FERTILITY AND STERILITY Copyright < 1976 The American Fertility Society Vol. 27, No.6, June 1976 Printed in V.SA. LUTEINIZING HORMONE-RELEASING HORMONE FOR INDUCTION OF FOLLICULAR MATURATION AND OVULATION IN WOMEN WITH INFERTILITY AND AMENORRHEA* SIMON R. HENDERSON, M.A., B.M., B.CR., M.R.C.O.G.,t JOHN BONNAR, M.D., F.R.C.O.G.J ANDREW MOORE, B.A., AND PAMELA C.B. MACKINNON, M.A., PH.D., M.B., B.S. Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, and Departments of Clinical Biochemistry and Human Anatomy, Oxford, England Five patients with primary infertility and secondary amenorrhea who did not respond to clomiphene with a gonadotropin or estrogen surge were treated with 500 WI of luteinizing hormone, follicle-stimulating hormone-releasing hormone (LH-RH), self-administered subcutaneously every 8 hours for 14 days. Of four patients who responded to this treatment, three showed follicular maturation, ovulation, and menses, although the luteal phase was deficient; in the fourth patient, follicular maturation and menses occurred without evidence of ovulation. For their second course of treatment these four patients were given LH-RH with the addition of human chorionic gonadotropin when the urinary estrogen levels indicated follicular maturation. All four patients responded with ovulation, an adequate luteal phase, and menses, without clinical indication of ovarian hyperstimulation. These results suggest that LH-RH may be a better alternative to human menopausal gonadotropin in the treatment of anovulatory infertility. Earlier reports of the use of luteinizing hormone, follicle-stimulating hormonereleasing hormone (LH-RH) in low doses to induce ovulation in anovulatory women have indicated limited success. I 3 However, there have been recent reports of follicular maturation, ovulation, and pregnancy in amenorrheic patients with anorexia nervosa following long-term treatment with LH-RH with or without the added stimulus of human chorionic gonadotropin (HCG).4, 5 The aim of this study was to examine the use of LH-RH alone and in comb i- Accepted December 24, 1975. *Supported in part by Grant G 973/809/C from the Medical Research Council (to P. C. B. M.). tmedical Research Council Senior Clinical Research Fellow, Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford. :j:present address: Rotunda Hospital, Dublin, Ireland. 621 nation with HCG for stimulating follicular maturation, ovulation, and normal luteal function in otherwise healthy but amenorrheic patients who complained of infertility. PATIENTS AND METHODS Five involuntarily infertile women, ages 23 to 31, with amenorrhea of 2 to 7 years' duration were examined thoroughly for gynecologic, endocrinologic, and neurologic disease. X-ray examinations of the skull and pituitary fossa were normal. Serum thyroxine and triiodothyronine levels were normal, as were 24-hour urinary 17 -hydroxycorticosteroid and 17-ketosteroid determinations. Basal urinary estrogen and plasma 17.B-estradiol levels were low, and serum prolactin levels were normal (range, 10 to 24 ng/md. After investigation, all five patients were given at
622 HENDERSON ET AL. June 1976 least one course of clomiphene (200 mg daily for 5 days), which failed to result in a significant elevation of plasma luteinizing hormone (LH) or folliclestimulating hormone (FSH) levels or a subsequent rise in basal body temperature (BBT), total urinary estrogens, or plasma 17,8-estradiol. Hormone Assays. LH and FSH were estimated by a double-antibody radioimmunoassay. Details of standards and quality controls used in this assay system have been reported previously.6 Plasma 17,8-estradiol and progesterone levels were also measured by radioimmunoassay using specific antisera. 7 Total urinary estrogens were measured, after extraction, by an automated spectrophotometric technique 8 ; the values just prior to ovulation ran~ed between 86 and 126 JLg/24 hours. With this method urinary estrogen values were generally 30 to 40% higher than those obtained with another technique. 9 LH-RH Regimen. The LH-RH test was performed on each patient following the diagnostic/therapeutic trial of clomiphene. An intravenous dose of 50 JLg of LH-RH (Hoechst Pharmaceuticals, Hounslow, Middlesex, United Kingdom) was given, and blood samples were obtained prior to injection and at various intervals thereafter. 10 The maximal plasma LH and FSH responses were calculated by determining the difference between the maximal postinjection and the mean preinjection concentrations of LH and FSH. The first therapeutic regimen consisted of 500 JLg of LH-RH every 8 hours, self-administered subcutaneously into a skin fold below the umbilicus. In the second course of treatment, HCG (5000 IU) was given intramuscularly after urinary estrogen levels had increased (> 100 JLg/24 hours) for 2 days. Additional 3000-IU doses of HCG were then given twice weekly to maintain the luteal phase. During both regimens, plasma was assayed twice weekly for LH, FSH, 17,8-estradiol, and progesterone content, and total urinary estrogens were determined on almost a daily basis during LH-RH treatment. RESULTS Treatment. Height, weight, mean basal LH and FSH levels, and LH and FSH responses at the time of the LH-RH test are shown in Table 1. All patients responded to LH-RH with a marked rise in plasma LH levels and with a smaller but significant rise in FSH. In two patients (patients 2 and 5) the basal plasma FSH values were higher than the LH levels, and the FSH increases were larger than those of the other patients. Results of the first course of treatment are shown in Table 2 and summarized in Table 3. Figure 1 (patient 1) shows the typical hormone and BBT patterns that occurred in three patients. The short duration of BBT elevation, prematurely low or undetectable levels of progesterone, and early onset of menses indicate TABLE 1. Plasma Gonadotropin Levels before and after an Intravenous Injection of 50 p.g of LH-RH Patient Height Weight LH FSH Basal Response" Basal Response" em kg miulml 1 158 48.5 9 51 7 8 2 165 55.8 4 38 9 11 3 155 50.6 11 49 3 3 4 168 53.6 15 155 10 9 5 163 49.0 3 32 7 19 "Maximal postinjection concentration minus basal concentration.
Vol. 27, No.6 LH-RH'INDUCED FOLLICULAR MATURATION AND OVULATION 623 TABLE 2. Induction of Follicular Maturation and Ovulation with LH-RH Peak urinary estrogen Day of Plasma progesterone" Duration of Patient Course temperature temperature Day of cycle Concentration rise First Second rise p.g124 hr nglml nglml days 1 1 10 235 12 15.5 <1.0 10 2 13 215 14 25.4 29.3 16 2 1 12 140 1.0 <1.0 2 13 110 14 21.8 18.6 14 3 1 11 120 13 12.6 5.1 9 2 11 220 12 20.1 14.5 13 4 1 12 150 13 23.5 6.8 11 2 11 160 12 24.3 20.5 14 5 1 7 85 1.0 <1.0 "Specimens collected during the I uteal phase (the first being obtained 1 week before the second). inadequate corpus luteal function in these three treatment cycles. In one patient (patient 2), good follicular maturation occurred as shown by the elevation of urinary estrogen and plasma 17,a-estradiol levels, but there was no evidence of ovulation when the BBT pattern and progesterone levels were examined (Fig. 2). In patient 5 the absence of urinary estrogen or plasma 17,a-estradiol elevation strongly suggested that follicular maturation had not occurred (Fig. 3). Results obtained using a combined LH-RH and HCG regimen are summarized in Tables 2 and 3. The hormonal profiles of patient 1 (Fig. 4) suggest that, since BBT and progesterone levels had risen before HCG was given, ovulation had already occurred. Similar hormone profiles were obtained in patient 3. However, in patients 2 and '4 there was no indication that ovulation had occurred prior to administration of HCG. Adequate luteal phases were obtained in all four patients treated with LH-RH followed by HCG, as judged by BBTs elevated for more than 13 days and progesterone levels of more than 8 ng/ml on two occasions 1 week apart (Table 2). The mean plasma LH and FSH concentrations 3 hours after a subcutaneous injection of 500 p,g of LH-RH twice weekly were very variable. However, mean plasma LH levels were significantly higher during the 2nd week of LH-RH treatment than during the 1st week, although the mean FSH levels remained unchanged. During the 2 weeks following the first course of LH-RH treatment, both the mean LH and FSH levels were significantly lower than the mean pretreatment levels. Follow-Up. During the 10 months following LH-RH treatment, patients 1 and 2 became pregnant after treatment with clomiphene, which had been used for inducing follicular maturation since additional supplies of LH-RH were not TABLE 3. Induction of Follicular Maturation and Ovulation with LH-RH Total no. Follicular phase" Luteal phase" LH-RH regimen of courses Ovulation'} Adequate Inadequate Adequate Inadequate First course (LH-RH) 5 4 1 3 0 3 Second course (LH-RH + HCG) 4 4 0 4 4 0 Total 9 8 1 7 4 3 a Follicular phase was judged adequate ifthe total urinary estrogen level was greater than 100 /-tg/24 hours. bovulation presumed by elevations of BBT and plasma progesterone level. "Luteal phase was judged adequate if the plasma progesterone level was greater than 8 ng/ml on two occasions 1 week apart.
624 HENDERSON ET AL. June 1976 c 37.5 LHRH 500119 tds sc. 37.0 [ BASAL BO~Y TEMPERATURE 36.5 [ Menses I22'0Zl 200 TOTAL URINARY ~ ESTROGENS ~ 100 ~ 650 I 4501-250 20 200.~ Plasma 1713 - estradiol -;f \~ 16.. E 150 12 E Plasma progesteron~' : 100 6 ~ -! ;/ \.... 50 4....... DAY 12 16 20 24 FrG. 1. Basal body temperature and total urinary estrogen, plasma 17f3-estradiol, and plasma progesterone levels before, during, and after LH-RH administration (patient 1, course 1). available; HCG had been given, when the urinary estrogens were high, to trigger ovulation and to maintain the luteal phase. Patient 3 reverted to spontaneous menstrual cycles 2 months after LH-RH treatment and became pregnant 4 months later without treatment. Patient 4 remained unresponsive to clomiphene, but she ovulated four times during eight courses of human menopausal gonadotropin and HCG. The last patient (patient 5) reverted to having a menstrual period every 3 months and did not wish to continue treatment for infertility. There were no side effects in any of the patients as a result of the LH-RH treatment. DISCUSSION The present studies indicate that follicular maturation can be achieved in other WIse healthy but amenorrheic women, who are unresponsive to clomiphene, using the LH-RH regimen first described by Mortimer et ap and Nillius et aly The patients treated in the previous t 36.8 I 36 5 LH RH 5001-'9 Ids 5C 36 2 BASAL BODY TEMPERATURE \ 50 I TOTAL URINARY ESTROGENS ~ 100 "- 50!l' 350 28 300 24 250 20. 200 \ 6 E E "- \ 50 12 '? \ 00 50........... ;~.:~:~ I Plosma 17a- estradiol /.~\ ;/.,... \\...,.)1' \ p,oge"",o"" Menses 12 \6 20 24 DAY FIG. 2. Basal body temperature and total urinary estrogen, plasma 17f3-estradiol, and plasma progesterone levels before, during, and after LH RH administration (patient 2, course 1).
Vol. 27, No.6 LH-RH INDUCED FOLLICULAR MATURATION AND OVULATION 625 'c :::J lh-rh 500119 tds sc. 36.2 [ BASAL BODY TEMPERATURE ~ 100 TOTAL URINARY ESTROGENS ~ ':Fooo ~nodoo E 100 ';;; 200 16 12 Plosma 17{3-estradlo) 8 ~ "-.',......... 4 CI...... C Plos ma. progesterone DA Y 12 16 20 24 FIG. 3. Basal body temperature and total urinary estrogen, plasma 17~-estradiol, and plasma progesterone levels before, during, and after LH RH administration (patient 5, course 1). studies using this regimen4, 5, 11 had either recovered or were recovering from anorexia nervosa, and these patients have abnormally high FSH responses to LH-RH in relation to the degree of weight 10ss.6 These high FSH responses to LH RH may be especially favorable for ini- tiating follicular growth and maturation. 4 However, the patients in the present study did not have anorexia nervosa and showed normal LH and FSH responses to LH-RH. In a similar study4 on patients with anorexia nervosa, a lowered FSH response to long-term LH RH treatment was found when the plasma 17,B-estradiol levels were rising. However, our data, based on less frequent blood sampling, failed to support these earlier observations. On the other hand, plasma LH levels 3 hours after LH-RH injections increased during the 2nd week of treatment compared with those during the 1 st week, suggesting that pituitary sensitivity to LH-RH had increased because of the rising levels of plasma 17,B-estradiol. In three patients (patients 1, 3, and 4), increases in estrogen levels were followed by ovulation, indicating that the hypothalamic and pituitary mechanisms involved in the LH surge were intact. c H CG HCG "1 36.5 LH-RH sao,,9 tds Ie. 3000 iou. Menses 36.0 BASAL BrDY TEMPERATURE 200 TOTAL URINARY ESTROGENS -".. 100 '" ~ 350 28 Plasma progesterone 300 24 250 20 200 16 E E ';;; 150 12 ";:a Q. C 100 50 0 Plasma 17f)-eltra - / 'dlol... l.......... 20 24 2B 32 DAY FIG. 4. Basal body temperature and total urinary estrogen, plasma 17~-estradiol, and plasma progesterone levels before, during, and after LH-RH and HCG administration (patient 1, course 2).
626 HENDERSON ET AL. June 1976 However, in patient 2 ovulation did not occur in spite of an adequate increase in estrogen levels, indicating hypothalamic-pituitary dysfunction. The lack of estrogen increase in patient 5 in spite of elevated levels of LH and FSH after LH-RH administration suggests either relatively insensitive ovaries or an abnormal production of LH and/or FSH. The luteal deficiency seen in patients 1, 3, and 4 following ovulation may be related to the low plasma LH and FSH levels found after cessation of LH-RH treatment, perhaps due to lack of endogenous LH-RH production. This association of low LH and FSH levels and deficient luteal phases provides additional evidence for the importance of small but critical levels of LH and FSH in the maintenance of the luteal phase. The rationale for the addition of HCG to the LH-RH regimen during the second course was to ensure that ovulation occurred. Subsequent injections of HCG were given to provide at least 14 days of high LH levels in order to maintain the corpus lute urn. Ovulation, as judged by elevated BBT and progesterone concentrations, occurred in all four patients treated with this regimen. However, these indices provide only presumptive evidence of ovulation, and conception, which is the only absolute proof of ovulation, did not occur in these patients in spite of excellent postcoital tests. There was no clinical evidence of ovarian hyperstimulation in the present study, perhaps because controlling systems within the pituitary prevented excessive release of gonadotropins. The conversion of a negative response to clomiphene into a positive response in two patients and the reversion to normal menstrual cycling in one patient following long-term LH-RH treatment suggests that the etiology for amenorrhea in these patients is chronic endogenous LH-RH deficiency. The possibility exists that, in these three amenorrheic patients, large doses of exogenous LH-RH restored to the pituitary its ability to respond to its steroid environment and to endogenous LH-RH. There is now a need for a trial of longterm LH-RH treatment in infertile women who are unresponsive to clomiphene since, from present evidence, LH-RH appears to offer the advantages of lack of hyperstimulation and decreased cost compared with human menopausal gonadotropin. In the future the use of analogs of LH-RH with greater potency and longer duration of action may reduce the number of injections required to induce follicular maturation. Acknowledgments. We thank Professor A. C. Turnbull for his interest and support, and Drs. G. A. Paulson, W. R. Butt, G. A. Niswender, and D. Exley for antisera used in the radioimmunoassays. We also thank Dr. W. Bogie (Hoechst Pharmaceutical, Hounslow, Middlesex, United Kingdom) for LH-RH (Hoe 471), Mr. R. Laynes for technical assistance, and Mrs. Jennie Fairweather for carrying out some of the 17,B-estradiol and progesterone assays. REFERENCES 1. Zarate A, Canales ES, Soria J, Gonzalez A, Schally A V, Kastin AJ: Further observations on the therapy of anovulatory infertility with synthetic luteinizing hormone-releasing hormone. Fertil Steril 25:3, 1974 2. Zanartu J, Dabancens A, Kastin AJ, Schally A V: Effect of synthetic hypothalamic gonadotropin-releasing hormone (FSHlLH-RH) in anovulatory sterility. Fertil SteriI25:160, 1974 3. Bonnar J, Henderson SR, Bogie W, Dutton A, Moore A, MacKinnon PCB: Induction of ovulation in women with synthetic gonadotrophin releasing hormone (LHIFSH-RH Hoe 471). Br J Obstet Gynaecol. In press 4. Nillius SJ, Wide L: Gonadotrophin-releasing hormone treatment for induction of follicular maturation and ovulation in amenorrhoeic women with anorexia nervosa. Br Med J 3:405, 1975 5. Mortimer CH, Besser GM, McNeilly AS: Gonadotrophin releasing hormone therapy in the induction of puberty, potency, spermatogenesis and ovulation in patients with hypothalamic-
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