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Evidence That GnRH Decreases with Gonadal Steroid Feedback but Increases with Age in Postmenopausal Women

Reproductive Endocrine Unit and the National Center for Infertility Research, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Janet E. Hall, M.D., Reproductive Endocrine Unit, Bartlett Hall Extension-5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: . jehall{at}partners.org

Abstract

Studies of the effects of gonadal steroid negative feedback and age on the hypothalamic-pituitary axis in postmenopausal women (PMW) have identified significant but inconsistent changes in gonadotropin dynamics. In the current study, we investigated the effect of gonadal steroid replacement and age on overall GnRH secretion estimated by using submaximal GnRH receptor blockade.

Twenty-four healthy PMW, 45–55 yr (n = 13) and 70–80 yr (n = 11), were studied. Subjects were studied at baseline (BL) on no hormone replacement therapy, after 1 month of transdermal estrogen (50 µg/d; E) and again after a further month of E and 7 d of transvaginal progesterone (100 mg bid; E + P). At each admission, blood was sampled every 30 min for 4 (BL and E) or 8 h (E + P) before and 10 h after sc administration of a submaximal dose (5 µg/kg) of the NAL-GLU GnRH antagonist ([Ac-D2Nal¹, D4ClPhe², DPal³, Arg⁵, DGlu(AA)⁶, DAla¹⁰] GnRH). Percent inhibition of LH was calculated by expressing the difference between the nadir following GnRH antagonist administration and the preantagonist baseline as a percent of the baseline.

Physiologic E and P levels were achieved with the appropriate hormone replacement regimens. Mean LH levels decreased from baseline with E alone and decreased further with E + P (81.4 ± 6.6, 68.2 ± 8.1 and 48.0 ± 4.3 IU/liter, respectively; P < 0.005). Percent inhibition of LH following submaximal GnRH receptor blockade decreased with age (57.6 ± 1.8% in young PMW vs. 51.4 ±2.2% in old PMW; P < 0.05) implying an increase in GnRH secretion with age. There was an increase in percent inhibition of LH in response to submaximal GnRH receptor blockade with E and a further increase with E + P (54.8 ±1.5%, 58.8 ± 1.9% and 69.9 ± 2.8%, respectively; P < 0.05), indicating a progressive decrease in endogenous GnRH secretion with gonadal steroid feedback. Mean LH and FSH levels were lower at baseline in old compared with young PMW. However, the effect of gonadal steroid feedback on endogenous GnRH secretion was similar in young and old PMW.

In conclusion: 1) The overall quantity of GnRH secretion increases with age as demonstrated by the progressive decrease in LH inhibition following submaximal GnRH antagonist administration with increasing age; 2) E negative feedback is associated with a decrease in GnRH secretion (as indicated by an increased percent inhibition of LH following submaximal GnRH receptor blockade); 3) E2 and P are associated with a further decease in overall amount of GnRH secreted; and 4) Age does not dampen the inhibition of hypothalamic GnRH secretion by E and P in PMW.

THERE IS INCREASING evidence that reproductive aging may involve alterations in hypothalamic and pituitary function in addition to the well-known loss of gonadal function (1, 2). Understanding both the physiology and site of action of ovarian steroid feedback and the age-related changes in the neuroendocrine system is imperative to discern the mechanisms of reproductive aging and, eventually, develop effective therapeutic options to manage this transition.

With ovarian follicle depletion at menopause, the hypothalamic and pituitary components of the reproductive axis are released from the negative feedback of ovarian steroids and inhibin, resulting in increases in LH and FSH (3, 4, 5). Loss of gonadal steroid feedback is associated with an increase in gonadotropin subunit expression and an increase in LH-containing gonadotropes in the rodent (see Ref. 6 for review). However, even in animal models it is unclear whether this change is primary or secondary to changes in hypothalamic input, as there is also an increase in expression of GnRH mRNA in castrate animals that is restored to intact levels with estrogen (E) treatment (7, 8, 9).

Gonadotropin secretion declines with age after menopause in some (2, 10, 11, 12, 13, 14), though not all, studies (15, 16), suggesting that gonadotropin secretion is influenced not only by gonadal feedback but also by age. Results of previous studies examining the effect of age on hypothalamic function that used LH as a marker of GnRH pulse frequency are inconsistent (12, 13, 14, 15, 16). However, recent studies using gonadotropin free -subunit (FAS) as a marker of pulsatile GnRH secretion have clearly shown a decrease in pulse frequency and amplitude with increasing age (2, 17). Only three studies have methodically evaluated the effect of age on E and progesterone (P) feedback in postmenopausal women (PMW) (13, 14, 17). All confirmed a negative feedback effect of E2 on gonadotropin secretion, but the influence of age on gonadotropin secretion was inconsistent between these studies, as were effects on LH pulse frequency.

In the current study, we sought to determine the independent effects of the response to gonadal steroid negative feedback and aging on the hypothalamus in PMW. Direct measurement of GnRH from the pituitary portal blood has been used in animal studies to quantitatively assess the physiology of GnRH secretion (18) but is not feasible in human studies. In addition, peripheral blood levels do not accurately reflect hypothalamic GnRH secretion (19). The frequency of pulsatile LH and FAS secretion has been used to reflect the frequency of pulsatile GnRH as an indicator of hypothalamic function in the human (2, 20, 21). However, LH and FAS pulse amplitude cannot provide a direct indication of the amount of GnRH secreted per pulse as additional factors including gonadal steroid feedback and preceding interpulse interval may influence the pituitary response to GnRH (20). Using a competitive GnRH antagonist, a semiquantitative estimate of the overall amount of GnRH secretion can be derived, as previously described (22). At submaximal doses, a known quantity of a GnRH antagonist competes with endogenous GnRH to bind to the GnRH receptor. LH can be used as the marker for GnRH as its secretion is controlled by GnRH alone (22). Based on established pharmacological principles, the degree to which LH is inhibited by administration of a known submaximal dose of a GnRH antagonist will be inversely proportional to the amount of GnRH being secreted. The use of this physiologic probe provides a semiquantitative measure of differences in the overall amount of GnRH secreted in response to gonadal steroid feedback in young and old PMW. Our results suggest that there is a hypothalamic site of negative feedback of both E2 and P and that there is an unexpected increase in the overall amount of GnRH associated with aging.

Subjects and Methods

Subjects

Young (aged 45–55 yr; n = 13) and old (aged 70–80 yr; n = 11) PMW were studied. All subjects were healthy and had experienced their last menstrual period a minimum of 12 months previously. Three old and two young subjects had undergone bilateral oophorectomy in the past, whereas the majority had undergone natural menopause. None of the women had taken hormone replacement for a minimum of 2 months before the study, nor were they on other medications known to interact with the neuroendocrine reproductive axis. PRL, TSH, complete blood count, liver function tests, renal function tests, and electrocardiogram were normal in all subjects. All subjects had documented normal mammograms within the last two years and no contraindications to E or P use. Subjects took ferrous gluconate 324 mg/d, starting 1 month before the initial study and for the duration of the study.

The study was approved by the Human Research Committee of the Massachusetts General Hospital, and signed informed consent was obtained from each subject before participation.

Experimental protocol

Eligible subjects were admitted to the Clinical Research Center of the Massachusetts General Hospital for three 14–18 h visits at 30-d intervals. After the first baseline (BL) visit on no hormone replacement, subjects were discharged wearing an E patch (50 µg/d) to be changed every 84 h per a prearranged schedule (E). They continued the E patch after the second visit and began using P suppositories (100 mg twice daily intravaginally) 7 d before and during the third admission (E + P). During each visit, blood was sampled every 30 min through an antecubital iv catheter for 4 h to assess baseline LH secretion. After studying the first 8 subjects, we found that 4 h of sampling was inadequate to accurately assess baseline LH secretion with E + P in all subjects, given the slowing of GnRH pulsatility in response to P (21, 23). Thus, the baseline frequent sampling was extended to 8 h for E + P in the remaining 14 subjects. In all subjects, sampling began 4 h after a dose of P. Immediately following baseline sampling, patients received a single submaximal dose of the NAL-GLU GnRH antagonist (5 µg/kg) by sc injection and sampling was continued for another 10 h. The NAL-GLU GnRH antagonist was formulated in sterile water with 5% glucose at a final concentration of 1 mg/ml, as previously described (22, 24).

All blood samples were analyzed for LH. Preantagonist baseline FSH levels were determined from a pool of equal aliquots of the baseline samples. Serum E2 and P levels were drawn at the beginning of each visit. Interim blood levels for E2 and/or P were also drawn between visits 1 and 2 to confirm E2 levels within the early-midfollicular phase range and between visits 2 and 3 to confirm P levels in the mid-luteal phase range. Two subjects (both young PMW) did not complete the study for personal reasons; one was studied at BL alone and the other at BL and E.

Assays

Serum LH, FSH, E2, and P levels were determined by immunoassay (AxSYM, Abbott Laboratories, Chicago, IL), as previously described (2, 25, 26). All samples from an individual subject’s study were measured in duplicate in the same assay. The interassay coefficient of variation for LH was 6.5–6.8% for LH levels ranging from 4.6–81.2 IU/liter. Gonadotropin values are expressed in units/liter as equivalents of the Second International Reference Preparation of human menopausal gonadotropins.

Data analysis

E and P effects on mean LH and FSH and percent changes from baseline were compared between young and old PMW using ANOVA for repeated measures with posthoc analysis by Newman-Keuls testing. To determine the LH response to submaximal GnRH receptor blockade with the NAL-GLU GnRH antagonist, the studies were divided into the baseline (pretreatment) 4-h or 8-h period and the subsequent postantagonist 10-h period. Baseline LH was calculated from the arithmetic mean of the 4 h or 8 h pretreatment LH measurements before administration of the NAL-GLU GnRH antagonist. The nadir following antagonist administration was calculated using a moving average (three points for the 30-min LH values). Suppression of LH by a fixed submaximal dose of GnRH antagonist was calculated as percent inhibition from baseline using the formula [(mean pretreatment baseline - nadir)/mean pretreatment baseline] x 100. The percent LH suppression results at visit three were excluded from the analysis for three subjects (all old PMW) due to the lack of LH pulses in the 4-h baseline sampling window.

To assess the effect of sex steroid feedback on overall amount of GnRH, percent LH inhibition with E and E + P was compared with baseline, using ANOVA. To assess the effect of age, mean LH, mean FSH, and percent LH inhibition at baseline were compared between young and old PMW using unpaired t tests. Results in the two groups of PMW were also compared with percent inhibition of LH in response to the identical dose of the NAL GLU GnRH antagonist (5 µg/kg) in normal women studied in the early follicular phase (n = 6) in previously published studies (22), using ANOVA. Linear regression analysis was also performed, assessing percent LH inhibition across all ages (combining all premenopausal and PMW) and controlling for E2 levels. This group of premenopausal women was selected as baseline E2 levels were similar to both groups of PMW.

A P value of <0.05 was considered to be statistically significant and results are expressed as the mean ± SEM.

Results

The mean body mass index of the participants was 25.4 ± 0.2 kg/m² (mean ± SEM; range 16.0–32.1). There was no difference in body mass index between young and old PMW or with E or E + P treatment. At baseline, low gonadal steroid levels and elevated gonadotropins confirmed menopausal status in all subjects (Table 1). At baseline, LH and FSH levels were lower in old compared with young PMW (P < 0.05). With E and P replacement, physiologic levels of E2 and P were achieved in both groups. Mean LH and FSH levels decreased significantly with the addition of gonadal steroids in both old and young PMW (P < 10^-5) (Table 1). There was an overall significant effect of age on LH secretion in response to the negative feedback effect of gonadal steroid replacement (P < 0.05). Specifically, the percent change in LH secretion was greater in older compared with younger PMW in response to E (28 ± 8% vs. 8 ± 8%, respectively) and E + P (48 ± 6% vs. 30 ± 3%, respectively). There was no effect of age on the negative feedback effect of gonadal steroids on FSH as there were similar percent changes in FSH secretion in young and old PMW in response to E and E + P.

Suppression of LH following 5 µg/kg of the NAL-GLU GnRH antagonist was evident in the young and old PMW in the absence of gonadal steroids and with E and E + P (Fig. 1). Percent inhibition of LH following submaximal GnRH receptor blockade was significantly greater in the younger compared with the older PMW in the absence of hormone replacement (P < 0.05), suggesting more endogenous GnRH secretion in the older compared with the younger PMW (Fig. 2). When comparing percent LH inhibition in young premenopausal women in the early follicular phase to young and old PMW, there was a strong trend in the decrease in percent inhibition of LH with increasing age (61.8 ± 6.1% vs. 57.6 ± 1.8% vs. 51.5 ± 2.1% for early follicular and young and old PMW, respectively P = 0.06). These results were confirmed by linear regression analysis which demonstrated a decline in percent inhibition of LH across all ages, adjusting for E2 levels (R = 0.48; P = 0.03).

View larger version (33K): [in this window] [in a new window]   Figure 1. Plasma LH (mean ± SEM) in young (n = 13) and old (n = 11) PMW at baseline and following E and a further month of E and P. The timing of administration of 5 µg/kg of the NAL-GLU GnRH antagonist is indicated by the vertical line.

View larger version (17K): [in this window] [in a new window]   Figure 2. Percent inhibition of LH following submaximal GnRH receptor blockade is decreased in old compared with young PMW at baseline, implying an increase in endogenous GnRH secretion in older women.

In PMW, addition of E2 resulted in an increase in percent inhibition of LH (P < 0.05). With E + P, there was a further increase in percent inhibition of LH (P < 0.0001). These results suggest that endogenous GnRH secretion is progressively decreased in the presence of E and E + P. The increase in percent inhibition of LH in response to gonadal steroid feedback was parallel in young and old PMW (Fig. 3), implying that inhibition of GnRH secretion by gonadal steroids does not change with aging in PMW.

View larger version (17K): [in this window] [in a new window]   Figure 3. Percent inhibition of LH in young () and old (•) PMW in response to gonadal steroid negative feedback indicating a significant increase in percent inhibition of LH with E alone and a further increase with E + P. These changes are consistent with a decrease in the overall amount of endogenous GnRH secreted in the presence of gonadal steroids. Note the parallel changes in younger and older PMW suggesting that steroid negative feedback on the overall amount of GnRH secreted is preserved with aging.

While the negative feedback effects of E and P on gonadotropin secretion have been well described in women, there is less information available regarding the hypothalamic vs. pituitary site of action of these effects, particularly those of E. Estimation of the overall quantity of endogenous GnRH secretion, using competitive GnRH receptor blockade with known submaximal doses of a GnRH antagonist, provides a model to allow us to determine whether there is a major hypothalamic site of gonadal steroid negative feedback. In PMW, it is also possible to isolate the independent effects of gonadal steroid negative feedback and aging on the neuroendocrine components of the reproductive axis.

The use of incomplete GnRH receptor blockade to estimate the amount of endogenous GnRH secretion based on the degree of LH suppression is possible because GnRH is the only known secretagogue for LH and because GnRH and the NAL-GLU GnRH antagonist bind to a single receptor type. Changes in percent inhibition of LH with aging or gonadal hormone status are unlikely to reflect changes in receptor affinity for GnRH or the antagonist as there is no change in GnRH receptor affinity over a wide range of both physiologic and pharmacologic conditions (22). Although both E and GnRH itself are known to influence GnRH receptor number (27, 28), any potential changes in either GnRH receptor number or postreceptor amplification of the GnRH signal will be present both before and after acute GnRH receptor blockade and will be accounted for by expressing the data as percent change from baseline. Previous studies have indicated that the NAL-GLU GnRH antagonist is rapidly absorbed after sc administration (29). While we included only healthy subjects and confirmed normal renal, hepatic, and thyroid function in all subjects, we cannot rule out the possibility that metabolism of the antagonist may have been altered with aging (30).

In the current studies, we examined the inhibition of LH in response to a single submaximal dose of the NAL-GLU GnRH antagonist to provide an estimate of the overall amount of endogenous GnRH based on our previous observation that the GnRH/GnRH antagonist dose-response relationship tested over four doses is identical between postmenopausal and normally cycling women (22, 31). This method does not permit actual quantitation of GnRH. It does, however, allow comparisons to be drawn regarding the ability of endogenous GnRH to compete with a fixed amount of antagonist in different physiological circumstances. From these data, inferences can be drawn regarding the relative amounts of endogenous GnRH being secreted between different populations and physiological conditions (22, 32).

The current studies provide evidence that aging is associated with a progressive increase in the overall amount of endogenous GnRH secreted. This increase in GnRH with age from premenopausal to older PMW occurs in the setting of similar sex steroid levels and is independent of changes in baseline LH levels. In estrogen-deficient women, a number of studies including our own, have demonstrated a decrease in gonadotropin pulse frequency with age that suggests a slowing of the GnRH pulse generator (2, 12, 13, 16, 17). Taken together, these studies, which show a decrease in GnRH pulse frequency, but an increase in overall amount of GnRH secreted imply that the quantity of GnRH secreted per pulse increases with age. While autopsy studies have suggested that GnRH secretion may be increased with age (33, 34), the effects of withdrawal of E negative feedback were not isolated from changes specifically due to aging. However, the number of neurons expressing POMC mRNA is decreased in the infundibular nucleus in PMW studied at autopsy (35). As administration of E with or without P had no effect on POMC gene expression in oophorectomized monkeys, it is suggested that this effect on POMC gene expression in PMW may well be due to age rather than hormonal status (36). The loss of POMC gene expression with aging may result in a loss of opioid mediated dampening of GnRH secretion with age, providing support for the age-associated increase in the overall amount of GnRH suggested by the current study.

Previous studies that have examined the effect of aging on the neuroendocrine components of the reproductive axis have almost uniformly documented a decrease in mean levels of LH, FSH, and FAS with age in PMW (2, 10, 11, 12, 13, 14, 17). These findings were confirmed in the present study. The decrease in mean gonadotropin levels with aging in the face of increased GnRH secretion suggests that the pituitary is less responsive to GnRH in older compared with younger PMW. In PMW, gonadotropin responses to exogenous GnRH administration have been shown to be impaired as a function of aging in one study (12), whereas another study has reported an increase in LH response to GnRH as a function of aging, but a decrease in LH response as a function of years post menopause (16). Further studies evaluating the independent age-related changes in pituitary sensitivity to GnRH will be required to resolve this issue. In these studies, it will be critical to control for preceding interpulse interval that is both increased with aging (17) and known to influence pituitary responsiveness to GnRH (20).

A hypothalamic site of ovarian steroid negative feedback in normal PMW is demonstrated in the current studies by a decrease in the overall amount of GnRH secreted with addition of E alone and in combination with P. Previous studies using a variety of intact animal models have provided evidence in support of a hypothalamic site of E negative feedback (6, 37, 38). In addition, E2 administration decreased GnRH mRNA levels in rat hypothalamic tissue slices (39) and has now been shown to down-regulate GnRH mRNA expression in GTI-7 GnRH neurons (40). Both indirect and direct mechanisms for E regulation of hypothalamic GnRH neurons have been proposed (see Ref. 41 for review).

P is thought to have a primary hypothalamic site of negative feedback that operates through slowing of the GnRH pulse generator (21, 23, 42), although E priming appears to be essential for up-regulation of P receptors (42, 43). Both direct and indirect effects on GnRH are likely (41, 44), as with the negative feedback effects of E.

Despite the evidence for a hypothalamic site of E and P negative feedback in animal studies, changes in GnRH dynamics with gonadal steroid feedback in PMW have been less clear. In steroid-deficient women, E administration was associated with an increase (45), decrease (14), or lack of change (46) in LH pulse frequency and no change in FAS pulse frequency (17). Addition of P uniformly suppressed GnRH pulse frequency using either LH (45) or FAS (17) as markers of GnRH secretion. In the present study, percent inhibition of LH in response to submaximal GnRH receptor blockade increased with E replacement implying that the overall amount of GnRH decreased in response to the negative feedback effect of E in PMW. As we found no decrease in GnRH pulse frequency with an identical E replacement regimen to that used in the current study (17), these data suggest that E is associated with a decrease in the amount of GnRH secreted with each pulse. Thus, a hypothalamic effect accounts, at least in part, for the decrease in gonadotropin secretion with E administration in PMW. A hypothalamic site of P negative feedback is supported by a concomitant decrease in GnRH pulse frequency (17) and overall amount. Interestingly, our previous studies had indicated that the quantity of GnRH was similar in the early and late follicular phase despite changing E2 levels (22). It is possible that the effect of E negative feedback on GnRH quantity was not apparent in these studies in the normal menstrual cycle due to the shorter duration of E exposure compared with that used in the current protocol.

A change in the responsiveness to E negative feedback at the hypothalamus with increasing age has been demonstrated in the rat (47). Only three studies have addressed this issue in steroid-deficient women and with conflicting results. There was a greater effect of E negative feedback on mean gonadotropin levels and LH pulse frequency in women with normal compared with early menopause in the studies of Santoro et al. (14). On the contrary, the effect of E negative feedback on mean LH and LH pulse frequency was less in old compared with young PMW in the studies of Rossmanith et al. (13). Our studies in old and young PMW indicated a greater effect of E negative feedback on mean LH with increasing age, but no age-related difference in FAS pulse frequency (17). Whether the effect of E negative feedback at the pituitary is greater in older women as implied by these studies must await further investigation. The only study to examine the suppressive effect of P on pulse frequency has indicated that this effect is not attenuated with aging (17). In the current studies, we again found a greater effect of E and E plus P on mean LH in older compared with younger PMW, but a parallel effect of gonadal steroid feedback on the overall amount of GnRH in older and younger PMW. These results imply that responsiveness to gonadal steroid negative feedback at the hypothalamus is maintained with aging.

In conclusion, using incomplete GnRH receptor blockade to investigate the overall amount of endogenous GnRH secreted, these studies imply that there is an increase in the overall amount of GnRH secretion with increasing age despite a previously demonstrated decrease in GnRH pulse frequency. The overall amount of endogenous GnRH secretion is decreased in association with E and P administration in PMW, indicative of a hypothalamic site of negative feedback of both gonadal steroids. Further, these results suggest that age does not dampen the responsiveness of the hypothalamus to the negative feedback effects of E and P.

Acknowledgments

We gratefully acknowledge the technicians of the Reproductive Endocrine Unit Core Laboratory under the direction of Patrick Sluss, Ph.D., for their superb technical contributions to this study, and the nurses from the General Clinical Research Unit for their excellent work. We also thank the women who participated in this study for their cooperation and commitment to this research

Footnotes

This work was supported by R01-AG-13241 and M01-RR-1066. Dr. Gill has received fellowship support from the British Columbia Endocrine Research Foundation and Parke-Davis Canada and support for this project from the NAMS/Solvay Pharmaceuticals, Inc. Clinical Research Fellowship Grant.

Abbreviations: BL, Baseline; E, estrogen; FAS, free -subunit; NAL-GLU GnRH antagonist, [Ac-D2Nal¹, D4ClPhe², DPal³, Arg⁵, DGlu(AA)⁶, DAla¹⁰] GnRH; P, progesterone; PMW, postmenopausal women.

Received April 2, 2001.

Accepted February 15, 2002.

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