± 1.0 140.2 ± 13.9 23.6 ± 13.2 ± 19.9 ± 7.0

3.7 1.3 2.5

91.2 ± 6.8 55.4 ± 4.5 315.4 ± 27.8

10.1 ± 1.7 6.3 ± 0.6 2.3 ± 0.1

81.4 ± 7.8 46.4 ± 3.9 175.2 ± 16.9

261.6 ± 26.1 121.5 ± 13.4 54.0 ± 11.4

14.0 ± 2.8 6.3 ± 0.2 4.2 ± 0.1

238.0 ± 24.8 101.7 ± 8.7 38.8 ± 8.7

aValJ.es are means ± standard error.

of the stimulated peak and the basal level (Table 1). This tendency was also noticed in serum. FSH response to LH-RH (Table 2). An obvious individual difference was no~iced in the serum LH response to the administration of syntheticLH-RH in patients with secondary amenorrhea. As shown in Figures 2 to 4, the basal levels of LH and FSH in patients with secondary amenorrhea were comparable to, or slightly higher than, those of normal subjects. The patterns of response to LH-RH were similar to those of normal women. However, the magnitude of response, particularly that of LH, differed markedly in each individual. Of the 32 patients, the serum LH responses of 14 were as high or higher than that of the normal follicular phase when expressed as the ratio of the stimulated peak level to the basal level. Eleven patients had responses comparable to that of the nor-

mal luteal phase, and five of these had responses between that of the normal luteal phase and that of postmenopausal women (Fig. 5; Table 1). In 2 of the 32 patients, the pituitary failed to respond to LH-RH (Fig. 4). The mean serum FSH levels in the amenorrheic patients in response to LH-RH were lower than those of normal subjects and higher than those of postmenopausal women. The FSH response seemed more delayed and prolonged in amenorrheic patients than in normal subjects (Fig. 5; Table 2). As shown in Table 3, among 26 infertile amenorrheic patients who were treated with clomiphene and subsequent injection of LH-RH for induction of ovulation, 12 had high, 10 moderate, and 4 low responses of serum LH to the test dose of synthetic LH-RH. Ovulation was induced in 10 of 12 patients (25 of 35 cycles) with high response, 6 of 10 patients (12 of 28 cycles) with moderate response,

TABLE 2. Serum FSH Response to LH-RH in Normal Women, Postmenopausal Women, and Patients with Secondary Amenorrhea" Subjects

No.

Basal level (B)

Stimulated peak value (S)

S:B ratio

5 5 5 14 11 5 2

11.5 ± 5.3 8.0 ± 0.9 116.8 ± 13.2 13.2 ± 14.0 ± 12.0 ± 14.0

aValues are means ± standard error.

1.3 1.5 2.5

FSH (S - B) mIUlml

mIUlml

Normal Follicular phase . Luteal phase Postmenopausal Secondary amenorrhea High response Moderate response Low response No response

~

33.6 ± 5.1 22.0 ± 3.1 199.8 ± 23.1 30.7 ± 31.9 ± 24.0 ±

2.4 2.9 3.7

3.1 ± 0.4 2.8 ± 0.1 1.7 ± 0.2

22.8 ± 3.5 14.0 ± 2.9 83.0 ± 19.0

2.5 ± 0.2 2.3 ± 0.1 2.2 ± 0.4

18.0 ± 18.0 ± 12.0 ±

1.8 1.6 3.6

January 1976

HUANG

68 UI

UI

1IIlU/1Il

mITJ/ml

200

4110

Ul-RH

1 300

100 200

' !

15

30

,

60 Minutes

PSI!

iftIu/ 50

FSI! mIU/ml

M« Minutes

o

5

15

30

60 Minutes

120

FIG. 2. Individual responses of LH and FSH to intravenous injection of25ILg of synthetic LH-RH in amenorrheic patients with high response.

and 1 of 4 patients (1 of 13 cycles) with low response. Five pregnancies occurred. No side effects of any kind were observed in either the patients or the two babies born to them. DISCUSSION

It is generally considered that in most patients with secondary amenorrhea the disturbance is located in the hypothalamus rather than in the pituitary. The results of this study are consistent with this point of view and, at the same time, are in agreement with other investigations4 , 5, 14 in that individual differences in the magnitude of the LH response to exogenous LH-RH are marked in these patients. In this study, 32 patients with secondary amenorrhea were divided into four

120

FIG. 3. Individual responses of LH and FSH to intravenous injection of 25 ILg of synthetic LH-RH in amenorrheic patients with moderate response.

groups according to their serum LH response to intravenous injection of 25 ILg of synthetic LH-RH. The patients whose stimulated LH peaks (expressed as a percentage of the control) were within the range of (mean ± 2 SE), or higher than, that of the late follicular phase of normal subjects were classified as "high responders" (750% and above); those within the range of that of the luteal phase of normal subjects as "moderate responders" (500 to 740%); those lower than that of the luteal phase but higher than that of postmenopausal women as "low responders" (250 to 490%); and those within the range of, or lower than, that of postmenopausal women as having "no response" (lower than 240%). By this classification, the most beneficial outcome was obtained in the patients with high response. The incidence of ovulation in patients in this group was not signifi-

Vol. 27, No.1

69

LH-RH INDUCTION OF OVULATION: PITUITARY RESPONSIVENESS

IJI IIlU/ml

IJI

Percent of control

100

_ _ Am.11

1000

1JI-R11

_ _ Am.'!

UI-RH 800

._Am.L

1

~-~F l>r--~L

600

400

200

--- ..

.

100

120

o

5

15

30

120

60 Minutes

FSI! mIU/

FSH

Percent of control

SO

300

"~~ _-~~,,::o,~ -;._.... "

o

5

... ,

15

30

60

120

Minutes

Minutes

FIG. 4. Individual responses of LH and FSH to intravenous injection of 25 f,Lg of synthetic LH-RH in amenorrheic patients with low response (--) and no response (---).

cantly different from that in patients with moderate response, but was significantly

FIG. 5. Effect ofLH-RH on serum levels ofLH and FSH, expressed as percentage of control level, in amenorrheic patients with high response fA,m.H.) (n = 14), moderate response fA,m.M) (n = 11), and low response fA,m.L) (n = 5) in late follicular phase (j') and luteal phase fL) of nonnal women (n = 5) and in postmenopausal women (}') (n = 5).

more frequently than in patients with low response. Thus the number of ovulating patients was not significantly different, but the difference in the number of ovulatory cycles was statistically significant

TABLE 3. Result of Ovulation Induction with Clomiphene and Subsequent Injection of LH-RH in Patients with Secondary Amenorrhea Ovulation

Treatment Serum LH re~n.. to LH-RH in amenorr eic patients

No. of

No. of

patients

cycles

No. of patients

High response (S:B 7,5 and above) Moderate response (~:B 5.0-7.4) Low response (S:B 2.5-4.9)

12 10 4

35 28 13

10 6 1

60.0"

Total

26

76

17

"Not significant. bp < 0.05 between high and moderate or low response (student's t-test). cp < 0.001 between high and low response. dp < 0.05 between moderate and low response.

%

No. of cycles

%

25.0"' b

25 12 1

71.4b. c 42.9b. d 7.7c. d

65.4

38

50.0

83.3"' b

HUANG

70

These results suggest that the pituitary reserve which is tested by the intravenous injection of synthetic LH-RH (25 f.Lg) may predict the outcome with such therapy. A higher incidence of ovulation may be expected in patients with higher response to the LH-RH test. Theoretically, this may also be true for the induction of ovulation with clomiphene in patients with anovulatory infertiIlty, since it is already known that clomiphene acts as an ovulation inducer by competing with endogenous estrogen at the level of hypothalamic-pituitary area to increase the release of gonadotropins. The pituitary reserve, therefore, plays an important role in the treatment of anovulation. It is believed that the variation in ovarian secretion of estrogen may cause the alteration in the pituitary response to LH-RH.3,4 Thus, those amenorrheic patients with an apparent high response to LH-RH may be considered to have more active ovarian estrogen secretion as well as a more abundant pituitary reserve than those with a low response. This may, simultaneously, indicate easier induction of ovulation in patients with high or moderate response to LH-RH than those with low or no response to LH-RH. The mean basal levels of serum gonadotropins, which were noted to be higher than those of normal subjects in this group of amenorrheic patients, suggest that some of them were associated with a mild degree of ovarian impairment. One of the patients with a high response, whose basal serum LH level was as high as 32 mIU/ml, ovulated by the LH-RH test and conceived after treatment. This fact may indicate the feasibility of inducing ovulation in patients with a mild degree of ovarian impairment. However, the limit of ovarian impairment for which such therapy may be effective requires further elucidation. SUMMARY

The pituitary reserve of gonadotropins

January 1976

was evaluated with intravenous injections of 25 f.Lg of synthetic luteinizing hormone-releasing hormone (LH-RH) in 32 patients with secondary amenorrhea. An obvious individual difference in the magnitude of the serum LH response was noted in these patients. Thus the patients were classified into four groups according to their pituitary responsiveness as expressed by the ratio of the serum LH stimulated peak to the basal level. Patients with values within the range of, or higher than, that of the normal follicular phase were classified as having high response (750% and above); within the range of that of the normal luteal phase as having moderate response (500 to 740%); between that of the normal luteal phase and that of postmenopausal women as having low response (250 to 490%); and within the range of, or lower than, that of postmenopausal women as having no response (240% and below). Among 26 clomiphene-failed patients in this series who were treated again with clomiphene and subsequent injection of LH-RH, 10 of 12 patients (25 of 35 cycles) with high response, 6 of 10 patients (12 of 28 cycles) with moderate response, and 1 of 4 patients (1 of 13 cycles) with low response ovulated. Five patients became pregnant. The results of this study indicate that in amenorrheic women the higher the pituitary response to LHRH the greater the chance of inducing ovulation. Patients with secondary amenorrhea may thus be classified by assessing their pituitary response to LH-RH, which may be useful in predicting the chance for the successful induction of ovulation. Acknowledgments. The author wishes to acknowledge the National Institute for Medical Research, London, and the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Md., for the generous supply of 2nd IRP-HMG and human pituitary gonadotropins, antisera, and LER-907, respectively. Synthetic LH-RH was kindly supplied by Farbwerke Hoechst AG, Frankfurt (Main)

Vol. 27, No.1

LH-RH INDUCTION OF OVULATION: PITUITARY RESPONSIVENESS

through Teh Hua Chemical & Parmaceutical Co., Ltd., Taiwan. I am grateful to Professor Ping-Yen Wei of this department for his encouragement and advice in this study. REFERENCES 1. Matsuo H, Baba Y, Nair RMG, Arimura A,

2.

3.

4.

5.

Schally AV: Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun 43:1334, 1971 Matsuo H, Arimura A, Nair RMG, Schally AV: The synthesis of porcine LH- and FSH-releasing hormone by solid phase method. Biochem Biophys Res Commun 45:654, 1971 Yen sse, Vandenberg G, Rebar R, Ebara Y: Variation of pituitary responsiveness to synthetic LRF during different phases of the menstrual cycle. J Clin Endocrinol Metab 35:931, 1972 Nillius SJ, Wide L: Variation in LH and FSH response to LH-releasing hormone during menstrual cycle. J Obstet Gynaecol Br Commonw 79:865, 1972 Yen sse, Rebar R, Vandenberg G, Judd H: Hypothalamic amenorrhea and hypogonadism: response to synthetic LRF. J Clin Endocrinol Metab 36:811, 1973

71

6. Nillius SJ, Wide L: The LH-releasing hormone test in 31 women with secondary amenorrhea. J Obstet Gynaecol Br Commonw 79:874, 1972 7. Keller PJ: Treatment of anovulation with synthetic luteinizing hormone-releasing hormone. Am J Obstet Gynecol 116:696, 1973 8. zarate A, Canales ES, Schally AV, AyalaValdes L, Kastin AJ: Successful induction of ovulation with synthetic luteinizing hormonereleasing hormone in anovulatory sterility. Fertil Steril 23:672, 1972 9. Huang K-E: Use of synthetic luteinizing hormone-releasing hormone in induction of ovulation in amenorrheic patients. Fertil Steril 26: 796, 1975 10. Seki K: Serum follicle-stimulating hormone and luteinizing hormone in women in various endocrinological states. Acta Obstet Gynaecol Jap 19:25,1972 11. Midgley AR Jr: Radioimmunoassay for human follicle-stimulating hormone. J Clin Endocrinol Metab 27:295, 1967 12. Midgley AR Jr: Radioimmunoassay: a method for human chorionic gonadotropin and luteinizing hormone. Endocrinology 79:10,1966 13. Noyes RW, Hertig AT, Rock J: Dating of the endometrial biopsy. Fertil Steril1:3, 1950 14. Aono T, Minagawa J, Kinugasa T, Miyake A, Kurachi K: The diagnostic signifICance of LHreleasing hormone test in patients with amenorrhea. Am J Obstet Gynecol119:740, 1974