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Reproductive Endocrine Unit, Reproductive Endocrine Sciences Center and National Center for Infertility Research, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114
Address correspondence and requests for reprints to: Kathryn A. Martin, Reproductive Endocrine Unit, Massachusetts General Hospital, Bartlett Hall Extension 5, 55 Fruit Street, Boston, Massachusetts 02114.
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Abstract |
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The frequency and amplitude of LH secretion was compared with that seen in the early follicular phase (EFP) of normally cycling women. The spectrum of abnormalities of LH pulses was 8% apulsatile, 27% low frequency/low amplitude, 8% low amplitude/normal frequency, 43% low frequency/normal amplitude, 14% normal frequency/normal amplitude. Of patients studied overnight, 45% demonstrated a pubertal pattern of augmented LH secretion during sleep. Of patients studied repeatedly, 75% demonstrated at least 2 different patterns of LH secretion, and 33% reverted at least once to a normal pattern of secretion. An increase in LH pulse frequency was seen in 12 of 15 subjects in response to naloxone (opioid receptor antagonist). Clonidine (alpha-2 adrenergic agonist) was associated with a decrease in mean LH in 3 of 3 subjects. An increase in LH pulse frequency was seen in 4 of 8 subjects in response to metoclopramide (dopamine receptor antagonist), but the response was not statistically significant. Baseline abnormalities in LH secretion did not appear to influence response to neurotransmitter modulation. Conclusions: 1) HA represents a spectrum of disordered GnRH secretion that can vary over time; 2) LH pulse patterns at baseline do not appear to influence the ability to respond to neurotransmitter modulation; 3) Opioid and adrenergic tone appear to influence the hypothalamic GnRH pulse generator in some individuals with HA.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Because direct measurement of hypothalamic GnRH in humans is not possible, studies have used LH as an index of hypothalamic GnRH secretion. Patients with congenital GnRH deficiency [idiopathic hypogonadotropic hypogonadism (IHH) or Kallmann’s syndrome when associated with anosmia] show a complete absence of pulsatile LH secretion, reflecting a complete absence of GnRH secretion (4). In contrast, previous neuroendocrine studies in patients with HA have demonstrated evidence of pulsatile LH secretion in most HA patients. Some report a lower mean frequency of LH pulses in HA patients than in normally cycling women (6, 7), while other studies have demonstrated a broader neuroendocrine spectrum, from complete absence of LH pulsations to normal-appearing secretion patterns (4, 5).
Because a variety of neurotransmitters influence hypothalamic GnRH secretion, defects in central neurotransmitter systems are thought to underlie some disordered patterns of GnRH release. Although one cannot directly study the impact of opioid tone on hypothalamic GnRH release, the observation that LH pulse frequency (7, 8) and amplitude (9) increase after opiate receptor blockade, suggests that an increase in opioid tone may contribute to HA in a subset of women. Other work in humans has explored the role of inhibition by the dopaminergic (10, 11, 12, 13, 14, 15) and noradrenergic systems (7, 15, 16) with conflicting results.
An important question is whether patterns of hypothalamic GnRH secretion vary over time in women, and whether underlying pulse patterns affect clinical presentation or response to neurotransmitters. Previous studies exploring the stability of secretion patterns over time used small numbers of subjects or suboptimal blood-sampling intervals (4, 5, 7). The variability in underlying patterns of GnRH secretion and the variable response to neurotransmitter modulation raises the question of whether response to specific neuromodulation may correlate with a specific pattern of GnRH secretion, for example slow frequency or lack of LH secretion with unresponsiveness to naloxone (17).
Between 1978 and 1998, we completed the largest study to date characterizing the pattern of GnRH secretion in women with HA. Fifty subjects participated in 73 frequent-sampling studies designed to determine the spectrum of GnRH secretory defects in HA, the variability in neurosecretory defects over time in individual subjects, and the relationship between neurosecretory pattern and response to neurotransmitter modulation.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Forty-nine women with HA participated in at least one frequent-sampling study. HA was defined as a history of secondary amenorrhea of at least 6 months duration with low or normal gonadotropins, or a history of primary amenorrhea with low or normal gonadotropins, evidence of some pubertal development, and normal central nervous system imaging. All subjects weighed between the 10th and 90th percentile for height (18), reported no current eating disorder, engaged in no excessive exercise [defined as greater than 20 miles/week of running or its equivalent (19)], and had no history of systemic illness, galactorrhea, hirsutism, or ovarian enlargement. All had normal baseline blood count, ferritin, thyroid function tests, prolactin, and free testosterone. Women with IHH or Kallmann’s syndrome (congenital GnRH deficiency) were not included in this study. The studies were approved by the Institutional Review Board at Massachusetts General Hospital, and informed consent was obtained from all subjects.
Baseline sampling study
Forty-nine patients participated in a total of 72 baseline studies. Fifty-eight of the 72 baseline studies included a period of night-time sampling. Twenty subjects participated in one baseline study only, 7 subjects participated in repeated baseline studies but no neurotransmitter studies, 17 subjects participated in one baseline study and one neurotransmitter study, and 5 subjects participated in repeated baseline studies and one neurotransmitter study. The baseline protocol for all studies after 1982 involved frequent blood sampling every 10 min, for 8–24 h. Five studies, which occurred before 1982, used a 20-min blood-sampling interval but were identical in all other respects. Patients assigned to a neurotransmitter study participated in a 12- to 18-h baseline protocol, after which the neurotransmitter was administered and blood drawn at 10-min intervals for the next 6–12 h. All neurotransmitter infusions began in the morning, between 0600 h and 1000 h.
Naloxone (Astra USA, Inc., Westboro, MA), an opiate receptor antagonist, was administered as a continuous iv infusion of 0.8 mg/h over 6 h, with the exception of 3 patients who received graded naloxone infusions of 0.2 mg/h, 0.8 mg/h, and 2.0 mg/h for 4 h each. The naloxone dose of 0.8 mg/h was chosen because it had proved capable of inducing blockade of endorphins and evoking increased GnRH in previous human studies (7, 20). Metoclopramide (A.H. Robbins Co., Richmond, VA), a dopamine receptor antagonist, was administered as a continuous iv infusion at 5 mg/h over 6 h. Adequate central dopaminergic blockade was insured by a measurable increase in prolactin levels in all subjects. The 5 mg/h dose of metoclopramide had previously been determined to induce blockade of dopamine receptors and alter GnRH pulse frequency in studies with humans and animals (7, 14, 21). Clonidine (Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT), an alpha-2 agonist that reduces central adrenergic output via an autoinhibitory feedback loop on norepinephrine-secreting cells, was administered as a single 0.15 mg oral dose. The dose of clonidine was chosen as that effective to stimulate GH secretion (22, 23) and to effectively suppress LH secretion in humans (15, 16).
Assays
Plasma LH, FSH, and estradiol (E2) concentrations were measured by RIA, as previously described (24, 25), and methodologies remained stable over the 20- yr duration of the study. All samples from an individual study were measured in the same assay. The intraassay coefficient of variation (CV) was estimated for each assay by replicate measurements of a pooled plasma sample.
Data analysis
Pulses were determined using a modification of the Santen and Bardin method (26) with the amplitude calculated as the difference between the peak and the preceding nadir for each pulse, as previously reported (27). Undetectable values were assigned the lowest measurable assay values. A pulse was defined as a peak consisting of at least 2 points, in which the highest point met the criteria of 2 IU/L and 3 times the intra-assay coefficient of variation (%CV) above the nadir point, and a second point met at least one of those criteria. The frequency is expressed per 24 h. Values obtained for study subjects were compared with values obtained using identical pulse analysis criteria for LH secretion during the early follicular phase (EFP) in 15 normally cycling women. All controls had regular menstrual cycles of 27–32 day duration in the previous 2 yr, a normal physical examination with no hirsuitism, acne, or galactorrhea, a body weight within 15% of normal according to the Sargent scale, and no current use of hormonal medications or history of excessive exercise (27, 28). The EFP was used for control data because the low estrogen and progesterone levels seen in HA most closely match the hormonal milieu present in the EFP of a normal menstrual cycle.
Patterns of LH secretion were analyzed and classified into the following categories: [nlist]
1) Apulsatile, defined as the complete absence of LH pulsations;
2) Low amplitude, defined as a normal LH pulse frequency but a mean LH pulse amplitude more than 1 SD below the mean amplitude in EFP controls;
3) Low frequency, defined as a normal LH pulse amplitude but a mean LH pulse frequency more than 1 SD below that in EFP controls;
4) Low frequency/low amplitude, defined as both frequency and amplitude of LH pulses more than 1 SD below that in EFP controls; and
5) Unclassified, defined as both frequency and amplitude of LH pulses within the normal range for women in the EFP.
The first baseline study was used for classification of patients who participated in multiple studies.
Observed LH pulse patterns were also analyzed for an increase in LH secretion during sleep. A pattern of sleep augmentation was defined as a mean amplitude of sleep-associated pulses greater than or equal to 1.5 times the mean amplitude of waking pulses.
Significance of observed differences in hormonal parameters, pulse characteristics, and responses to neurotransmitters was determined using paired t tests. Values are expressed as the mean ± SE unless specified, and the two-sided 0.05 level is construed as significant unless otherwise noted.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Baseline clinical characteristics of subjects at the time of study
are shown in Table 1
. Subjects who
participated in neurotransmitter studies or repeated baseline studies
did not differ in clinical characteristics from the group as a
whole.
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).
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Table 2 demonstrates mean values for E2,
FSH, mean LH, LH pulse frequency, and LH pulse amplitude for the
different LH pulse classification groups and normal women in the EFP.
As expected, based on the definition of LH pattern classification
criteria, many parameters are significantly lower than those seen in
control women.
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provides an example
of sleep augmentation in an individual study. Those who showed sleep
augmentation did not differ in age or mean BMI from the total sample
and did not show a consistent pattern of disordered GnRH release.
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Twelve HA patients participated in 2–8 studies each, for a total
of 39 studies. Time between studies ranged from 1 month to 11 yr.
During this interval, none of the 12 patients had a weight change
greater than 10% of body weight, and no patient had spontaneous
resumption of menses. Mean E2 levels stayed constant across the
studies. Seventy-five percent of patients demonstrated at least 2
different patterns of LH secretion on repeat studies, and 33% reverted
at least once to an unclassified pattern. The variability of
LH secretion patterns held when data was analyzed for daytime only or
nighttime only. Figure 3 shows studies
from one patient who had a low frequency/low amplitude
pattern at baseline, an unclassified pattern 1 month after
baseline, and a low frequency pattern 8 months after
baseline. Data during sleep was available in 2 or more studies for 10
of the 12 patients—5 consistently demonstrated sleep augmentation, 2
had no evidence of sleep augmentation in any study, and 3 demonstrated
sleep augmentation in some studies but not others.
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Twelve of the 15 subjects treated with naloxone responded
with an increase in pulse frequency. The increase in LH pulse frequency
seen in the group overall was significant, from a preinfusion mean of
7.58 ± 1.54 pulses/24 h to a mean pulse frequency during naloxone
infusion of 10.25 ± 2.17 pulses/24 h (P < 0.05).
Before naloxone infusion, the mean pulse frequency of HA subjects was
more than 1 SD below the mean pulse frequency of normal
women in the EFP. During infusion, however, mean pulse frequency rose
into the normal range. Mean pulse amplitude and mean LH levels did not
change during naloxone infusion. Figure 4
illustrates a responsive and an unresponsive study with naloxone
infusion. Mean E2 for the group was 39.1 ± 7.1 pg/mL. Naloxone
responders did not have a uniform underlying neuroendocrine pattern (1
apulsatile, 5 low frequency/low amplitude, 1
low amplitude, 3 low frequency, and 2
unclassified). Naloxone nonresponders also showed a spectrum
of patterns of LH pulse secretion (1 each of apulsatile,
low frequency, and low frequency/low amplitude).
Nonresponders did not differ significantly from responders in BMI, mean
baseline E2 levels, or mean baseline LH levels.
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Four of the 8 subjects treated with metoclopramide responded to
metoclopramide infusion with an increase in LH pulse frequency.
However, no significant changes in mean LH pulse frequency, pulse
amplitude, or mean LH levels were seen in the group overall. Figure 5 illustrates a responsive and an
unresponsive study with metoclopramide infusion. Mean E2 for the group
was 30.0 ± 4.5 pg/mL. Similar to the observations with naloxone,
metoclopramide responders did not have a consistent baseline pattern of
LH secretion (2 low frequency/low amplitude, 2
low frequency). Metoclopramide nonresponders also showed
variable baseline patterns of LH secretion (1 apulsatile, 2
low frequency, 1 low frequency/low
amplitude).
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All 3 patients who participated in clonidine studies displayed
evidence of decreased LH secretion after clonidine administration. Mean
LH decreased significantly from a mean of 6.18 ± 1.18 IU/L to
3.84 ± 1.22 IU/L (P < 0.05). Both frequency and
amplitude decreased, but did not reach statistical significance. Figure 6 illustrates a typical response to
clonidine administration. E2 was below the detectable limit of the
assay for the group. Baseline LH secretion pattern did not appear to
affect the ability to respond to clonidine (2 low frequency,
1 unclassified).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patients displayed different LH secretion patterns when studied at different time points during their period of amenorrhea. Seventy-five percent of patients studied repeatedly met criteria for at least two different classifications, and 33% reverted at least once to an unclassified pattern. The variability of LH secretion abnormalities observed in this large sample of patients studied over time confirms earlier data reported in smaller series that showed similar variability (5, 29). This may imply that most HA patients do not have a static defect in GnRH secretion, but rather have changing patterns of GnRH secretion that vary over time and are insufficient to support ovulation or folliculogenesis.
Because 75% of all subjects studied on more than one occasion demonstrated changes in LH secretory patterns, it is reasonable to assume that a substantial proportion of our HA population would show similar variation if studied multiple times. If patients are thought to have changing, not static defects, then women who meet different classification criteria on a single study may nonetheless have similar hypothalamic dysfunction. In this patient population, no differences were found in age, duration of amenorrhea, or E2 levels between subjects with different observed GnRH abnormalities. Patients who displayed a low frequency pattern did have a lower mean BMI than women with low amplitude and low frequency/low amplitude patterns. However, when subjects who demonstrated low frequency patterns on their first baseline studies were studied on multiple occasions, they demonstrated all neuroendocrine patterns in the absence of any significant weight change. The existence of multiple secretion patterns seen over time in women who showed no associated clinical changes supports the idea that different observed patterns may represent different time points in a single illness, not different illnesses.
Subjects’ responses to opiate receptor blockade with naloxone indicate that increased opioid tone may play a role in the etiology of HA in a subgroup of patients. Work with immortalized human GnRH-secreting neuron cell lines has shown that these cells possess opiate receptors (30), and experimental work in animals and humans has demonstrated a pre-ovulatory peak and post-ovulatory trough in beta-endorphin levels during normal menstrual cycles, suggesting a role for endogenous opioids in the control of GnRH release (31, 32, 33, 34). Short-term opiate receptor blockade increases the frequency of LH secretion in humans (7, 8, 9), and recent research indicates that prolonged opiate receptor blockade may induce menstrual resumption in women with HA (35) and in animals with stress-induced amenorrhea (36). Our results are consistent with previous human studies, suggesting that increased opioid tone may contribute to amenorrhea in a subset of patients. Previous work by Armeanu et al. (17) indicated that a subset of patients with a stable low amplitude or apulsatile pattern did not respond to naloxone infusion. However, our findings indicate that patients with all types of baseline abnormalities are capable of responding to naloxone.
Although this study was not designed to measure the sensitivity of the GnRH-secreting neurons to opiate blockade in women with HA, one can speculate that the development of HA and the fluctuating patterns of LH secretion observed in patients may reflect a hypersensitivity of GnRH-secreting neurons to changing levels of opioid tone. Lifestyle variables commonly associated with HA, such as exercise, stress, and weight loss, can induce transient changes on plasma beta-endorphin levels (37, 38, 39, 40, 41, 42), which may impact the GnRH-secreting neurons in the hypothalamus. Research indicates that brief exposure to morphine ranging from 16 h to 4 days is sufficient to alter the basal secretory activity of GnRH-secreting neurons (43, 44) and their response to gonadal steroids (34). If a transient increase in endogenous opioids, such as that caused by stress, weight loss, or exercise training, can significantly change secretion patterns in a hypersensitive individual, then the observed changes in GnRH secretion pattern over time may reflect a hypothalamic response to changing levels of opioid tone in these individuals.
Unlike blockade of opiate receptors, blockade of dopamine receptors in the women studied here did not have a clear effect on LH secretion. Research in multiple animal models and immortalized human cell lines indicates that dopamine may stimulate hypothalamic GnRH release (45, 46) while simultaneously inhibiting pituitary LH release (47, 48, 49). Therefore studies in humans, which are forced to use pituitary-secreted LH as a marker of hypothalamic GnRH activity, may not be able to adequately distinguish pituitary from hypothalamic effects of dopamine. The lack of consistent findings in human studies (10, 11, 12, 13, 14, 15), including ours, supports the possibility that pituitary LH secretion may not be an adequate reflection of dopaminergic effects on the hypothalamic-pituitary axis.
Recent animal data has demonstrated that noradrenergic signals stimulate GnRH release in rabbits (50) and immortalized human GnRH-releasing cell lines (46). Previous work in normally menstruating women demonstrated a decrease in LH secretion after norepinephrine suppression with clonidine (15, 16), and the HA subjects in our study also responded to clonidine administration with a significant decrease in mean LH. The finding that clonidine lowered mean LH in our subjects may imply that norepinephrine stimulates LH release in humans as it does in lower mammals. However, these interactions appear to be complex. Experiments in pig pituitary glands (51) have demonstrated an interaction between opiates and dopamine in the control of GnRH release, and the work of Nazian et al. (46) with immortalized hypothalamic neurons indicates that opiates decrease the responsiveness of GnRH-releasing neurons to beta-adrenergic stimulation. The interaction between norepinephrine and opiates seen in animals, if true for humans, might indicate a role for endogenous opiates in suppressing norepinephrine-mediated GnRH release in women with HA.
In conclusion, in a large group of women with HA, we have observed a broad spectrum of GnRH secretion abnormalities. Importantly, the abnormal GnRH secretion is not stable, but varies over time within an individual. The observed response to neurotransmitter modulation indicates a possible role for opiate modulation in the etiology of this disorder, while the role of central adrenergic tone in the maintenance of GnRH pulsatility remains to be elucidated. Further investigation is needed to explore potential differences in sensitivity to opioid tone between eumenorrheic women and women with HA.
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Footnotes |
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Received February 18, 1999.
Revised March 29, 1999.
Accepted March 31, 1999.
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References |
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