The synthesis and release of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gland is primarily under the control of gonadotropin-releasing hormone (GnRH). There is some empirical evidence in the rat for the existence of a separate follicle-stimulating releasing hormone principle yet to be isolated.^{1, 2} In the primate, however, GnRH induces the synthesis and release of both LH and FSH from pituitary gonadotrophs. A decapeptide (Glu ¹-His ²-Trp ³-Ser ⁴-Tyr ⁵-Gly ⁶-Leu ⁷-Arg ⁸-Pro ⁹-Gly ¹⁰-amide) GnRH of arcuate nucleus origin reaches the pituitary gonadotrophs through the portal vessels. This chapter reviews the pituitary response to GnRH and its modulation by sex steroids throughout the menstrual cycle and in pathologic states.

The GnRH Gene

The structure of the peptide encoded by the GnRH gene is shown in Figure 1. The gene encodes for a signal peptide sequence, the GnRH sequence itself, and for a posttranscriptional processing signal (three amino acids long). In addition, it encodes for a 56-amino acid peptide that has been termed GnRH-associated peptide (GAP).³ The structure of GAP is similar in the human, rat, and mouse, and because it is present in the same neurons as GnRH in the primate brain, it is believed to be cosecreted with GnRH.⁴ Although less potent, GAP, like GnRH stimulates the in vitro release of gonadotropins. Perhaps more important than its effect on gonadotropin release, GAP inhibits prolactin release from rat anterior pituitary cell cultures. However, when administered systemically to rats, GAP exhibits both an inhibitory activity on the pituitary release of prolactin and a secretagogue action on FSH.⁵ The physiologic role of GAP has not been defined. For instance, unlike dopamine, the putative prolactin inhibitory factor, GAP is unable to inhibit prolactin secretion by human pituitary adenoma cells in culture.⁶

Another compound under study is a 13-amino acid long peptide within GAP that has been termed precursor human GnRH(14–26) (see Fig. 1). This smaller peptide stimulates the secretion of gonadotropins when applied to baboon and human pituitary cell cultures.^{7, 8} Nevertheless, unlike GAP, precursor human GnRH(14–26) has no effect on prolactin secretion. Interestingly, its action on the release of gonadotropins is not inhibited by GnRH antagonists and, therefore, does not appear to be mediated via the pituitary GnRH receptor. It is tempting to speculate that sustained precursor human GnRH(14–26) release may be responsible for the residual secretion of immunoreactive, but not bioactive, gonadotropins in GnRH antagonist-treated men or women.^{9, 10}

GnRH Secretion

GnRH is secreted in a pulsatile fashion that has been described in nearly every mammal.¹¹ This phenomenon depends on a pulse generator with intrinsic rhythmicity¹² located in the area of the arcuate nucleus of the mediobasal hypothalamus. Each pulsatile secretory episode of LH results from a corresponding hypothalamic discharge of GnRH.¹³ In contrast, although GnRH is necessary for FSH secretion, GnRH and FSH pulses are not always temporally related.¹⁴ LH does not interfere with the “pulse generator” (short feedback).¹²

The GnRH Receptor

GnRH binds to specific membrane receptors at the level of the pituitary gonadotroph from which it disassociates rapidly.¹⁵ Once unbound, GnRH is highly susceptible to proteases in the hypothalamus, pituitary, and blood.¹⁶ Upon the binding of GnRH, microaggregation of receptors and internalization occurs, a phenomenon that is markedly reduced in vitro in the absence of calcium.¹⁷ The binding of GnRH to its receptor results in two cellular responses (Fig. 2). On the one hand, a rapid influx of Ca⁺⁺ from the extracellular pool into the cells takes place. This ion, in turn, activates a calcium-binding protein, calmodulin. On the other hand, the binding of GnRH to its receptors stimulates the cellular production of specific membrane-associated lipid-like diacylglycerols, which, acting as a second messenger, activate the enzyme protein kinase C (PKC). Independently, activated calmodulin and activated PKC can promote the release of gonadotropins. However, the formation of diacylglycerols amplifies the action of Ca⁺⁺-calmodulin, thereby synergistically enhancing the release of gonadotropins.¹⁸

In recent years, the human GnRH receptor (GnRHr) has been cloned,¹⁹ as has been accomplished in the rat,^{20, 21} mouse,²² and sheep.²³ The human GnRHr cDNA is 1330 nucleotides long, and contains an apparent poly (A⁺)- tail at the 3' end.¹⁹ The open reading frame of this gene encodes for a 328 amino acid protein receptor, the structure of which is illustrated in Figure 3. Analysis of the peptide sequence suggests the presence of seven hydrophobic transmembrane domains, as well as several potential phosphorylation and glycosylation sites. Particularly noteworthy is the fact that the GnRHr is unique among the 7-transmembrane domain, G-protein coupled receptors in that it lacks a C-terminal cytoplasmic domain. It has been suggested that the absence of this domain may be related to the propensity of the GnRHr to desensitize.¹⁹

Cloning of the GnRH receptor has enabled investigators to probe various tissues for the presence of mRNA corresponding to the GnRH receptor. GnRHr transcripts are expressed in the pituitary,¹⁹ pituitary adenomas,²⁴ ovary, breast, testis, prostate,¹⁹ and granulosa-luteal cells from patients undergoing in vitro fertilization procedures.²⁵ The human GnRHr gene produces an 800 bp polymerase chain reaction product found in multiple tissues, and a 5.0 kb mRNA on northern analysis. Interestingly, GnRHr mRNA expression in granulosa-theca cells is upregulated by GnRH, suggesting a potential paracrine role for GnRH in the ovary. In summary, the recent cloning of the human GnRHr gene has provided new avenues to better understand the mechanisms underlying GnRHr function.

Modulation of the GnRH Pulse Generator by Sex Steroids

The relative importance of the modulation of the pulse generator by sex hormones has not been well-defined. Sex steroids modulate the pulse generator directly and indirectly. For example, both testosterone and progesterone can each decelerate GnRH pulse frequency, a phenomenon mediated, in part, by the opioidergic system.^{26, 27} A role for estradiol is suggested by the observation that in those treated with estradiol, the female rhesus monkeys, as well as preovulatory women, display increased GnRH amplitude and frequency.^{28, 29} Nonetheless, estradiol does not seem to play a major physiologic role in the modulation of the “pulse generator.” In fact, ovulation can be induced in women with hypothalamic failure using GnRH delivered in a fixed pulsatile pattern. Interestingly, estradiol decreases pulse generator activity in castrated female monkeys.³⁰ This observation points to a putative, yet indeterminate, ovarian factor that at least partially protects the neural system from the influence of estrogen. During the luteal phase, the influence of estrogens and progesterone on the pulse generator, rather than on the pituitary responsiveness to GnRH, results in a slower LH pulse frequency.³¹ Taken together, the data presented suggest that the most important modulating effect of sex steroids is exerted at the pituitary level. Estrogens and progesterone, however, facilitate the actual release of GnRH from the hypothalamus.⁹

The sustained release of LH and FSH depends on the pulsatile stimulation of the gonadotroph by GnRH. For instance, within a narrow pulse range, increases in GnRH pulse frequency result in a reduction in gonadotropin secretion, regardless of the magnitude of the pulse (Fig. 4).³² This paradoxic effect is believed to result from a reduction (down-regulation) in the number of pituitary GnRH receptors.^{17, 33} At lower frequencies, however, a self-priming effect operates as well.

Accordingly, the provision of GnRH heightens the pituitary response to a subsequent similar stimulus.

In the ovariectomized and hypothalamus-pituitary disconnected ewe, a decrease in GnRH pulse frequency results in decreased LH pulse amplitude, secondary to a reduction in the amount of releasable LH.³⁴ The frequency of GnRH pulses has a qualitative effect as well. For instance, in monkeys, changes in the frequency of GnRH pulses alters the FSH/LH ratio, whereas changes in the amplitude of the pulses have a minor effect.³⁵ For example, the pulsatile administration (every 2 hours) of GnRH to hypogonadotropic men coincides with the release of LH and the α-subunit common to both gonadotropins, but not with the release of FSH. When GnRH is administered every 30 minutes, the release of α-subunits becomes erratic and baseline LH increases, although the coincidence with α-subunit pulses is lost. In addition, FSH decreases to barely detectable levels, resulting in an increased LH/FSH ratio.³⁶ In contrast to the critical importance of the frequency of GnRH pulses, changes in the amplitude seem to have a lesser role.¹¹ Nevertheless, in ovariectomized sheep, the pulse amplitudes of GnRH and LH are highly correlated.³⁷

In women of reproductive age, a single bolus of exogenous GnRH elicits an acute release of LH and, to a lesser extent, of FSH. When GnRH is given at 2-hour intervals, a dose-response relationship exists between GnRH (from at least 10 mg and up to 300 mg doses) and LH.³⁸ The response is detected 5 minutes after intravenous injection of GnRH and reaches its peak between 30 to 45 minutes later. In response to GnRH, the pituitary may release prolactin as well. Increased circulating prolactin following a 4-hour infusion of GnRH was first reported in a group of patients suffering from anorexia nervosa, who had gained weight during the week preceding the test.³⁹ A similar effect was described in normal women, in whom the finding was more pronounced during the peri-ovulatory period.⁴⁰ Because the prolactin increment following infusion of GnRH correlates with both the FSH response⁴¹ and with the LH response,⁴² the observed increase is believed to be the consequence of a paracrine interaction between gonadotropin and prolactin. However, elevations of prolactin in response to GnRH administration have been documented in 7 of 10 women during the midtrimester of gestation, despite the lack of gonadotropin response to GnRH. This nonspecific prolactin response could also have been explained by the positive effect of high circulating levels of estrogens during gestation on prolactin production and release, thus sensitizing the lactotroph to nonspecific stimuli.⁴³ Moreover, GnRH sensitizes the lactotrophs to thyrotropin-releasing hormone action in normal women.⁴⁴ Finally, GnRH may bind to somatotrophic and thyrotrophic cells as well, resulting in actions not yet clarified.¹⁵

Self-Priming Action of GnRH

Gonadotropin-releasing hormone induces two separate phenomena in the pituitary. On the one hand, GnRH primes (sensitizes) the gonadotroph to subsequent stimulation by increasing the number of its own receptors (Fig. 5). A similar phenomenon of increased pituitary responsiveness to GnRH is involved in the maturation of the hypothalamic-hypophyseal axis during puberty,⁴⁵ a phenomenon that is used to diagnose true precocious puberty. On the other hand, GnRH triggers the release of LH and FSH. Both the “sensitization” and the “release” effects are dose-dependent, but functionally separate with the lower doses promoting priming, the higher doses favoring release. The responses of FSH parallel those of LH but on a much smaller scale.⁴⁶

One way by which the endocrine milieu modulates the pituitary response to GnRH is by regulating the number of GnRH receptors rather than by altering their affinity to GnRH. Indeed, in the rat, pituitary responsiveness correlates well with the number of GnRH receptors.¹⁸ In this connection, estradiol seems necessary to unmask the self-priming effect of GnRH.⁴⁷ In the rat, low levels of prolactin are also necessary for GnRH to increase its own receptor number. Nevertheless, a different mechanism may be at play as well because both bromocriptine, a dopaminergic agent that decreases prolactin release, or estradiol alone can sensitize the pituitary to GnRH without necessarily increasing GnRH receptors.⁴⁸

More recently, it has been demonstrated that inhibitors of PKC may inhibit the self-priming actions of GnRH.⁴⁹ Furthermore, it has been postulated the effects of the sex steroids on the pituitary response may be directly related to an effect on PKC, independent of LH and Ca⁺⁺ influx.⁵⁰

Effect of Estrogens on the Pituitary Response to GnRH

In women, estrogens, in addition to unmasking GnRH self-priming, appear to protect from pituitary desensitization. In this connection, estrogen is antagonized by progesterone.⁵¹ Moreover, estradiol, at physiologic doses, increases the pituitary response to GnRH.⁵² In contrast, the provision of pharmacologic doses of estradiol to normally cycling women, resulting in serum levels of 700 to 1200 pg/mL, induces a marked diminution in pituitary responsiveness to even high doses (300 mg) of GnRH.⁵³ The modulation of the pituitary response to GnRH by estradiol is duration-dependent. For instance, the administration of estradiol at doses resulting in serum levels of 100 to 200 pg/mL enhances the LH response to 100 mg of GnRH, provided the preceding exposure to estrogen was at least 84 hours in duration.⁵⁴ The pituitary responsiveness is concentration-dependent and duration-dependent. Thus, LH and FSH response to GnRH is proportional to the dose of estradiol administered for 6 days. Minimal estradiol concentrations, resulting in increased gonadotropin responsiveness to 100 mg of GnRH, are 90 pg/mL for LH and 145 pg/mL for FSH.⁵⁵ Interestingly, the administration of estradiol benzoate to normal women at a dose resulting in serum estradiol concentrations similar to those seen at midcycle can trigger an LH surge similar to that observed prior to ovulation. The above studies, however, do not accurately reflect the in vivo situation because the GnRH doses evaluated are not physiologic and the peptide is not given in a pulsatile fashion. The ovariectomized monkey with hypothalamic lesions in which gonadotropin secretion is re-established by chronic pulsatile GnRH administration serves as a better model to study the role of estrogen in modulating pituitary sensitivity to GnRH. The administration of estradiol to such animals results in an initial decline in gonadotropin levels, followed by a discharge of LH and FSH. This suggests that estradiol exerts both a negative and a positive feedback action at the level of the pituitary gland.⁵⁶

To explain the observed effects of GnRH on the pituitary release of gonadotropins, the existence of two pools of gonadotropins has been postulated,³⁸ namely, a readily “releasable” pool and a “reserve” pool. The former reflects pituitary “sensitivity” to a discrete GnRH stimulus. The latter represents intracellular gonadotropin not immediately available for release. The so-called capacity of the pituitary is defined as the amount of gonadotropins released in response to a larger and, therefore, more prolonged stimulation with GnRH. In this context, pituitary capacity consists of the releasable and reserve pools, as well as the amount of newly synthesized LH as the pituitary is continuously challenged.⁵⁷

To assess the sensitivity of the gonadotroph or the status of the “releasable pool,” submaximal doses of GnRH must be used. Because the plasma clearance of exogenous GnRH is reduced by at least 50% when the dose is increased from 10 to 100 mg,⁵⁸ the stimulus provided by relatively high doses of GnRH can be long enough to evoke a release from the reserve pool as well. When 10 mg of GnRH is administered every 2 hours for 10 hours, the LH response to the first dose reflects the releasable pool. The integrated concentrations of gonadotropins over time or the area under the LH and FSH curve during the 10-hour period reflects the reserve pool because the initial releasable pool is considered exhausted. Interpreted in this manner, the administration of estradiol benzoate to mimic follicular phase levels of estradiol results in increased releasable and reserve pools.⁵⁹ Estrogens, however, seem preferentially to increase the reserve pool. For instance, as the follicular phase progresses and estradiol levels increase, the disparity between the response to 10 mg (releasable pool response) and the response to 150 mg (releasable plus reserve pools) increases in favor of the latter.⁶⁰ In contrast, hypogonadal women have increased sensitivity to GnRH,^{59, 60} particularly if the baseline estradiol levels are less than 15 pg/mL.⁶¹ Such patients respond similarly to low or high doses of GnRH, indicating the absence of an adequate reserve pool.⁶⁰ This condition is reversed by the administration of estradiol,⁶¹ again confirming that estradiol preferentially increases the reserve pool. Similarly, increased sensitivity is seen in postmenopausal patients. When estradiol is replaced in these women, the acute releasable pool is decreased at the second day of replacement but recovers on the fifth day, describing a U-shaped curve. The reserve pool (measured as the pituitary response to the second and third 10 mg dose of GnRH given every 2 hours) increases on the fifth day of estrogen replacement.⁶² The addition of progesterone at the end of the estradiol treatment further amplifies the response to a GnRH stimulus. As mentioned before, in addition to the effects described, estrogen may influence the pituitary response in more ways than one. For instance, estrogen inhibits pituitary desensitization due to the down-regulation of receptors that follows the continuous administration of GnRH, an effect that is antagonized by progesterone.⁵¹ Finally, estrogen administration to oophorectomized ewes has produced a LH pulse profile, suggesting a more prolonged LH response to each GnRH pulse.⁶³ A similar observation was made in women during the periovulatory period.²⁹

Effect of Progesterone on the Pituitary Response to GnRH

Progesterone given after the administration of estrogens further amplifies the estrogen-enhanced responsiveness of the pituitary to GnRH.^{59, 64} In fact, progesterone, but not 17α-hydroxyprogesterone, is able to trigger an LH and FSH surge in most normal estrogen-primed women.^{64, 65, 66} Indeed, the small preovulatory rise of progesterone may play a role in the generation of the midcycle LH surge. The above notwithstanding, progesterone antagonizes estrogen as it reduces the extent of pituitary desensitization in response to a continuous GnRH infusion.⁵¹ Interestingly, combination oral contraceptives (containing both estrogens and progestins) do not appear to affect the pituitary sensitivity to GnRH.⁶⁷

Other Modulating Factors of the Pituitary Response to GnRH

The FSH and LH response to GnRH may be modulated by dopamine as well. Hyperprolactinemic women, presumably dopamine deficient, display an increased LH and FSH response to GnRH. Exogenous dopamine infusion decreases this response in these patients as it does in normal women.⁶⁸

Neuropeptide Y, a putative modulator of GnRH release,⁶⁹ has been shown to enhance GnRH-stimulated LH release.^{70, 71}

Gonadotropin surge attenuating factor (GnSAF) is a proposed modulator of the midcycle gonadotropin surge, which is found in follicular fluid.⁷² It has been suggested that GnSAF attenuates both the primed and unprimed response of the pituitary to GnRH.^{73, 74} One investigator has gone so far as to speculate that the self-priming phenomenon is, in fact, a result of the interplay between GnRH and GnSAF.⁷⁵ Nonetheless, the very existence of GnSAF and the physiologic role for this agent are unclear.

Inhibin has been shown to inhibit the GnRH-stimulated secretion of FSH, as well as to diminish the LH pulse amplitude,⁷⁶ independent from the GnRH stimulation protocol.⁷⁷

Acute intake of alcohol appears to diminish the response of LH and free α-subunit, but not FSH to the GnRH stimulus in men.⁷⁸

Age brings about an impaired LH secretory capacity in men. While baseline parameters remain unchanged, the bioactive/immunoactive ratio of LH is decreased in response to an exogenous GnRH stimulus in older men.⁷⁹ Additionally, older postmenopausal women have a decreased sensitivity to estrogen feedback as evidenced by the response to a clomiphene citrate challenge than do recently postmenopausal women.⁸⁰ The significance of these findings remain to be determined.

Finally, short-term fasting increases the sensitivity of the gonadotroph to GnRH in men and women, an action independent of the level of plasma glucose.⁸¹

Changes in the Pituitary Response to GnRH During the Menstrual Cycle

Two mechanisms may play a role in the control of the hypothalamic-pituitary axis, which culminates in the midcycle LH surge and subsequent ovulation. The first, as previously described, is the modulation of the pituitary response to GnRH by sex steroids. The second may be the influence of these steroids on the GnRH pulse generator. Although the GnRH pulse generator appears to play only a permissive role in gonadotropin release, the gonadotropin pulse pattern varies during the cycle according to the hormonal status (i.e., high frequency, low amplitude LH pulses during the follicular phase and low frequency, high amplitude LH pulses during the luteal phase). The female rhesus monkey exposed to estradiol benzoate displays increased GnRH amplitude before and during an estradiol-triggered LH surge compared with nonestrogen exposed controls. It is, therefore, conceivable that, although not essential, an estradiol-triggered midcycle GnRH surge may play a role in the generation of the midcycle LH surge.²⁸ Similarly, the high levels of preovulatory estradiol may be responsible for the preovulatory increase in pulse frequency and amplitude of LH and thus, by inference, of GnRH, as observed in normal women.²⁹ By the same token, during the luteal phase, the luteal reaction in LH pulsatility appears to result from the action of estradiol and progesterone at the level of the GnRH pulse generator, rather than the pituitary gonadotrophs.³¹

Aside from steroidal interactions at the level of the hypothalamus, gonadotropin release is modulated by altering the pituitary response. Before demonstrating the influence of sex steroids on the pituitary response, the effect of 100 to 150 mg of GnRH was shown to vary during the menstrual cycle. While a more pronounced response is described during the late follicular phase, various reports disagree over whether the response is greater in the luteal phase^{82, 83} or in the late follicular phase.⁸⁴

It is evident that the maximal sensitivity to GnRH occurs at midcycle, a phenomenon modulated mainly by the circulating levels of estradiol. As the follicular phase progresses, there is a change in the pituitary response to large (150 mg), but not to smaller (10 mg), doses of GnRH,³⁸ indicating that estradiol exerts a preferential effect on the reserve pool. Additionally, the self-priming effect of GnRH may be an important determinant of pituitary sensitivity. Thus, the administration of 10 mg of GnRH at midcycle results in a tenfold increase in pituitary sensitivity.³⁸ A U-shaped curve best describes the pituitary response to GnRH during the follicular phase (Fig. 6). During the early follicular stage, when circulating estradiol levels are at their lowest, both the releasable (sensitivity) and reserve pools appear depleted. However, at even lower estradiol concentrations (i.e., hypogonadism), pituitary sensitivity and capacity appear to increase,^{61, 62} perhaps due to an increased GnRH output, resulting in a self-priming effect. On the other hand, levels of estradiol consistent with the midfollicular and late-follicular phase increase pituitary sensitivity. More important, however, is the stimulatory effect of estrogens on the gonadotrophic capacity. Consequently, the observed negative feedback action of estrogens on the pituitary is exerted more at the level of gonadotropin release rather than at the level of gonadotropin synthesis.⁵⁶ During the follicular phase, transfer of gonadotropins from the reserve to the releasable pool may occur (Fig. 7). The LH surge thus may be the consequence of multiple factors including estrogens acting in concert with perhaps the amplifying action of progesterone^{59, 64, 65, 66, 85}; the estrogen-induced self-priming effect of GnRH⁸⁶; the prolongation of GnRH-dependent LH pulses in the presence of estradiol³⁴; and the possible, but not crucial, estrogen effect on GnRH release.²⁸

In recent years, a great debate has raged over whether or not GnRH plays a pivotal role in the midcycle gonadotropin surge. In support of this notion is the demonstration that GnRH antagonists can effectively prevent the midcycle LH surge.^{87, 88, 89, 90} Additionally, there exists the finding that the frequency of multiactivity unit volleys emanating from the region of the hypothalamus decreases just before the LH surge, a phenomenon most likely related to the preovulatory E₂ surge.^{91, 92} In this regard, however, evidence also exits refuting a role for GnRH in the midcycle surge. For instance, it has been known for some time that women with hypogonadotropic hypogonadism treated with pulsatile GnRH do not require additional GnRH for the LH surge to occur.⁹³ More importantly, GnRH pulse frequency (as evidenced by LH and free α-subunit pulse frequency) does not change at midcycle.⁹⁴ Clearly, the role of GnRH in the midcycle gonadotropin surge is not well understood at this time.

In summary, during the follicular phase, estradiol amplifies GnRH action at the pituitary level by increasing the reserve pool while at the same time inhibiting the release of gonadotropins. In the late follicular phase, however, increasing amounts of estradiol may directly increase both GnRH pulse frequency and amplitude and the transfer of LH from the reserve into the releasable pool. This results in the paroxysmal LH surge despite the inhibitory action of estradiol on LH release.

Pregnancy and Puerperium

The total content of pituitary LH may decrease during pregnancy, the lowest values being noted at term.⁹⁵ This finding is consistent with the observation of very low levels of circulating pituitary gonadotropins and a marked suppression of the gonadotropin response to GnRH by as early as 8 weeks of gestation.^{43, 96} Since pituitary insensitivity to GnRH persists for the first 2 to 3 weeks after delivery, despite the abrupt decline of estrogens and progesterone during the puerperium, the lack of response after birth may reflect the time needed for the recovery in the synthesis of gonadotropins. The puerperium is characterized by a period of relative pituitary refractoriness, followed by increased responsiveness to GnRH after the third week for FSH and the fifth to eighth weeks for LH.⁹⁷ The lack of gonadotropic response may play a major role in postpartum amenorrhea. Others have shown that lactating women, when compared with their midfollicular phase counterparts, display no LH pulses; lower basal circulating levels of LH; a similar LH response to GnRH, and a significantly higher response of FSH (relative to LH) in the face of a comparable stimulus.⁹⁸ Interpreted in light of studies in the subhuman primate wherein decreased GnRH pulsatility results in lower LH and higher FSH levels,²⁸ the described response in puerperal women can perhaps be attributed to the slower frequency of GnRH release.

Hypothalamic Amenorrhea

Women with congenital absence of GnRH present a blunted pituitary response to GnRH, indicating low reserve and releasable pools.⁹⁹ However, the low response to a single GnRH bolus could be due to the absence of GnRH receptors. Therefore, repeated doses of GnRH or steroid priming is required to bring about a normal response.¹⁰⁰

Patients with secondary amenorrhea of suprapituitary origin display low to normal gonadotropin levels in the absence of organic pituitary lesions. Adult patients with very low circulating levels of estradiol (less than 15 pg/mL) show an exaggerated response to low doses of GnRH (10 mg) as compared with those with higher estradiol levels consistent with those observed in normal women in the early follicular phase.⁶¹ Although it is clear that these women have an altered response to estradiol, the pituitary gonadotrophs are fully capable of responding to ovulation induction with pulsatile GnRH.

Hyperprolactinemic Amenorrhea

The pituitary response to GnRH in hyperprolactinemia will, as might be expected, obviously depend on the cause of the abnormal condition. Thus, an expansive macroadenoma may compromise the pituitary severely enough to cause pituitary failure and, therefore, a lack of response to GnRH or other releasing factors. In cases of hyperprolactinemia in which the pituitary is otherwise largely functional (i.e., microadenoma, idiopathic hyperprolactinemia), an increase in both the releasable and reserve pools of FSH, with a decrease in the circulating levels of both LH and FSH has been demonstrated.^{64, 101} These findings were attributed to the hypoestrogenism, secondary to the hyperprolactinemia, and the consequent decrease in the negative feedback effect of estradiol on FSH release. The administration of bromocriptine, a dopaminergic agent capable of correcting the hyperprolactinemia, dopamine itself, or the surgical excision of a microadenoma reversed these findings. Given hyperprolactinemic women with normal sellar tomograms, no correlation exists between the degree of hyperprolactinemia and the response to GnRH, which is similar to that of normal controls. This suggests that prolactin does not interfere with the pituitary response to GnRH,¹⁰² but that the hypogonadotropic state is caused by a dopamine deficiency.^{64, 103}

Anovulation and Dysfunctional Uterine Bleeding

Normoestrogenic women with dysfunctional uterine bleeding and adolescents with anovulation have normal responses to GnRH.^{104, 105}

Polycystic Ovarian Disease

Patients with polycystic ovarian disease have an exaggerated LH and FSH response to GnRH,^{38, 106} a response correlated with the basal level of circulating LH.^{107, 108} While some investigators have found a decreased response to repetitive (every 2 hours) GnRH administration (10 mg),¹⁰⁸ others have found increasing LH responses to repeat doses of 25 mg of GnRH.¹⁰⁷ The increased pituitary sensitivity and the increase in the basal circulating levels of LH may be related to the increased circulating estrogen levels.¹⁰⁷ Compared with normal women in the follicular phase, women with polycystic ovarian disease respond to 150 mg of GnRH with increased LH and FSH release.¹⁰⁸ However, the FSH response does not change when GnRH is increased from a low (10 mg) to a high (150 mg) dose, perhaps due to the preferential inhibitory action of estrogen on FSH release.^{109, 110} More recently, it has been demonstrated that the frequency of the GnRH pulse generator is elevated in polycystic ovarian disease patients,¹¹¹ as evidenced by elevations in pulsatile LH and free α-subunit release.¹¹² In addition, through the use of a GnRH antagonist (Nal-Glu), it has been shown that these patients do not experience an increase in the amplitude of GnRH pulses.¹¹² These data lend support to the hypothesis that an increase in the GnRH pulse frequency leads to a partial desensitization of the gonadotrope, which, in turn, is responsible for the abnormal gonadotropin secretory dynamics seen in polycystic ovarian disease.¹¹¹

Isolated Gonadotropin Deficiency

By definition, patients with isolated gonadotropin deficiency may lack any response to GnRH. However, the diagnosis requires prolonged intermittent GnRH stimulation because some patients with hypothalamic hypogonadotropic hypogonadism and others with delayed puberty will not respond to GnRH, due to lack of a prior self-priming effect of GnRH.^{100, 113}

Isosexual Precocious Puberty

Physiologic puberty is associated with increasing pituitary responsiveness to GnRH.⁴⁶ This is because of the self-priming action of the decapeptide and increased estrogenic exposure, resulting in an adult type response. Conversely, precocious puberty secondary to exogenous or autonomous estrogen exposure is generally characterized by suppressed responses to GnRH.

Hypergonadotropic Hypogonadism

Women with this condition (i.e., Turner's syndrome) manifest increased pituitary sensitivity when circulating levels of estradiol are very low.¹¹⁴ This response is similar to the one seen in patients with hypothalamic amenorrhea.⁶¹

The GnRH Challenge Test

The administration of GnRH to women with hypogonadotropic amenorrhea can differentiate patients with hypothalamic hypogonadism from those with isolated pituitary gonadotropin failure. The latter will not respond, whereas those with hypothalamic hypogonadism will respond, provided the stimulus is repetitive enough to “awaken” (possibly through up-regulation of pituitary receptors to GnRH) the biosynthesis and release capabilities of the gonadotroph. In addition, the gonadotropin response to intravenous administration of GnRH (100 to 150 mg) over 30 seconds may segregate patients with central (true) from secondary (pseudo) precocious puberty.¹¹⁵ Thus, as a result of prior exposure to pulsatile GnRH, girls with true precocious puberty will have an adult-like response. Conversely, those whose development is due to nonhypothalamic-pituitary-induced steroidogenesis will not respond. Incidentally, the administration of GnRH will not affect the action of other releasing factors on the pituitary. In fact, a combined sequential anterior pituitary function test using corticotropin-releasing hormone, GnRH, growth hormone-releasing hormone, and thyrotropin-releasing hormone has been described.¹¹⁶

In the delicate balance characteristic of the hypothalamic-pituitary-ovarian axis, sex steroids seem to exert the most important modulatory action because their influence changes during each cycle, while the pituitary responds to what appears to be a permissive role for the hypothalamic GnRH pulse generator.

The elucidation of the structure and sequence of GnRH quickly led to efforts to synthesize more potent and long-acting analogs of the hormone.

Many analogs have now been synthesized, some acting as agonists, others having predominantly antagonistic properties.¹¹⁷ The potential for further development and the indications for clinical use continue to increase, making an understanding of the physiology and pharmacology of the compounds important for the clinician.

GnRH Agonists

Gonadotropin-releasing hormone agonists were developed in the hope of yielding compounds with potential ovulation-promoting properties. However, it was soon learned that treatment with GnRH agonists, as with continuous high-dose infusions of native GnRH, cause a biphasic response in gonadotropin release from the pituitary and a paradoxic gonadal suppression.^{32, 118} Thus, administration of GnRH agonists of prolonged action results in paradoxic antireproductive effects, which ultimately lead to a hypogonadal state. Gonadal steroids approach undetectable levels after 1 to 3 weeks of treatment; a similar time is required for recovery.

As with native GnRH, the pituitary response to acute administration of GnRH agonists is variable, depending on the dose of agonist used and the phase in the menstrual cycle during which the compound is administered. It has been shown that the pituitary responds more briskly and to lower doses of GnRH agonists in the late follicular phase compared with the early follicular phase (Fig. 8).¹¹⁹ This can be explained by the priming of the pituitary with GnRH in the early follicular phase, resulting in an increased releasable pool of gonadotropin in the high-estrogen phase of the cycle. Higher dosages of GnRH agonists not only cause an acute rise in gonadotropins but also a sustained elevation over several hours to days. Similarly, the duration of the gonadotropin rise appears to be longer in the early follicular phase than in the late follicular phase (13 to 36 hours and 36 to 48 hours, respectively) (see Fig. 8).¹¹⁹ ln the midluteal phase, the response to an identical dose of the same GnRH agonist is almost identical to that observed in the early follicular phase.¹²⁰ It is apparent that the administration of GnRH agonists mimics the infusion of high doses of naturally occurring GnRH, which stimulate an increase in LH and FSH levels for the first 4 to 6 hours, followed by a sustained decline to normal levels if GnRH is discontinued or to low to nondetectable levels if administration continues.¹²¹ The GnRH-induced shutdown of pituitary function is felt to be due to “desensitization” and “down-regulation” of the pituitary gonadotroph. Desensitization refers to an uncoupling of the GnRH receptor from the secretory signal (i.e., gonadotropin release), whereas down-regulation is thought to result from a decreased number of available membrane receptors for GnRH following internalization of the GnRH-receptor complexes.¹²²

Structure of GnRH-Analogs

Peptidases in the pituitary gland and hypothalamus rapidly degrade naturally occurring GnRH by cleaving the decapeptide molecule between amino acids Gly⁶ to Leu⁷ and at the Pro⁹ to Gly¹⁰ bond (Fig. 9).^{123, 124} Thus, the circulating half-life of GnRH is relatively short (2 to 8 minutes). It is believed that the bond between amino acids 6 and 7 is the most vulnerable to degradation by pituitary endopeptidases because of its location at the type IIb turn (hairpin turn) of the GnRH molecule.^{123, 124} Adequate hormone-to-receptor binding is thought to be due to the configuration of the molecule that is maintained by amino acids (Glu^l, Gly⁶, Gly¹⁰). GnRH agonists were developed to increase potency over naturally occurring GnRH through enhanced binding affinity to the GnRH receptor and decreased enzymatic proteolysis.^123,125,126 Substitutions by metabolically stable residues in the above-mentioned positions (amino acids 6 and 10) result in increased potency and prolonged half-life.¹²² It also has been suggested that protection of the agonists from renal elimination by albumin binding may also account for their prolonged action.¹²⁷

GnRH Antagonists

Unlike GnRH agonists, which act by decreasing receptor availability and uncoupling, GnRH antagonists act by competing with GnRH for receptor sites, never causing a stimulatory signal. Therefore, antagonists have the advantage of a rapid onset of action without the initial stimulatory burst of gonadotropins. The pituitary response to GnRH antagonists differs in several ways when compared with their agonistic counterparts. The circulating levels of LH and FSH decline rapidly after the administration of a GnRH antagonist (maximal reduction occurring at 4 and 9 hours after an intravenous dose, respectively)¹²⁸ and their pulse amplitudes are lowered (Fig. 10). Circulating gonadotropins return to basal levels and resume pulsatile release between 10 and 24 hours following GnRH antagonist administration. Contrary to the response to the agonists, the response to GnRH antagonists does not seem to vary during the menstrual cycle. The maximal decline of FSH, however, is slightly more profound in the midluteal phase.¹²⁹ There appears to be a dose-dependent relationship between the GnRH antagonist administered and the LH response, a phenomenon also seen with GnRH agonists. Higher doses of GnRH antagonists act faster to abolish LH pulsation.¹³⁰ The duration of LH suppression is dose-dependent as well.¹³⁰ Nevertheless, suppression of plasma immunoreactive FSH was found to be insignificant at all doses and not dose-dependent. Thus, LH release is more profoundly inhibited than FSH, suggesting that a GnRH-independent releasing factor for FSH may operate as well.¹³⁰

Interestingly, the antagonists were actually synthesized before the agonists.¹³¹ The idea of preventing ovulation by blocking the release of gonadotropins was initially exciting but later proved disappointing due to difficulties in developing a clinically useful GnRH antagonist.¹³² Indeed, the earlier generations of GnRH antagonists stimulated histamine release from mast cells, and therefore had anaphylactoid potential.^133,134 Furthermore, some have agonistic properties.¹²⁸ Particularly strenuous demands are put on GnRH antagonists for clinical use because as little as 10% receptor occupancy by GnRH can cause LH release,^122,135 requiring the antagonist to be present continually at the receptor to block native GnRH binding. To accomplish their inhibitory action, antagonists are endowed with a long half-life and a high receptor affinity.¹³²

In recent years, gonadotropin-releasing hormone antagonists that elicit minimal histamine release have been evaluated for clinical use. The potential benefits of an agent that lacks the initial stimulatory phase of the response to the agonist are self-evident. In clinical studies, GnRH antagonists have been shown capable of preventing premature LH surges in women undergoing ovulation induction.^87,136,137 These agents have also been studied in men for the treatment of sex steroid-dependent disorders, such as benign prostatic hypertrophy.¹³⁸ The promising data suggest the enormous potential for the application of GnRH antagonists in disorders in which GnRH agonists are currently indicated, including endometriosis and uterine leiomyomata,¹³⁹ as well as the possibility of their use in the ovarian hyperstimulation syndrome, and breast and ovarian cancer.¹⁴⁰

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