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While GnRH neurons likely have an intrinsic pulsatile secretory pattern (1), many
neurotransmitters may modify the GnRH secretory pattern. The two predominant
pathways include the actions of catecholamines and the endogenous opioids (figure 1; 2-
4) though dopamine and other neurotransmitters such as NPY and galanin may be
involved (5-8). The bulk of data examining the role of catecholamines on the reproductive
axis come from studies conducted in rodent models. In the presence of estradiol, intra-
cerebral ventricular administration of norepinephrine (pulsatile) stimulates LH secretion
(9) whereas treatment with phenoxybenzamine, a potent blocker of catecholamine alpha
receptors, inhibits GnRH and gonadotrophin release (10,11). Opioids exert an inhibitory
effect on GnRH release. Treatment with morphine analogues reduces gonadotrophin
release in primates and humans while administration of opioid antagonists such as
naloxone increases LH (12,13). Indeed, CNS opioid tone may serve to relay, at least in
part, feedback inhibition from gonadal steroids on GnRH secretory patterns and be an
essential component of dynamic gonadotrophin release during the menstrual cycle.
1
Figure 1. Schematic of feedback control of
hypothalamic-pituitary-ovarian axis.
NE=norepinephrine, E=epinephrine, DA=dopamine and
EOP=endogenous opioids. From Marshall JC: Regulation
of gonadotropin secretion. In DeGroot LJ (Eds):
Endocrinology (ed3). Philadelphia, Saunders Press,
1995. pp 1993-2007
The nature of the GnRH and gonadotrophin secretory pattern is a pulsatile burst.
Each LH pulse likely reflects a prior GnRH pulse. Direct measurement of GnRH
secretory kinetics is not feasible in humans due to the dilution of GnRH (and
other hypothalamic factors) from the hypophyseal-portal vessels to the systemic
circulation. Hence, the patterns of GnRH release are inferred from data on LH
release. However, data from sheep, rodents and other primates confirm this
relationship between GnRH and gonadotrophins (14-16).
GnRH also has a well recognized "self-priming" effect (17,18). Using various
GnRH deficient models, it has been clearly documented that LH secretion
increases with sequential GnRH pulses. Regulation of GnRH responsiveness is at
least in part via changes in the numbers of GnRH receptors. Data reported from
animal models suggest that GnRH receptor numbers decline in the absence of
GnRH or following treatment with a GnRH antagonist (19-21). Conversely,
2
pulsatile GnRH restores GnRH receptor expression in a dose, frequency and time-
dependent fashion. Loss of receptor expression can occur in the face of either
continuous or very high amplitude GnRH, likely reflecting at least one component
of the phenomenon of "desensitization" (22). In addition, changes in second
messenger systems such as protein kinase A, protein kinase C and
calcium/calmodulin appear important in modulating both GnRH signaling and
perhaps desensitization (23-26).
Pioneering research from Knobil and colleagues revealed the critical nature of the
pulsatile secretory patterns needed for reproductive function (27,28). Using
castrated monkeys with hypothalamic lesions to render the animals GnRH
deficient, it was reported that only intermittent GnRH maintained LH secretion
while continuous GnRH was associated with a rapid decline in LH release.
Moreover, the nature of the pulsatile GnRH signal conferred an additional
regulatory mechanism. Fast frequency pulsatile GnRH favored LH release while
slower frequency pulses resulted in a relatively higher FSH secretion. These
findings are supported by data in other animal models of exogenous GnRH
treatment suggesting that the genes encoding the gonadotrophin subunits are
regulated in a similar pattern to LH and FSH release. Thus, the characteristics of
the hypothalamic signal are a critical component to reproduction and, when
appropriate regulation of GnRH is dysfunctional such as in Polycystic Ovarian
Syndrome (to be discussed later in this chapter), alterations in fertility and
gonadal functions may ensue.
1. Pubertal Maturation
Immediately after birth, and for the first few months of life, gonadotrophin
secretion, and presumably hypothalamic release of GnRH, is increased (29).
Neither the mechanism behind the hypergonadotropism, nor the subsequent
transition to lower levels of gonadotrophin secretion, is certain. Perhaps
stimulation from maternal steroid hormones such as estradiol induces GnRH
release. Thereafter, within the first year of life through the time of puberty, both
boys and girls have low levels of gonadotrophins and gonadal hormones.
GnRH/LH pulses can be detected in prepubertal children, albeit having low
amplitude and slow frequency (every 3-4 hours) patterns of release.
In children, pulsatile LH release can be detected using highly sensitive modern hormone
assay systems and mathematical techniques to properly identify pulsatile patterns of
secretion (30-37). However, pulses are of very small amplitude and slow frequency. The
onset of pubertal maturation is heralded by the development of a diurnal pattern of LH
secretion (Figure 2, left panels).
3
Figure 2. Plasma LH patterns through 24 hours in
normal versus hyperandrogenemic girls in early versus
late puberty. In early puberty, there is an increase in LH
pulsation observed during sleep; which is present, but
abnormal in hyperandrogenemic girls. Later in puberty,
this pattern is firmly established in normals, but
completely disordered in hyperandrogenemic girls.
2. Menstrual Cycle
By convention, the menstrual cycle is divided into two phases: beginning with the first
day of menses and extending through 12-16 days duration is the follicular phase.
Development of the dominant follicle with increasing gonadotrophin and estradiol
4
secretion, culminating in the mid-cycle gonadotrophin surge, broadly characterizes this
phase. During the luteal phase, there is reduced gonadotrophin secretion in the presence of
a corpus luteum and elevated progesterone and inhibin levels that, in turn, also decline
with the failure of fertilization resulting in menstruation. It is the complex coordination of
the human menstrual cycle that reflects the true dynamic nature of the
hypothalamic/pituitary/gonadal axis (Figure 3; 38-40).
The initial portion of the follicular phase is characterized by a relatively high ratio
of FSH to LH (41). This is likely the result of a slow GnRH pulse frequency as
evidenced by LH release with an approximately 90 minute interpulse interval.
This pattern is most prominent during the night-time hours. Data in rodents and
primates clearly detail the frequency dependence of gonadotrope function with
slow frequency GnRH favors FSH synthesis and secretion while fast frequency
GnRH signals selectively increases LH . This period of FSH drive is critical for the
recruitment and maturation of ovarian follicles. FSH induces granulosa cell
expression of both LH receptors and levels of the enzyme aromatase, needed for
5
the synthesis of estradiol (42-44). By the middle of the follicular phase, LH pulse
frequency has increased to a nearly hourly pattern. With this, LH levels rise and
ovarian release of estradiol increases significantly toward mid-cycle. The increase
in estradiol, and ovarian production of the inhibins (dimeric peptides that
selectively inhibit FSH synthesis and secretion) reduce FSH levels, perhaps
playing an important role in limiting the final maturation of the non-dominant
follicles (45-50).
At the end of the follicular phase, or mid-cycle, the increasing levels of estradiol
result in an enhancement of LH responsiveness, thereby inducing the LH surge.
Also, progesterone levels begin to increase and may further augment the LH
responses to the ongoing GnRH stimulus (51-53). During the surge, LH levels
remain increased for 36-48 hours, during which time ovulation occurs, estradiol
levels decline and luteinization of the follicle results in increasing production of
progesterone. This increase in progesterone plays a critical role in regulating
GnRH release by decreasing GnRH pulse frequency (every 2-5 hours). The
actions of progesterone are mediated at least in part via endogenous opioid
production as opioid antagonist treatment increases GnRH secretion during the
luteal phase (54-57).
In the absence of fertilization, the corpus luteum regresses and gonadal steroid
and peptide production declines. As the inhibitory actions of progesterone are
removed, GnRH pulse frequency increases and LH pulsatility returns to a near
hourly pattern. The fall in inhibin and estradiol result in a rise in FSH secretion.
Thus, the beginning of follicular recruitment for the subsequent menstrual cycle
actually begins during the later portion of the prior luteal phase/menstrual cycle.
6
PATHOLOGY OF THE HYPOTHALAMIC- PITUITARY- GONADAL AXIS
Epidemiology
Among women with PCOS, the risk for co-morbidities such as obesity, diabetes
(gestational and type 2), hyperlipidemia, cardiovascular disease, and endometrial
cancer are increased. Obesity is found in 40-60% of women with PCOS,
depending on ethnicity and geography, and is intimately linked to other co-
morbidities associated with PCOS (62,63). The risk of developing diabetes is
higher in obese individuals with PCOS when compared to lean women.
Hypertension is associated with PCOS in obese, but not lean individuals (64).
Significantly higher levels of LDL have been demonstrated in women with PCOS
when compared to controls (65). The risk for myocardial infarction has been
found to be approximately seven times that of the normal population in one
study, however, this figure was not adjusted for BMI(66).
Decreased fertility and early pregnancy loss are common in patients with PCOS.
The incidence of infertility is estimated at a mean of 74% (range 35-94%) (67).
The rate of spontaneous abortion is approximately 30%, double that of
pregnancy loss in normal women (68). However, conception and delivery rates
are improving with recent developments in treating women with PCOS (see
below).
Pathophysiology
7
excess ovarian androgen production. Treating these women with a GnRH agonist
to desensitize LH secretion leads to decreased levels of LH, testosterone, and
androstenedione (69).
The loss of normal ovarian cyclicity may be self-perpetuating. After the mid-cycle
LH surge, production of ovarian progesterone (P) by the corpus luteum will, in
normal women, provide feedback to the hypothalamus and foster the initiation of
a subsequent menstrual cycle. Indeed, administration of exogenous progesterone
to women with PCOS causes a selective increase in FSH and folliculogenesis (85).
As an additional mechanism in women with PCOS, an impaired sensitivity of the
hypothalamus to P has been demonstrated in plasma P concentrations less than
10 ng/ml, but LH secretion was able to be suppressed with higher P
concentrations (13-15 ng/ml)(86). This suppression of LH is also seen in women
in PCOS who have recently ovulated. From these data, it is hypothesized that
during pubertal maturation, a vicious cycle of: (1) decreased hypothalamic
sensitivity to the low levels of P seen during puberty (2) persistent rapid GnRH
pulsatility leading to excessive LH secretion (and LH stimulated ovarian androgen
production) (3) impaired folliculogenesis and anovulation (4) absence of normal
luteal progesterone production and hence no feedback to the hypothalamus,
leads to the spectrum of symptoms seen in PCOS (figure 2).
8
demonstrated that these women are insulin resistant and have elevated insulin
levels. However, insulin administration during a hyperinsulinemic, euglycemic
clamp has been shown to have no affect on gonadotrophin secretion (87).
Hyperandrogenemia has also been suggested to be the cause of decreased
sensitivity to P. Supporting this theory is data showing that treatment of PCOS
women with the anti-androgen flutamide restores GnRH inhibition by low doses of
P (88).
The paracrine factors activin, inhibin and follistatin have also been suggested to
play a role in the pathogenesis of PCOS, however the contribution of these
proteins to the disease process remains to be elucidated.
Diagnosis
In the past, the diagnosis of PCOS has been complicated by the wide variability of
presenting symptoms. Some experts have used the criterion of the presence of
polycystic ovaries on ultrasound for diagnosis. In fact, polycystic-appearing
ovaries on ultrasound are found in 16-25% of normally cycling, non-
hyperandrogenemic women (89), and not all women with clinical symptoms
suggestive of PCOS have polycystic ovaries on ultrasound. As mentioned above,
some series have found that the presence of elevated LH levels in women with
this disorder is as low as 35%. Obesity, hirsutism and acne are found with
varying frequency, depending on the population studied. Due to this, the NIH
consensus conference on PCOS in 1990 determined that the diagnostic criteria
require only evidence for chronic anovulation and hyperandrogenemia, and the
exclusion of other related disorders such as hyperprolactinemia, thyroid
dysfunction, late-onset congenital adrenal hyperplasia or androgen secreting
tumors (90).
Treatment
The treatment for PCOS is dependent upon the concerns of the individual patient.
In all women who are obese, weight loss should be strongly encouraged, as this
will often ameliorate many symptoms, as well as promote fertility and decrease
the risk of diabetes and hypertension.
For patients who do not desire fertility, maintenance of regular menses either by
the administration of medroxyprogesterone or combination oral contraceptives
will lower the risk of endometrial hyperplasia and carcinoma. It has also been
suggested that oral contraceptives may reduce the risk of ovarian cancer in
patients with PCOS (63).
Hirsutism
Oral contraceptives will often lower LH and testosterone levels and decrease
hirsutism and acne (91). As an alternative or additional approach, blockade of
9
androgen action at the pilosebaceous unit, spironolactone and cyproterone are
the two most common anti-androgens used in the treatment of hirsutism (92-
94), though cyproterone is not available in the US.
Infertility
Restoring fertility in women with PCOS has changed dramatically in recent years.
Clomiphene citrate has been used as an inducer of ovulation for many years and
is believed to act by blocking the effects of estrogen on the hypothalamus. Loss
of estrogen inhibition results in increased GnRH release, causing increased levels
of FSH and follicular development. One common sequelae of clomiphene therapy
is that of functional ovarian cysts. The physician should allow these to regress
before initiating a subsequent course of clomiphene. Injectable gonadotrophins
have also been used to induce ovulation but carry the risk of ovarian
hyperstimulation and multiple gestation (95).
Treatment with metformin to reduce insulin resistance has been shown to have
great promise in treatment of infertile PCOS patients. Insulin increases LH-
induced ovarian androgen production and that treatment with diazoxide (which
blocks insulin secretion from the b-cell) results in marked decrease in
testosterone levels in PCOS women (96). In women with PCOS, metformin
improves insulin sensitivity (97) and has also been found to improve menstrual
cyclicity. A number of studies have now been published demonstrating improved
rates of ovulation and reduction of androgens in obese women with PCOS (97-
102). In one study by Moghetti, et al, a significant reduction in LH and serum
androgens was seen during the 6 months of treatment and these effects were
maintained for an additional 6 months of open-label treatment (99). Trials
investigating pre-treatment of PCOS women with 5 weeks of metformin have
demonstrated an eight-fold increase in spontaneous ovulation and a tenfold
increase in clomiphene-induced ovulation (102). Furthermore, it has been
suggested that hyperstimulation by injected gonadotrophins may be decreased if
women are pretreated with metformin (98)
Less data is available for the safety and efficacy of other insulin sensitizers such
as the thiazolidinediones and D-chiro inositol, but early studies suggest that
these treatments decrease the androgen milieu and enhance ovulatory function.
2. Hypogonadotropic Hypogonadism
10
hypogonadism). The distinction between these disorders can usually be made my
measurement of follicle stimulating hormone (FSH) and luteinizing hormone (LH).
In hypogonadotropic hypogonadism, both the FSH and LH will tend to be
inappropriately low given the subnormal sex-steroid levels.
GnRH neurons originate outside the central nervous system in the olfactory
placode and migrate along the olfactory nerve, up the nasal septum and through
the cribriform plate to the forebrain where they find their place in the arcuate
nucleus of the hypothalamus. GnRH has been demonstrated in the human brain
as early as 4.5 weeks of gestation. LH and FSH are first measurable in the
embryonic brain at 10 weeks of gestation and in the peripheral blood at 12 weeks
(105). The levels of LH and FSH continue to rise peaking at mid-gestation and
falling thereafter. Although GnRH secretion is clearly present throughout fetal
development, the early testicular testosterone production required in utero for
normal sexual and genital differentiation in males is maintained by stimulation
from maternal human chorionic gonadotropic hormone (hCG). However, GnRH
secretion is thought to be necessary late in fetal development and in the early
neonatal period for complete descend of the testis and growth of the external
genitalia. Thus, patients with hypogonadotropic hypogonadism may present with
cryptorchidism and /or microphallus in the neonatal period as a result of
inappropriately low and sex-steroid levels during fetal development (104, 106-
108).
11
sensorineural hearing loss, synkinesia, oculomotor abnormalities, red-green color
blindness and cerebellar ataxia (106,108,110,111). Shortened metacarpals, pes
cavus deformity of the feet and renal agenesis have also been described
(106,110,112).
Both IHH and KS are characterized by: i) complete or partial absence of pulsatile
LH secretion (113,114); ii) normal gonadotrophin secretion after exogenous
physiologic GnRH administration (113-115); iii) normal levels of other
hypothalamic-pituitary hormones; iv) normal radiographic imaging of the
hypothalamus and pituitary. While the pathogenesis of GnRH deficiency appears
to be quite similar, there are several documented patterns of inheritance that
may contribute to the variability in presentation and associated abnormalities in
different subjects (106,109,116). GnRH deficiency has been associated with
autosomal dominant, autosomal recessive and X-linked patterns of inheritance.
Furthermore, family members of probands with KS have been found to have IHH
without olfactory defects suggesting that the expressed phenotype for any one
genetic abnormality may be quite varied (106,109).
The KS gene (KAL gene) has been localized to Xp22.3. The protein encoded by
the KAL gene has been termed anosmin (117,118). The use of anti-GnRH
antibodies in a 19-week human fetus with X-linked KS showed that GnRH
neurons migrate from the olfactory placode and cross the perforations in the
cribriform plate however, they appear to arrest at the dorsal aspect of the plate
without reaching the forebrain or their normal location in the arcuate nucleus of
the hypothalamus (119). It has therefore been postulated that anosmin may be
crucial for the normal entrance of the GnRH neurons into the olfactory bulb
and/or for the establishment of neuronal interactions between the incoming
olfactory axons and the central neurons of the bulb.
Nonetheless, autosomal genes have also been found to be responsible for GnRH
deficiency and it is postulated that these mutations may indeed account for the
majority of cases (6109). The identities of the genes responsible for GnRH
deficiency have not been elucidated. No particular mutations of the GnRH gene
itself have been described in humans. In contrast, mutations of the GnRH
receptor have been identified and the phenotype of the affected subjects varies
widely (120-122). Patients have been described with normal pubertal but small
testis and abnormal semen analysis while others have been found to have a more
complete gonadotrophin deficiency with primary amenorrhea and infertility and
delayed puberty with absence of secondary sexual characteristics and cryptorchid
testes.
12
agonist with a complete normalization of the pattern of gonadotrophin secretion.
Furthermore, classical factors leading to functional GnRH deficiency such as
stress, excessive exercise or weight loss are absent in these subjects. This
acquired GnRH deficiency appears to be irreversible.
A series of patients have been described with normal testicular size in the setting
of eunuchoid body proportions and secondary sexual characteristics. Men with
this syndrome have normal spermatogenesis but have inadequate serum
testosterone levels to achieve full secondary virilization (124). They are similar to
patients with acquired IHH in that they have normal testicular size but differ in
that they have preserved spermatogenesis and can usually be treated with either
hCG or testosterone alone (125). This disorder is felt to result from insufficient
GnRH secretion in that these patients have a normal response to GnRH agonist
but impaired LH secretion in the setting of clomiphene citrate use (126). In
addition, as has been described in a subset of patients with IHH or KS (113,114),
frequent blood sampling in patients with fertile eunuch syndrome reveals a
pattern of nocturnal LH and testosterone secretion similar to that observed during
puberty (127).
B. Delayed puberty
The prevalence of delayed puberty in relatives of patients with IHH has been
estimated at 12% that is in excess of the estimated 1% for the general
population (109). These subjects had otherwise normal puberty suggesting
therefore that delayed puberty may represent the mildest form of GnRH
deficiency.
Abnormal b-subunit of LH
LH, FSH, hCG and thyroid stimulating hormone (TSH) belong to the family of
glycoprotein hormones. These hormones have a heterodimeric structure
consisting of a noncovalently associated common a subunit and a unique b
subunit. This heterodimerization is crucial for normal hormonal action. The b
subunit is responsible for the interaction with hormone-specific receptors and for
signal transduction.
Four genetic variants have been identified in the b subunit of LH however only
one of these has been found to result in altered activity of the hormone. This
mutant LH resulted from a missense mutation in codon 54 causing a Gln to Arg
substitution leading to normal serum immunoreactive LH levels but impaired
bioactivity (128). The initial case was described in a 17 year-old man with
delayed puberty, two-fold elevation of LH, normal FSH levels and decreased
13
serum testosterone. There are several infertile men described in the family of this
proband however all the females studied were normal.
A common LH variant has also been identified in which there are two amino acid
changing mutations in the b subunit of the LH gene at codon 8 (Trp for Arg) and
codon 15 (Ile for Thr) (129,130). The latter is responsible for introducing an
altered glycosylation site resulting in abnormal detection by monoclonal
antibodies. The prevalence of this variant LH varies greatly among populations
with a carrier frequency as high as 53.5% in Australian aborigines (131).
Functional differences have been demonstrated between variant and wild type
LH. Variant LH has been shown to have a shorter half-life in the circulation (132).
On the other hand, there appears to be a 40% higher promoter activity of the
gene that may partially compensate for the shortened half-life (133).
Homozygotes for the LH variant have been found to have recurrent spontaneous
abortions, menstrual irregularities, infertility and polycystic ovarian syndrome in
a Japanese population (134,135). However, the frequency of LH variant was
found to be lower in a PCOS population in Finland and the Netherlands (136).
Men presenting with inactivating mutations of the LH receptor may have variable
phenotypes ranging from feminization of the external genitalia to small testis and
delayed development of secondary sexual characteristics. In contrast, females
with inactivating mutations have normal secondary sexual characteristics but
have amenorrhea associated with increased LH concentration (139,140).
4. Hypothalamic Amenorrhea
14
pulsatile secretion of GnRH from the hypothalamus. GnRH pulsatile release is
under the regulation of a pulse generator located in the arcuate nucleus in the
medial central hypothalamus. The pulse generator is sensitive to a number of
metabolic factors including stress, caloric deficits and particularly weight loss.
Disruption of the normal GnRH secretion in HA is oftentimes first manifested by a
luteal phase defect with low peak progesterone levels and with luteinization
occurring without ovulation. The LH pulse frequency appears to be increased in
athletes with a luteal phase defect. Furthermore, strenuous exercise has also
been associated with a prolonged follicular phase and an abnormal midcycle LH
peak resulting in low estrogen levels and intermittent suppression of menstrual
cycles.
Exercise
Nutrition
It is believed that an energy deficit resulting from restrained caloric intake and
specifically low number of fat calories leads to a negative energy balance in
athletes, anorectics and women with FHA. This energy imbalance leads to a
15
reduced metabolic rate and metabolic adaptations including reduced
triiodothyronine (T3) (151,152), leptin(152,153) and insulin-like growth factor
(IGF-I) levels (152) and increased mean 24-hour cortisol concentration (154). In
addition, the cortisol and corticotropin (ACTH) response to corticotropin releasing
hormone (CRH) is blunted in patients with FHA suggesting that the increases in
24-h cortisol concentration results from increases in CRH production despite
intact cortisol feedback mechanisms. CRH can inhibit the GnRH-gonadotrophin
axis leading to appropriate suppression of the reproductive system during states
of starvation.
Studies of women with FHA compared to BMI matched controls revealed a lower
fat body mass (152,155) and 50% less fat and twice as much fiber consumption
when compared to normal controls (155). Furthermore, decreased LH pulse
frequency with or without changes in pulse amplitude resulted in lower 24-hour
LH concentrations and an increased FSH/LH ratio (152-155). Such changes in LH
secretion were completely reversed in exercising women when the caloric intake
was increased to meet the energy demands despite continued exercise thus
suggesting, that the nutrient restriction and not the increased physical activity
per se results in the observed gonadotrophin abnormalities (152).
Psychogenic Factors
Psychogenic stress is believed to result in abnormal GnRH secretion and has been
postulated as the cause of FHA. Studies using eating disorder and depression
inventories have identified higher scores in women with HA however these are
both in the subclinical range (155). However, as mentioned above, there also
appear to be confounding nutritional derangement in patients with FHA
suggesting that the true etiology of this disorder is likely to be multifactorial.
Prader-Willi Syndrome
Structural Abnormalities
Any tumor or cyst of the hypothalamus or pituitary may lead to abnormal GnRH
or pituitary gonadotrophin secretion secondary to structural impairment of the
normal gland. Malignant tumors of the central nervous system are more likely to
affect the hypothalamus (e.g. meningiomas) than the pituitary however
16
metastatic disease form the breast, lung or prostate may present with pituitary
dysfunction. Furthermore, prolactin elevation from lactotroph tumors or pituitary
stalk deviation from mass effect may also result in hypogonadotropic
hypogonadism due to prolactin inhibition of gonadotrophin secretion. Similarly,
infiltrative diseases such as sarcoidosis and Langerhans cell histiocytosis;
hemochromatosis-related iron deposition in the gonadotrophs; and tuberculous
meningitis may all result in secondary hypogonadism.
Drugs
Critical Illness
17