Endocrine Control Of Menstruation

Endocrine Control of Menstruation David Stanford, M.D.

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I.

Overview: the Hypothalamic/pituitary/ovarian/endometrial Axis A.

One of four principal endocrine systems

B.

Not necessary for survival

C. Primary roles

II.

1.

Reproduction

2.

Maintenance of estrogen-dependent tissues

3.

Growth and development

The Hypothalamic Gonadotropin-releasing Hormone Pulse Generator A.

Located in the arcuate nucleus

B.

Receives multiple inputs 1.

Dopaminergic

2.

Noradrenergic

3.

Opioidergic

4.

Possibly "cortical"

5.

Possibly "metabolic"

C. Gonadotropin-releasing hormone (GnRH) pulses released at "basal" rate of 1 pulse/hour D. Transport to anterior pituitary via portal blood system III. The Anterior Pituitary A.

Site of pulsatile gonadotropin secretion in response to GnRH

B.

Up-regulation vs down-regulation

C. The luteinizing hormone LH surge 1.

Possible mechanisms

2.

Mystery of positive feedback

D. Modulators of pulse frequency: the progesterone/endorphin axis IV.

The Ovary A.

The two-cell theory of ovarian steroidogenesis 1.

LH interacts with theca cells and yields androgen production

2.

Follicle-stimulating hormone (FSH) interacts with granulosa cells and yields aromatization of androgens to estrogens

B.

Ovulation

The hypothalamo-pituitary ovarian endometrial axis or the reproductive axis for short is one of the four principal endocrine systems that traverses the anterior pituitary gland. Two of them, the adrenal axis and the thyroid axis, are absolutely necessary in the short-term for survival. The other two, the reproductive axis and the growth hormone axis, are not. But it is important to bear in mind that even if a woman is not interested in reproduction, the normal function of her reproductive axis is essential for her health because there are a number of other estrogen dependent tissues which, if deprived of estrogen over an extended period of time, will pay a price. Of course, the important ones are cardiovascular health, osteoporosis, central nervous system, female genital tract and probably skin. Turning first to luteinizing hormone, LH levels across the menstrual cycle are generally fairly stable if you look at mean levels on a particular day. As we will talk about in a few more minutes, there is actually a fine structure to LH levels which is characterized by the presence of pulses which occur about once an hour in the follicular phase. But in terms of mean levels across the day, LH levels are pretty much the same across the menstrual cycle with the exception of this one extraordinary feature which occurs at mid cycle which, of course, is the LH surge - the signal for ovulation. The pattern for FSH has slightly different features which are worth noting. FSH levels have a clear decline between the beginning of the menstrual cycle and ovulation. There is a little bit of an FSH surge in mid cycle and we are not really sure whether or not this has any physiologic significance. During the luteal phase, FHS levels are very, very low and finally at the very end of the luteal phase with the decline in sex steroid levels, FSH levels begin to increase. This increase actually is known to be significant because it has been shown that really at this point, when FSH levels being to rise, you for the first time see gonadotropin dependent follicular recruitment for the following cycle. We know that the reason for this difference in behavior between the two gonadotropins is that FSH is much more sensitive to negative feedback by estradiol and, in fact, all these features can be explained by what is happening with estradiol levels. Estradiol just prior to ovulation, estrogen levels rise fairly high to between 250300 picograms/ml. It is in response to this steady increase in estradiol levels that the FSH levels decline. Following ovulation, there is a fairly substantial decline in estradiol levels although it is probably not quite as dramatic as is shown schematically on this slide. This does have a little bit of clinical relevance. This decline, is not unusual for women who are having perfectly normal menstrual cycles to have a little bit of mid cycle spotting. The reason for the mid cycle spotting is that they can get a little bit of an estrogen withdrawal bleed right here. With the establishment of the corpus luteum, of course, estradiol levels rise and then fall. Progesterone levels are quite low during the follicular phase. They are uniformly less than 1 nanogram/ml in the serum. Just prior to ovulation, progesterone levels rise a little bit and that rise turns out to be part of the constellation of signals which causes the LH surge. Right around the time of ovulation, serum levels of progesterone cross 1 nanogram/ml. Again, with the establishment of the corpus luteum, progesterone levels rise substantially peaking in the mid luteal phase and then declining.

C. Luteinization of the follicle leads to progesterone production D. Local (paracrine) control mechanisms

V.

1.

Insulin-like growth factor-1

2.

Inhibin: an endocrine/paracrine hybrid

3.

Others a.

Epidermal growth factor

b.

Transforming growth factor-"

c.

Fibroblast growth factor

d.

Platelet-derived growth factor

e.

Angiogenic growth factors

The Endometrium A.

Only physiologic role is to support implantation

B.

Estrogen serves to stimulate endometrial proliferation

C. Progesterone inhibits estrogen-induced proliferation and induces

Just as an aside regarding the utility of measuring estradiol levels or progesterone levels in normal cycling women who aren't taking fertility drugs, about the only question you can really answer with a serum progesterone level is whether or not your patient has ovulated within the last 14 days. It is important to bear in mind that if you are worried about whether or nor your patient has an adequate luteal phase, if you think she might have a luteal phase defect that might be causing her to miscarry or to not become pregnant, you have to do an endometrial biopsy. There is a very, very poor relationship between circulating levels of progesterone and the presence of luteal phase deficiency. It is also not particularly useful to measure progesterone levels in women who are pregnant who you may be concerned might be threatening to miscarry and the like. You cannot identify women who will benefit from supplemental progesterone therapy once they are pregnant by measuring their serum progesterone levels. Everything we know about the biology of the luteal phase tells us that if a woman is going to benefit from progesterone supplementation, she probably needs to start it shortly after ovulation. Estradiol levels are generally not very useful in women of reproductive age who aren't being followed in some sort of ovulation induction regimen. If you have a patient with severe hypothalamic amenorrhea, you might measure an estrogen level but generally it is not a very useful test in terms of discriminating between

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additional

structural

changes;

this

leads

to

secretory

endometrium D. Withdrawal of hormonal support following the demise of the corpus luteum results in menstruation

different kinds of chronic and ovulation states. The first half of the cycle can be called either the follicular phase or the proliferative phase depending upon your bias. The interval between ovulation and menstruation in human beings is pretty much a biological constant. It averages quite close to 13½-14 days. Anything less than 12 should probably be considered abnormal. Anything more than 16 and the patient is probably pregnant.

The interval between the onset of the LH surge and the actual moment of ovulation is approximately 36 hours +/- 3 or 4. In contrast to the luteal phase, the interval between the first day of the period and ovulation is variable both from patient to patient and from cycle to cycle in the same patient. In my view, anything less than 10 days for the follicular phase is probably abnormal and anything more than 21 is probably abnormal. The vast majority of normal cycling women who don't have endocrine problems have menstrual cycle intervals between 26 and 31 days. The central nervous system gets inputs from a lot of places but very importantly it gets them from the environment. The arcuate nucleus and the median eminence from a place called the GNRH pulse generator, releases episodically a gonadotropin releasing hormone which is a ten amino acid polypeptide which traverses the portal blood system and impinges on the gonadotrope cells of the anterior pituitary. Subsequently, the gonadotrope cells release the two gonadotropins, LH and FSH, which in turn drive the ovary. The ovary, in addition to releasing an oocyte once a month, produces a number of sex steroids. The most important of them, of course, are estrogen and progesterone. These two hormones not only are responsible for priming the uterus and preparing the endometrium for implantation but they engage in important negative feedback loops at both the anterior pituitary and hypothalamic levels. In humans, there is only one gonadotropin releasing hormone. There is some evidence from other species that there might be a separate releasing factor for FSH but that has never been seen in humans. There is clearly a peptide known as inhibin which is a gonadal peptide which seems to be involved in a endocrine type of negative feedback loop on FSH alone. There is some controversy about whether circulating levels of inhibin are really what matters but the current consensus is that inhibin, secreted by either ovary or the testis in man, feeds back to the anterior pituitary and selectively suppresses FSH secretion. The hypothalamic pulse generator, is located in the arcuate nucleus at the base of the brain. It gets multiple inputs. It gets inputs from the cerebral cortex. It gets inputs from the midbrain, primarily noradrenergic inputs. It may get serotonergic inputs from other parts of the brain. It is very, very complex. The most interesting parameter about the pulse generator that can vary is the pulse frequency and this appears to be a function of the sex steroid milieu. The GNRH molecules reach the anterior pituitary by means of the portal blood system. In the early '50s, it was widely assumed that the anterior pituitary was hardwired to the brain by direct neural connections. No one knew anything about the portal blood system and certainly no one knew anything about hypothalamic releasing or inhibiting factors. Around this time, a British scientist named Jeffrey Harris proposed, on relatively little evidence, that the way the brain controlled anterior pituitary function was by these very means. By releasing certain chemical inhibitors or stimulators which traversed the portal blood system and ran the anterior pituitary. Unfortunately British science, particularly in the '50s, was a very conservative establishment and because of the fact that he didn't have sufficient evidence to support this hypothesis, he was very severely criticized for even talking about this. Ultimately, he became an alcoholic and died of cirrhosis of the liver but not before he was vindicated. The portal blood system basically starts with the superior hypophyseal artery which ramifies into this lacy venous plexus and carries releasing and inhibiting factors from the base of the brain, from the hypothalamus to the anterior pituitary. 3

The arcuate nuclei are shaped kind of like thick potato chips. There is one on either side of the third ventricle. They kind of cradle it. The way we think of the arcuate nucleus is that it contains three families of neurons which all kind of talk to each other. This is extremely complicated. It is obviously something that is somewhat difficult to study, particularly in humans. But our understanding is that in the first place we have GNRH neurons which make the GNRH itself and dump in into the portal blood system. But residing alongside of the GNRH neurons are two other families of neurons, one of which makes dopamine which is a neurotransmitter and the other which makes beta-endorphin. The three of these kind of intercommunicate and result in the final output of the GNRH. As I mentioned earlier, there are inputs that sort of modulate this overall system which originate either in the midbrain or other parts of the brain. These three families of neurons are kind of interacting in some unspecified way but the output is a train of pulses of gonadotropin releasing hormone which occur again in the follicular phase about once an hour. They traverse the portal blood system, they impinge upon the gonadotrophs and what we know from animal experiments is that there is a very, very clear one-to-one relationship between GNRH pulses and gonadotropin pulses in the periphery. LH has a much shorter half-life than FSH and because of that it is very easy to see LH pulses in the periphery if you do frequent blood sampling experiments. The reason you can see them is because the LH is released as a pulse and it is cleared very rapidly. In contrast, FSH has a number of extra sialic acid residues and consequently it has a very long half-life. So even if you do very frequent blood sampling, if you measure FSH, you don't see these sharp pulses. But at least with respect to LH, they are quite easy to see and we now have very sophisticated computer programs which count them for us in an objective way. During the follicular phase, the frequency of the pulse generator is about one pulse per hour. In the luteal phase the pulses become much taller and much less frequent and these two changes kind of balance each other out and mean LH levels stay the same in the luteal phase as they were in the follicular phase. When this phenomenon was first studied, it was widely assumed that it had some significance in terms of the physiology of the corpus luteum. A lot of time and energy was spent sort of elucidating what was going on with this. It was discovered that if you treat a woman in the follicular phase with progesterone, her pulses slow down and get bigger. It was determined that this is a progesterone mediated effect and the timing kind of makes sense. You don't really see this change evolving until progesterone is being secreted in significant amounts in the luteal phase and if you study pulse frequencies in women who are chronic anovulators. A second series of experiments illuminated another aspect of this observation. If in the luteal phase in a normal cycling woman, you infuse naloxone which blocks endogenous endorphins, then you rapidly convert the pulse pattern to a follicular phase pattern. Likewise, if you give naloxone to that patient who you have given progesterone to in the follicular phase, you can block the progesterone mediated effect. This significant alteration in pulse frequency of both GNRH and, by proxy, LH is it is caused by progesterone and it is mediated by this beta-endorphin system in the brain. There are women with the disorder called isolated gonadotropin deficiency which is actually a misnomer. It is actually an isolated deficiency of gonadotropin releasing hormone. They have perfectly normal anterior pituitary glands and it is possible to treat these women with pulsatile gonadotropin releasing hormone. You can supply them with a little artificial hypothalamus. You can put an IV in an arm vein and they walk around for two weeks getting little pulses of GNRH once an hour. What was discovered once these patients were getting pregnant was that if they forgot to take off their pulse generators and they continued to be treated with the GNRH pulsatile therapy through the luteal phase with the same frequency and dosage, it had absolutely no bearing on corpus luteum function or their chances of getting pregnant. It may be that this is the endocrinologic version of the appendix. It is just something that evolved a long time that is still there but has no major bearing on our current physiology. The anterior pituitary is where pulsatile gonadotropin secretion originates from.

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The pattern of hormone release from the target tissue, which in this case is the gonadotrope cells and what is happening with the binding site concentration of the trophic hormone. We have GNRH binding sites on the gonadotrope cells of the anterior pituitary gland. The term up-regulation refers to the phenomenon whereby pulsatile secretion of the trophic hormone allows the target tissue to work its best. What happens when GNRH is secreted in a pulsatile manner is that the number of GNRH binding sites rises to a maximum and stays there. Because of this, the target tissue is able to, in a continuous manner, release pulse after pulse after pulse of the hormone - in this case LH. In contrast, it has been found that if you infuse a steady bombardment of GNRH nonstop, what you see is an initial rise in the number of binding sites on the gonadotrope cells but then they disappear and this phenomenon whereby the receptors for GNRH disappear under this relentless stimulation from GNRH is called down-regulation. What you see in terms of target organ response is that LH levels initially rise and then decline to very low levels to the point where, for all intensive purposes, normal levels of gonadotropin that are required for ovarian function are no longer being released. Once this phenomenon was identified, pharmaceutical companies jumped on it as a potential tool for developing drugs which could very selectively shut off the reproductive axis. The strategy they used was to take the native GNRH molecule and modify it at several positions so that in essence you had a super agonist. You had a molecule that looked just like GNRH but bound very avidly to the GNRH receptor and we now have a series of drugs which work this way with names like Lupron, Naprelan and Synarel. They are extremely useful if you are taking care of patients who have diseases that get worse when there is estrogen around. We also use these GNRH agonists in patients who are undergoing in vitro fertilization and embryo transfer but that is really a different context. We don't understand all of the details of the LH surge and how it occurs, there are a couple of things that we do know. The LH surge only occurs if serum estradiol levels have been elevated to a sufficient level for a sufficient period of time. We know that time interval has to be a few days, probably two or three days and we know that the level above which the estradiol level has to be is probably above 150 or 200 picograms/mL. The observation that these prolonged high levels of estradiol lead to this sudden outpouring of LH in the form of the LH surge is referred to as positive feedback. What is happening beginning right before menstruation is that a cluster, a group of follicles, are chosen basically as candidates for dominance in the next cycle under gonadotropin stimulation. Usually, this cluster at this point consists of somewhere between 6 and 8 follicles. As one goes through the follicular phase, one by one these follicles become atretic until at the very end you are left with a single dominant follicle. Experiments in rhesus monkeys which have 28 day cycles which are very similar to those of women, indicate that if you do selective vein sampling from the two ovaries, you can actually detect, on the basis of estradiol levels coming from that ovary, which ovary is going to give you the dominant follicle by day 5. So it may actually be destined as early as the 5th day of the menstrual cycle which is going to be the winning follicle. There are a number of theories about how the dominant follicle gets selected and in a couple of slides I will show you the one that seems to have the most appeal. Primordial follicles are quite small. They are lined with a single layer of granulosa cells and as one moves through the follicular phase, they become larger and once you get into the mid-follicular phase, you get antrum formation. You get the development of a fluid pocket which you can actually see on ultrasound. When you get to the point right before ovulation, you are dealing with a very macroscopic structure. A normal ovary is about 1 x 3 x 4 cm in dimension. It is sort of a football-shaped structure, an almond-shaped structure, and the 5

dominant follicle is 2 cm across. If you look at a ovary at midcycle at the time of laparoscopy and you see a dominant follicle, it looks pretty big. Because sometimes a corpus luteum can look like something that is abnormal and the temptation is to sort of fool around with it, poke it and prod it and that is one thing you do not want to do because a corpus luteum is a very vascular structure. The important features of dominant follicles here are a large antrum, which is really easy to see on ultrasound, and the oocyte itself with its own personal investment of granulosa cells. The inner layer of cells are the granulosa cells and this outer investment of cells moving out into the stroma are the thecal cells. The best way to think about a growing dominant follicle is that it is comprised of two concentric spheres which are stuck to each other by a basement membrane. The inner cells are the granulosa cells and the outer cells on the outside of the basement membrane are the thecal cells. What we now understand about the way steroidogenesis takes place in the ovary is what is referred to as the two-cell theory. Thecal cells seem to be primarily under the control of LH. LH binds to LH receptors in the thecal cell. Then under a cyclic-AMP mediated process, it turns cholesterol, which is the mother of all steroids into androgens, principally androstenedione and testosterone. These androgens migrate across the basement membrane by simple diffusion and then under FSH stimulation within the granulosa cells again by means of a cyclic-AMP mediated phenomenon, the androgens are aromatized to estrogens. As one proceeds through the follicular phase, several things happen. In response to rising levels of estradiol, in the first place, the granulosa cells become more plentiful. They undergo mitosis. There are more of them. Secondly, and very importantly, these rising estradiol levels induce the development of additional FSH receptors in the granulosa cells. Regarding how the dominant follicle gets selected, it has been proposed that the following is the mechanism. Perhaps early in the cycle, there is one follicle that happens to be at that particular moment the biggest. It is making a little more estrogen than its competitors and it has a few more FSH receptors. It is making more estradiol, it is able to endow itself with more FSH receptors than any of the other follicles. Then, as these rising estradiol levels suppress circulating FSH levels, in essence it starves out its competition. It can get by on lower levels of circulating FSH because its got the most receptors. What happens when we use fertility drugs which contain FSH to induce multiple ovulation is to in essence override this system by maintaining constant high levels of FSH in the circulation. Towards the end of the follicular phase, these rising estradiol levels induce the production of LH receptors on the granulosa cells. This is a change that is in anticipation of the conversion of the granulosa cells into the luteinized cells which will form the corpus luteum. It is probably the case that you need both gonadotropins in both the follicular and the luteal phases. But as a practical matter, in the follicular phase the gonadotropin that you need the most is FSH. You need very little LH to give you the amount of androgen that you need to grow a dominant follicle. You really need FSH the most in the first half of the cycle. In the luteal phase, the gonadotropin that you need the most to keep a corpus luteum going is LH. In practice, if for example, we want to support corpus luteum function in a patient who doesn't make gonadotropins, what we will ordinarily do is give her a shot of hCG every three days to provide her with some sort of LH support. So there is kind of an asymmetry in the importance of the gonadotropins in the two halves of the menstrual cycle. The LH surge leads to the actual rupture of the dominant follicle and release of the oocyte is very complex. There is a lot more to it than just a bolus of LH traveling through the bloodstream, eroding a hole in the dominant follicle and allowing the oocyte to emerge. Apparently, the stimulation of this entire complex by LH results in an entire cascade of different consequences which are probably all related in one way or another to normal ovulation. A number of 6

peptides get made locally including things like oocyte maturation inhibitor, collagenases, prostaglandins and the like and they all seem to work together to allow both follicular rupture and the timely maturation of the developing oocyte. The endometrium only has one biologic purpose and that is to support implantation. It doesn't do anything else. The endometrium, at the very beginning of the menstrual cycle when menstrual bleeding is still occurring, heals up and stops bleeding in response to estradiol. Over the next 14 days, the thickness of the endometrium goes from just a millimeter or two up to about, if you look at a stripe on vaginal probe ultrasound, probably 6 or even 7 mm. Once progesterone starts getting secreted in substantial amounts, that height is largely fixed. What happens by in large in response to progesterone is that the histology changes into secretory endometrium. At a deeper level, the secretory endometrium seems to develop the capability to make other peptides that it couldn't ordinarily make. A good example is prolactin.

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