0021-972X/91/7303-0511$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 73, No. 3 Printed in U.S.A.
Effect of Superovulation with Human Menopausal Gonadotropins on Growth Hormone Levels in Women* ELLEN E. WILSON, R. ANN WORD, WILLIAM BYRD, AND BRUCE R. CARR Division of Reproductive Endocrinology and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
ABSTRACT. GH synthesis and secretion are influenced by several factors, including age, body weight, and sex steroid hormones. Endogenous and exogenous estrogens influence the circulating levels of GH. The purpose of the present investigation was to define the relationship between serum GH and estradiol levels during the follicular phase in women with normal ovulatory menstrual cycles compared with that in women undergoing superovulation with human menopausal gonadotropins (hMG) alone or hMG plus GnRH agonists during treatment for infertility. Serum GH and estradiol levels were determined by immunoassay in eight women during the follicular phase of a spontaneous natural cycle (group I). Thirty women underwent ovulation induction with hMG alone (group II), and 30 women received GnRH agonists followed by hMG (group III). During the follicular phase estradiol levels increased gradually in group
T
HERE is considerable evidence that endogenous as well as exogenous sex steroids augment GH synthesis and secretion. Basal and stimulated GH levels in men and women have been shown to correlate with circulating estrogen levels under a variety of circumstances. For example, a significant increase in GH secretion occurs during normal or precocious pubertal development (13). Pulsatile GH activity in girls with Turner's syndrome is augmented by estradiol therapy (4). Postmenopausal women treated with exogenous estrogen have increased basal and GH-releasing hormone-stimulated GH secretion (5). Basal plasma GH levels rise significantly in men treated with estrogens and fall to basal levels after cessation of therapy (6). Likewise, exogenous estrogen therapy has been shown to augment GH levels in baboons and steers (7, 8). On the other hand, GnRH agonists, which diminish estrogen levels, appear to inhibit GH release in adult women during treatment for endometriosis or leiomyomas and in children treated with GnRH
I and reached a peak estradiol level of 1.19 ± 0.2 nmol/L (mean ± SEM). AS expected, estradiol levels rose faster and reached higher levels in groups II and III (5.44 ± 0.62 and 8.73 ± 0.91 nmol/L, respectively). Whereas serum GH levels increased minimally in group I, reaching a peak level of 2.54 ± 1.15 nmol/L, serum GH concentrations increased markedly after day 8 in groups II and III, reaching peak levels of 8.70 ± 1.58 and 7.54 ± 1.12 nmol/L, respectively (P < 0.01). Basal to peak GH levels were higher in groups II and III than in group I. In summary, there are modest increases in GH levels during the follicular phase of the normal menstrual cycle, but the levels are markedly increased during superovulation with hMG or hMG plus GnRH agonists, and parallel increases in estradiol levels. (J Clin Endocrinol Metab 73: 511-515,1991)
agonists for precocious p u b e r t y (2, 9, 10).
Women treated with human menopausal gonadotropins (hMG) for superovulation provide an excellent population to study the influence of high endogenous estrogen levels on GH levels. Previous investigations have suggested an important role for GH in the genesis of developing follicles in infertile women receiving superovulation treatment (11-14). In the present investigation we examined the effect of superovulation by hMG on GH levels and sought to determine the effect of the addition of GnRH agonists to hMG on circulating GH levels. The effect of superovulation with or without GnRH agonists on GH and estradiol levels was then compared to the changes during the follicular phase in normal ovulatory women. Materials and Methods Subjects Group I (control group) consisted of eight healthy female volunteers with regular cyclic menses who were not taking any medications. All women in groups II and III had undergone a complete infertility evaluation before entering the study. Ovulatory status was documented by basal body temperature charts and midluteal phase progesterone levels and/or by endometrial
Received November 8,1990. Address all correspondence and requests for reprints to: Bruce R. Carr, M.D., Paul C. MacDonald Professor of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9032. * This work was supported in part by Grant HD-07190.
511
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
WILSON ET AL.
512
biopsy. These patients were infertile due to male factors, endometriosis, tubal disease, or unexplained infertility. Women in group II (n = 30) underwent superovulation with hMG alone. After follicular stimulation, they underwent either intrauterine insemination or in vitro fertilization (IVF). Women in group III (n = 30) underwent superovulation with hMG after GnRH agonist suppression for IVF. Study design Serum samples were obtained from cycle days 1-3 until the time of ovulation. Women in group II were treated with hMG (Pergonal, Serono, Randolf, MA) beginning with one or two ampules a day (day 3) and variable doses thereafter depending on the patient's estradiol response. hCG (Profasi, Serono) was administered to induce ovulation. Women in group III were treated with a GnRH agonist (leuprolide acetate, TAP Pharmaceuticals, Chicago, IL) sc to inhibit gonadotropin secretion (1 mg/day for a minimum of 14 days). Women in group III were then treated with three ampules of hMG for 3 days starting on day 3 and variable doses of hMG thereafter depending on the patient's estradiol response. GnRH agonist treatment was continued during follicular stimulation (0.5 mg/day) until the administration of hCG. Ovulation was induced by 5000IU hCG when two or more follicles reached 18 mm in diameter, as determined by sonography. Hormone assays Serum samples were separated, aliquoted, and frozen until the time of assay. Serum GH was measured with a double antibody 125I RIA (Diagnostic Products Corp., Los Angeles, CA). The intraassay variation for GH was 5.9%, and the interassay variation was 8.3%. 17/3-Estradiol was quantified using a nonextracted, solid phase 125I RIA (Diagnostic Products Corp.) of serum samples. The intraassay variation was 5.1%, and the interassay variation was 8.0%. All assays were performed in duplicate, and all samples from each subject were measured in a single assay. Statistical analysis Differences in peak hormone levels were measured by oneway analysis of variance. Basal and peak GH levels were compared using Student's t test. P < 0.05 was considered significant.
Results A summary of the clinical and laboratory data is presented in Table 1. Eight women were studied during a natural cycle (group I), 30 women underwent hMG stimulation for intrauterine insemination or IVF (group II), and 30 women underwent GnRH analog suppression followed by hMG stimulation for IVF (group III). Group I (control group) consisted of healthy female volunteers with regular cyclic menses and midluteal progesterone values greater than 31.8 nmol/L. Groups II and III consisted of infertile women who had undergone a complete
JCE & M • 1991 Vol 73 • No 3
infertility evaluation before entering the study. Mean ages were 28.3, 33.4, and 34.9 yr, and mean body weights were 54.0, 60.3, and 60.1 kg in groups I, II, and III, respectively. The mean age and body weight were not statistically different between the groups. The total number of ampules of hMG given to group II (mean ± SEM, 23.8 ± 2.94) was not statistically different from that given to group III (27.5 ± 0.7). Basal estradiol levels were 0.05 ± 0.02, 0.22 ± 0.09, and 0.11 ± 0.30 nmol/L in groups I, II, and III, respectively. Peak estradiol levels were significantly greater in group III (mean ± SEM, 8.73 ± 0.91 nmol/L) than in group II (5.44 ± 0.62 nmol/L) or group I (1.19 ± 0.21 nmol/L). Peak estradiol levels were also significantly greater in group II than in group I. The length of the follicular phase and peak levels of estradiol in all groups were variable. Data were normalized by assigning the peak estradiol value of each patient to day 10 regardless of the length of their stimulation cycle. The increase in estradiol levels as a function of time is illustrated in Fig. 1. Peak estradiol values increased modestly in group I. As expected, the greatest increase in estradiol levels were seen in group III, and intermediate levels were seen in patients in group II. The basal to peak estradiol levels in each of the three groups is presented in Fig. 2. Each group demonstrated a significant increase in peak estradiol levels compared to basal levels. Basal GH levels were 1.28 ± 0.87, 0.80 ± 0.41, and 1.70 ± 0.25 nmol/L in groups I, II, and III, respectively. As presented in Table 1, the peak GH levels were 2.54 ± 11.5, 8.7 ± 1.58, and 7.54 ± 1.2 nmol/L in groups I, II, and III, respectively. The increase in GH levels during the follicular phase of the menstrual cycle normalized to the peak GH level to day 10 is illustrated in Fig. 3. The GH levels varied and did not increase until approximately day 8, when the levels of estradiol were increased in all groups. The increase in GH levels was modest in group I patients, but in groups II and III GH increased markedly before injection of hCG. The basal to peak GH increments are presented in Fig. 4. GH levels increased from basal to peak in all three groups, but the increase in GH levels in group I was not statistically significant. In contrast, the basal to peak GH increments in groups II and III were statistically significant (P < 0.01).
Discussion Several investigators have reported that increases in estradiol levels are associated with increased GH release (4-8). It has also been shown that inhibition of estradiol secretion by treatment with GnRH agonists results in diminished GH levels and/or GH-releasing hormonestimulated GH release (2, 9,10). In this investigation we sought to determine the changes in GH levels during the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
SUPEROVULATION AND GH LEVELS
513
TABLE 1. Patient characteristics and peak GH and estradiol levels Protocol
Number of patients
Age"
Weight" (kg)
8
28.3 ± 2.8
54.0 ± 5.2
30
33.4 ± 1.0
60.3 ± 3.1
30
34.9 ± 1.0
60.1 ± 1.2
Group I (natural cycle) Group II (hMG) Group III (hMG-GNRH agonist) 0
Total amps of pergonal"
Peak estradiol (nmol/L)a
Peak GH (nmol/L)°
1.19 ± 0.21c'd
2.54 ± 1.15*
23.8 ± 2.9
5.44 ± 0.62w
8.70 ± 1.58^
27.5 ± 0.7
8.73 ± 0.916'6
7.54 ± 1.12'
Mean ± SE.
DAYS
II
in
FOLLICULAR STIMULATION
FlG. 1. Follicular phase estradiol levels in unstimulated and superovulated women. Daily estradiol levels in superovulated and unstimulated women were measured during the follicular phase of the menstrual cycle. Since the length of the follicular phase was different in each of the women studied, the data were normalized by adjusting the peak day of estradiol to day 10. Each data point represents the mean (±SEM) estradiol levels, expressed in nanomoles per L. • , Group I (unstimulated) patients; • , group II (hMG); A, group III (hMG-leuprolide acetate).
FlG. 2. Follicular phase basal and maximal peak levels of estradiol in superovulated and unstimulated women. Each bar represents the mean ± SEM. • , Serum estradiol levels on day 1 of follicular stimulation with hMG or day 3 of the unstimulated normal cycle. • , Peak estradiol response in these women. Group I represents the normal unstimulated control women, group II women were treated with hMG alone, and group III women were treated with GnRH agonist plus hMG. In paired t tests * * is significantly different from * (P = 0.005).
follicular phase in women experiencing normal ovulatory cycles compared to those in women undergoing superovulation with hMG or hMG plus GnRH agonist. During normal cycles (group I) estrogen and GH levels increased modestly, but the increase in GH levels was not statistically significant. In superovulated women (groups II and III), however, the increase in estrogen and GH levels was dramatic. The marked GH response in hMG-stimulated cycles may reflect the higher levels of estradiol achieved. Temporally, gradual increases in estradiol levels precede the sharp rise in serum GH, suggesting a possible threshold effect of estradiol for pituitary GH secretion. The inhibitory effects of the GnRH agonists on GH secretion can be overcome by treatment with hMG, which, in fact, resulted in higher levels of estradiol
and similar levels of GH in patients in group III compared to those in group II. Thus, the present investigation suggests that estradiol is a primary regulator of GH release by the pituitary in women. Several growth factors and neuropeptides have been proposed to modulate follicular growth and hormone secretion. Intraovarian insulin-like growth factor-I (IGFI) is believed to be important in follicular growth and is up-regulated by GH release from the pituitary (15). IGFI and GH enhance FSH-induced differentiation of cultured rat granulosa cells (16,17). In addition, it has been reported that the addition of GH to human granulosa cells in vitro was associated with an increase in estradiol secretion (18). It is possible that the rise of estradiol levels during
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
WILSON ET AL.
514
6
8
10
DAYS
FIG. 3. Daily GH levels in superovulated and unstimulated women during the follicular phase of the menstrual cycle. Since the length of the follicular phase was different in each woman studied, the data were normalized by adjusting the peak day of GH secretion to day 10. Each data point represents the mean (±SEM) GH levels, expressed in nanomoles per L. • , Group I (unstimulated); • , group II (hMG alone); A, group III (hMG plus leuprolide acetate).
JCE & M • 1991 Vol 73 • No 3
stimulation in women with ovaries that are relatively resistant to hMG therapy. These studies demonstrated a reduction in the number of ampules of hMG required to induce ovulation and improved follicular development in women treated with hMG plus GH (11-14). The mechanism or pathway by which estradiol increases GH release is not clearly understood. It is possible that sex steroids exert a direct influence on the somatotrophs of the pituitary. Estrogens have been shown to increase GH mRNA levels in rat pituitary tissues (19, 20). Alternately, estrogen may increase GH release at a suprapituitary site, involving alterations in the release of GH-releasing hormone and somatostatin (21-23). In rats, estrogen therapy enhances the GH-releasing action of hypothalamic extracts (24). Circulating estrogens appear to increase hypothalamic opioid activity as well as central «2-adrenergic receptor affinity, which may lead to increased GH release (21, 25-29). The effect of GnRH agonists on lowering GH levels may occur as a result of lowering estrogen levels, direct pituitary suppression, or both. In summary, the results of the present investigation and those of others demonstrate that estrogen levels are correlated with an increase in the release of GH. We demonstrated that superovulation with hMG was associated with increased serum GH levels compared to those in nonstimulated normal cycles.
References
II
III
FOLLICULAR STIMULATION
FiG. 4. Basal and maximal peak levels of GH in superovulated and normal unstimulated women. Each bar represents the mean ± SEM. D, GH levels on day 1 of follicular stimulation by hMG or day 3 of the unstimulated natural cycles. • , Peak GH response in those women. Group I represents the normal unstimulated women, group II women were treated with hMG alone, and group III women were treated with GnRH agonist plus hMG. In paired t tests, * * is significantly different from • (P = 0.005).
normal cycles as well as in superovulated cycles of women treated with hMG results in an increase in GH levels which may thereby act directly or indirectly (by IGF-I) on the ovarian follicle to further augment follicle growth and estrogen secretion. There is evidence that GH therapy can augment the ovarian response to gonadotropic
1. Mauras N, Blizzard RM, Link K, Johnson HL, Rogol AD, Veldhuis JD. Augmentation of growth hormone secretion during puberty: evidence for a pulse amplitude-modulated phenomenon. J Clin Endocrinol Metab. 1987;64:596-601. 2. Mansfield MJ, Rudin CR, Crigler JF, et al. Changes in growth and serum growth hormone and plasma somatomedin-C levels during suppression of gonadal sex steroid secretion in girls with central precocious puberty. J Clin Endocrinol Metab. l988;66:3-9. 3. Miller JD, Tannenbaum GS, Colle E, Guyda HJ. Daytime pulsatile growth hormone secretion during childhood and adolescence. J Clin Endocrinol Metab. 1982;55:989-91. 4. Mauras N, Rogol AD, Veldhuis JD. Specific, time-dependent actions of low-dose ethinyl estradiol administration on the episodic release of growth hormone, follicle-stimulating hormone, and luteinizing hormone in prepubertal girls with Turner's syndrome. J Clin Endocrinol Metab. 1989;69:1053-8. 5. Dawson-Hughes B, Stern D, Goldman J, Reichlin S. Regulation of growth hormone and somatomedin C secretion in postmenopausal women: effect of physiological estrogen replacement. J Clin Endocrinol Metab. 1986;63:424-32. 6. Wiedemann E, Schwartz E, Frantz AG. Acute and chronic estrogen effects upon serum somatomedin activity, growth hormone, and prolactin in man. J Clin Endocrinol Metab. 1976;42:942-52. 7. Copeland KC, Johnson DM, Kuehl TH, Castracane VD. Estrogen stimulates growth hormone and somatomedin C in castrate and intact female baboons. J Clin Endocrinol Metab. 1984;58:698-703. 8. Breier BH, Gluckman PD, Bass JJ. Influence of nutritional status and oestradiol-17/3 on plasma growth hormone, insulin-like growth factors I and II and the response to exogenous growth hormone in young steers. J Endocrinol. 1988; 118:243-50. 9. Harris DA, Van Vliet G, Egli CA, et al. Somatomedin-C in normal puberty and in true precocious puberty before and after treatment with a potent luteinizing hormone-releasing hormone agonist. J
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
SUPEROVULATION AND GH LEVELS Clin Endocrinol Metab. 1985;61:152-9. 10. Word RA, Odom MJ, Byrd W, Carr B. The effect of gonadotropinreleasing agonists on growth hormone secretion in adult premenopausal women. Fertil Steril. 1990;54:73-8. 11. Homburg R, Eshel A, Abdalla HI, Jacobs HS. Growth hormone facilitates ovulation induction by gonadotrophins. Clin Endocrinol (Oxf). 1988;29:113-7. 12. Blumenfeld Z, Lunenfeld B. The potentiating effect of growth hormone on follicle stimulation with human menopausal gonadotropin in a panhypopituitary patient. Fertil Steril. 1989;52:328-31. 13. Volpe A, Coukos G, Artini PG, et al. Pregnancy following combined growth hormone-pulsatile GnRH treatment in a patient with hypothalamic amenorrhoea. Hum Reprod. 1990;5:345-7. 14. Homburg R, West C, Torresani T, Jacobs HS. Cotreatment with human growth hormone and gonadotropins for induction of ovulation: a controlled clinical trial. Fertil Steril. 1990;53:254-60. 15. Davoren JB, Hsueh AJW. Growth hormone increases ovarian levels of immunoreactive somatomedin C/insulin-like growth factor I in vivo. Endocrinology. 1986; 118:888-90. 16. Adashi EY, Resnick CE, Svoboda ME, van Wyk JJ. Somatomedin C enhances induction of LH receptors by FSH in cultured rat granulosa cells. Endocrinology. 1985; 116:2369-75. 17. Jia X-C, Kalmijn J, Hsueh AJW. Growth hormone enhances follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology. 1986;118:1401-9. 18. Mason HD, Beard RW, Franks S. Stimulation of estradiol production by growth hormone (hGH) in cultured human granulosa cells [Abstract]. Proc of the 72nd Annual Meet of The Endocrine Soc. 1990;195. 19. Lloyd RV, Cano M, Landefeld TD. The effects of estrogens on tumor growth and on prolactin and growth hormone mRNA expres-
515
sion in rat pituitary tissues. Am J Pathol. 1988; 133:397-406. 20. Jin L, Song J, Lloyd RV. Estrogen stimulates both prolactin and growth hormone mRNAs expression in the MtT/F4 transplantable pituitary tumor. Proc Soc Exp Biol Med. 1989;192:225-9. 21. Phipps WR, Campbell BF, Pescovitz OH. Effect of naloxone on the growth hormone response to clonidine in normal women during the mid-luteal phase. Psychoneuroendocrinology. 1989;14:137-43. 22. Evans WS, Krieg RJ, Limber ER, Kaiser DL, Thorner MO. Effects of in vivo gonadal hormone environment on in vitro hGRF-40stimulated GH release. Am J Physiol. 1985;249:E276-80. 23. Ross RJM, Grossman A, Davies PSW, Savage MO, Besser GM. Stilboestrol pretreatment of children with short stature does not affect the growth hormone response to growth hormone-releasing hormone. Clin Endocrinol (Oxf). 1987;27:155-61. 24. Malacara JM, Valverde-R C, Reichlin S, Bollinger J. Elevation of plasma radioimmunoassayable growth hormone in the rat induced by porcine hypothalamic extract. Endocrinology. 1972;91:1189-92. 25. Szabo M, Frohman LA. Effects of porcine stalk median eminence and prostaglandin E2 on rat growth hormone secretion in vivo and their inhibition by somatostatin. Endocrinology. 1975;96:955. 26. Wardlaw SL, Wehrenberg WB, Ferin M, Antunes JL, Frantz AG. Effect of sex steroids on j8-endorphin in hypophyseal portal blood. J Clin Endocrinol Metab. 1982;55:877-81. 27. Wehrenberg WB, Wardlaw SL, Frantz AG, Ferin M. /3-Endorphin in hypophyseal portal blood: variations throughout the menstrual cycle. Endocrinology. 1982;111:879-81. 28. Stubbs WA, Delitala G, Jones A, et al. Hormonal and metabolic responses to an encephalin analogue in normal man. Lancet. 1978;2:1225-7. 29. Delitala G, Grossman A, Besser M. Differential effects of opiate peptides and alkaloids on anterior pituitary hormone secretion. Neuroendocrinology. 1983;37:275-9.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
Vol. 73, No. 3 Printed in U.S.A.
Effect of Superovulation with Human Menopausal Gonadotropins on Growth Hormone Levels in Women* ELLEN E. WILSON, R. ANN WORD, WILLIAM BYRD, AND BRUCE R. CARR Division of Reproductive Endocrinology and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
ABSTRACT. GH synthesis and secretion are influenced by several factors, including age, body weight, and sex steroid hormones. Endogenous and exogenous estrogens influence the circulating levels of GH. The purpose of the present investigation was to define the relationship between serum GH and estradiol levels during the follicular phase in women with normal ovulatory menstrual cycles compared with that in women undergoing superovulation with human menopausal gonadotropins (hMG) alone or hMG plus GnRH agonists during treatment for infertility. Serum GH and estradiol levels were determined by immunoassay in eight women during the follicular phase of a spontaneous natural cycle (group I). Thirty women underwent ovulation induction with hMG alone (group II), and 30 women received GnRH agonists followed by hMG (group III). During the follicular phase estradiol levels increased gradually in group
T
HERE is considerable evidence that endogenous as well as exogenous sex steroids augment GH synthesis and secretion. Basal and stimulated GH levels in men and women have been shown to correlate with circulating estrogen levels under a variety of circumstances. For example, a significant increase in GH secretion occurs during normal or precocious pubertal development (13). Pulsatile GH activity in girls with Turner's syndrome is augmented by estradiol therapy (4). Postmenopausal women treated with exogenous estrogen have increased basal and GH-releasing hormone-stimulated GH secretion (5). Basal plasma GH levels rise significantly in men treated with estrogens and fall to basal levels after cessation of therapy (6). Likewise, exogenous estrogen therapy has been shown to augment GH levels in baboons and steers (7, 8). On the other hand, GnRH agonists, which diminish estrogen levels, appear to inhibit GH release in adult women during treatment for endometriosis or leiomyomas and in children treated with GnRH
I and reached a peak estradiol level of 1.19 ± 0.2 nmol/L (mean ± SEM). AS expected, estradiol levels rose faster and reached higher levels in groups II and III (5.44 ± 0.62 and 8.73 ± 0.91 nmol/L, respectively). Whereas serum GH levels increased minimally in group I, reaching a peak level of 2.54 ± 1.15 nmol/L, serum GH concentrations increased markedly after day 8 in groups II and III, reaching peak levels of 8.70 ± 1.58 and 7.54 ± 1.12 nmol/L, respectively (P < 0.01). Basal to peak GH levels were higher in groups II and III than in group I. In summary, there are modest increases in GH levels during the follicular phase of the normal menstrual cycle, but the levels are markedly increased during superovulation with hMG or hMG plus GnRH agonists, and parallel increases in estradiol levels. (J Clin Endocrinol Metab 73: 511-515,1991)
agonists for precocious p u b e r t y (2, 9, 10).
Women treated with human menopausal gonadotropins (hMG) for superovulation provide an excellent population to study the influence of high endogenous estrogen levels on GH levels. Previous investigations have suggested an important role for GH in the genesis of developing follicles in infertile women receiving superovulation treatment (11-14). In the present investigation we examined the effect of superovulation by hMG on GH levels and sought to determine the effect of the addition of GnRH agonists to hMG on circulating GH levels. The effect of superovulation with or without GnRH agonists on GH and estradiol levels was then compared to the changes during the follicular phase in normal ovulatory women. Materials and Methods Subjects Group I (control group) consisted of eight healthy female volunteers with regular cyclic menses who were not taking any medications. All women in groups II and III had undergone a complete infertility evaluation before entering the study. Ovulatory status was documented by basal body temperature charts and midluteal phase progesterone levels and/or by endometrial
Received November 8,1990. Address all correspondence and requests for reprints to: Bruce R. Carr, M.D., Paul C. MacDonald Professor of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9032. * This work was supported in part by Grant HD-07190.
511
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
WILSON ET AL.
512
biopsy. These patients were infertile due to male factors, endometriosis, tubal disease, or unexplained infertility. Women in group II (n = 30) underwent superovulation with hMG alone. After follicular stimulation, they underwent either intrauterine insemination or in vitro fertilization (IVF). Women in group III (n = 30) underwent superovulation with hMG after GnRH agonist suppression for IVF. Study design Serum samples were obtained from cycle days 1-3 until the time of ovulation. Women in group II were treated with hMG (Pergonal, Serono, Randolf, MA) beginning with one or two ampules a day (day 3) and variable doses thereafter depending on the patient's estradiol response. hCG (Profasi, Serono) was administered to induce ovulation. Women in group III were treated with a GnRH agonist (leuprolide acetate, TAP Pharmaceuticals, Chicago, IL) sc to inhibit gonadotropin secretion (1 mg/day for a minimum of 14 days). Women in group III were then treated with three ampules of hMG for 3 days starting on day 3 and variable doses of hMG thereafter depending on the patient's estradiol response. GnRH agonist treatment was continued during follicular stimulation (0.5 mg/day) until the administration of hCG. Ovulation was induced by 5000IU hCG when two or more follicles reached 18 mm in diameter, as determined by sonography. Hormone assays Serum samples were separated, aliquoted, and frozen until the time of assay. Serum GH was measured with a double antibody 125I RIA (Diagnostic Products Corp., Los Angeles, CA). The intraassay variation for GH was 5.9%, and the interassay variation was 8.3%. 17/3-Estradiol was quantified using a nonextracted, solid phase 125I RIA (Diagnostic Products Corp.) of serum samples. The intraassay variation was 5.1%, and the interassay variation was 8.0%. All assays were performed in duplicate, and all samples from each subject were measured in a single assay. Statistical analysis Differences in peak hormone levels were measured by oneway analysis of variance. Basal and peak GH levels were compared using Student's t test. P < 0.05 was considered significant.
Results A summary of the clinical and laboratory data is presented in Table 1. Eight women were studied during a natural cycle (group I), 30 women underwent hMG stimulation for intrauterine insemination or IVF (group II), and 30 women underwent GnRH analog suppression followed by hMG stimulation for IVF (group III). Group I (control group) consisted of healthy female volunteers with regular cyclic menses and midluteal progesterone values greater than 31.8 nmol/L. Groups II and III consisted of infertile women who had undergone a complete
JCE & M • 1991 Vol 73 • No 3
infertility evaluation before entering the study. Mean ages were 28.3, 33.4, and 34.9 yr, and mean body weights were 54.0, 60.3, and 60.1 kg in groups I, II, and III, respectively. The mean age and body weight were not statistically different between the groups. The total number of ampules of hMG given to group II (mean ± SEM, 23.8 ± 2.94) was not statistically different from that given to group III (27.5 ± 0.7). Basal estradiol levels were 0.05 ± 0.02, 0.22 ± 0.09, and 0.11 ± 0.30 nmol/L in groups I, II, and III, respectively. Peak estradiol levels were significantly greater in group III (mean ± SEM, 8.73 ± 0.91 nmol/L) than in group II (5.44 ± 0.62 nmol/L) or group I (1.19 ± 0.21 nmol/L). Peak estradiol levels were also significantly greater in group II than in group I. The length of the follicular phase and peak levels of estradiol in all groups were variable. Data were normalized by assigning the peak estradiol value of each patient to day 10 regardless of the length of their stimulation cycle. The increase in estradiol levels as a function of time is illustrated in Fig. 1. Peak estradiol values increased modestly in group I. As expected, the greatest increase in estradiol levels were seen in group III, and intermediate levels were seen in patients in group II. The basal to peak estradiol levels in each of the three groups is presented in Fig. 2. Each group demonstrated a significant increase in peak estradiol levels compared to basal levels. Basal GH levels were 1.28 ± 0.87, 0.80 ± 0.41, and 1.70 ± 0.25 nmol/L in groups I, II, and III, respectively. As presented in Table 1, the peak GH levels were 2.54 ± 11.5, 8.7 ± 1.58, and 7.54 ± 1.2 nmol/L in groups I, II, and III, respectively. The increase in GH levels during the follicular phase of the menstrual cycle normalized to the peak GH level to day 10 is illustrated in Fig. 3. The GH levels varied and did not increase until approximately day 8, when the levels of estradiol were increased in all groups. The increase in GH levels was modest in group I patients, but in groups II and III GH increased markedly before injection of hCG. The basal to peak GH increments are presented in Fig. 4. GH levels increased from basal to peak in all three groups, but the increase in GH levels in group I was not statistically significant. In contrast, the basal to peak GH increments in groups II and III were statistically significant (P < 0.01).
Discussion Several investigators have reported that increases in estradiol levels are associated with increased GH release (4-8). It has also been shown that inhibition of estradiol secretion by treatment with GnRH agonists results in diminished GH levels and/or GH-releasing hormonestimulated GH release (2, 9,10). In this investigation we sought to determine the changes in GH levels during the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
SUPEROVULATION AND GH LEVELS
513
TABLE 1. Patient characteristics and peak GH and estradiol levels Protocol
Number of patients
Age"
Weight" (kg)
8
28.3 ± 2.8
54.0 ± 5.2
30
33.4 ± 1.0
60.3 ± 3.1
30
34.9 ± 1.0
60.1 ± 1.2
Group I (natural cycle) Group II (hMG) Group III (hMG-GNRH agonist) 0
Total amps of pergonal"
Peak estradiol (nmol/L)a
Peak GH (nmol/L)°
1.19 ± 0.21c'd
2.54 ± 1.15*
23.8 ± 2.9
5.44 ± 0.62w
8.70 ± 1.58^
27.5 ± 0.7
8.73 ± 0.916'6
7.54 ± 1.12'
Mean ± SE.
DAYS
II
in
FOLLICULAR STIMULATION
FlG. 1. Follicular phase estradiol levels in unstimulated and superovulated women. Daily estradiol levels in superovulated and unstimulated women were measured during the follicular phase of the menstrual cycle. Since the length of the follicular phase was different in each of the women studied, the data were normalized by adjusting the peak day of estradiol to day 10. Each data point represents the mean (±SEM) estradiol levels, expressed in nanomoles per L. • , Group I (unstimulated) patients; • , group II (hMG); A, group III (hMG-leuprolide acetate).
FlG. 2. Follicular phase basal and maximal peak levels of estradiol in superovulated and unstimulated women. Each bar represents the mean ± SEM. • , Serum estradiol levels on day 1 of follicular stimulation with hMG or day 3 of the unstimulated normal cycle. • , Peak estradiol response in these women. Group I represents the normal unstimulated control women, group II women were treated with hMG alone, and group III women were treated with GnRH agonist plus hMG. In paired t tests * * is significantly different from * (P = 0.005).
follicular phase in women experiencing normal ovulatory cycles compared to those in women undergoing superovulation with hMG or hMG plus GnRH agonist. During normal cycles (group I) estrogen and GH levels increased modestly, but the increase in GH levels was not statistically significant. In superovulated women (groups II and III), however, the increase in estrogen and GH levels was dramatic. The marked GH response in hMG-stimulated cycles may reflect the higher levels of estradiol achieved. Temporally, gradual increases in estradiol levels precede the sharp rise in serum GH, suggesting a possible threshold effect of estradiol for pituitary GH secretion. The inhibitory effects of the GnRH agonists on GH secretion can be overcome by treatment with hMG, which, in fact, resulted in higher levels of estradiol
and similar levels of GH in patients in group III compared to those in group II. Thus, the present investigation suggests that estradiol is a primary regulator of GH release by the pituitary in women. Several growth factors and neuropeptides have been proposed to modulate follicular growth and hormone secretion. Intraovarian insulin-like growth factor-I (IGFI) is believed to be important in follicular growth and is up-regulated by GH release from the pituitary (15). IGFI and GH enhance FSH-induced differentiation of cultured rat granulosa cells (16,17). In addition, it has been reported that the addition of GH to human granulosa cells in vitro was associated with an increase in estradiol secretion (18). It is possible that the rise of estradiol levels during
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
WILSON ET AL.
514
6
8
10
DAYS
FIG. 3. Daily GH levels in superovulated and unstimulated women during the follicular phase of the menstrual cycle. Since the length of the follicular phase was different in each woman studied, the data were normalized by adjusting the peak day of GH secretion to day 10. Each data point represents the mean (±SEM) GH levels, expressed in nanomoles per L. • , Group I (unstimulated); • , group II (hMG alone); A, group III (hMG plus leuprolide acetate).
JCE & M • 1991 Vol 73 • No 3
stimulation in women with ovaries that are relatively resistant to hMG therapy. These studies demonstrated a reduction in the number of ampules of hMG required to induce ovulation and improved follicular development in women treated with hMG plus GH (11-14). The mechanism or pathway by which estradiol increases GH release is not clearly understood. It is possible that sex steroids exert a direct influence on the somatotrophs of the pituitary. Estrogens have been shown to increase GH mRNA levels in rat pituitary tissues (19, 20). Alternately, estrogen may increase GH release at a suprapituitary site, involving alterations in the release of GH-releasing hormone and somatostatin (21-23). In rats, estrogen therapy enhances the GH-releasing action of hypothalamic extracts (24). Circulating estrogens appear to increase hypothalamic opioid activity as well as central «2-adrenergic receptor affinity, which may lead to increased GH release (21, 25-29). The effect of GnRH agonists on lowering GH levels may occur as a result of lowering estrogen levels, direct pituitary suppression, or both. In summary, the results of the present investigation and those of others demonstrate that estrogen levels are correlated with an increase in the release of GH. We demonstrated that superovulation with hMG was associated with increased serum GH levels compared to those in nonstimulated normal cycles.
References
II
III
FOLLICULAR STIMULATION
FiG. 4. Basal and maximal peak levels of GH in superovulated and normal unstimulated women. Each bar represents the mean ± SEM. D, GH levels on day 1 of follicular stimulation by hMG or day 3 of the unstimulated natural cycles. • , Peak GH response in those women. Group I represents the normal unstimulated women, group II women were treated with hMG alone, and group III women were treated with GnRH agonist plus hMG. In paired t tests, * * is significantly different from • (P = 0.005).
normal cycles as well as in superovulated cycles of women treated with hMG results in an increase in GH levels which may thereby act directly or indirectly (by IGF-I) on the ovarian follicle to further augment follicle growth and estrogen secretion. There is evidence that GH therapy can augment the ovarian response to gonadotropic
1. Mauras N, Blizzard RM, Link K, Johnson HL, Rogol AD, Veldhuis JD. Augmentation of growth hormone secretion during puberty: evidence for a pulse amplitude-modulated phenomenon. J Clin Endocrinol Metab. 1987;64:596-601. 2. Mansfield MJ, Rudin CR, Crigler JF, et al. Changes in growth and serum growth hormone and plasma somatomedin-C levels during suppression of gonadal sex steroid secretion in girls with central precocious puberty. J Clin Endocrinol Metab. l988;66:3-9. 3. Miller JD, Tannenbaum GS, Colle E, Guyda HJ. Daytime pulsatile growth hormone secretion during childhood and adolescence. J Clin Endocrinol Metab. 1982;55:989-91. 4. Mauras N, Rogol AD, Veldhuis JD. Specific, time-dependent actions of low-dose ethinyl estradiol administration on the episodic release of growth hormone, follicle-stimulating hormone, and luteinizing hormone in prepubertal girls with Turner's syndrome. J Clin Endocrinol Metab. 1989;69:1053-8. 5. Dawson-Hughes B, Stern D, Goldman J, Reichlin S. Regulation of growth hormone and somatomedin C secretion in postmenopausal women: effect of physiological estrogen replacement. J Clin Endocrinol Metab. 1986;63:424-32. 6. Wiedemann E, Schwartz E, Frantz AG. Acute and chronic estrogen effects upon serum somatomedin activity, growth hormone, and prolactin in man. J Clin Endocrinol Metab. 1976;42:942-52. 7. Copeland KC, Johnson DM, Kuehl TH, Castracane VD. Estrogen stimulates growth hormone and somatomedin C in castrate and intact female baboons. J Clin Endocrinol Metab. 1984;58:698-703. 8. Breier BH, Gluckman PD, Bass JJ. Influence of nutritional status and oestradiol-17/3 on plasma growth hormone, insulin-like growth factors I and II and the response to exogenous growth hormone in young steers. J Endocrinol. 1988; 118:243-50. 9. Harris DA, Van Vliet G, Egli CA, et al. Somatomedin-C in normal puberty and in true precocious puberty before and after treatment with a potent luteinizing hormone-releasing hormone agonist. J
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
SUPEROVULATION AND GH LEVELS Clin Endocrinol Metab. 1985;61:152-9. 10. Word RA, Odom MJ, Byrd W, Carr B. The effect of gonadotropinreleasing agonists on growth hormone secretion in adult premenopausal women. Fertil Steril. 1990;54:73-8. 11. Homburg R, Eshel A, Abdalla HI, Jacobs HS. Growth hormone facilitates ovulation induction by gonadotrophins. Clin Endocrinol (Oxf). 1988;29:113-7. 12. Blumenfeld Z, Lunenfeld B. The potentiating effect of growth hormone on follicle stimulation with human menopausal gonadotropin in a panhypopituitary patient. Fertil Steril. 1989;52:328-31. 13. Volpe A, Coukos G, Artini PG, et al. Pregnancy following combined growth hormone-pulsatile GnRH treatment in a patient with hypothalamic amenorrhoea. Hum Reprod. 1990;5:345-7. 14. Homburg R, West C, Torresani T, Jacobs HS. Cotreatment with human growth hormone and gonadotropins for induction of ovulation: a controlled clinical trial. Fertil Steril. 1990;53:254-60. 15. Davoren JB, Hsueh AJW. Growth hormone increases ovarian levels of immunoreactive somatomedin C/insulin-like growth factor I in vivo. Endocrinology. 1986; 118:888-90. 16. Adashi EY, Resnick CE, Svoboda ME, van Wyk JJ. Somatomedin C enhances induction of LH receptors by FSH in cultured rat granulosa cells. Endocrinology. 1985; 116:2369-75. 17. Jia X-C, Kalmijn J, Hsueh AJW. Growth hormone enhances follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology. 1986;118:1401-9. 18. Mason HD, Beard RW, Franks S. Stimulation of estradiol production by growth hormone (hGH) in cultured human granulosa cells [Abstract]. Proc of the 72nd Annual Meet of The Endocrine Soc. 1990;195. 19. Lloyd RV, Cano M, Landefeld TD. The effects of estrogens on tumor growth and on prolactin and growth hormone mRNA expres-
515
sion in rat pituitary tissues. Am J Pathol. 1988; 133:397-406. 20. Jin L, Song J, Lloyd RV. Estrogen stimulates both prolactin and growth hormone mRNAs expression in the MtT/F4 transplantable pituitary tumor. Proc Soc Exp Biol Med. 1989;192:225-9. 21. Phipps WR, Campbell BF, Pescovitz OH. Effect of naloxone on the growth hormone response to clonidine in normal women during the mid-luteal phase. Psychoneuroendocrinology. 1989;14:137-43. 22. Evans WS, Krieg RJ, Limber ER, Kaiser DL, Thorner MO. Effects of in vivo gonadal hormone environment on in vitro hGRF-40stimulated GH release. Am J Physiol. 1985;249:E276-80. 23. Ross RJM, Grossman A, Davies PSW, Savage MO, Besser GM. Stilboestrol pretreatment of children with short stature does not affect the growth hormone response to growth hormone-releasing hormone. Clin Endocrinol (Oxf). 1987;27:155-61. 24. Malacara JM, Valverde-R C, Reichlin S, Bollinger J. Elevation of plasma radioimmunoassayable growth hormone in the rat induced by porcine hypothalamic extract. Endocrinology. 1972;91:1189-92. 25. Szabo M, Frohman LA. Effects of porcine stalk median eminence and prostaglandin E2 on rat growth hormone secretion in vivo and their inhibition by somatostatin. Endocrinology. 1975;96:955. 26. Wardlaw SL, Wehrenberg WB, Ferin M, Antunes JL, Frantz AG. Effect of sex steroids on j8-endorphin in hypophyseal portal blood. J Clin Endocrinol Metab. 1982;55:877-81. 27. Wehrenberg WB, Wardlaw SL, Frantz AG, Ferin M. /3-Endorphin in hypophyseal portal blood: variations throughout the menstrual cycle. Endocrinology. 1982;111:879-81. 28. Stubbs WA, Delitala G, Jones A, et al. Hormonal and metabolic responses to an encephalin analogue in normal man. Lancet. 1978;2:1225-7. 29. Delitala G, Grossman A, Besser M. Differential effects of opiate peptides and alkaloids on anterior pituitary hormone secretion. Neuroendocrinology. 1983;37:275-9.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 05 June 2015. at 08:14 For personal use only. No other uses without permission. . All rights reserved.
