0021-972X/83/5704-0797$02.

00/0 Journal of Clinical Endocrinology and Metabolism Copyright 1983 by The Endocrine Society

Vol. 57, No. 4 Printed in U.S.A.

Induction of Midcycle Gonadotropin Surge by Ovarian Steroids in Women: A Critical Evaluation*

J. H. LlUt AND S. S. C. YEN*
Department of Reproductive Medicine, School of Medicine (T-002), University of California, San Diego, La Jolla, California 92093

ABSTRACT. Despite major efforts, the roles of estradiol (E2) and progesterone (P4) as signals for triggering the midcycle gonadotropin surge in women remain incompletely defined. In the present study, a preovulatory configuration of E2 and P4 levels was achieved during the midfollicular phase of normal women and in hypogondal women (with E2 implants) by stepwise incremental infusions of E2 and P4. During the 96 h of E2 infusion alone, reproducible and concomitant surges of LH and FSH occurred after latency periods of (mean SE) 58 6.9 and 68.2 2.6 h in midfollicular and hypogonadal subjects, respectively. The mean duration of the E2-induced surge was shorter for both normal midfollicular (22.7 3.9 h) and hypogonadal women (21.8 2.5 h) than is the spontaneous midcylce surge (48 h).

When P 4 infusion was superimposed 48 h after the initiation of E2 infusion, a LH/FSH surge that approximated the dimension of the spontaneous surge was achieved in both groups of women. For the hypogonadal women, but not midfollicular women, P4 infusion also advanced the surge onset by 14.7 h (P < 0.001). Our data represent the first unequivocal demonstration that incremental E2 sustained for about 60 h constitutes the ovarian signal for initiating the midcycle gonadotropin surge and that a small increment of P4 secreted by the preovulatory follicle is required to establish the normal dimension of the surge. Thus, P4, although essential for creating a normal gonadotropic surge, operates by synergizing the obligatory action of E2. (J Clin Endocrinol Metab 57: 797, 1983)

URING the past decade, numerous studies have described the negative and positive feedback actions of Ovarian steroids on gonadotropin secretion in women (1). However, unequivocal evidence that the increment in circulating estradiol (E2) from the dominant follicle is the sole signal for triggering the gonadotropin surge at midcycle in women, as in nonhuman primates (2-4), remains to be established. Attempts to reproduce surges in humans using pharmacological or physiological doses of various estrogens have yielded disparate results, with several studies reporting small concomitant LH and FSH release and others only LH release (5-10). Critical evaluation of these studies reveals that the LH surge induced by exogenous estrogen is smaller in dimension and in many instances resembles a rebound of LH rather than a surge. Induction of a gonadotropin surge comparable to the normal spontaneous surge has yet to be convincingly demonstrated. While infrequent sampling may preclude accurate measurement of the dimension, the absence of a concomitant FSH surge observed in
Received March 9,1983. * This work was supported by NIH Grant HD-12303 and in part by UCSD General Clinical Research Center NIH Grant RR-00827. This research was conducted in part by the Clayton Foundation for Research, California Division. t Fellow in Reproductive Endocrinology. $ Senior Clayton Foundation Investigator. To whom all correspondence and requests for reprints should be addressed.

several estrogen studies (9,10) introduces further uncertainty that the positive feedback effect of E2 is solely responsible for triggering the normal preovulatory surge of gonadotropin in women. Alternatively, the ability of a relatively small amount of progesterone (P4) to elicit a rapid increment of both LH and FSH after estrogen priming has been clearly demonstrated (8-14). Furthermore, small doses of P 4 augment the FSH and LH responses to GnRH (9, 15, 16). The secretion of P 4 by human granulosa cells from the preovulatory follicle is well established (17). Earlier reports showed a rise in P 4 at (18, 19) or before the LH peak (20, 21), but subsequent studies have established that circulating P 4 rises significantly about 12 h before the surge onset (22, 23). Thus, the ovarian signal(s) for the induction of the appropriate gonadotropin surge at midcycle in women requires reevaluation. In the present experiments, the differential roles of E2 and P 4 were examined using a protocol that permitted a relatively precise demonstration of the timing and dimensions of the gonadotropic surge as induced by incremental E2 alone and of the augmenting role of P 4 in both cycling and hypogonadal women. Materials and Methods
Five normal cycling women, aged 25-36 yr, and five hypogonadal women (four ovariectomized and one premature ovar797

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ian failure), aged 25-37 yr, participated in this study. None of the subjects had received any medication for at least 1 month before the study. This project was approved by the Human Investigations Committee of the University of California, San Diego, and informed consent was obtained from each volunteer. Experiments consisting of E2 and combined E2 with P 4 infusions to simulate the preovulatory circulating steroid conditions were performed in cycling and hypogonadal women. The E2 preparation used for the infusion experiments has been described previously (24). P4 stock solution was prepared by dissolving 10 mg P 4 in 2 ml absolute ethanol to which were added 7 ml propylene glycol and 1 ml distilled water (final concentration, 1 mg/ml). Sterilization was achieved by passage through a Millipore filter (Millipore Corp., Bedford, MA). Preparation of the P4 infusion solution was identical to that of the E2 infusion. Normal cycling women (cycle length, 27-31 days) were studied during the midfollicular phase of their cycles (days 7-9). Their mean (SE) baseline E2 level was 74.6 10.8 pg/ml. All experiments were begun at 0800 h. After two baseline samples were obtained at 15-min intervals, E2 was administered by a portable autoinfusion pump (Autosyringe, Hookset NH) for 96 h at incremental rates. It was determined previously that late follicular phase E2 levels can be achieved and maintained by the following rates of infusion (expressed as micrograms per kg/h): 0.2 for 12 h, 0.27 for 12 h, 0.33 for 12 h, and 0.4 for 60 h. Similarly, periovulatory P4 levels were attained by an infusion rate (micrograms per kg/h) of 1.5 for 24 h, followed by a rate of 2.0 for 24 h. Blood samples were obtained through an indwelling venous catheter in the opposite arm every 12 h for the first 48 h and then every 2 h during the last 48 h. In combined E2 and P 4 experiments, P 4 infusion was superimposed 48 h after initiation of the E2 infusion and continued through the last 48 h of the study. Hypogondal women received sc implants of an E2 pellet (25 mg; Schering Corp., Kenilworth, NJ). The procedures for E2 and E2 with P 4 infusion were identical to those described for normal cycling women. Experiments were performed between the third and sixth months after E2 implants, when the mean (SE) serum E2 levels were 78.6 10.3 and 50.7 4.4 pg/ml for the E2 and the E2 with P4 studies, respectively (Fig. 1). Serum levels of LH, FSH, E2, and P 4 were measured by RIA (16, 25-27). The initiation of the surge was defined as the first LH value exceeding the mean + 2 SDs of the two preceding values. The time of onset of the surge served as the temporal base (time zero) for expressing time-dependent changes in hormone levels. Analyses were performed using Student's t test.

E > E

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Results
In response to the E2 infusion alone, circulating E2 in midfollicular phase women increased from 65 13 to 300 29.1 pg/ml over the first 12 h and reached a relatively constant level of 365 87 pg/ml after 48 h (Fig. 2). LH and FSH levels were relatively stable until 58 6.9 h after initiation of E2 infusion (E2 concentration, 410 42.7 pg/ml), when a surge of both LH and

FSH occurred in all five women. The duration of the ascending limb of the surge (from onset to peak) was 12 8.7 h, with a descending limb lasting 10.7 h. The mean duration of the gonadotropin surge was 22.7 3.9 h, with a range of 18-32 h. Peak LH and FSH levels were 79.3 18.7 and 15 2.4 mlU/ml, respectively. After the surge, brief second rises in LH (A, 24.5 11.5 mlU/ml) and FSH (A, 4.6 2.7 mlU/ml) were observed. When P 4 was superimposed (Fig. 3), serum P 4 levels

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FIG. 3. Mean (SE) serum LH, FSH, E2, and P 4 concentrations in response to incremental E2 infusions superimposed by P 4 infusion in four midfollicular phase subjects.

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rose rapidly from 0.36 0.1 to 1.2 ng/ml during the first 4 h. Thereafter, a slower increment was achieved, with the serum P 4 level reaching 2.5 ng/ml at the end of the experiment. Incremental E2 levels were comparable to those in the first study. LH and FSH levels remained fairly constant until 59 4.1 h after initiation of E2 infusion and 11 h after beginning the P 4 infusion, when a simultaneous LH and FSH surge occurred in all women. Serum levels of E2 and P 4 were 315 55 pg/ml and 1.7 0.5 ng/ml, respectively, at the time of surge onset. Peak LH and FSH levels of 97.5 42.1 mlU/ml (range, 75.7-222 mlU/ml) and 13.9 0.8 mlU/ml, respectively, were attained. The mean duration of the surge was 36.5 h, with an ascending limb of 17.5 h and a descending limb of 19 h. In response to E2 infusion alone in five hypogonadal women (with E2 implants), the basal E2 levels increased from 78.6 10.3 pg/ml to a plateau of 443.7 68.3 pg/ ml and remained relatively constant thereafter (Fig. 4). With rising E2 levels, both LH (P < 0.05) and FSH (P < 0.05) levels declined. Twenty-four hours after initiation of E2 infusion, there was a brief increment in LH levels in two women. Thereafter, LH and FSH continued their decline until 68.2 2.6 h after initiation of E2 infusion, when a simultaneous LH and FSH surge occurred in all women. At the time of surge initiation, the mean E2 level was 430 105 pg/ml. Peak LH and FSH levels were 109 18 and 44.1 9.8 mlU/ml, respectively. The mean

FIG. 4. Mean (SE) serum LH, FSH, and E2 concentrations in response to incremental E2 infusion in five hypogonadal subjects with E2 implants.

duration of the surge was 21.8 2.5 h, with a short ascending limb (8.6 1.3 h) and a descending limb of 13.2 h. A shoulder effect, with release of both LH and FSH after the surge, was again observed. In separate experiments, when P 4 infusion was superimposed, serum E2 and P 4 levels increased in a fashion similar to that found in the study performed in midfollicular women (Fig. 5). Both LH and FSH declined after E2 infusion, but the decrease was statistically significant only for FSH (P < 0.02). LH and FSH surges occurred 53.5 4.4 h after beginning the E2 infusion and 5.5 h after beginning the P 4 infusion. E2 and P 4 levels were 530 63 pg/ml and 1.2 0.1 ng/ml, respectively, at the time of surge initiation. The total duration of the LH surge was 44.5 2.9 h, with an ascending limb of 27 h and a descending limb of 17.5 h. Peak LH and FSH levels were 215 36.7 and 69.2 21 mlU/ml, respectively. There was marked episodic LH release in individual women (Fig. 6), accounting for the large SE in LH concentrations. The differences in LH and FSH surge dynamics in response to E2 infusion alone and E2 with P 4 infusion in hypogonadal and midfollicular phase women are shown in Fig. 7. The addition of P 4 significantly prolonged the surge duration in both midfollicular (P < 0.05) and

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JCE & M 1983 Vol 57 No 4 80

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FIG. 7. Comparison of the LH and FSH surge dynamics (mean SE) between midfollicular phase and hypogonadal subjects during E2 and combined E2 with P4 infusions. A Max, The time from surge onset to peak. Latent phase refers to the time from the initiation of E2 infusion to the onset of the surge. *, P < 0.05; **, P < 0.001.

HOURS FIG. 6. Episodic fluctuations during the surges of LH and FSH in one hypogonadal subject in response to incremental E2 and P4 infusion.

hypogonadal (P < 0.001) women. In hypogonadal women, a shortened latent period (A, 14.7 h; P < 0.001) for surge initiation as well as an increased duration (A, 18.4 h; P < 0.05) of the ascending limb were also evident.

Discussion
In the present study, through the infusion of incremental doses of E2 and P4, we have succeeded in creating

a circulating steroid environment in both midfollicular phase and hypogonadal women, the latter with E2 implants, that mimics the preovulatory phase of the normal menstrual cycle (19-23, 28-32). Hormonal dynamics at midcycle, both quantitatively and qualitatively, described by Hoff et al. (23) were used as a reference for comparison of experimentally elicited surges of gonadotropins. Our study has convincingly demonstrated that incremental E2 alone can reproducibly elicit simultaneous surges of LH and FSH in both midfollicular and hypogonadal women. This finding is in general agreement

ROLE OF E2 AND P4 IN MIDCYLE SURGE with observations made by Yen and Tsai (5) and Monroe et al. (6), but is in contrast to the findings of Chang and Jaffe (9) and March et al. (10) in which incremental E2 (via daily im injections of E2 benzoate) induced LH release without an accompanying FSH rise. A satisfactory explanation to account for the failure to elicit concomitant FSH release in these studies is not apparent. After the surge, a brief second rise in LH or a shoulder effect occurred in both midfollicular and hypogonadal women during the E2 infusion. This biphasic pattern of LH release has been occasionally observed during the normal spontaneous midcycle surge (33). In both midfollicular and hypogonadal women, the initiation of the gonadotropin surge required 58-68 h of incremental E2 priming, which closely approximates the period between the appearance of the accelerated rise in circulating E2 and the onset of the spontaneous midcycle surge in normal women (23). However, the surge duration of 21-24 h induced by incremental E2 was clearly shorter than the 48 h spontaneous midcycle surge (19, 22, 23). This difference may be due to the maintenance of high E2 levels in the present experimental procedure, whereas the spontaneous midcycle surge is accompanied by an acute decline of E2 levels 6 h after surge onset (23). Furthermore, the shortened surge duration observed with E2 infusion alone may reflect the absence of amplification by P 4 . The present study demonstrates that superimposed P 4 infusion producing circulating levels resembling those found before and during the onset of the spontaneous midcycle surge (1.2-1.9 ng/ml) (22, 23) caused the induced gonadotropin surge duration of both LH and FSH to increase significantly to 36.5 h in midfollicular phase women and 44.5 h in hypogonadal women. This increased duration primarily reflects the prolongation of the ascending limb of the surge. In addition, the surge magnitude was amplified and the latent period was shortened in hypogonadal women but not in midfollicular women. These disparate responses may be explained by the differences that may exist in the neuroendocrine status of the hypothalamic-pituitary system between normal midfollicular women and hypogonadal women with E2 implants. In conclusion, the data provided by this study establish that in women, incremental E2 is the ovarian signal that triggers the onset of the LH-FSH surge and that P4, in the levels found during the preovulatory phase of the cycle, amplifies the duration of the surge and augments the effect of E2 rather than serving an obligatory role. Thus, preovulatory levels of P 4 may be required for the full expression of the surge in women.

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References
1. Yen SSC 1982 Neuroendocrine regulation of gonadotropin and prolactin secretion in women: disorders in reproduction. In: Vaitukaitis JL (ed) Clinical Reproductive Neuroendocrinology. Elsevier, New York, pp 137-176 2. Goodman RL, Knobil E 1981 The sites of action of ovarian steroids in the regulation of LH secretion. Neuroendocrinology 32:57 3. Wildt L, Hausler A, Hutchison JS, Marshall G, Knobil E 1981 Estradiol as a gonadotropin releasing hormone in the rhesus monkey. Endocrinology 108:2011 4. Ferin M, Rosenblatt H, Carmel PW, Antunes JL, Van de Wiele RL 1979 Estrogen-induced gonadotropin surges in female rhesus monkeys after pituitary stalk section. Endocrinology 104:50 5. Yen SSC, Tsai CC 1972 Acute gonadotropin release induced by exogenous estradiol during the midfollicular phase of the menstrual cycle. J Clin Endocrinol Metab 34:298 6. Monroe SE, Jaffe RB, Midgley AR 1972 Regulation of human gonadotropins. XII. Increase in serum gonadotropins in response to estradiol. J Clin Endocrinol Metab 34:342 7. Leyendecker G, Wardlaw S, Nocke W 1972 Experimental studies on the endocrine regulations during the periovulatory phase of the human menstrual cycle. Acta Endocrinol (Copenh) 71:160 8. Aono T, Miyake A, Kinugasa T, Kurachi K 1976 Progesterone advancement of estrogen-induced luteinizing hormone release during the midfollicular phase in normal cyclic women. J Endocrinol 71:451 9. Chang RJ, Jaffe RB 1978 Progesterone effects on gonadotropin release in women pretreated with estradiol. J Clin Endocrinol Metab 47:119 10. March CM, Gobelsmann U, Nakamura RM, Mishell DR1979 Roles of estradiol and progesterone in eliciting the midcycle luteinizing hormone and follicle-stimulating hormone surges. J Clin Endocrinol Metab 49:507 11. Odell WD, Swerdloff RS 1968 Progestogen induced luteinizing and follicle stimulating hormone surge in postmenopausal women: a simulated ovulatory peak. Proc Natl Acad Sci USA 61:529 12. Nillius SJ, Wide L1971 Effects of progesterone on the serum levels of FSH and LH in postmenopausal women treated with estrogen. Acta Endocrinol (Copenh) 67:362 13. Leyendecker G, Wildt L, Gips H, Nocke W, Plotz E 1976 Experimental studies on the positive feedback effect of progesterone, 17ahydroxyprogesterone, and 20adihydroprogesterone on the pituitary release of LH and FSH in the human female. Arch Gynaekol 221:29 14. Rakoff JS, Rigg LA, Yen SSC 1978 The impairment of progesterone-induced pituitary release of prolactin and gonadotropin in patients with hypothalamic chronic anovulation. Am J Obstet Gynecol 130:807 15. Shaw RW, Butt WR, London DR 1975 The effect of progesterone on LH and FSH response to LHRH in normal women. Clin Endocrinol (Oxf) 4:543 16. Lasley BL, Wang CF, Yen SSC 1975 The effects of estrogen and progesterone on the functional capacity of the gonadotrophs. J Clin Endocrinol Metab 41:820 17. McNatty KP, Makris A, deGrazia C, Osathanondh R, Ryan KS 1979 The production of progesterone, androgens and estrogens by human granulosa cells, thecal tissue and stromal tissue from human ovaries in vitro. J Clin Endocrinol Metab 49:687 18. Johansson EDB, Wide L 1979 Periovulatory levels of plasma progesterone and luteinizing hormone in women. Acta Endocrinol (Copenh) 62:88 19. Thorneycroft IH, Syribyatta B, Tom WK, Nakamura RM, Mishell DR 1974 Measurement of serum LH, FSH, progesterone, 17hydroxyprogesterone and estradiol-170 levels at 4 hour intervals during the periovulatory phase of the menstrual cycle. J Clin Endocrinol Metab 39:754 20. Yussman MA, Taymor ML 1970 Serum levels of follicle stimulatory hormone and of plasma progesterone related to ovulation by corpus luteum biopsy. J Clin Endocrinol Metab 30:396 21. Abraham GE, Odell WD, Swerdloff RS, Hopper K 1972 Simultaneous radioimmunoassay of plasma FSH, LH, progesterone, 17-

Acknowledgments
We acknowledge the technical assistance provided by P. Malcom, J. Aurand, and J. Wong. Our special appreciation goes to Dr. A. Lein for his critical review and to Dr. R. Rebar for patient referrals.

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ease. Am J Obstet Gynecol 121:496 28. Ross GT, Cargille CM, Lipsett MB, Rayford PL, Marshall SR, Strott CA, Rodbard D 1970 Pituitary and gonadal hormones in women during spontaneous and induced ovulatory cycles. Recent Prog Horm Res 26:1 29. Mishell DR Jr, Nakamura RM, Crosignani PG, Stone S, Kharma K, Nagata Y, Thorneycroft IH 1971 Serum gonadotropin and steroid patterns during the normal menstrual cycle. Am J Obstet Gynecol 111:60 30. Moghissi KS, Syner FN, Evans TN 1972 A composite picture of the menstrual cycle. Am J Obstet Gynecol 114:405 31. Korenman SG, Sherman BM 1973 Further studies of gonadotropin and estradiol secretion during the preovulatory phase of the menstrual cycle. J Clin Endocrinol Metab 36:1205 32. Landgren BM, Aedo AR, Nunez M, Cekan SZ, Diczfalusy E 1977 Studies on the pattern of circulating steroids in the menstrual cycle. Acta Endocrinol (Copenh) 84:620 33. Thomas K, Walckiers R, Ferin J 1970 Biphasic pattern of LH midcycle discharge. J Clin Endocrinol Metab 30:269

IV International Congress on Prolactin

The IV International Congress on Prolactin will be held June 27 to 29, 1984 in Charlottesville, Virginia, U.S.A. The program will consist of invited lectures and oral and poster presentations chosen on the basis of submitted abstracts. For further information please contact: Robert M. MacLeod, Ph.D. IV International Congress on Prolactin University of Virginia School of Medicine Charlottesville, VA 22908, U.S.A.