Neuroendocrine Control of the Menstrual Cycle

The Reproductive Axis

Normal reproductive function in women involves repetitive cycles of follicle development, ovulation, and preparation of the endometrium for implantation should conception occur in that cycle. This pattern of regular ovulatory cycles is achieved through precise functional and temporal integration of stimulatory and inhibitory signals from the hypothalamus, the pituitary, and the ovary ( Fig. 7.1 ). The reproductive system functions in a classic endocrine mode. The master hormone, gonadotropin-releasing hormone (GnRH), is secreted in a pulsatile fashion from the hypothalamus into the pituitary portal venous system. GnRH regulates the synthesis and subsequent release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from gonadotropes within the anterior pituitary into the circulation. FSH and LH stimulate the recruitment and development of ovarian follicles, ovulation, corpus luteum formation, and the coordinated secretion of estradiol, progesterone, inhibin A, and inhibin B. A key component of this system is the modulatory effect of ovarian steroids and inhibins on gonadotropin secretion. Ovarian steroids impact the amplitude and/or frequency of GnRH secretion through their effects on KNDy neurons, discussed below (also see Chapter 1 ), that act upstream of GnRH neurons in the hypothalamus. In addition, ovarian steroids and inhibins act directly at the pituitary level. Negative feedback restraint of FSH secretion is critical to the development of the single mature oocyte that characterizes human reproductive cycles. In addition to negative feedback controls, the menstrual cycle is unique among endocrine systems in its dependence on estrogen-positive feedback to produce the preovulatory LH surge that is essential for ovulation.

Fig. 7.1, Neuroendocrine control of reproduction requires the pulsatile secretion of gonadotropin-releasing hormone ( GnRH ) released into the pituitary portal system to stimulate the synthesis and secretion of luteinizing hormone ( LH ) and follicle-stimulating hormone ( FSH ) from pituitary gonadotropes.

Neuroendocrine Components of the Reproductive Axis

Genetic findings from patients with congenital deficiencies in gonadotropin secretion have significantly advanced our understanding of the ontogeny and upstream regulation of GnRH .
Kisspeptin, neurokinin B, and dynorphin are important regulators of GnRH synthesis and secretion and transduce gonadal steroid feedback to GnRH neurons .
The differential control of LH and FSH requires the integration of GnRH pulse amplitude and frequency with direct pituitary feedback from estradiol and inhibins .
Ovarian negative feedback on LH and FSH is primarily, but not exclusively, mediated through kisspeptin control of GnRH secretion with the additional pituitary effect of the inhibin/activin/follistatin system on FSH .
Ovarian positive feedback in women and nonhuman primates is mediated primarily at the pituitary level with the permissive involvement of GnRH; in rodents increased kisspeptin-mediated GnRH stimulation is required in addition to direct pituitary effect .

Gonadotropin-Releasing Hormones

Luteinizing-releasing hormone (LHRH) was isolated, characterized, and synthesized in 1971. The central role of this decapeptide in the propagation of the species makes it fitting that Drs. Schally and Guillemin received the Nobel Prize in Physiology and Medicine in 1977 for its isolation. It was expected that separate releasing hormones for LH and FSH would be discovered. However, subsequent studies provided evidence that both LH and FSH are secreted in response to LHRH, resulting in the common use of the term gonadotropin-releasing hormone (GnRH) for the decapeptide originally referred to as LHRH.

GnRH neurons differentiate in the olfactory placode, cross the cribriform plate into the forebrain, and migrate to the medial basal hypothalamus, where they establish connections with the pituitary portal system in the median eminence as part of the hypothalamic tuberoinfundibular system. The initial leg of this migratory journey occurs along the scaffold of olfactory, vomeronasal, and terminal nerves. The importance of this developmental pathway is evidenced in patients with Bosma arhinia microphthalmia syndrome. In this syndrome, individuals are born without a nose, are anosmic, and fail to go through puberty due to hypogonadotropic hypogonadism. In humans there are approximately 7000 GnRH expressing neurons in areas of the brain linked to gonadotropin regulation. Unlike neurons secreting other hypothalamic releasing factors, GnRH neurons do not exist in a defined nucleus but are scattered throughout the medial basal hypothalamus, with additional scattered neurons in the preoptic area.

Over the past three decades, genetic studies in patients with idiopathic hypogonadotropic hypogonadism (IHH) with concomitant disruption of the olfactory system resulting in anosmia (Kallmann Syndrome; KS) or without anosmia (normosmic idiopathic hypogonadotropic hypogonadism; nIHH), have resulted in unprecedented growth in our understanding of the complex neuroendocrine control of reproduction. Mutations in well over 50 genes have now been discovered in this rare patient population. Validation of their function in animal and cell systems indicates that these can be broadly classified into four groups. A number of genes have been discovered that are involved in the early migration and axonal guidance of GnRH neurons on the path to their eventual home in the hypothalamus, some of which are associated with other developmental defects. This group of genes includes Kallmann 1 ( KAL1 ) now known as anosmin 1 (ANOS1), chromodomain helicase DNA binding protein 7 (CHG7), sex-determining region of Y-box 10 ( SOX10 ), semaphorin-3A (SEMA3A), ^, fasciculation and elongation protein zeta family zinc finger 1 ( FEZF1 ), fibronectin leucine-rich transmembrane protein 3 ( FLRT 3), IL-17 receptor D ( IL17RD ), and structural maintenance of chromosomes flexible hinge domain-containing protein 1 ( SMCHD1 ), among others. Genes involved in the control of GnRH secretion include kisspeptin and its receptor ( KISS1/KISS1R ), tachykinin 3 and its receptor ( TAC3/TACR3 ), gonadotropin-releasing hormone1 ( GNRH1 ), ^, and dosage-sensitive sex reversal 1 ( DAX1 ), also known as nuclear receptor subfamily 0, group B, member 1 ( NROB1 ). Genes that appear to play a role in both GnRH ontogeny and function include fibroblast growth factor 8 and its receptor fibroblast growth factor receptor 1 (FGF8/FGFR1), prokineticin 2 and its receptor (PROK2/PROKR2), ^, heparin sulfate 6-O-sulfotransferase 1 (HS6ST1), WD repeat domain 11 (WDR11), AXL receptor tyrosine kinase ( AXL ), NMDA receptor synaptonuclear signaling and neuronal migration factor ( NSMF ), dual specificity phosphatase 6 ( DUSP6 ), sprouty homolog 4 ( SPRY4 ), and fibroblast growth factor 17 ( FGF17). Finally, the genes involved in gonadotrope stimulation that have been discovered to date in association with IHH include only DAX1 and gonadotropin-releasing hormone receptor ( GNRHR ). As more patients are identified and studied, this list will undoubtedly continue to grow.

Pulsatile Secretion of GnRH

A prominent feature of the reproductive system is the absolute requirement for pulsatile secretion of GnRH into the pituitary portal system for normal gonadotropin secretion. The now classic studies of Knobil and colleagues in hypothalamic-lesioned monkeys receiving GnRH first showed that intermittent stimulation of the pituitary results in secretion of LH and FSH, while constant GnRH stimulation is associated with suppression of gonadotropin levels. Isolated GnRH neurons exhibit an intrinsic pulsatility, but there is also a significant body of research indicating that external influences modify and coordinate the secretion of GnRH, influencing both the amplitude and frequency of pulsatile GnRH secretion.

Neuromodulators of GnRH Secretion

While a number of neurotransmitters are involved in the control of GnRH secretion in animal species, only a few have been shown to have an effect on humans. Although there is evidence for a stimulatory role of the α-adrenergic system in several animal models, it is much less likely that it plays a role in the control of the human menstrual cycle. The role of the dopaminergic system remains controversial, but studies that have documented an increase in LH pulse frequency in response to a dopamine antagonist in women with hypothalamic amenorrhea suggest that dopamine may inhibit GnRH secretion in women. ^,

Kisspeptin

Knock-out models suggest that there is considerable redundancy in the systems that ultimately control GnRH secretion; however, it is now firmly established that the kisspeptin pathway is a key upstream modifier of GnRH secretion. As with the genes that are now known to control the developmental migration of GnRH neurons, a role for kisspeptin in reproduction was initially discovered by the combination of genetic studies in patients with IHH which identified mutations in the gene encoding the kisspeptin receptor ( KISS1R , formerly known as G-protein coupled receptor 54 [ GPR54 ]) and knock-out mouse models. ^, Kisspeptin is an extremely powerful stimulator of LH, an action that is blocked by a GnRH antagonist, indicating that the effect of kisspeptin on LH is mediated through control of GnRH secretion. ^, The kisspeptin system is thought to play a dominant role in the onset of puberty and mediates estrogen and progesterone negative feedback in the median eminence. Studies of kisspeptin administration in women, performed using different isoforms and either subcutaneous, intravenous bolus, or intravenous infusion modes of administration, have demonstrated a marked difference in LH response depending on cycle phase and hormonal status. The response to kisspeptin is consistently robust in the late follicular, preovulatory, and luteal phases of the menstrual cycle and in postmenopausal women, while there is some inconsistency in the early follicular phase with a lower, and in some cases absent, LH response to kisspeptin The LH response to continuous kisspeptin was similarly low in postmenopausal women but increased after two weeks of estrogen replacement in a dose-dependent fashion, an effect that appears to be mediated at both the pituitary and the hypothalamic level.

In rodents and sheep, there is ample evidence that positive feedback is manifest in the hypothalamus as well as the pituitary with the generation of a marked increase in GnRH at the time of the midcycle gonadotropin surge. ^, Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) have now been implicated in this estrogen-positive feedback on GnRH secretion in rodents. The relationship of the AVPV to the suprachiasmatic nucleus (SCN) in the rodent provides a potential mechanism for the known circadian timing of the proestrus surge in the rodent.

Despite compelling evidence in lower animal species, it is likely that kisspeptin control of GnRH does not play a role in the midcycle surge in women; studies in women demonstrate a paucity of kisspeptin neurons in an analogous hypothalamic region and evidence discussed below demonstrates that a GnRH surge is not required for the generation of a midcycle LH surge in GnRH-deficient women and further suggests that a GnRH surge is not present in normal women.

Neurokinin B

Neurokinin B (NKB), which is encoded by the tachykinin 3 gene (TAC3) and its cognate receptor, NK3R, encoded by TACR3, have also been implicated in the normal control of GnRH secretion through genetic studies in patients with IHH. NKB stimulates LH secretion, acting upstream of the GnRH neuron. ^, TAC3 and KISS are colocalized in the human as well as in other species, particularly in the arcuate/median eminence. There is significant evidence that the effect of NKB on GnRH is exerted primarily through kisspeptin. NKB agonists stimulate gonadotropin secretion while NKB3 antagonists inhibit LH secretion, but do not appear to do so in the setting of high estrogen, as in the preovulatory phase Interestingly, the NKB3/NK3R system also plays a role in hot flashes in estrogen-deficient states.

Endogenous Opioids/Dynorphin

There is substantial evidence for the involvement of endorphins in transducing the negative feedback effects of progesterone on pulsatile GnRH secretion from studies using the opioid receptor blocker, naloxone, in women. ^, However, naloxone binds not only to the mu receptor but also to the kappa and gamma receptors and thus these early studies could not provide mechanistic specificity. Dynorphin which binds to the kappa-opioid receptor has now been identified as the key mediator of progesterone negative feedback.

KNDy Neurons

In a variety of animal species and humans it has been shown that kisspeptin, NKB, and dynorphin are coexpressed in cells in the arcuate nucleus/median eminence that are now referred to as KNDy neurons. These neurons express estrogen, progesterone, and androgen receptors and mediate gonadal steroid negative feedback on GnRH secretion with increasing evidence that they are also involved in the initiation and termination of GnRH secretion that results in its pulsatile secretion. Gamma-amino butyric acid (GABA) may also be involved in mediating estrogen-negative feedback on GnRH secretion, particularly in the arcuate/median eminence. ^,

RFamide-Related Peptides

RFaimde-related peptides (RFRP) are the mammalian orthologues of gonadotropin inhibitory hormone (GnIH) which was first discovered in the hypothalami of the quail. In humans, RFRP-1 and RFRP-3 neurons send axonal projections to GnRH neurons. ^, RFRPs are secreted into the pituitary portal system and their receptor, G-protein coupled receptor 147 (GPR147), is present on gonadotropes as well as in the hypothalamus Taken together with functional data from animal and cellular systems, these findings suggest that RFRPs function at both the hypothalamus and pituitary to regulate the secretion of LH and FSH. Interestingly, there is also evidence that RFRPs increase food intake in sheep without reducing energy expenditure. There is currently limited data to address the role of these peptides in humans. A three-hour infusion of custom synthesized GnIH resulted in a modest suppression of LH secretion in postmenopausal women but failed to inhibit LH secretion in response to pulses of kisspeptin-10 in men. Additional studies will be required to ascertain its physiology and potential therapeutic role in men and women.

Sleep and Circadian Effects on GnRH Secretion in Women

Endocrine systems are profoundly influenced by both sleep and endogenous circadian rhythms, which are intrinsic rhythms that persist in the absence of sleep or other environmental cues. Diurnal (day and night) rhythms of LH and gonadal steroids have been well described in men and women. However, studies in which sleep and other environmental cues were controlled have failed to demonstrate an endogenous circadian rhythm of LH or FSH in early follicular phase women ( Fig. 7.2 ) and in postmenopausal women, despite the presence of robust circadian rhythms of temperature, cortisol, and TSH. ^,

Fig. 7.2, Mean + sem of temperature, FSH, LH, FAS, and TSH levels in early follicular phase women (n = 11) during a constant routine of light, position, wake, and nutritional intake over a 24-hour period.

In contrast, there is compelling evidence that sleep directly affects the pulsatile secretion of LH and presumably that of GnRH. Studies that have separated the effects of sleep from time of day demonstrate that during puberty in boys and girls, pulsatile LH secretion is increased during sleep. ^, More recent studies indicate that LH pulses are most commonly preceded by slow wave sleep (SWS) and further studies have shown that even with repeated sleep interruption, 20 min of accumulated sleep is associated with LH pulse onset in this population. These studies suggest that factors associated with SWS stimulate GnRH secretion or that there is an upstream regulator of both GnRH secretion and deep sleep in puberty.

Paradoxically, with the maturation of the reproductive system and the onset of ovulatory menstrual cycles, there is a notable slowing of pulsatile LH secretion at night in the early follicular phase of the cycle. ^, Sleep reversal studies in women have demonstrated that the early follicular phase of nighttime slowing is due to sleep rather than the time of day. Importantly, within sleep, brief periods of wakefulness are associated with the onset of LH pulses ( Fig. 7.3 ), while SWS is inhibitory to LH pulses.

Fig. 7.3, As indicated in the left panel, sleep is specifically associated with slowing of luteinizing hormone ( LH ) pulses.

Data relating luteal phase characteristics to early follicular phase nighttime pulse frequency suggests that prior progesterone exposure sensitizes the GnRH pulse generator to the inhibitory effects of sleep in the early follicular phase. In this regard, it is of interest that GnRH pulse frequency, as determined by the pulsatile secretion of the gonadotropin-free alpha subunit (FAS), is also slower during sleep than wake in postmenopausal women whose gonadal steroid levels are similar to those in early puberty, where sleep is stimulatory. Although the effect of sleep is much less than in normally cycling women, this finding suggests that the inhibitory effect of sleep on LH pulse frequency is not only related to prior progesterone exposure but that other factors relating to the development of a mature reproductive axis are involved.

Gonadotropin-Producing Cells of the Pituitary

LH and FSH are synthesized in gonadotropes, which comprise between 7% and 15% of the cells in the pituitary. Immunohistochemical studies in the rat indicate that approximately 70% of gonadotropes stain for both LH and FSH, while the remainder stains for LH or FSH in approximately equal numbers. As the animals approach the day of the gonadotropin surge in proestrus, monohormonal cells begin to express both βLH and βFSH, while a population of cells that express growth hormone (GH) also express the gonadotropin subunits.

LH and FSH are glycoprotein hormones whose polypeptide and polysaccharide components are essential for their activity. Biosynthesis of intact gonadotropins involves (1) translation of βLH, βFSH, and the common gonadotropin α-subunit; (2) posttranslational modification and folding; (3) combination of the β- and α-subunits; and (4) modification of the oligosaccharide residues on LH and FSH as they traverse the Golgi. FSH is synthesized under the dual control of GnRH and the activin/inhibin/follistatin system and is secreted primarily in a constitutive manner with little storage. In contrast, LH is packaged into granules and stored. LH and the gonadotropin-free α subunit (FAS) are then secreted in response to GnRH stimulation of the gonadotrope.

Gonadotropn Isoforms

Multiple isoforms of LH and FSH, differing in their carbohydrate structure and charge, coexist in both pituitary and serum. When combined with a β-subunit, the α-subunit has two glycosylation sites. FSHβ also has two potential glycosylation sites, while LHβ has a single potential site. This results in the secretion of FSHtri and FSHquatro and LHdi and LHtri. Terminal sulfonated and/or sialylated residues on these glycoforms add further isoform heterogeneity to secreted gonadotropins. More basic forms of both LH and FSH yield a greater in vitro potency, but a shorter half-life in the circulation, while the opposite is true for less basic forms. The greater number of sialic acid residues on FSH prolongs its half-life, whereas the greater number of sulfonated N-acetyl-galactosamine (GalNAc) asparagine-linked oligosaccharides on LH is associated with more rapid clearance due to binding to a specific hepatic receptor. Sulfonation and sialylation of LH and FSH vary across the menstrual cycle and in the absence of gonadal steroids; postmenopausal women have a greater preponderance of sialylated forms of both LH and FSH. The number of sulfonated and sialylated residues on LH and FSH is tightly linked to hormone clearance in women and, by inference, to bioactivity. Thus, the disappearance of LH following GnRH receptor blockade with a potent GnRH antagonist is significantly prolonged in postmenopausal women compared to women in the follicular phase and at the midcycle surge (MCS), while the disappearance of FAS is unaffected by the absence of gonadal function ( Table 7.1 ). Changes in the isoform composition of FSH in the normal menstrual cycle are likely to augment the effect of the rise in FSH on follicle recruitment and development during the luteal-follicular transition through a decrease in clearance; in contrast isoform changes that increase clearance would curtail the potential effect of the increase in FSH on follicle recruitment at midcycle.

Table 7.1

The Half-Life of LH, But Not the Gonadotropin Free α Subunit (FAS), Is Influenced by the Gonadal Steroid Milieu

From Sharpless JL, Supko JG, Martin KA, Hall JE. Disappearance of endogenous luteinizing hormone is prolonged in postmenopausal women. J Clin Endocrinol Metab . 1999;84[2]:688–694.

	LH		Gonadotropin Free α Subunit
	Baseline (IU/L) Mean ± SEM	T1/2 (minutes) Mean ± SEM	Baseline (pg/mL) Mean ± SEM	T1/2 (minutes) Mean ± SEM
Postmenopausal	62 ± 3	139 ± 35	774 ± 45	51 ± 26
EFP, LFP, ELP	10 ± 1	57 ± 28	266 ± 44	41 ± 12
MCS	56 ± 11	78 ± 20	627 ± 122	41 ± 19

LH , luteinizing hormone; FSH , follicle-stimulating hormone.

Effect of Obesity

There is also evidence that gonadotropin secretion is modulated by a factor or factors related to obesity. Serum levels of LH are inversely related to body mass index (BMI) in normal women and in women with polycystic ovary syndrome (PCOS). Further studies have indicated that the inhibitory effect of obesity on LH secretion in PCOS is not mediated at the hypothalamus but is associated with a decrease in both the pituitary response to GnRH and the half-life of endogenous, but not exogenous, LH. ^, The latter finding is consistent with the increase in sulfonated isoforms of LH and FSH as a function of increasing BMI in women with PCOS. Recent studies have shown that in obese men and obese women without PCOS, the combination of an insulin and lipid/heparin infusion which results in increased free fatty acids and triglycerides, suppresses LH and FSH. Although the characteristics of pulsatile LH secretion were not measured in this study, the studies described above would suggest that the resultant decrease in LH and FSH is mediated at the pituitary.

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