The frequency of pulsatile luteinizing hormone-releasing hormone treatment and luteinizing hormone and follicle-stimulating hormone secretion in women with amenorrhea of suprapituitary origin*†

FERTILITY AND STERILITY

Vol. 51, No.3, March 1989

Copyright e 1989 The American Fertility Society

Printed in U.S.A.

The frequency of pulsatile luteinizing hormone-releasing hormone treatment and luteinizing hormone and folliclestimulating hormone secretion in women with amenorrhea of suprapituitary origin*t

Cornelis B. Lambalk, Ph.D.:j:§ II Joop Schoemaker, Ph.D.§ G. Peter van Rees, Ph.D.:j: Hannie A.M. J. van Dieten:j: University of Leiden, Leiden; Academic Hospital of the Vrije Universiteit, Amsterdam, The Netherlands

The influence of luteinizing hormone-releasing hormone (LH-RH) pulse frequency on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was studied in hypogonadotropic hypogonadal women. They received three regimens of 5 days of pulsatile LH-RH (5 !Lg/pulse) given at 30-, 90-, or 180-minute intervals, with at least 6 weeks between treatments. On day 1, LH and FSH increased in proportion to the LH-RH pulse frequency. After 5 days of treatment with the 30- and 90-minute intervals, LH was still elevated, but FSH had returned to pretreatment levels together with a decline of the FSH response. The LH response only declined during treatment with the 30-minute pulse interval. During each treatment, estradiol (E 2 ) increased. Explanations for dissociation between LH and FSH secretion during treatment with higher LH-RH pulse frequencies could be: (1) desensitization ofFSH rather than LH secretion on LH-RH; (2) a differential effect of E 2 on LH and FSH; (3) nonsteroidal ovarian factors selectively regulating LH and/or FSH release. Fertil Steril51:416, 1989

The pituitary gland needs to be stimulated in a pulsatile fashion by luteinizing hormone-releasing hormone (LH-RH) in order to maintain luteinizing hormone (LH) and follicle-stimulating hormone Received May 24, 1988; revised and accepted November 17, 1988. * Supported by a grant from the Foundation for Medical Research (MEDIGON), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). t Presented in part at the Sixth Reinier de Graaf Symposium, Nijmegen, The Netherlands, August 27 to 29, 1987. :j: Department of Pharmacology, Sylvius Laboratories, Uni· versity of Leiden. § Department of Obstetrics and Gynaecology, Division of Reproductive Endocrinology and Fertility, Academic Hospital of the Vrije Universiteit. II Reprint requests: Cornelis B. Lambalk, Ph.D., Department of Obstetrics and Gynaecology, Academic Hospital of the Vrije Universiteit, P.O. Box 7057, 1007MB Amsterdam, The Netherlands. 416

Lambalk et al.

LH-RH pulse rate and gonadotropin levels

(FSH) secretion. 1 Continuous LH-RH infusions desensitize the pituitary gland in releasing gonadotropins. 2•3 Although LH-RH controls the secretion of both gonadotropins, it has been demonstrated that the LH-RH pulse frequency influences LH and FSH secretion differentially. An LH-RH pulse given every hour maintains the release of LH and FSH in the ovariectomized hypothalamus-lesioned monkey. Faster frequencies such as three pulses per hour reduce the release of both gonadotropins in plasma, whereas slower frequencies such as one pulse per 3 hours cause a decline of LH and a paradoxical rise of FSH. 4 In men with elevated plasma FSH, the LH pulse frequency was lower than in men with normal FSH plasma levels, suggesting that a lower LH-RH frequency favors FSH secretion, provided that the pulsatile LH pattern is a full reflection of endogenous LH-RH secretion. 5 If the frequency of pulsatile LH-RH administration to Fertility and Sterility

Table 1

Subject

Clinical and Endocrine Data

Age

Percent ideal body weight

yrs

Initial LH level (MRC68/40)

Initial FSH level (MRC68/39)

Initial E 2 level

Ujl

Ujl

pmoljl

Type of amenorrhea

1 2

21 32

82 91

4.6 4.3

8.3 7.6

121 87

Primary Primary

3 4 5

30 22 24

76 93 118

3.3 1.8 1.8

4.8 3.0 4.8

117 46 57

Secondary Primary Primary

hypogonadotropic men is reduced, serum FSH levels increase. 6 Spratt et al. 7 observed a progressive rise of plasma LH and no changes in FSH when the frequency of pulsatile LH-RH treatment in hypogonadotropic men was increased. Pulsatile LH-RH administration is now widely used for the induction of ovulation. 8 •9 The development of follicles and ovulation depends on circulating levels of LH and FSH. If a differential response of both gonadotropins is LH-RH pulse frequency coded, then the frequency of pulsatile LH-RH treatment may be an important determinant of follicular development and ovulation rate. The aim of the present study was to establish whether in the human female, in the presence of intact gonads, the individual concentrations of LH and FSH could be changed by varying the frequency of stimulation with pulsatile LH-RH. Therefore, we investigated the influence of the frequency of pulsatile LH- RH treatment on LH and FSH secretion in women whose LH-RH secretion is virtually absent.

U/L

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Subjects

Vol. 51, No.3, March 1989

Weight-loss-related amenorrhea Idiopathic hypogonadotropic hypogonadism Weight-loss-related amenorrhea Kallmann's syndrome Idiopathic hypogonadotropic hypogonadism

taken every 10 minutes during a period of 6 hours. None of the subjects showed LH pulsatility (Fig. 1). We chose a fixed dose of 5 Jlg of LH-RH in order to compare our results with results of extensive pharmacokinetic studies that had been carried out with this do!'je. 10 A period of 5 days of treatment (from Monday through Friday) had been chosen based on practical and ethical considerations. The experiment was approved of by the subcommittee for ethics of research on human subjects of the hospital. Informed consent was obtained from all women. In the morning of the first day of each treatment,

MATERIALS AND METHODS

Five women with amenorrhea of suprapituitary origin participated in the study. Table 1 summarizes their clinical and some of their endocrine data. Mean plasma LH and FSH concentrations before treatment were 3.2 ±0.6 U/1 standard error of the mean (SEM) and 5.7 ± 1.0 U/1, respectively. The women were selected from the patient population of the Division of Reproductive Endocrinology and Fertility of our hospital. The criterion for selection was the virtual absence of pulsatile LH secretion. This is measured routinely in all amenorrheic patients by estimating LH in blood samples

Diagnosis

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Figure 1 Plasma patterns of five women with amenorrhea of suprapituitary origin as measured in blood samples taken every 10 minutes during a period of 6 hours. Lambalk et al.

LH-RH pulse rate and gonadotropin levels

417

a cannula (Abbocath, 20-gauge, Abbott Hospitals Inc., North Chicago, IL) was inserted into a convenient forearm vein. This cannula was used for frequent blood sampling and for pulsatile administration of LH-RH during the 5 days of treatment.

Plasma E 2 was measured in the first sample of each experimental day with the use of a commercially available RIA kit (lnstitut fiir Reaktorforschung, Wiirlingen, Switzerland). The intra-assay and interassay coefficients of variation were 4% and 11%, respectively.

Pulsatile LB-RH Treatment

In a randomly chosen order, each subject received three treatment regimens of 5 days with pulsatile LH-RH given by means of a portable infusiOii pump (Autosyringe, Travenol Laboratories, Hooksett, NH) with intervals of 30, 90, or 180 minutes. The LH-RH dose was 5 ~g/pulse (50 ~1). None of the subjects had been treated with any hormone for more than 1 year before this study. The minimum period between the different treatment periods was at least 6 weeks. Blood Sampling

On day 1 and day 5, blood was sampled every 10 minutes during 6.5 hours (from 8.30 A.M. until3.00 P.M.). These periods constituted the first and last periods of 6 hours of pulsatile LH-RH treatment, which was started at 9.00 A.M. on day 1 and ended at 3.00 P.M. on day 5. During the blood sampling periods, the pulses of LH-RH (5 ~gin 1 ml of saline) were given by hand via the cannula, in order to ensure proper timing of the LH-RH pulses. Blood was centrifuged, and plasma was separated and frozen at -2o·c until assayed.

Analysis of Data

The individual means of LH and FSH concentrations of the samples taken during the last 3 hours of each first and last day of each treatment period were calculated. Furthermore, plasma values of both hormones before the LH-RH injections at 12.00 A.M. on day 1 and day 5 of all treatment periods were used. As a measure for the LH and FSH responsiveness, the increment was taken that occurred 30 minutes after the LH-RH injections at 12.00 A.M. of each first and last day. Significance of differences between means was estimated with a two-way analysis of variance followed by the paired t-test. In the event of variance being proportional to the means, data were transformed logarithmically before making statistical calculations. Linear and log-linear coefficients of correlation (r) were calculated for the various parameters of gonadotropin secretion and plasma estradiol (E 2 ) concentrations during treatment. In the statistical tests, significance was considered to exist if one-sided P values were ~0.05. RESULTS

Hormone Measurements

Luteinizing hormone and FSH were measured in duplicate with the use of a double-antibody solidphase radioimmunoassay (RIA; Amerlex, Amersham, UK). Each subject's hormone concentrations of each treatment were measured in a single assay. Luteinizing hormone was expressed in U /1 MRC (Medical Research Counsil) 68/40, the lower limit of detection being 0.8 U /1. The intra-assay coefficient of variation, calculated as described previously, 11 ranged from 4.0% to 9.1% for values between 0.8 and 15.0 U /1 and from 4.4% to 6.0% for values between 15.1 and 30.0 U /1. At the level of 4.0 U /1 the interassay c9efficient of variation was 17.0%. Follicle-stimulating hormone was expressed in U /1 MRC 68/39, with a lower limit of detection of 1.0 U/1. The intra-assay coefficient of variation ranged from 2.8% to 8.8% for values between 1.0 and 15.0 U/1. The interassay coefficient of variation was 9.3%, calculated at the level of 5.0 U/1. 418

Lambalk et al.

LH-RH pulse rate and gonadotropin levels

LHandFSH

Figure 2 shows the mean plasma patterns of LH and FSH of the five subjects during the first and last 6 hours of the three periods of 5 days on which pulsatile LH-RH was given, each time with a different frequency. Repeated LH-RH injections of 5 ~g resulted in immediate secretion of both hormones after starting treatment on day 1, irrespective of the pulse frequency. On the last day oftreatment (day 5), secretion of both gonadotropins still occurred in response to every LH-RH injection if intervals of 180 and 90 minutes were used. During the first 6 hours of treatment on day 1, both gonadotropins increased in proportion to the frequency used. During the treatment with the 30-minute pulse interval, a continued rise was observed. This was reflected in a significantly higher mean plasma concentration of LH and FSH as compared with the 180-minute pulse interval treatment (Fig. 3). Fertility and Sterility

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Mean plasma FSH concentrations kept even more in pace with increasing pulse frequencies. On day 5, a dissociation between the LH and FSH secretion was observed. Plasma FSH concentrations were not related any more to the LH-RH pulse frequencies used and had all returned to the same low level as before treatment. In contrast, LH plasma levels on day 5 were still significantly higher during treatment with the highest frequency (Fig. 2). Mean plasma LH concentrations Vol. 51, No.3, March 1989

had not changed when compared with day 1 (Fig. 3). Follicle-stimulating hormone levels were not significantly increased any more during treatment with either frequency of LH- RH treatment. We also compared the plasma values of both gonadotropins in the samples taken before the LH-RH injections at 12.00 A.M. on each first and last treatment day. Changes in this parameter were about the same as that of the mean of the concentrations in the samples of the last 3 hours. A further differLambalk et al.

LH-RH pulse rate and gonadotropin levels

419

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Figure 3 Mean and SEM of some parameters for plasma LH (left panel) and FSH (right panel) of the five subjects on the first and last day of each treatment period with pulsatile LHRH. The interval between the 5 ILg LH-RH injections is stated at the bottom of each bar. (a) significantly different from values during treatment with other frequencies (two-way analysis of variance [ANOVA] and paired t-test); (b) significantly different from corresponding value on the first day of treatment (paired t-test); (c) significantly different from value during LH-RH treatment every 30 minutes (two-way ANOVA and paired ttest).

entiation between these "basal" concentrations of LH could be made during the treatment with the 90- and 180-minute intervals on day 5 (Fig. 3). The mean increments of LH and FSH to the injections at 12.00 A.M. also are given in Figure 3. On the first day of the three treatments, no differences were observed between the LH responses, nor between the FSH responses. After 5 days of treatment, the highest LH and FSH responses were observed during the treatment with the largest pulse interval of 180 minutes. Only the amplitudes of FSH during treatment with the 30- and 90-minute pulse interval significantly declined on day 5 as compared with those on day 1. Estradiol

Plasma E 2 increased slightly but significantly during each of the three treatments (Table 2). Sig420

Lambalk et al.

nificantly higher levels were reached during treatment with the LH-RH pulse intervals of 30 and 90 minutes. A negative linear correlation (r = -0.57) could be calculated between initial E 2 concentrations on day 1 and the increase of plasma E 2 during each treatment, regardless of the frequency. There was no relation between FSH values on day 5 before the LH-RH injection at 12.00 A.M. and changes in E 2 • Remarkably, the response of FSH on day 5 was negatively correlated in a log-linear way to both the plasma E 2 of this day (r = -0. 71; Fig. 4A) and the increase of plasma E 2 during each treatment (r = -0.45; Fig. 4B). In contrast, no such correlations could be made for LH.

LH-RH pulse rate and gonadotropin levels

All women reacted immediately and repeatedly to the Ll:f-RH injections given on day 1 by secreting both gonadotropins. When LH-RH was given at intervals of 90 or 180 minutes, not only was a clear pulsatile LH response observed, but this also was seen with FSH. Similar observations were made after 5 days of treatment. This indicates that pulsatile LH-RH can regulate pulsatile FSH secretion in women. The capability of pulsatile LH-RH to regulate pulsatile FSH secretion is often questioned. In women without functioning ovaries (postmenopause, gonadal dysgenesis) pulsatile FSH secretion often is observed to be asynchronous from LH pulses. Injections of 20 J.Lg LH-RH in these women resulted in pulses of LH but not of FSHP It could be that a clear pulsatile reaction to LH-RH can only be observed when gonads are present, as is the case with the subjects in the present study. While on the first day of each treatment with Table 2 Mean and SEM of Plasma E 2 of Five Subjects on the First and Last Day of Each Treatment with Pulsatile LH-RH MeanE2 andSEM (n = 5) Interval between LH-RH injections

Day1

Day5

min

pmol/1

pmol/1

180 90 30

83± 17 89± 18 86± 15

147 ± 12a.b 246 ± 68b 250± 49b

• Significantly different compared with values during treatment with other LH-RH pulse frequencies. b Significantly different from corresponding value on the first day of the same treatment. Fertility and Sterility

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Figure 4 The log-linear correlation between the response of plasma FSH to the LH-RH injection at 12.00 A.M. on the last day (day 5) of each treatment period with pulsatile LH-RH (n = 15) versus plasma estradiol concentrations on this day (A) and versus the change of E 2 during each treatment(B).

pulsatile LH-RH, LH and FSH reacted quite similarly, a differential response was observed after 5 days of treatment with different frequencies. This was characterized by still elevated secretion of LH and a return of FSH to pretreatment levels during treatment with LH-RH pulse intervals of 30 and 90 minutes. Other investigators have reported an initial rise of FSH followed by a decline during prolonged pulsatile LH-RH treatment for the induction of puberty. 13 Our observations are also in agreement with those of Spratt et al. 7 For this differential reaction of LH and FSH, some explanations can be offered. Firstly, although LH-RH was given intermittently, some degree of desensitization may have occurred that affected the secretion ofFSH earlier or more strongly than that of LH. Indeed, the FSH responsiveness to LHRH was diminished considerably after 5 days of treatment with the 30- and 90-minute pulse intervals. Conversely, the LH response was only diminished after the 30-minute pulse interval treatment compared with after treatment with the 180-minute pulse interval. We have shown previously that pulsatile LHRH, given to postmenopausal women, is able to induce a state of partial desensitization of both LH and FSH. 12 Others have shown that continuous infusion of LH-RH to postmenopausal women results in a more rapid and stronger desensitization of FSH than of LH. 14 A similar observation was made if continuous LH-RH was administered to ovariectomized rats. 2 It could be that more frequent LH-RH pulses result in a higher grade of desensitization just because more LH-RH is administered and thus, due to accumulation, a more or less continuous stimulation is approached. Handelsman and Swerdloff10 showed that intravenous injections of 5p,g LH-RH lead to accumulation if the Vol. 51, No.3, March 1989

interval between injections is shorter than 45 minutes. Accumulation would therefore only explain the desensitization during the 30-minute pulse interval regimen and not the desensitization during treatment with the 90-minute pulse interval. Analternative explanation for desensitization would be that shorter pulse intervals between LH-RH injections may not allow the pituitary gland to fully recover from the short-term refractoriness occurring after each single LH-RH injection. This phenomenon has been shown to exist in women with amenorrhea of suprapituitary origin. 15 A second explanation for the differential secretion of LH and FSH could be that gonadal factors exert a differential feedback on both gonadotropins. Estradiol may preferentially suppress the FSH response to LH-RH. 16•17 Indeed, in our experiments, plasma E 2 increased more during treatment with 90- and 30-minute pulse intervals. Moreover, a significant negative correlation was observed between the FSH responsiveness on day 5 and the rise of E 2 during treatment, as well as the E 2 plasma levels on day 5. No such correlation could be made for LH. Nevertheless, these observations have to be interpreted with care. Estradiol may not be the only factor involved. We have to consider another part of gonadal feedback that is nonsteroidal. Already developing follicles may have started to produce inhibin, a glycoprotein that preferentially inhibits the secretion ofFSH. However, in vitro experiments have shown that inhibin-containing follicular fluid mainly attenuates LH-RH-independent release ofFSH and still enables LH-RH -stimulated release of FSH. 18 The observation that lower LH-RH pulse frequencies increase plasma FSH levels was not confirmed in the present experiments.4 •6 We conclude that in women with amenorrhea of Lambalk et al.

LH-RH pulse rate and gonadotropin levels

421

suprapituitary origin, a differential response of LH and FSH to different frequencies of pulsatile LHRH treatment can be observed. This concerns sustained elevation of LH and a return of FSH to pretreatment levels during treatment with higher LHRH pulse frequencies, rather than an increase of FSH during treatment with lower frequencies. In the human female, regulatory mechanisms are apparently rapidly activated to keep plasma FSH levels within narrow limits. The selective return ofFSH to pretreatment levels resulted from low responsiveness to LH-RH. This may have been caused by the pulsatile LHRH treatment and/or initiated ovarian feedback.

Acknowledgments. We thank Mr. Fedde Scheele and Mrs. Ted J. M. De Vries Robles-Korsen for their skillful assistance in conducting the experiments and Misses Ellen M. Heidema and Rolien H. F. de Ru for their help in preparing the manu· script.

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REFERENCES 1. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E: Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631, 1978 2. Lambalk CB, Van Rees GP, Schoemaker J, De Koning J, Van Dieten JAMJ: The influence of bovine Follicular Fluid (bFF), phenobarbital (PB) and continuous LHRH on pulsatile FSH and LH in the ovariectomized (OVX) rat. In Lambalk CB: Pulsatile LHRH and Gonadotropin Secretion, Thesis, Leiden, The Netherlands, 1988, p 125 3. Schuiling GA, De Koning J, Zurcher AF, Gnodde HP, Van Rees GP: Induction of LH surges by continuous infusion of LHRH. Neuroendocrinology 20:151, 1976 4. Wildt L, Hausler A, Marshall G, Hutchison JS,Plant TM, Belchetz PR, Knobil E: Frequency and amplitude of gonadotropin-releasing hormone stimulation and gonadotropin secretion in the Rhesus monkey. Endocrinology 109:376, 1981 5. Gross KM, Matsumoto AM, Southworth MB, Bremner W J: Evidence for decreased luteinizing hormone-releasing hormone pulse frequency in men with selective elevations of follicle-stimulating hormone. J Clin Endocrinol Metab 60:197, 1985 6. Gross KM, Matsutomo AM, Bremner W J: Differential control of luteinizing hormone and follicle-stimulating hormone secretion by luteinizing hormone-releasing hormone

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18.

pulse frequency in man. J Clin Endocrinol Metab 64:675, 1987 Spratt DI, Finkelstein JS, Butler JP, Badger TM, Crowley WF Jr: Effects of increasing the frequency of low doses of gonadotropin-releasing hormone (GnRH) on gonadotropin secretion in GnRH -deficient men. J Clin Endocrinol Me tab 64:1179, 1987 Crowley WF Jr, Filicori M, Spratt DI, Santoro NF: The physiology of gonadotropin-releasing hormone (GnRH) secretion in men and women. Recent Prog Horm Res 41:473, 1985 Schoemaker J, Van Kessel H, Simons AHA, Korsen TJM: Induction of first cycles in primary hypothalamic amenorrhea with pulsatile luteinizing hormone-releasing hormone: a mirror of female puberal development. Fertil Steril 48: 2041, 1987 Handelsman DJ, Swerdloff RS: Pharmacokinetics of gonadotropin-releasing hormone and its analogs. Endocr Rev 7:95,1986 Lambalk CB, De Koning J, Van Kessel H, Van Rees GP, Schoemaker J: Calculation of intra-assay variations per assay and its relevance to LH pulse detection. IRCS Med Sci 13:1183, 1985 Lambalk CB, Van Kessel H, De Koning J, Van Dieten JAMJ, V!m Rees GP, Schoemaker J: Pulsatile LHRH treatment alters pituitary sensitivity for LHRH and the spontaneous LH pulse frequency in women without ovarian activity. In Research on Gynecological Endocrinology, Edited by AR Genazzani. Carnford, England, Parthenon Publishing, 1986, p 393 Delemarre-van de W aal HA: Reflections on pulse interval in GnRH induction of male puberty. In Pulsatile GnRH, Edited by HJT Coelingh Bennink, AA Dogterom, RE Lappohn, R Rolland, J Schoemaker. Haarlem, The Netherlands, Ferring Publication, 1986, p 235 Heber D, Swerdloff RS: Down-regulation of pituitary gonadotropin secretion in postmenopausal females by continuous gonadotropin-releasing hormone administration. J Clin Endocrinol Metab 52:171, 1981 Lambalk CB, Schoemaker J, Van Rees GP, Van Kessel H, De Koning J, Van Dieten JAMJ, De Vries Robles-Korsen TJM: Short-term pituitary desensitization to luteinizing hormone-releasing hormone (LH-RH) after pulsatile LHRH administration in women with amenorrhea of suprapituitary origin. Fertil Steril47:385, 1987 Marshall JC, Case GD, Valk TW, Corley KP, Sauder SE, Kelch RP: Selective inhibition of follicle-stimulating hormone secretion by estradiol. J Clin Invest 71:248, 1983 Yen SSC, Vandenberg G, Siler TM: Modulation of pituitary responsiveness to LRF by estrogen. J Clin Endocrinol Metab 39:170, 1974 Jenner AAJ, De Koning J, Van Rees GP: Effect ofinhibinlike activity on LH-RH-stimulated release of FSH by pituitary glands from female rats in vitro. Life Sci 32:1091, 1983

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