Mechanisms Regulating the Menstrual Cycle in Women1

Mechanisms Regulating the Menstrual Cycle in Women 1 RAYMOND L. VANDE WIELE, 2 J E F F BOGUMIL, INGE DYRENFURTH, MICHEL FERIN, RAPHAEL JEWELEWICZ, 3 MICHELLE WARREN, 3 TAWFIK RIZKALLAH, 4 AND GEORGE MIKHAIL 5 Department of Obstetrics and Gynecology, and the International Institute for the Study of Human Reproduction, College of Physicians and Surgeons of Columbia University, New York City, New York

I. Introduction Recently, remarkable progress has been made in the treatment of the patient with anovulation. Within a few years, a syndrome that heretofore was a major irreducible cause of infertility has almost ceased to exist as a therapeutic problem. Great as the satisfaction over these advances ought to be, it should not detract from the realization that the ultimate therapeutic aim, the restoration of spontaneously cyclic ovarian function, still eludes us. Indeed following induction of ovulation, be it by gonadotropins (Gemzell et al, 1968; Vande Wiele and Turksoy, 1965), or Clomid (Kistner, 1968), whenever therapy is stopped, the patient reverts to her original abnormal ovarian function. The only therapeutic modality that is a frequent exception to this rule, surgical resection of the ovaries, cures only a fraction of the patients with anovulation, and the mechanism by which it does so, remains an utter mystery. It is unlikely that much progress will be made toward this aim of restoring spontaneously cyclic function in amenorrheic women until more is known about normal reproductive function. Much information is available about individual events in the human ovarian cycle, but until very recently, little if any progress has been made toward an integration of the various elements in this extremely complex sequence of events. The problem is a formidable one, and its solution could not have been attempted before the recent breakthrough in analytic techniques. Radioimmunoassays and variants thereof have made it possible to follow, in the same individual, on a daily or even more frequent basis, the many changes in hormonal levels that have to be taken into account if one is to understand the regulation of the menstrual cycle. To be considered are the changes in FSH, LH, estradiol, estrone, progesterone, and androstenedione to mention 1

Supported in part by U.S. Public Health Service grant HD-02996. Career Scientist, Health Research Council of the City of New York. 3 U.S.P.H.S. Trainee. 4 Present address: Woman's Hospital, St. Luke's Hospital Center, New York, New York. 5 Present address: Southwestern School of Medicine, University of Texas, Dallas, Texas, 2

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only the most important ones. The real difficulty however lies not so much in the analytic task, big as it may be, but arises from the fact that at all times the changes in these hormones are coupled, each of them controlling as well as being controlled by the others. The complexity of such a situation is extreme, and it is evident that conventional methods fail when applied to the understanding of changes in such a complicated network of interacting processes. To meet these difficulties, and with the help of members of the Engineering Department of Columbia University, we have made extensive use of the principles and methods of systems analysis. Attempts to apply the "systems" approach to the study of reproductive processes have been reported before (Rapoport, 1952; Thompson et al, 1969; Schwartz, 1969). Most of the methods used in our studies have been published in detail. FSH and LH were measured by radioimmunoassay (Midgley, 1966, 1967). Pro­ gesterone by the radioligand method (Neill et al., 1967). To measure plasma estradiol and estrone, we used a radioimmunoassay, recently developed in our laboratory, employing antibodies that were made by the application of the hapten principle (Mikhail et al., 1970). In a few instances, plasma estrogens were measured by the method of Abraham (1969a). This presentation is divided into four sections. The first section deals with the events in the early and late proliferati ve period of the menstrual cycle, the main emphasis being on the relationship between FSH and estrogens. In the second section, evidence will be presented indicating that the rising titer of estrogens is the trigger of the preovulatory LH release. In the third section are discussed studies that relate to the control of the secretion of progester­ one by the corpus luteum and of the duration of the postovulatory period. In the fourth section, a short description will be given of the manner in which the available physiological information may be incorporated into a model of the menstrual cycle. Finally, some results of computer simulation studies based on this model will be briefly described. II. The Preovulatory Period In terms of plasma estrogens, the preovulatory period can be divided into two phases. In the first phase, which lasts until 6 or 7 days before the LH surge, their level is low and virtually constant. During the second phase, estrogens rise, first slowly, then very rapidly, reaching a maximum the day before or the day of the LH surge. Two cycles that are typical for a series of 18 normal cycles studied by us (Dyrenfurth et al., 1970), are illustrated in Fig. 1. Similar results were reported by Abraham (1969a,b) and by Korenman et al. (1969a,b). The changes in the urine are not as marked but have the same general configuration (H. Burger et al., 1968). In addition to these quantitative changes, there is a change in the ratio of plasma estradiol to estrone, this ratio being lower in the early than in the later

M E C H A N I S M S REGULATING T H E MENSTRUAL

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CYCLE

part of the cycle. This finding confirms an earlier observation by Baird and Guevara (1969), who used a double-isotope derivative method to measure plasma estradiol and estrone. The explanation for this change in ratio most likely lies in the dual origin of plasma estrogens. In the normal female, with intact adrenal function, a significant fraction of plasma estrone is derived from the peripheral conversion of androstenedione secreted by the adrenal 700

W.F.

600· 500 E,-E 2 (pg/ml)

400300-1 200 H

100-1 0

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FIG. 1. Plasma levels of estradiol and estrone in two normal women with regular menstrual cycles.

(MacDonald et al., 1967); (Baird et al., 1968). At the time of menstruation and immediately thereafter, this fraction is relatively important since the contribution by the ovary at this time is small. The growing follicle, how­ ever, as shown by studies of ovarian vein blood (Lloyd et al., 1969; Mikhail, 1967), secretes mainly estradiol and only very small, if any, significant amounts of estrone, thereby increasing the estradiol to estrone ratio. The most important relationship at this time of the cycle is obviously that between estrogens and gonadotropins. It is likely that, in this respect, the changes in FSH are more important than the changes in LH. Studies in which amenorrheic patients have been given gonadotropins to stimulate ovarian function indeed indicate that, provided the FSH-LH ratio remains

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RAYMOND L. VANDE WIELE ET AL.

within certain limits, it is the dose of FSH rather than that of LH that is the important quantitative determinant in the response of the ovarian follicle (Jacobson and Marshall, 1969). In Fig. 2 is shown a scattergram of the levels of plasma estradiol on various days prior to the LH surge. We have not calculated the slope of the estrogen curve since more data remain to be collected. The general shape of the curve, however, is evident upon simple E 2 (pg/ml)

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inspection. Above the values for plasma estradiol, we have indicated the range of the FSH levels corresponding to that part of the cycle. A reciprocal relationship between the two variables is evident and obviously not un­ expected. After all, the push-pull theory of the FSH:estrogen relationship was first enunciated 40 years ago (Moore and Price, 1930; Brouha and Simonnet, 1930; Lamport, 1940). It is of interest to note that, although the negative feedback effect of estrogens has been known for several decades, there are no precise data on the temporal and quantitative aspects of this relationship. Qualitative studies abound (Chester-Jones and Bael, 1962; Flerko, 1966; Donovan, 1966; Harris and Campbell, 1966), but, as far as we are aware, there is no study in which estradiol has been given, by continuous infusion, in amounts that

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MECHANISMS REGULATING THE MENSTRUAL CYCLE

approach physiological rates of secretion and during which FSH or LH has been measured at frequent enough intervals to make it possible to evaluate the exact timing of the change in gonadotropin levels. Neither has an attempt been made to determine by such a design the smallest dose of estradiol, per unit of time, that will produce a significant change in FSH or the maximum depression of FSH that can be obtained. 2500-1

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FIG. 3. LH and FSH levels in postmenopausal women to whom estradiol was admin­ istered by continuous infusion.

We have preliminary answers to some of these questions. To menopausal women, we have administered continuous infusions of estradiol and estrone at rates that approach physiological rates of secretion. FSH and LH levels were determined at 6-hour intervals. In Fig. 3 are shown the results of two experiments during which estradiol was infused at rates of 100 and 150 ìg/24 hours. These rates correspond to secretory rates in the early proliferative phase (Vande Wiele et al., 1968). In both cases, the depression in FSH and LH levels was statistically significant. An infusion of estradiol at 50 ìg/24 hours produced borderline significant results whereas an infusion of estrone at 600 ìg/day failed to produce significant changes in either FSH or LH.

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More important perhaps, the decrease in gonadotropins was evident within 6 hours after the beginning of the infusion. For computer simulation studies, it is necessary to know the function that describes the estradiol-FSH relationship. The data we have from our continuous infusion studies are clearly insufficient for this purpose since we

i

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y 2 S 4000.e-° 0 5 y ^) + 3 5 0 . e - a 0 0 3 3 y ( 4 ) + 5 0 0 FIG. 4. Levels of FSH as a function of the levels of estradiol. For the calculation of data, see text.

have explored only a small fraction of the total range of estrogen secretions. Provided one is satisfied with an approximation, this function can be cal­ culated from data in the literature. In doing this it must be assumed that any change in FSH resulting from a change in the estrogens occurs rapidly, i.e., within less than half a day, an assumption that appears warranted even from our preliminary data with continuous infusions of estrogens. A graphic repre­ sentation of this function is shown in Fig. 4. The FSH data are those published by Cargille et al. (1969); the estrogen data are ours. When the ovarian secretion of estrogens is zero, as in castrates or in menopausal women, FSH is maximal. On the other hand, available data indicate that

M E C H A N I S M S REGULATING T H E MENSTRUAL CYCLE

69

very large amounts of estrogens, in excess of any amount ever made by the ovary still will not depress FSH levels to zero (Odell et al., 1968). In plotting the graph, we have assumed that beyond 400 picograms of estradiol per milliliter there is no further depression of FSH; 50 to 800 pg/ml is the range found in our studies of the menstrual cycle (Dyrenfurth et al., 1970). The slope of the curve is steepest when estrogen levels are below the minimum levels seen during the normal menstrual cycle, and in this area of the graph, small changes in estrogens will produce large changes in FSH. On the other hand, during the normal menstrual cycle, even major changes in estrogens produce only small changes in FSH. It must be stressed that the graph in Fig. 4 is a composite of data from various sources and that the position of the elbow in the curve is only approximate. Studies are clearly indicated to determine its exact location. For the computer simulation studies, we have used the function illustrated in Fig. 4, a mathematical approximation of which is shown at the bottom of the figure. In considering changes in FSH as a result of a negative feedback mech­ anism, we have disregarded the effect of androgens, progesterone, and other steroids. Available evidence indicates that androgens even at high concentra­ tions have only a minor effect on FSH levels (Buchholz, 1959). We have, however, preliminary evidence that progesterone administered in continuous infusions at rates that approximate rates of secretion during the normal postovulatory period depress FSH levels. It may therefore become necessary to include the negative feedback role of progesterone in simulation studies of the postovulatory period. After considering the effect of estrogens upon FSH secretion, the inverse aspect of this relationship remains to be discussed. As is evident from Fig. 4, the effect of FSH on estrogen secretion is complex and is obviously not a simple proportional one. At the beginning of the cycle, when FSH levels are high, there is little change in the level of estradiol, and the greatest rate of increase in estradiol corresponds to a minimum in the level of FSH. The curve behaves as if, with increasing maturation, the follicle becomes progres­ sively more sensitive to the effect of FSH. To study the relationship between FSH and estradiol, a convenient model may be found in the amenorrheic woman treated with constant doses of FSH. In such patients, endogenous gonadotropic secretion is small or nil, and changes in FSH as a result of a negative feedback mechanism can, there­ fore, be ignored. In Fig. 5 is illustrated a series of studies in patients treated by us with human menopausal gonadotropins (HMG). The similarity be­ tween the estrogen curves in these patients and those in normally cyclic women is striking. After a latent period which is variable in length, estrogen secretion starts and then within 6 or 7 days rises precipitously to ovulatory levels. Fig. 6 illustrates two more such studies, and in these cases, in addition

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to plasma levels of estrogens, we also measured plasma levels of FSH as further confirmation of the constant level of stimulation. Similar results were reported by J. B. Brown et al. (1969) and by Butler (1969). It is clear that the response of the ovary to the gonadotropins is not a simple function of the level of FSH. As the follicle grows, additional factors

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DAYS OF TREATMENT FIG. 5. Urinary estrogens in patients treated with human menopausal gonadotropins (HMG). The amount of HMG administered is indicated by the height of the shaded

come into operation that potentiate the response of the follicle to the stim­ ulatory effect of FSH. Physiological mechanisms are known that could explain this behavior. Estradiol itself, by a local effect, stimulates the growth of the ovarian follicle even in the absence of FSH, and also potentiates its response to gonadotropins. This effect has been documented by the adminis­ tration of estrogens, either systemically or locally, to hypophysectomized animals (Ingram, 1959; Croes et al., 1959; Meyer and Bradbury, 1960; Bradbury, 1961), and even in organ cultures of growing rat follicles (Kullander, 1961). The reverse effect, one of inhibition, is seen with androgens. Androgens, again by a local mechanism, inhibit follicular growth and de­ crease the sensitivity of the follicle to gonadotropin stimulation (Payne et al., 1956). Such an inhibitory effect of androgens was demonstrated even in

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KAYMOND L. VANDE W I E L E ET AL.

humans by the intraovarian injection of a long-acting testosterone ester in amounts that, when administered systemically, did not disturb the menstrual cycle (Hoffmann and Meger, 1965). The intraovarian effect of androgens and its possibly important role in the menstrual cycle, has not been suffi­ ciently appreciated, to some degree because not enough attention has been given to the cyclic changes in ovarian androgen secretion in the human. Partly, this is due to the fact that androgen levels in peripheral blood do not Early to mid follicular

FIG. 7. Steroids in ovarian venous plasma of 18 women with normal menstrual cycles (Lloyd et al, 1969).

change dramatically during the cycle. Androstenedione and other plasma androgens derive to an important degree, either directly or indirectly from adrenal secretions. Consequently, changes in the peripheral blood resulting from cyclic secretions of androgens by the ovary will be masked by this adrenal contribution (Vande Wiele et al., 1968). The magnitude of the changes in ovarian androgen production is evident from Fig. 7, which illus­ trates the results of an unpublished study of androgen levels in ovarian vein blood (Lloyd et al., 1969). Androstenedione levels in ovarian vein blood sharply increase in the late follicular period, decrease slightly during the early luteal, to reach a second peak during the mid-luteal period. Smaller changes in testosterone and dehydroisoandrosterone secretion parallel those of androstenedione. Similar studies have been reported by Mikhail (1967). These important intraovarian fluctuations in the concentrations of andros­ tenedione may become important determinants in the growth of the ovarian follicle. It remains to be shown that androstenedione, in concentrations that cor­ respond to the intraovarian concentrations observed during the normal

M E C H A N I S M S REGULATING T H E MENSTRUAL CYCLE

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menstrual cycle, exerts an effect on the growing follicles. Up to now, all experimental designs have ignored this important quantitative aspect of the relationship between androgens and the follicle. Experiments designed to answer this important question are now in progress. In modeling the effects of gonadotropins on estrogen levels, we have taken these complex local effects of estrogens and androgens into account. Figure 8 shows some of the equations we have used in our computer studies to describe the factors controlling the changes in estrogen and androgen levels. These equations introduce a novel and still somewhat hazy concept—the z ( 1 ) = Measure of largest follicle xa) = F D [ G E - y ( 2 ) · ? ( 8 ) + E F - y ( 4 ) - A F 1 ^ ( 5 ) ] ^ ( 1 ) _ FM-*» ( 1 ) FSH-LH Est. Androg. # ( â ) = Average measure of other follicles *(6) = F D [ G E - y ( 2 ) . y ( 8 ) + E F - y ( 4 ) - A F e - y ( 5 ) ] * 2 ( e ) - FM-*8 ( e ) FSH-LH Est. Androg. y ( 4 ) = Plasma level of estradiol* y ( 4 ) = { l [ x ( 1 ) - 0.01] 2 + 25[* ( â ) - 0.008] 2 } F E - y ( 2 ) + AE FSH y ( 5 ) == Concentration of androstenedione* y ( 5 ) = {10[* ( 1 ) - 0.01]2 + 25[÷ ( â ) - 0.008] 2 } F A - y ( 2 ) + AA * For x(1) > 0.01 a n d * ( 6 ) > 0.008 FIG. 8. Equations used in the computer studies to simulate the changes in plasma estrogen (Est.) and androgen (Androg.) levels during the preovulatory period.

measure of the follicle—a concept which is introduced to represent the changing sensitivity of the follicle as maturation proceeds. X\, the measure of the follicle about to ovulate, as well as xe, the mean measure of the other follicles, are complex functions of many factors, including FSH and LH effects, a positive term to account for the estrogen and a negative term to account for the androgen effects. The negative terms of x1 to the third power are necessary to prevent the measure of the follicle from reaching explosive values. y±, and y-}) the levels of estrogens and androstenedione respectively, are functions of x1 and xQ and of FSH. As is evident from the shape of the equation, estrogen or androgen secretion will not start to rise until xx and #6 are larger than 0.01 and 0.008, respectively. I I I . The Role of Estrogens and Progesterone as Triggers of the Ovulatory L H Release Both estrogens and progesterone have been implicated as triggers of the ovulatory LH surge. In the immature rat, Hohlweg (1934) demonstrated that the administration of estrogens results in the formation of corpora lutea.

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RAYMOND L. VANDE W I E L E ET AL.

Many studies amplified this initial observation, and the evidence in favor of estrogens as the trigger of LH release in rats was recently reviewed by Shirley et al. (1968). Goding et al. (1969), using a radioimmunoassay to measure plasma LH, demonstrated that in the sheep, the administration of estradiol-17ß in amounts as small as 10 ìg will produce a surge of LH. The LH levels peaked 9 hours after the injection of the estrogen and in many instances, the peak was indistinguishable from that preceding spontaneous ovulation. In the human, also, there is evidence that estrogens trigger LH release. W. E. Brown et al. (1953), and later other investigators (Zondek, 1954; Kupperman et al., 1958), reported that in many anovulatory women, ovula­ tion can be induced by the administration of estrogens. Other workers, how­ ever, were less successful with this treatment (Bickenbach and Döring, 1959; M. Burger, 1960; Probst and Beller, 1961). On the other hand, increases in urinary gonadotropins regularly follow the administration of estrogens as reported by various investigators (Vorys et al., 1965; Kaiser et al., 1966), most recently by Stevens and Vorys (1967). Using a radioimmunoassay to measure LH and FSH in blood, Swerdloff and Odell (1969) presented evidence that synthetic estrogens will produce repeated bursts of LH but not of FSH. We have recently reevaluated the role of intravenous estrogens on LH re­ lease. Fig. 9 shows the results of four such studies in three women who were anovulatory, but presented clinical evidence of estrogen production. Follow­ ing the intravenous administration of 20 mg of Premarin, there was a signif­ icant rise in plasma LH which in two out of the three patients was of the magnitude seen during the spontaneous ovulatory LH surge. The interval between the administration of the estrogen and the rise in LH, 36 hours, was surprisingly long and remarkably constant. In none of these patients was ovulation induced, as evidenced by the absence of a biphasic temperature chart or sustained changes in the blood progesterone. These results again illustrate the necessity of looking simultaneously at all parameters of the reproductive system. If we had tested only for ovulation, the effect of the administration of Premarin would have been thought to be nil. This absence of ovulation is not surprising. In the normal individual, estrogen secretion and follicular maturation are coupled, and the LH surge will not occur until the follicle is competent to respond to ovulatory stimulus of LH. Following the injection of Premarin, the LH surge occurs, however, prematurely and, unless fortuitously the estrogen had been administered at a time when an almost mature follicle was present, ovulation should not be expected to occur. Progesterone, under certain conditions, will trigger LH release, but under others it will postpone ovulation (Everett, 1948). It has been shown to

MECHANISMS REGULATING THE MENSTRUAL CYCLE

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induce or to advance ovulation in several animal species as well as in women (Flerko, 1966; Fraps and Dury, 1943; Nallar et al, 1966). More recently, Odell and Swerdloff (1968), studying plasma FSH and LH levels in castrates and menopausal women whose elevated gonadotropic levels had been de­ pressed by the administration of estrogens, showed that progesterone in PREMARIN (20mg i.v.)

PROG. (ng/ml)

FIG. 9. LH and progesterone levels in anovulatory women following administration of 20 mg of Premarin.

intravenous

amounts as small as 10 mg administered intramuscularly, will produce a sudden and transient rise in LH and FSH. Similar results have been reported by Thomas and Ferin (1969). Before either estrogens or progesterone can be implicated as triggers of the ovulatory LH surge during the normal menstrual cycle, it is necessary to examine the temporal and quantitative relationships between the plasma concentrations of estradiol and progesterone and of LH. In a group of young women with normal cycles, we have measured plasma levels of estradiol, estrone, progesterone, FSH, and LH throughout one or more complete

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RAYMOND L. VANDE WIELE ET AL.

menstrual cycles (Dyrenfurth et al, 1970). Figures 10 and 11 illustrate the results of two such studies and make it possible to compare the changes in estradiol and in progesterone in relation to the LH peak. It is evident that estrogens reach a maximum either before or at the time of the LH peak, S.R.

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FIG. 10. Simultaneous determinations of LH, FSH, estradiol, progesterone, and basal body temperature in a woman with normal ovulatory menstrual cycles.

whereas plasma progesterone does not show any significant changes until the LH surge is well on the way. Figure 12 shows levels of LH and of progester­ one during three consecutive cycles of a normal young woman. Clearly there is no change in the concentration of plasma progesterone that could serve as a signal to the hypothalamus or the pituitary for the release of LH. The same relationship between plasma levels of estrogens and the LH surge was reported by Abraham (1969b) and Korenman et al. (1969a). Another line of evidence implicating estrogens rather than progesterone

M E C H A N I S M S REGULATING T H E MENSTRUAL

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77

as the trigger for the LH surge during the normal menstrual cycle, derives from studies in women in whom ovulation was induced by indirect means. In an occasional patient treated with gonadotropins, ovulation will occur during the FSH phase of the treatment and prior to the administration of the chorionic gonadotropin, the agent which is usually required to induce ovulaMENSES |

PROG. (ng/ml)

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FIG. 11. Simultaneous determinations of LH, FSH, estradiol, progesterone, and basal body temperature in a woman with normal ovulatory menstrual cycles.

tion. In several such cases studied by us, ovulation, and presumably therefore the endogenous LH release, was preceded by an increase in the urinary estro­ gens without any concomitant increase in pregnanediol. Figure 13 illustrates a study in a patient in whom ovulation was induced by the administration of Clomid. The same sequence as that seen in the spontaneously ovulating women is clearly indicated. There is an increase in estrogens, most likely the result of the early increase of FSH (Ross et al., 1970). The LH release follows the rise in estrogens and definitely precedes any change in blood

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RAYMOND L. VANDE WIELE ET AL.

progesterone. Similar results have been recently published by Mauvais-Jarvis (1969). Although these considerations strongly favor the estradiol rather than the progesterone theory of LH release, evidence remains circumstantial. Further­ more, they do not rule out the possibility that other steroids may be involved as triggers of the LH surge. Strott and Lipsett (1968) reported that prior to

60 A PLASMA PROG. (ng/ml)

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PLASMA PROG. (ng/ml)

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PLASMA PROG. (ng/ml)

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FIG. 12. Plasma LH and progesterone levels during three consecutive cycles in a woman with normal ovulatory menstrual cycles.

the LH surge there is an increase in 17a-hydroxyprogesterone. Earlier in this discussion, we presented evidence that prior to the LH release the ovary secretes significant amounts of androgens, mainly androstenedione, but also small amounts of dehydroisoandrosterone and testosterone. Direct evidence for the estrogen theory of LH surge may be found in experiments we recently carried out using antibodies to estrogens and to progesterone. Since these antibodies have no biological activity, other than by binding the steroids in the circulation, and thereby preventing them from reaching their target organs, they are uniquely useful as tools in assessing the role of steroids in certain physiological processes. Figure 14 illustrates the inhibition by antibodies to estradiol of the peripheral effect of estradiol in a classical bioassay, the uterine weight assay system. Most important, as

M E C H A N I S M S REGULATING T H E MENSTRUAL

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79

shown in the figure, antibodies to estrogen will not inhibit the biological effects of stilbestrol, a synthetic estrogen having a molecular structure that differs from that of the natural estrogens (Ferin et al., 1968). The half-life of the antibody is rather short, and its effect fades within 2 or 3 days unless repeated injections are given. We have carried out rather exhaustive studies Clomid (lOOmg/d) 3 < E

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to evaluate the specificity of these antibodies to estradiol. The biological activity of estradiol is not inhibited by the administration of antibodies to testosterone or to progesterone. Antibodies to estradiol, on the other hand, will not inactivate the biological effect of androgen or progesterone (Ferin et al., 1968, 1969a,b). Proof of the specificity of the antibodies to estrogen has also been obtained by studies in the radioimmunoassay system (Mikhail et al, 1970). We have used two experimental models to study the role of estrogens in the initiation of the LH surge. One model was the immature rat treated with pregnant mare serum (PMS) (Ferin et al., 1969a); the other, the 4-day-

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RAYMOND L. VANDE WIELE ET AL.

cycle mature rat. The experiments involving this latter model have been recently published (Ferin et al., 1969b) and are illustrated in Fig. 15. In the 4-day-cycle animal, LH release occurs in the afternoon of proestrus. The LH discharge is preceded by a rise in the estrogens in the ovarian vein as well as in the peripheral blood, as recently shown by Yoshinaga et al. (1969). As expected, this increase in estrogen levels is accompanied by an increase in uterine weight and a very typical ballooning of the uterus, due to accumula­ tion of fluid, the timing of which is indicated in the figure. Following LH E 2 (/xg)

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0-

FIG. 14. Inactivation of the biological effect of exogenous estrogens by antibodies to l7(3-estradiol. (DES-diethylstilbestrol).

release, progesterone and progesterol levels increase, resulting in a relaxation of the cervical sphincter (Armstrong, 1968; Yoshinaga et al., 1969). The fluid is released from the uterus and the ballooning disappears. Ovulation occurs 12 hours after the LH release, 2 days of diestrus follow and the cycle starts over again. When antibodies to estradiol are administered, the effect of the rise in estrogens is nullified as indicated by the absence of uterine ballooning and the disappearance of cornified cells from the vagina. There is no LH release as we were actually able to determine, through the courtesy of Dr. Midgley, and there was no ovulation. As the action of the antiestrogens fades, there is restoration of estrogen activity, ballooning reappears, and a new cycle is reinsti tu ted. Interestingly enough, to block ovulation, the antibody has to be administered not later than 12 hours prior to the expected time of LH release. This long interval between the actual LH release and the trigger effect of estrogens is fascinating and in agreement with the above-mentioned experiments of Goding et al. (1969) in the sheep, and with the 36-hour interval between the administration of intravenous Premarin and the discharge of LH. The absence of ovulation is not due to a

M E C H A N I S M S REGULATING T H E MENSTRUAL

CYCLE

81

direct effect of the antibody on the ovary since ovulation was restored by the administration of human chorionic gonadotropin at the time that LH would have normally been released. Similarly, replacing the effect of the endogenous estrogens by the administration of stilbestrol, whose activity is not inhibited by antibodies to estradiol, restored ovulation. These results # RATS OVULATING

X 1

|

r BOTI

D2 |

P

LH

|

D

|

P

|

*

AT EXPECTED TIME

LH ova

* 1

E

D,

ova

| (e)

1 Bai. 1 ► D2 | P | E

Di

Dz |

LH ova

(e)

rÉ á Ð* (e)

LH

1

|

♦E

ÃÂáú |

D|

Dz

p

|

tlonti-E2l LH ova

I D2 | P | ( e )

2 8 / /28

º3

LH ova

D,

ÃÂáÔº 1 Dz 1 P 1 E

D|

Dz |

D

D 1 D 1D

D

D

D,

_P.2 1

■ 4 lanti-E2l Η'Γ.Κ LH ova

* I

I D2

ÃÂÏÐ P ► 1 E

A lanti-Ez+DES· LH ova

1 BaT 1 1 Dz | P | E

|

LH ova

Di

1 1 iE

DzÐáÔº | P |

13

[antt-pj

FIG. 15. Effect of antibodies to estradiol and progesterone in 4-day-cyclic rats. Bal, ballooning of uterus ; LH, LH release ; P, proestrus (basal cells in vaginal smears) ; E, estrus (cornified cells in vaginal smears) ; D1 and D 2 , first and second day of diestrus (leucocytes in vaginal smears); (e), atypical estrus (cornified cells and leucocytes). Crossed arrows indicate, that no LH or no ova were released.

were in sharp contrast to the experiments in which we administered anti­ bodies to progesterone. In this case, LH discharge and ovulation occurred normally, yet there was proof that the biological effect of progesterone was inactivated since in the animals treated with antiprogesterone, the ballooning of the uterus, which normally disappears as a result of the appearance of progesterone, following the release of LH, persisted. The evidence presented does not rule out the possibility of a synergistic effect of small amounts of progesterone or other steroids on LH release but certainly does implicate estrogens as the most important, if not unique, trigger. The discussion is not germane to the possibility that progesterone

82

RAYMOND L. VANDE W I E L E ET AL.

or other steroids may have an effect inside the ovarian follicle on the processes leading to ovulation. There is evidence for instance, that progester­ one is present in small amounts in the human preovulatory follicle (Zander et al., 1958). The discussion relates solely to the role of progesterone reach­ ing the central nervous system via the peripheral circulation. If estrogens appear to trigger LH release, the question arises whether their effect is a threshold phenomenon, i.e., LH being "dumped" as soon as the concentration of estrogen reaches a critical level, or whether the hypo­ thalamus or pituitary is activated by an estrogen titer which is to exert its effect over a certain period of time. We have no satisfactory answer to this question. Our experiments with Premarin appear to indicate that a threshold concentration of estrogens is sufficient to trigger LH release but do not rule out the possibility that the level of this threshold may be a function of the time of stimulation, or even of the rate of change of estrogen levels. It is interesting that, in animals, the centers of the positive and the negative feed­ back are distinct (McCann and Porter, 1969). The existence of such distinct centers that appear to have different thresholds of activation raises many interesting physiological and pathological possibilities. We have incorporated this positive feedback role of estrogen on LH in our model for digital computer simulation. We have assumed that the LH surge is set off by a threshold concentration of plasma estrogens, which for the normal cycle is set at 500 pg/ml. The part of the model that deals with the ovulatory process obviously includes many other decision functions. There is, for instance, a 24-hour delay between the time at which estrogens reach threshold values for the LH surge and the actual LH surge. We have also assumed that LH release can occur only when the LH concentration in the pituitary exceeds a certain minimal value. Consequently, it became necessary to incorporate a separate sequence describing the changes in the concentration of pituitary LH. Another component of the model couples follicular growth and LH surge so that ovulation cannot occur unless the follicle has reached a certain size at the time the LH surge comes about. IV. The Postovulatory Period In the human, the events from the beginning of the LH surge to the actual rupture of the follicle are poorly known. In animals, they have been pains­ takingly studied. There is a sudden and important growth of the follicle, formation of a stigma, and finally, ovulation (Blandau, 1966). The interval between the LH surge and ovulation varies according to species, from a few hours to a day or more (Blandau, 1966; Johansson et al., 1968). The characteristics of the LH and FSH peak have been reviewed most recently by Ross et al. (1970). In the same article they also discussed the changes in progesterone and 17a-hydroxyprogesterone during the normal and abnormal

M E C H A N I S M S REGULATING T H E MENSTRUAL CYCLE

83

postovulatory phase. Their findings are essentially in agreement with ours. Baird and Guevara (1969), Abraham (1969a,b), Korenman et al. (1969a), and we (Dyrenfurth et al., 1970) recently have measured estrogens in the plasma during the postovulatory period and found changes corresponding to those previously described in the urine (J. B. Brown and Matthew, 1962). The changes in androgen secretion at the time of, and following, ovulation have been reviewed by us (Vande Wiele et al., 1968). Recently, data on the levels of androgen in the ovarian vein during the postovulatory period have become available. (Mikhail, 1967; Lloyd et al., 1969). Our main interest lies in the regulation of the life span of the corpus luteum and its secretion of progesterone. Short (1964) and Anderson et al. (1969), in presentations to this conference group, reviewed the role of various factors controlling the function of the corpus luteum in animals. Other excellent reviews of this subject have recently appeared (Rothchild, 1965, 1966), and there is no need for one more appraisal of this very com­ plicated subject. It seems well established that, even in the rat, there is no single luteotropic hormone. Prolactin, LH, FSH, and estrogens all appear necessary for normal corpus luteum function, but the relative importance of the role of each of these hormones varies from species to species. In addition to luteotropic stimuli, luteolytic factors have been identified, and in several species, including the guinea pig, sow, and others, this luteolytic process appears to originate in the uterus. In the human, there is no evidence in favor of, and in fact only evidence against, the operation of luteolytic uterine factors (Beling et al., 1970). Recent studies of the function of the corpus luteum following induction of ovulation by sequential therapy with human menopausal gonadotropin (HMG) and human chorionic gonadotropin (HCG) have led to the sugges­ tion that, in the human, once ovulation has occurred, the life span of the corpus luteum and its progesterone secretion are largely independent of the pituitary (Vande Wiele and Turksoy, 1965). Indeed, after the induction of ovulation even by a single injection of HCG, the duration of the postovulatory period is normal and blood progesterone follows a course that mimics that seen during the normal spontaneous cycle. Further evidence for such au­ tonomy of the corpus luteum, at least from LH and FSH, may be found in a comparison of the levels of FSH and LH and of progesterone during the postovulatory phase. Following the ovulatory surge, LH concentrations decrease rapidly to levels that in fact are lower than those in the preovulatory phase (Cargille et al., 1969). Progesterone starts to rise while LH is decreas­ ing, and the drop in progesterone prior to menstruation is not preceded by a further drop in the LH level; a sequence of events certainly not in favor of LH as a luteotropic agent. Recently, we have carried out a series of studies dealing with the function

84

RAYMOND L. VANDE WIELE ET AL.

of the corpus luteum in hypophysectomized women. These patients are ideally suitable for such studies since only in this type of patient is it possible to rule out synergistic effects of other pituitary hormones. Residual pituitary function in hypophysectomized patients is not unknown, but exhaustive studies in our patients, before they were included in the group, argue against this possibility. Figure 16 illustrates two such studies which were typical for the whole group. One patient, V., was hypophysectomized before puberty Pergonal

G.3

(2-4amp/d)

Pergonal ( 2 a m p / d ) HCG ( 5 0 0 0 I U/d)

12

20

28

36

44

52

60

I

5

13

17

21

25

29

DAYS OF T R E A T M E N T REPLACEMENT THERAPY

Thyroid : 240 mg/d Cortisone: 37.5mg/d

Thyroid: Cortisone:

120 mg/d 37.5mg/d

FIG. 16. Urinary steroids in two hypophysectomized patients treated with human menopausal and chorionic gonadotropins (HMG and HCG).

because of visual disturbances due to a large craniopharyngioma. After treat­ ment with HMG to induce follicular maturation, ovulation was induced with HCG. The excretion of pregnanediol during the postovulatory period was completely normal, and the length of the postovulatory phase was 13 days. The other patient, G.3, was hypophysectomized for acromegaly at the age of 28. Again, the postovulatory period was completely normal. The conclusion to be derived from these studies, that after induction of ovulation corpus luteum function proceeds normally without any further pituitary stimulation, is not necessarily applicable to spontaneous cycles since ovulation was induced with HCG, not with LH. The luteotropic effect of HCG in humans is well established (W. E. Brown and Bradbury, 1947; De Watteville, 1948; Segaloff et al., 1951; Goldzieher and Wooley, 1957;

M E C H A N I S M S REGULATING T H E MENSTRUAL

CYCLE

85

Palmer, 1957; Müller, 1961; Geller, 1967) and, in fact, has been the basis for the development of tests to evaluate the functional capacity of the corpus luteum (Jayle, 1967). In view of the long half-life of HCG, this luteotropic effect may persist for several days (Rizkallah et al., 1969). Recently, due to the courtesy of the National Pituitary Agency, we have been able to obtain enough pituitary LH to repeat these experiments, this Pergonal (2-6amp./d)

S.B. I |HCG(IO,OOOIU/CI)J

5 0 0 "I PLASMA 4 0 0 ♦J ESTROGENS (pg/ml) 300 200 100 0 2 0 0 -| URINARY ESTROGEN (/xg/ml) 100 ^

1 „

0 50 40 30 20 10 0 20 15 10 5 0

lo

ð

oo „

ME,yJ-

r-rμ к

SES

ç-ÃÃÐ 1M1 IIIÐÔÉIh ð 1

! Ð [~U

-çÔÐ hn-,

ô_ð Ð 1 M M r 1

º PLASMA H PROGESTERONE (ng/ml)

! [

H

1

Im

1

H

'

Ã

1

1T

T

o

] PREGNANEDIOL H

o „ „T_j_n 1 1 1 1 1 1 1 i-i 1

(mg/d)

j 1

i

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pk

10 14 18 22 26 DAYS OF TREATMENT

30

-

34

FIG. 17. Induction of ovulation in a hypophysectomized patient (SB) by treatment with HMG and HCG.

time inducing ovulation with LH. We have carried out 10 such experiments. In Fig. 17, is shown a course of HMG and HCG in a patient who, at the age of 14, had undergone a hypophysectomy to remove a craniophargioma. Blood estrogens and progesterone prior to and after the induction of ovula­ tion by a single injection of HCG are shown. The levels of urinary estrogens and pregnanediol parallel the blood levels. The postovulatory phase was normal in all its characteristics. Treatment was then repeated, but this time ovulation was induced with LH. The results are shown in Fig. 18 (left). LH, 800 IU, was administered every 8 hours for a total of 3 injections. As shown in Fig. 18, this dose of LH produced plasma levels that were similar to those

86

RAYMOND L. VANDE W I E L E ET AL.

seen during the spontaneous ovulatory LH surge. There was an initial rise in estrogens and progesterone in the blood; the rise, however, was not sus­ tained. Within 5 days the levels had dropped to very low values, and the patient menstruated 6 days after the administration of LH. The experiments were repeated in 2 more patients: one was a patient who had undergone radiation for a pituitary tumor; the other patient had amenorrhea associated HMG (6amp./d)

MENSES

HMG (6amp./d) g g j j j j ^ ^ ^ ! ^ ^ ^ KWM^^

S.tμ. MENSES ♦""" 3x1200 IU HLH lU/d

T-

50 40 30 20 10 0

PLASMA PROGESTERONE ( ng/ml)

,ô , V T V 13

, * ," r T T,T V*i I

17 21 25 I 5 9 13 DAYS OF TREATMENT

17

21

25

29

33

37

41

FIG. 18. Induction of ovulation in patient SB, following pretreatment with human menopausal gonadotropin (HMG). Left : Ovulation was induced by HLH ; right : ovulation was induced by HLH, but HLH was continued at 400 IU per day.

with negligible gonadotropins but otherwise normal pituitary function. The results were identical to those in patient S.B. We were ready to accept the theory that, in addition to LH, another pituitary factor having luteotropic activity, conceivably prolactin, was neces­ sary for normal corpus luteum function. Before considering this hypothesis, we wanted to exclude the possibility that corpus luteum function depends on the continuous presence of low levels of LH as seen during the normal postovulatory phase, a possibility that seemed very unlikely but had to be ruled out. The same patient who served in the previous study served again as a willing subject. The results are shown in Fig. 18 (right). In this cycle, the injections of ovulatory doses of LH were followed by daily injections of

87

MECHANISMS REGULATING THE MENSTRUAL CYCLE

400 IU of LH. This dose was chosen because it was calculated that such a dose should approximate the secretory rate of LH during the normal postovulatory phase. This time estrogens and progesterone in the blood remained elevated, and the patient did not menstruate until 17 days after the initial administration of LH. ......"?* P. if.°.?F:^!.,

H MG (4omp./d)

21 25 I 5 9 DAYS OF TREATMENT

25

29

33

37

FIG. 19. Induction of ovulation in patient IS, using human luteinizing hormone (HLH) as ovulatory agent.

Figure 19 illustrates two studies in a patient with amenorrhea associated with low gonadotropins but otherwise normal pituitary function. In the first study when only an ovulatory burst of LH was administered, estrogens and progesterone stayed up only for approximately 5 days and the patient menstruated 7 days after the initial LH injection. In the subsequent study, the ovulatory injection of LH was followed by daily injections of 400 IU of LH. Estrogens and progesterone in the blood were detectable until 18 days after the initial administration of LH, and the patient started to menstruate the next day. The last patient we treated was a patient with an irradiated pituitary tumor, and the results are shown in Fig. 20. After the initial LH administra­ tion, LH was continued for 18 days. Twelve days after the ovulatory LH,

88

RAYMOND L. VANDE W I E L E ET AL.

there was a secondary increase in the LH level which within a few days rose to early pregnancy levels. Nineteen days after ovulation, a pregnancy test was positive, and this pregnancy is progressing normally. This work is still in progress but even at this stage, some conclusions appear to be warranted: HLH (400 lU/d)

PLASMA LH (ng/ml)

PLASMA PROG. (ng/ml)

1969, Julyl

29 Aug.l

FIG. 20. Treatment of patient MK with human menopausal gonadotropin, followed by human luteinizing hormone. This patient conceived during this therapy.

1. Pituitary LH in amounts ranging from 800 to 1200 IU administered every 8 hours for a total of 3 doses will induce ovulation in amenorrheic women properly prepared by the administration of HMG. 2. Normal function of the corpus luteum requires the continuous presence of small amounts of LH. In the absence of such stimulation, progesterone and estrogens will not reach normal values and the functional life of the corpus luteum is limited to a few days.

M E C H A N I S M S REGULATING T H E MENSTRUAL CYCLE

89

3. In patients given low dose LH for more than 7 days, the postovulatory period was extended to 17, 18, and 17 days. In our experience with patients treated with chorionic gonadotropin, such a long postovulatory period is extremely infrequent and these results suggest that LH, in fact, may some­ what prolong the functional life of the corpus luteum. In the one patient given LH beyond day 14, while LH was continued, the progesterone and estrogen levels dropped and she menstruated on day 18. If confirmed, these results would indicate that the function of the corpus luteum will cease after 14 days unless there is a secondary stimulus to revive it. HCG secreted by the implanted ovum may be this stimulus. V. Mechanisms of Regulation of the Menstrual Cycle We would like to propose the following scheme to explain the cyclic nature of the menstrual cycle. At the time of menstruation, FSH is at a relatively high level. This high level appears to reach a maximum prior to the onset of menstruation, and is assumed to be the restult of the low levels of steroids at the end of the preceding postovulatory phase. Stimulation by FSH results in follicular growth but initially with no or only mimimal estrogen secretion. Six or seven days later the estrogens start to rise initially slowly, then very rapidly. The rate of change in the secretion of the estrogens is a complicated function; it depends on the blood levels of gonadotropins and, probably more importantly, on the intraovarian levels of estrogens and androgens. When plasma estrogens reach a threshold value there is an explosive discharge of LH from the pituitary resulting in ovulation provided the morphological changes in the follicle have kept pace with the changes in the hormonal levels. After ovulation occurs, small amounts of LH are necessary for normal function of the corpus luteum and, unless there is secondary stimulus to the corpus luteum (due to the secretion of chorionic gonadotropin by the im­ planted egg), steroid levels start to decrease 7-8 days after ovulation and a new cycle starts. It is possible, as we have postulated, for the maturation of the follicle and the secretion of estrogen, that local factors also play a role during the postovulatory phase. It is conceivable, for instance, that in the postovulatory phase, follicular growth is inhibited by the high intraovarian levels of progesterone and androstenedione, and that new follicular growth will not start until these levels have returned to base values. As our concepts about the regulation of the menstrual cycle developed, we have used this information to construct a series of mathematical models that could serve as the basis for computer simulation studies. One such model, dealing with the events up to the time of ovulation is illustrated in Fig. 21. Physiological justification for many of the features in this model has been presented in the preceding text. As in all models of complicated biological systems, compromises or educated guesses must be made about the nature

90

RAYMOND L. VANDE WIELE ET AL.

Symbol Table for Model of Menstrual Cycle Yv Provision for exogenously supplied hormones Y2, Plasma FSH (ng LER 907/ml) YI, Plasma LH (ng LER 907/ml) Y4, Plasma estradiol (pg/ml) F 5 , Plasma androgens (ng/100 ml Ä 4 ) Y6, Plasma progesterone (unused) F 7 , Ovulatory transition index Xv Measure of largest follicle X 2 , Hypophysial LH content X%, Plasma LH level due to surge mechanism (ng LER 907/ml) X 4 , Luteal state (unused) X 5 , Luteal state (unused) X 6 , measure of smaller follicles X7, Ovulatory transition state Coefficient List : AA, Adrenal androgens AE, Non-follicular estradiol AF1, Androgen inhibition of large follicle AF6, Androgen inhibition of small follicle EF2, Estradiol stimulation of follicle FA, Follicular androgens FE, Follicular estradiol FM, Metabolic limitation on follicle GF, Follicular growth sensitivity to gonadotropin Functional relationship between variables are expressed by equations. The connecting cables are a visual aid showing these same relationships.

Decision functions

Yes = Path followed if inequalities true No =Path followed if inequalities false

Transfer functions

FIG. 21. Example of a model used for the computer simulation studies.

I V 3 >500/

I

No

<

Yes

YY

N o A
Yes

( and j \X< 4 0 0 /

à Logic Unit

Cl y{t)_

J JxJ)=-50X3W

dtXl'

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g^lOxf-50^

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^ x (/) = 0

dtx*

°

^÷^éïïï or

c

AIA (FSH) Exogenous Hormones

2=4000e 2

(-Ï.Ïä^)

(-0.0033>4) +350e + 500

(LH) (-0.05)4) K=2000e 3 (-0.0033)4) + I75e + 250 + Xx

(Estradiol) Ç=AE +(Xf+25Xf)(FE)V2

kk kk

(Androgens) /5=AA +(IOX,2+25X|)(FA)r2

Progesterone

Z7=(0, +1, -10)

Yes

No/x7>0.05 Y7>0 j

x

^

S1

°Pe^s.ope

_4j/|Dayj\

»

Time

X3 = Contribution of LH surge to net LH plasma level

îrti ×Ð

X2 = 0 ^ 2 = 1000

jr

dt*<

U

çL yw dt** = jGF(y2)(y3)+EF2()4) -AF1(^)ÎX,(f)2-FM(X^3) d_ Ëvit) dt 6 \GF(YZ){YZ)+EF2(Y4) -AF6(y5)!xl0-FM(X^3)

\dt

' "

c/r

x2=-iox2 Time

'

k-X6( 0 --4X6( '

Time

MECHANISMS REGULATING THE MENSTRUAL CYCLE

P

1000 P

*-

91

1000

·——■FSH

LH

u. 500

[THRESHOLD FOR LH SURGE-E2 500 pg/ml j

LH

}*^<* 1

0

I

2

3

4

5

'

'

»

'

i

6

7

8

9

10 II

-i —

1

L θ

li

12 13 14 15

FIG. 22. Computer "output" of the curves for FSH, estradiol and LH levels during the preovulatory period. Threshold for release of pituitary LH was set at a plasma level of 500 pg of estradiol per milliliter.

of certain of the linkages in the model. In even more instances, quantitative information about the functional relationships among variables is lacking or incomplete. The major value of such modeling lies in the requirement inherent in the techniques to express explicitly all linkages between the variables under con­ sideration in the model, as well as to derive equations that, while compatible with the physiological information, yield realistic curves in computer simula­ tions. Often such simulation reveals internal inconsistencies that had not been realized, or yields new insight to physiological processes. An example of the usefulness of such studies is shown in Figs. 22 and 23.

1000

1000

500

FIG. 23. Same as Fig. 22. Threshold for LH surge was set at 400 pg of estradiol per milliliter. Note the double peak in the curve for LH.

92

RAYMOND L. VANDE W I E L E ET AL.

Figure 22 presents the computer "output" for the curves of FSH, estradiol, and the surge part of the LH curve. The tonic part of the LH curve was not modeled in this simulation. After repeated simulation and adjustment in the various coefficients, the curves shown in the figure were obtained. These curves may be considered to be a satisfactory representation of the changes observed during the normal menstrual cycle. In the simulation shown in Fig. 22 the threshold concentration of estradiol, to trigger the ovulatory LH release, was set at 500 pg/ml. We were interested in evaluating the results that would ensue from small increases or decreases of this threshold concentration of estradiol, and in the subsequent simulation shown in Fig. 23 this variable was set at 400 pg/ml. The results were quite unexpected. Ovulation still occurred, but only after a second surge of LH. Analysis of the calculations for the other variables (not shown in the figures) revealed the mechanisms by which such a double peak in LH could originate. Under normal conditions follicular maturation and estrogen secretion are coupled, and this feature is part of the model. If the threshold concentration of estradiol that is necessary to trigger LH release is set at a lower level, the LH surge will occur before the follicle is competent, and will not be followed by ovulation. Consequently, estrogens continue to rise and as soon as the pituitary stores reach threshold volume, the pituitary fires again. This time follicular maturation is complete and ovulation occurs. A C KNO WLEDGMENTS

The authors would like to express their appreciation to Mr. C. Thompson of the Cutter Laboratories, Berkeley, California for a generous supply of Pergonal, and to the National Pituitary Agency and the Endocrine Study Section for FSH, LH, and antisera to these hormones. REFERENCES

Abraham, G. E. (1969a). / . Clin Endocrinol. Metab. 29, 866. Abraham, G. E. (1969b). Program 51st Meeting Am. Endocrine Soc, New York p. 115. Anderson, L. L., Bland, K. P., and Melampy, R. M. (1969). Recent Progr. Hormone Res. 25, 57. Armstrong, D. T. (1968). Am. J. Physiol. 214, 764. Baird, D. T., and Guevara, A. (1969). J. Clin. Endocrinol. Metab. 29, 149. Baird, D. T., Horton, R., Longscope, C , and Tait, J. F. (1968). Perspectives Biol. Med. 11, 384. Beling, C. G., Marcus, S. L., and Markham, S. M. (1970). J. Clin. Endocrinol. Metab. 30, 30. Bickenbach, W., and Döring, K. (1959). "Die Sterilität der Frau." Thieme, Stuttgart. Blandau, R. J. (1966). In "Ovulation" (R. B. Greenblatt, ed.), pp. 3-16. Lippincott, Philadelphia, Pennsylvania. Bradbury, J. T. (1Ç61). Endocrinology 68, 115. Brouha, L., and Simonnet, H. (1930). Proc. 2nd Intern. Congr. for Sex Res. London, 1931 p. 339. Oliver and Boyd, London. Brown, J. B., and Matthew, G. D. (1962). Recent Progr. Hormone Res. 18, 337.

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Brown, J. B., Evans, J. H., Adey, F. D., Taft, H. P., and Townsend, L. (1969). / . Obstet. Gynaecol. Brit. Commonwealth 76, 289. Brown, W. E., and Bradbury, J. T. (1947). Am. J. Obstet. Gynecol. 53, 749. Brown, W. E., Bradbury, J. T., and Juncek, E. C. (1953). Am. J. Obstet. Gynecol. 65, 733. Buchholz, R. (1959). Geburtsh. Frauenheilk. 19, 852. Burger, H., Catt, K., and Brown, J. (1968). J. Clin. Endocrinol. Metab. 28, 1508. Burger, M. (1960). Z. Geburtshilfe [Gynaekol.], Beilageh. 155, 83. Butler, J. K. (1969). Proc. Roy. Soc. Med. 62, 1. Cargille, C. M., Ross, G. T., and Yoshimi, T. (1969). J. Clin. Endocrinol. Metab. 29, 12. Chester Jones, I., and Bael, J. N. (1962). In "The Ovary" (S. Zuckerman, ed.), Vol. 1, p. 361. Academic Press, New York. Croes, R. S. E., Paesi, F. J. A., and dejongh, S. E. (1959). Acta Endocrinol. 32, 399. De Watteville, H. (1948). Gynaecologia 126, 207. Donovan, B. T. (1966). In "The Pituitary Gland" (G. W. Harris and B. T. Donovan, eds.), Vol. 2, p. 49. Univ. of California Press, Berkeley, California. Dyrenfurth, I., Mikhail, G., Jewelewicz, R., Ferin, M., Warren, M., Rizkallah, T., and Vande Wiele, R. L. (1970). / . Clin. Endocrinol. Metab. (in preparation). Everett, J. W. (1948). Endocrinology, 43, 389. Ferin, M., Zimmering, P. E., Lieberman, S., and Vande Wiele, R. L. (1968). En­ docrinology 83, 565. Ferin, M., Zimmering, P. E., and Vande Wiele, R. L. (1969a). Endocrinology 84, 893. Ferin, M., Tempone, A., Zimmering, P. E., and Vande Wiele, R. L. (1969b). Endocri­ nology 85, 1070. Flerko, B. (1966). N euro endocrinology (N.Y.) 1, 613-668. Fraps, R. H., and Dury, A. (1943). Anat. Record 87, 442. Geller, S. (1967). In "Endocrine Functions of the Ovary" (M. F. Jayle, ed.), p. 397. Pergamon Press, Oxford. Gemzell, C. A., Roos, P., and Loeffler, F. E. (1968). In "Progress in Infertility" (S. J. Behrman and R. W. Kistner, eds.), p. 375. Little, Brown, Boston, Massachusetts. Goding, J. R., Catt, K. J., Brown, J. M., Kaltenbach, C. C , Cummings, I. A., and Molle, B. J. (1969). Endocrinology 85, 133. Goldzieher, J. W., and Wooley, H. L. (1957). Progr. Gynecol. 3, 253. Harris, G. W., and Campbell, H. J. (1966). In "The Pituitary Gland" (G. W. Harris and B. T. Donovan, eds.), Vol. 2, p. 99. Univ. of California Press, Berkeley, Caifornia. Hilliard, J., Hayward, J., and Sawyer, C. (1964). Endocrinology 75, 957. Hoffman, F., and Meger, C. (1965). Geburtsh. Frauenheilk. 25, 1132. Hohlweg, W. (1934). Klin. Wochschr. 13, 92. Ingram, D. L. (1959). / . Endocrinol. 19, 123. Jacobson, A., and Marshall, J. R. (1969). Fertility Sterility 20, 171. Jayle, M. F. (1967). In "Endocrine Functions of the Ovary" (M. F. Jayle, ed.), p. 377. Pergamon Press, Oxford. Johansson, E. D. B., Neill, J. D., and Knobil, E. (1968). Endocrinology 82, 143. Kaiser, J., Wide, L., and Gemzell, C. (1966). Acta Obstet. Gynecol. Scand. 45, 53. Kistner, R. W. (1968). In "Progress in Infertility" (S. J. Behrman and R. W. Kitstner, eds.), p. 327. Little, Brown, Boston, Massachusetts. Korenman, S. G., Perrin, L. E., and McCallum, T. P. (1969a). / . Clin. Endocrinol. Metab. 29, 879.

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Korenman, S. G., Perrin, L. E., and Rao, B. R. (1969b). Program 51st Meeting En­ docrine Soc, New York p. 116. Kullander, S. S. (1961). Acta Endocrinol. 38, 598. Kupperman, H., Epstine, J., Blatt, M., and Stone, A. (1958). Am. J. Obstet. Gynecol. 75, 301. Lamport, H. (1940). Endocrinology 27, 673. Lloyd, C. W., Lobotsky, J., Weisz, J., Baird, D., McCracken, J., Puga, J., Pupkin, M., Zanatru, J., Seron, M., and Guerrera, R. (1969). Personal communication. McCann,, S. M., and Porter, J. C. (1969). Physiol. Rev. 49, 241. MacDonald, P. C , Rombaut, R., and Siiteri, P. K. (1967). / . Clin. Endocrinol. Metab. 27, 1103. Mauvais-Jarvis, P. (1969). In "Colloques de la société nationale pour l'étude de la stérilité de la fécundité" (J Ferin, ed.), Masson, Paris. Meyer, C. J., and Bradbury, J. T. (1960). Endocrinology 66, 121. Midgley, A. R. (1966). Endocrinology 79, 10. Midgley, A. R. (1967). / . Clin. Endocrinol. Metab. 27, 295. Mikhail, G. (1967). Clin Obstet. Gynecol. 10, 29. Mikhail, G., Wu, C , Ferin, M., and Vande Wiele, R. L. (1970). Steroids 15, 333. Moore, C , and Price, D. (1930). Proc. Soc. Exptl. Biol. M ιd. 28, 38. Müller, P. (1961). Gynaecologia 152, 341. Nallar, R., Antunes-Rodrigues, J., and McCann, S. M. (1966). Endocrinology 79, 907. Neill, J. D., Johansson, E. D. B., Datta, J. K., and Knobil, E. (1967). J. Clin. En­ docrinol. Metab. 27, 1167. Odell, W. D., and Swerdloff, R. S. (1968). Proc. Nati Acad. Sci. U.S. 61, 529. Odell, W. D., Parlow, A. F., Cargille, C. M., and Ross, G T. (1968). / . Clin. Invest. 47, 2551. Palmer, A. (1957). Fertility Sterility 8, 220. Payne, R. W., Hellbaun, A. A., and Owens, J. N. (1956). Endocrinology 59, 306. Probst, V., and Beller, F. K. (1961). Geburtsh. Frauenheilk. 21, 969. Rapoport, A. (1952). Bull. Math. Biophys. 14, 171. Rizkallah, T., Gurpide, E., and Vande Wiele, R. L. (1969). J. Clin. Endocrinol. Metab. 29, 92. Ross, G. T., Cargille, C. M., Lipsett, M. B., Rayford, P. L., Marshall, J. R., Strott, C. A., and Rodbard, D. (1970). Recent Progr. Hormone Res. 26, 1. Rothchild, I. (1965). Vitamins Hormones 23, 209. Rothchild, I. (1966). / . Reprod. Fertility Suppl. 1, 49. Schwartz, N. B. (1969). Recent Progr. Hormone Res. 25, 1. Segalo«, A., Sternberg, W. H., and Gaskill, C. J. (1951). / . Clin. Endocrinol. Metab. 11, 936. Shirley, B., Wolinsky, J., and Schwartz, N. B. (1968). Endocrinology 82, 959. Short, R. V. (1964). Recent Progr. Hormone Res. 20, 303. Stevens, V. C , and Vorys, N. (1967). Obstet. Gynecol. Surv. 22, 781. Strott, C. A., and Lipsett, M. B. (1968). / . Clin. Endocrinol. Metab. 28, 1426. Swerdloff, R. S., and Odell, W. D. (1969). / . Clin. Endocrinol. Metab. 29, 157. Thomas K., and Ferin, J. (1969). Personal communication. Thompson, H. E., Horgan, J. D., and Delfs, E. (1969). Bio-Physical J. 9, 278. Vande Wiele, R. L., and Turksoy, R. N. (1965). / . Clin Endocrinol. Metab. 25, 369. Vande Wiele, R. L., Dyrenfurth, I., and Gurpide, E. (1968). Clin. Endocrinol. 11, 603. Vorys, N., Ullery, J., and Stevens, V. C. (1965). Am. J. Obstet. Gynecol. 93, 641. Yoshinaga, K., Hawkins, R. A., and Stocker, J. F. (1969]. Endocrinology 85, 103.

MECHANISMS

REGULATING

THE MENSTRUAL

CYCLE

Zander, J., Forbes, T. R., von Munstermann, A. M., and Neher, R. Endocrinol. Metab. 18, 337. Zondek, B. (1954). Recent Progr. Hormone Res. 10, 395.

95

(1958). / . Clin.

DISCUSSION

N. B. Schwartz : The dual experimental and analytical approach has been very useful for us, too, in looking at the rat reproductive cycle. Last year I spoke on the physio­ logical model [Recent Progr. Hormone Res. 25, 1 (1969)]. I would like now to show the beginning of a computer simulation of the rat estrous cycle. The modeling that we have been doing is with the IBM 360 using the Continuous Systems Modeling Program. We have been collaborating with Mr. Paul Walsh, Fields System Center Representative at the Chicago IBM office. tìETA - LOSS RAIL Ci- LH A2- NEGATIVE FEEDBACK OF ES ON LH PRODUCTION. A0- SET POINT SECRETION RATE OF LH ALPHA2- LOSS RAIE OF ES M- POSITIVE EFFECT OF ES ON ITS RATE OF PRODUCTION. A3- GAIN RATE OF ES IN THE PRESENCE OF LH LHCP- LH CONCENTRATION IN THE CRITICAL PERIOD ESCP- ES CONCENTRATION IN THE CRITICAL PERIOD THES- THRESHOLD VALUE OF ES FOR ALLOWING SORGE TO OCCUR CT- TIME OF THE CRITICAL PERIOD AS MEASURED ON THE 24-HOUR CLOCK CLÜATA- DATA POINTS FOR THE SURGE AS A FUNCTION OF THE CLOCK CCl'N- A CONVERSION CONSTANT FOR EXPERIMENTING WITH CLOCK VALUES

FIG. A.

Identification of parameters utilized.

Figure A identifies the parameters we have included. Some of the values for these parameters are respectable data from the literature, but some of them have been made up and tested for various values in the computer simulation. ES, represents estrogen; LH, luteinizing hormone in the plasma. Since in this particular model we are deahng with the rat, we have included a "clock." The "CL data" define this 24-hour clock. As you will recall, the LH surge in the rat is controlled not just by estrogen, but by a central nervous system (CNS) function that starts off at 1400 hours. DYNAMIC ÖLH = - A 2 * E S ■ AC ■ SURGE LHDÜT = - B E T A * L H «♦ BLH ESUOT = -ALPHA2 * ES * A 3 * L H ■ M * E S * E S * S I G N A L L H 1 * lNTGRL(LHCP,LHDOT) ES1 = I N T G R L i ESCP.fcSDOT) LH= A M A X 1 ( 0 . , L H 1 ) ES = A M A X 1 ( 0 . , E S 1 ) PROCfcü SURGE,SIGNAL = S S ( E S , T H E S , C L O C K ) IF(SURGE) 10,10,2 10 I F ( ES - THES ) 1 , 2 , 2

1 SURGE = 0. SIGNAL = 1. GO TO 3 2 SURGE - CLOCK IF { ( SIGNAL .LE. 0.0 ) .OR. ( SURGE .GE. .1 ) ) 3 CONTINUE ENDPRG CLUCK = CCUN * AFGEN(CLDATA,AMOO(TIME + CT V 24.))

SIGNAL = 0.

FIG. B. Dynamic relations between system variables (CSMP).

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RAYMOND L. VANDE W I E L E ET AL.

Figure B shows the way in which these parameters and variables are put together in a dynamic formulation. Plasma LH and ES are generated by the top relationships. The bottom part of the program defines the surge system, indicating that if the time of day is 1400 hours it is "okay" for the system to go if estrogen is also high enough (exceeds THES). The "go" in this case means that "SIGNAL" becomes zero; the "SURGE" equals the values defined by "CL DATA." IBM 3 6 0 CSMP ZERO HOURS =16.00 OF PROESTRUS

3.0

140 120

2.0

100 80 60

1.0

40 -|20

i

0

0 o = Estrogen • =LH

g 3.0

140 x" _i

120 2.0

100 80 60

1.0 h

40 20 24

FIG. C.

48

72 HOURS

120

0

Computer simulation of estrogen and LH plasma levels over 130 hours.

Figure C represents the first 130 hours of two simulations. The time zero is at 1600 hours on the day of proestrus of a given cycle. In the upper curve, M is set at 966.275 (M being the factor by which estrogen is controlling its own rate of production). As you can see, 4-day cycles are generated. In the bottom part of Fig. C, the M equals 966.000 and the computer generates 5-day cycles. I wish to emphasize that this programming is still preliminary. E. Rosemberg: I am grateful for having had the privilege of seeing Dr. Vande Wiele's data related to the administration of human pituitary LH. As I recall, this material was used in hypophysectomized women. If you referred to the effect of HLH in hypophysectomized patients, one of the same patients received HMG and HCG, showing during this particular course of treatment a normal life span for the corpora lutea. However, when this patient received HLH, 800 IU, three injections during a 24-hour period instead of HCG, the life span of the corpora lutea was short. In order to correct

M E C H A N I S M S REGULATING T H E MENSTRUAL CYCLE

97

this situation you changed the experimental design in that HLH was given the day after withdrawal of HMG at a dose of 1200 IU, three injections during a 24-hour period. Then you continued the administration of H L H at a dose of 400 IU for 14-days. How can you explain the difference between the effects of HCG given as a single dose and the fact that to obtain the same results HLH had to be administered continuously in order to maintain a normal life span of the corpora lutea? R. L. Vande Wiele: An important cause for the difference in the effects of HCG and of LH lies, I think, in the difference between their half-life in the circulation. In a recent study [Rizkallah, T. et al., J. Clin. Endocrinol. Metab. 29, 92 (1969)], we found that following the intravenous administration of HCG its plasma disappearance curve shows an initial fast component with a half-life of 5.6 hours and a later slow component with a half-life of 24 hours. The half-life, on the other hand, of LH is much shorter [Köhler, P. O., Ross, G. T., and Odell, W. D., J. Clin. Invest. 47, 38 (1968)]. In the same article, we calculated that, after a single injection of 5000 IU of HCG, the plasma concentration of HCG is obtained which for many days remains higher than the equivalent maximum concentration of LH during the preovulatory surge. E. Rosemberg: I still think that the fact that HCG has a longer half-life than HLH does not provide the complete explanation. I am sure that there must be something else unless HCG has properties unknown to us at the present time. R. L. Vande Wiele: This is quite possible. HCG is a placental hormone, and it is very likely that it differs from LH in characteristics other than metabolism. J. R. Marshall: We have recently studied the effect of varying doses of HCG on corpus luteum function in women who have had ovulation induced with HMG and a single injection of HCG. Variations in HCG significantly affect both the levels of plasma progesterone and the duration of the interval from HCG injection to the onset of menses. Thus, HCG dose determines both magnitude and duration of corpus luteum function. R. L. Vande Wiele: It is well established that HCG, when administered in the postovulatory phase, will stimulate progesterone secretion, and if given in large enough doses will prolong the postovulatory phase. Brown and Bradbury already in 1947 showed such a luteotropic effect of large doses of HCG [Brown, W. E., and Bradbury, J. T., Am. J. Obstet. Gynecol. 53, 749 (1947)]. In fact Jayle has developed a test to evaluate the functional integrity of the corpus luteum which is based on this luteotropic effect (Jayle, M.D., "Endocrine Function of the Ovary." Pergamon Press, Oxford, 1967). C. P. Channing: In preliminary studies using hypophysectomized monkeys, Dr. Ernst Knobil and his colleagues have been able to induce formation of multiple corpora lutea with Pergonal alone. The corpora lutea thus formed secreted progesterone for up to about 10 days. A large subcutaneous dose of Pergonal in gelatin with a ratio of LH to FSH activity of approximately one was administered subcutaneously, following pretreat­ ment with the same preparation to induce follicular development. Of interest also is the finding by Drs. Neill and Knobil {Federation Proc. 28, 422, abstr. 2871 (1964)] that the length of progestin secretion rate of the corpus luteum of the intact rhesus monkey cannot be prolonged by exogenous HCG beyond that observed in normal pregnancy. Studies conducted in my own laboratory demonstrate that cultures of human granulosa cells can secrete progestins for up to 10-14 days in the total absence of pituitary hormones, strengthening the concept that under the proper condition the human corpus luteum in vivo as well as in vitro can secrete progestins for the normal life span without the continued presence of pituitary hormones. R. L. Vande Wiele: I have never been able to induce ovulation in hypophy-

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RAYMOND L. VANDE WIELE ET AL.

sectomized women with Pergonal alone. In humans it is also impossible to prolong the life span of the corpus luteum for much more than a few days beyond the normal 14 days, even when large amounts of chorionic gonadotropin are administered [Brown, W. E., and Bradbury, J. T., Am. J. Obstet. Gynecol. 53, 749 (1947)]. M. Holzbauer: We have recently started experiments on dogs in which we are collecting simultaneously the venous effluent from the ovaries and from the left adrenal gland. Our aim is to obtain information on the effect of acute hypophysectomy and of subsequent infusion into the jugular vein of different pituitary hormones on the rates at which steroids are secreted from these two endocrine glands. Figure D illustrates the Dog 4 5 5 (Mct-cstrus) Progesterone secretion from both ovaries

Hypophysectomy

INFUSION of L H : I O

20

>jg/min/kg

FIG. D. Secretion of progesterone into ovarian venous blood before and after hy­ pophysectomy and during the infusion of luteinizing hormone. Dog, $ , 14.8 kg, chloralose anesthesia. Ordinate: Micrograms of progesterone secreted by both ovaries during periods of 10 minutes. Abscissa: Time course of the experiment; black bars: blood collection periods. LH: luteinizing hormone (NIH-LH-B-4, bovine) infused into left jugular vein. secretion of progesterone from the ovaries of a dog in metestrus. Blood samples were collected for periods of 10 minutes before hypophysectomy and during the infusion of luteinizing hormone (NIH-LH-B-4). Two hours after hypophysectomy the secretion of progesterone by the ovary was decreased about 60% ; about one-half hour after the start of an infusion of LH in the jugular vein, it had returned to prehypophysectomy rates. Figure E shows the secretion rates of different steroids by the adrenal gland of the same dog. Two hours after hypophysectomy the secretion of cortisol and corticosterone was decreased by 60%; the secretion of the adrenal androgens and of some precursor steroids was decreased by 90% or more. Infusion of LH caused only a slight rise in adrenal steroid secretion. The amount of progesterone secreted over a period of 10 minutes by the left adrenal before hypophysectomy was 1.7 ìg. During the same period both ovaries of this dog secreted 19.0 ìg of progesterone. The fall of progesterone secretion by the ovary after acute hypophysectomy indicates that the corpus luteum of the dog requires

99

MECHANISMS REGULATING THE MENSTRUAL CYCLE

a pituitary stimulus to maintain its full secretory capacity. This is in good agreement with Dr. Vande Wiele's observations on the human ovary. S. G. Korenman: We have also found that the plasma level of estradiol frequently peaks a day before the LH peak, and one wonders why the follicle slows its secretion of estradiol without any substantial change in the level of gonadotropins. We have been blaming the falling FSH on the increasing level of estradiol at that time. I would like to Dog

455

Met-estrus

S t e r o i d s e c r e t i o n from the left

adrenal gland

lOOH

>jg/IOmin F +B

ANDROGENS PROGESTERONE IIOOH-PROG. PREGNENOLONE É3¼Ï Hypophysectomy

I4-OÖ

:

I4 30"

INFUSION of LH:

4 0 0

2-25 OI8 O 37 0-24

!

> I 6 0 0 hr 2 0 jjg/min/kg

FIG. E. Secretion of steroids by the left adrenal gland. Same dog as in Fig. D. Venous effluent from left adrenal gland collected during the same periods as ovarian blood samples. Ordinate: Secretion rates expressed as percent of prehypophysectomy secretion. Abscissa: Time course of the experiment. □, sum of cortisol and corticosterone, x, sum of androstenedione, llß-OH-androstenedione, and adrenosterone. · , progesterone, 0 ' llß-OH-progesterone, Ä : pregnenolone. suggest that perhaps a biosynthetic capacity change of these cells may occur just prior to ovulation. Dr. Channing's statement seems to have some relation to this. With regard to your computer simulation, I noted that neither you nor Dr. Schwartz has specifically referred to the possibility that the response of an increasing number of hormone-secreting cells to a fixed concentration of stimulant might very well be an in­ crease in secretion. An increased number of cells responding to the threshold level of gonadotropin would be expected to produce increasing levels of ovarian steroids. B. V. Caldwell: I believe that LH and estradiol peaks may not necessarily be related. We gave a sequence of progesterone and estradiol to eight ovariectomized sheep, and in all cases there was a normal behavioral response and an LH peak. We then actively im­ munized two of these animals against estradiol and injected the same series of steroids.

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In the immunized animals no behavioral estrous or LH peak was recorded. We then did the same experiment using diethylstilbestrol and again found an LH peak and a normal behavioral response in both the control and immunized animals, indicating that it was the neutralization of the exogenous estradiol that inhibited the behavioral response and LH peak when antibodies to estradiol were present in the animals. R. T Chatterton: I think that perhaps the question of why the estradiol synthesis decreases in the presence of continued LH may be partially answered by a paper we published in Endocrinology in February, 1969. During the diestrous phase of the rat cycle, LH was able to stimulate estradiol synthesis in vitro, but with ovaries removed later in proestrus and early estrus, LH had a markedly inhibitory effect on estradiol synthesis. R. L. Vande Wiele: This is an interesting observation that could account for the fact that plasma estrogens start to drop before LH reaches maximum values and presum­ ably, therefore, before ovulation has occurred. It is an important observation since it relates the drop in estrogens to a direct effect of the rising titer of LH, rather than to the actual rupture of the follicle. R. T . C h a t t e r t o n : In vitro the ovaries incubated without added LH synthesized more estrogen than the same ovaries incubated in the presence of LH at proestrus. K. J. Ryan: We have to bear in mind that the ovarian follicle does rupture at the time of ovulation and that steroid metabolism may be affected. Short has measured ovarian vein blood very close to the period of ovulation and has demonstrated a marked shift from estrogen production to progesterone soon after follicular rupture. The estro­ gens do not rise again until later in the luteal phase. W. D. Odell: In thinking about preovulatory increases in estradiol as a signal for the LH peak over the last year or so, we have continued to be bothered by a couple of events which you have reinforced today, Dr. Vande Wiele. In the first place, Dr. Swerdloff and I have shown, and you have verified, that administration of estrogen to eugonadal women produces LH elevations. However, consider the shapes of the LH peaks; they are broad and frequently jagged, not resembling an ovulatory peak. Perhaps more important, one does not observe a rise in FSH concomitantly with the LH peaks. As we and a number of other investigators have shown, the ovulatory peak consists of concomitant peaks of LH and FSH. In our published study of induction of "ovulatory-like" peaks in castrated and postmenopausal women [Proc. Nati. Acad. Sci. U.S. 61, S29 (1968)] with sequential estrogen-progesterone treatment, LH and FSH both peaked sharply and exactly mimicked the ovulatory surge. Double LH peaks were occasionally observed as you have described also to occur in normal women on occasion. Perhaps in analogy to the old debates that diabetes mellitus is a glucose overproduction versus an underutilization disease, both the rise in estradiol and 17-hydroxyprogesterone are involved as signals of the ovulatory peak; the 17-hydroxyprogesterone to induce the FSH peak concomitantly with the LH peak, and estradiol, of course, to further stimulate the LH peak. R. L. Vande Wiele: It is correct that the width of the LH peak in the patients to whom we administered intravenous Premarin exceeded that seen during the spontaneous LH surge. I would like to emphasize that the effect of a single administration of Premarin can certainly not be equated to the effects of the rising titer of estradiol during the normal preovulatory period. We are aware of this deficiency in our ex­ perimental design and plan to replace the intravenous administration of Premarin by the administration of estradiol in short-term continuous intravenous infusions at rates that approach the rates of secretion immediately prior to LH surge. Goding et al. [Endocri­ nology 65, 133 (1969)] have done this and found that the LH peak they obtained

MECHANISMS REGULATING THE MENSTRUAL CYCLE

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mimicked closely the LH peak seen during the spontaneous preovulatory LH surge. Your suggestion that a combination of two signals—one of estradiol, the other of l7a-hydroxyprogesterone—is an interesting one and cannot be excluded by the experimental design we used. A. Albert: If HCG and H L H have different effects on luteal function and on the luteal interval, there must be reasons for this more basic than differences in circulatory half-lives. Dr. Vande Wiele, in view of your remarks on the multiplicity of factors affecting luteal function, would you consider it possible that when intense luteal function is needed (such as in very early pregnancy) the luteotropic action of HCG is mediated not only by LH-like, but also by its FSH-like, qualities. R. L. Vande Wiele: I have no information about this possibility, which is certainly an interesting one. I think it would be difficult to design an experiment either to prove or disprove this possibility. A. R. Midgley: At the Second Annual Meeting of the Society for the Study of Reproduction, Dr. Beals reported that pseudo-pseudopregnant rat ovaries take up an extremely large amount of HCG while there is only slight uptake of rat LH, FSH, and prolactin. This suggests the HCG possesses unique properties not due to intrinsic FSH or LH activity. The luteotropic activity of HCG may depend not upon serum levels, but rather upon the ability of ovarian tissue in different functional states to bind and retain the hormone. Thus, prior to the development of the full pseudo-pseudopregnant state, there is a very low uptake of labeled HCG by ovarian tissue, while at the time of full luteinization there is a very large uptake. I wonder why you ignored consideration of the functional role of LH at times other than midcycle. You chose to examine the relationships solely between estradiol and FSH, whereas LH probably plays a very large role in the regulation of estrogens. Actually your own data regarding FSH were contrary to your conclusions regarding the data of others in that your concentrations of FSH were constant throughout the follicular and luteal phases. R. L. Vande Wiele: I mentioned in the text why, in our modeling of the pre­ ovulatory period, we gave more attention to the changes in FSH levels. In fact in the simulation we kept the tonic LH at a constant value. We felt that the data of Jacobson and Marshall were convincing enough to let us do this. Furthermore, at the time we were working on this part of the model there was still considerable doubt about the exact curve of LH. In the two normal cycles I described, the FSH levels were not constant. In many of the cycles we studied (but not in all), there was a high level of FSH in the preovulatory period. For the simulation, we used the FSH data of the Bethesda group. S. M. Husain: We have treated mature rats with various estrogens and progestins for a week and have attempted to induce ovulation in these rats with PMS and HCG. Rats pretreated with estrogens invariably responded by ovulating a significantly higher number of ova compared to the controls [Husain, S. M., Rev. Can. Biol. 28, 137 (1969)]. On the other hand, significantly fewer eggs were obtained in the progestin-pretreated rats. However, some rats pretreated with certain progestins, e.g., norethynodrel (contain­ ing inherent estrogenicity), did ovulate a high number of ova. This is also true of rats pretreated with progestin-estrogen combinations. These data led us to conclude that in our experiments estrogens facilitated gonadotropin-induced ovulation, whereas progestins inhibited it. K. Savard: An underlying theme running through today's discussion is the course of estrogens in the ovaries of the patients you have studied. We all admit that during the luteal phase the most likely source is the corpus luteum, if only by virtue of its mass of steroidogenic cells. I wonder whether this might not influence our thinking on the pre-

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ovulatory source, and direct it to the one follicle which is destined to ovulate. Is it not possible that the many follicles which react to gonadotropin but do not ovulate could be secreting estrogen and other steroids too, not only those in the ovary which ovulâtes, but in the contralateral one as well? Your measurements of peripheral estrogen apparently reflect only total ovarian estrogen, irrespective of the intraovarian source. R. L. Vande Wiele: I completely agree that the many follicles that grow during the preovulatory period, but do not ovulate, do secrete estrogens and possibly other steroids as well. I do not know how one could determine what fraction of the plasma estradiol is derived from these small follicles. In our simulation studies, we model the contribution to the peripheral levels of steroids by the small follicles separately from that of the largest follicle, the one that will ovulate. You may remember that in the equations for the measurement of the follicle we have a term xv which is the measure of the largest follicle, and xQ which is the average measure of the other growing follicles. C. D . Kochakian: Would you elaborate on the role of the changes in testosterone and androstenedione in the ovarian cycle? Are the changes just a reflection of the fact that these steroids are precursors of estrogen or do they have a direct effect? If they are just precursors, then the observation of Dr. Ryan that saturated androgens are not converted to estrogens might help to answer some of these questions. R. L. Vande Wiele: In our modeling of the menstrual cycle we assume the effect of testosterone and androstenedione to be an androgenic effect that by its local effect inhibits the growth of the follicle. D. T. Armstrong: Pursuing the question of whether follicles not destined to ovulate may play some physiological role, I wonder whether these follicles might play a luteotropic role during the lu teal phase of the cycle. Is it possible that the luteotropic effect of LH in the human, as in the rabbit, may be mediated indirectly via the ability of LH to stimulate estrogen secretion from such follicles, with the estrogen being the actual luteotropic agent? Such an explanation would be consistent with Dr. Korenman's observation, as discussed above, of reduced estrogen secretion in subjects with short luteal phases and with Dr. Ross' observations presented at this conference that such subjects tend to have lower than normal plasma FSH levels. R. L. Vande Wiele: This is an interesting possibility. However, I do not see how we can either prove or disprove it in the human. H. Friesen: Have you studied the effect of administering HPL in the postovulatory period to see whether it had any effect on progesterone secretion or the life span of the corpus luteum? Or should we infer from your data that LH is the sole luteotropic factor? R. L. Vande Wiele: Up to now we have not administered HPL in the postovulatory period. We have obtained a small amount of HPL from the Lederle Company, and we plan to carry out these experiments. It is difficult to answer the second part of your question. Our studies show that LH is sufficient to produce the postovulatory changes that are necessary for normal implantation since one of our patients conceived. For this reason I am ready to believe that LH is the sole luteotropic factor during that part of the postovulatory period. On the other hand, it is likely that the secondary boost to the function of the corpus luteum in patients who conceive results from the secretion of HCG by the implanted ovum. During that part then of the postovulatory period LH may not be the sole luteotropic hormone. K. Sterling: May I ask for further information concerning the difference between the actions of HCG and LH? From your comments, I gather that HCG has not only a longer half time but luteotropic activity as well. May I a^o ask about the present status of knowledge concerning secretion of prolactin during the progestational phase of the

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cycle? Would you be willing to speculate further concerning the problem of maintenance of the corpus luteum during the progestational phase of the cycle? R. L. Vande Wiele: I am aware of two studies of prolactin levels in the menstrual cycle [Simkin, B., and Arie, R., (1963) Proc. Soc. Exptl. Biol. Med. 113, 486 (1963); Gati, I., Doszpod, J., Preisz, J. Acta Physiol Acad. Sci. Hung. 32, 115 (1967)]. They gave conflicting results, and there were doubts about the specificity of the methods used in these studies. I may add that it is still doubtful whether, in the human, there is a specific prolactin. J. W. McArthur: Your observations concerning the hormonal requirements for a normal postovulatory phase are supported by the findings of Dr. J. B. Brown and his collaborators. They have reported [/. Obstet. Gynaecol. Brit. Commonwealth 76, 289 (1969)] that if the HPG treatment of women with anovulatory sterility is followed by a large ovulatory dose of HCG, say 10,000 IU, a luteal phase of normal length is obtained. However, if doses of 3000 IU or below are administered it is necessary to give repeated supplementary injections of the order of 500 IU during the luteal phase in order to assure normal luteal function. F. Maloof : In view of the recent data showing that clomiphene inhibits the binding of estrogen at its receptor sites (uterus, anterior pituitary), have you tried to inhibit the LH surge in midcycle, supposedly due to estrogens by clomiphene? From your work with clomiphene, do you have any specific ideas how clomiphene itself produces the rise in serum LH? R. L. Vande Wiele: We have not tried to inhibit the LH surge in midcycle by the administration of Clomid. Other than the ones that are currently in the medical literature, we have no specific ideas about the mode of action of Clomid.