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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
There is now evidence for alterations in the neuroendocrine control of the reproductive axis with aging, but its sensitivity to gonadal steroid negative feedback remains controversial. To examine the independent effect of age and gonadal steroid negative feedback, younger (45–55 yr; n = 7) and older (70–80 yr; n = 6) postmenopausal women (PMW) were studied at baseline on no HRT, after 1 month of transdermal estrogen (50 µg/d; E) and again after a further month of E and 7 d of transvaginal progesterone (P) (100 mg bid; E + P). At each admission, blood was sampled every 5 min for 8 h for measurement of gonadotropin free 
-subunit (FAS), which was used as a marker of GnRH pulse frequency. LH and FSH were measured in pooled samples.
Midfollicular and midluteal phase levels of E2 and P were achieved during the E and E + P treatments and were not different between younger and older PMW. There was a negative feedback effect of E and E + P on mean LH (P < 0.0001) and an additional effect of age (P < 0.003), with older women having lower values throughout. Mean FSH was also decreased with E and E + P (P < 0.0001) and was consistently lower in the older women (P < 0.05). Mean FAS levels decreased with hormonal treatment (P < 0.0001) and age (P < 0.001), but the effect of hormonal treatment was attenuated in the older group (P < 0.005). FAS pulse frequency was unchanged with addition of E, but dramatically decreased with E + P (P < 0.002). Both hormonal replacement (P < 0.05) and age (P < 0.005) decreased FAS pulse amplitude, an effect that was attributable entirely to E as there was no additional change with E + P.
These studies indicate that: 1) both age and gonadal steroids independently decrease mean LH, FSH, and FAS in PMW; 2) responsiveness to steroid negative feedback on FAS is attenuated with aging in absolute but not relative terms, whereas the effect on mean levels of LH and FSH is clearly preserved; and 3) FAS pulse frequency is unchanged with E2 administration but decreases dramatically with addition of P in both old and young PMW.
BETWEEN 1900 AND 1991, life expectancy at birth for women increased from 48 yr to nearly 79 yr (1). As a result, issues regarding the quality of extended life expectancy assume greater significance, and it becomes increasingly important that we strive to understand the biological processes that control the aging process. Despite the changes in longevity, the average age of menopause has remained at approximately 51 yr, and the average woman now spends a third of her life after menopause. The reproductive axis provides a unique opportunity to examine aspects of neuronal function by measurement of hormone levels in peripheral blood. In women, it is important to separate changes due to aging per se from those that are secondary to the marked hormonal changes that occur with menopause. Information derived from these studies will not only provide insights into aging of the reproductive system, but will also provide insight into the degree to which age-related neuroendocrine changes can be reversed by physiologic replacement of gonadal steroids.
Loss of ovarian function with menopause results in release of the negative feedback effect of gonadal steroids and peptides on the hypothalamic and pituitary components of the reproductive axis resulting in a dramatic increase in LH, FSH, and free -subunit (FAS) (2, 3, 4, 5, 6, 7). Evidence supports a decrease in gonadotropin secretion with age following menopause in most (7, 8, 9, 10, 11, 12), but not all studies (13, 14), indicative of an age-related decline in hypothalamic and/or pituitary function. Using FAS as a marker of GnRH secretion, we have previously demonstrated a decline in GnRH pulse frequency with aging after menopause (7), consistent with the results of some (10, 11, 14), but not all (12) studies in which LH was used as a marker of GnRH secretion. Despite this decrease in GnRH pulse frequency, there is a progressive age-related increase in the overall amount of GnRH secreted as assessed by the LH response to incomplete GnRH receptor blockade (15).
While animal studies suggested a decrease in the effects of steroid negative feedback on the hypothalamus and pituitary with aging (16), studies in women have been more controversial. Autopsy studies have demonstrated an increase in GnRH expression in postmenopausal compared with premenopausal women (17). However, this study did not isolate the effect of loss of gonadal feedback from aging, per se. Only two studies have systematically assessed the effect of age on estrogen (E) feedback in estrogen-deficient women (11, 12). Both studies demonstrated a negative feedback effect of E on LH and FSH and no effect on LH pulse amplitude. However, the effect of E replacement on LH pulse frequency was inconsistent between the two, as was the relative impact of age on E negative feedback.
It has previously been shown that FAS is superior to LH as a surrogate marker of GnRH pulse frequency in PMW due to the fast pulse frequencies encountered in the absence of gonadal feedback and the prolongation of LH clearance following menopause (7, 18, 19). In addition, these pulse frequencies require a shorter sampling interval than used in previous studies. We hypothesized that application of these methodologies would resolve previous discrepancies in the age-related effects of E negative feedback on GnRH pulse frequency. These studies would also permit us to examine the added effect of progesterone (P) as a function of age, which had not been addressed in previous studies.
Subjects and Methods
Subjects
Study subjects were healthy PMW (young, aged 45–55 yr; n = 8, and older, aged 70–80 yr; n = 6), who had experienced their last menstrual period at least 12 months previously. Three subjects in the older PMW group had undergone bilateral oophorectomy in the past, whereas the remainder had 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. All subjects were required to take ferrous gluconate 324 mg/d, starting 1 month before the first frequent sampling study and for the duration of the study. PRL, TSH, complete blood count, electrocardiogram, and mammogram were normal in all subjects and none had any contraindications to HRT.
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
Subjects were admitted to the General Clinical Research Center of the Massachusetts General Hospital on three occasions at 30-d intervals. During each admission, blood was sampled through an antecubital iv catheter every 5 min for 8 h from 2300 h to 0700 h. Subjects were studied at the same time of day at baseline, after 1 month of E replacement (Estraderm patch, 50 µg/d; E), and after a further month of E replacement and 7 d of transvaginal P (P suppositories 100 mg twice daily; E + P), designed to achieve physiological gonadal steroid levels consistent with mid-follicular phase and mid-luteal phase levels, respectively. All blood samples were analyzed for FAS. Serum LH and FSH levels were determined from a pool of equal aliquots of serum from each of the frequent blood samples. Serum E2 and P levels were measured at the beginning and end of each frequent sampling period.
Assays
Serum LH, FSH, FAS, E2, and P levels were determined by immunoassays, as previously described (7, 20). All samples from an individual subject’s study were measured in duplicate in the same assay. Gonadotropin values are expressed in U/liter as equivalents of the Second International Reference Preparation of human menopausal gonadotropins. Assay performance was monitored using control sera. Based on repeated measures of controls, the interassay coefficient of variation (CV) for LH was 6.5–6.8% for LH levels ranging from 4.6 to 81.2 IU/liter, for FSH was 4.2–6.0% for FSH levels ranging from 5.1 to 75.2 IU/liter. The interassay CV for FAS was 9.4–20.0% for FAS levels ranging from 163.4 to 595.4 ng/liter. Samples with FAS levels higher than the assay range were diluted and remeasured. To determine the intraassay CV for pulse identification, pools were constituted from the 5-min samples for each study, and aliquots were measured repeatedly throughout the FAS assay. The intraassay coefficient of variation for FAS was 5.5 ± 1.7%.
Data analysis
Pulsatile secretion of FAS was analyzed using the Cluster pulse detection algorithm (21). This algorithm maximizes the detection of true positive pulses and minimizes the detection of false positive pulses based on in vivo validation studies using similar pulse frequencies (18) and is equivalent to the modification of the Santen & Bardin program used in this laboratory in previous studies (18, 22, 23). The parameters chosen were a nadir and peak size of 2 x 2 with a t statistic value of 2.0 for both upstroke and downstroke. A regional CV for FAS was used in the pulse analysis to achieve a more robust assessment of the CV over the wide range of serum levels observed in this study. The regional CV was derived from the mean CV values for FAS from all of the samples assessed in the study and categorized into seven groupings of FAS concentrations.
Tests for normality were conducted on the mean hormone levels and FAS pulse frequency and amplitude. Those factors not normally distributed were logarithmically transformed before the analysis. Baseline mean hormone levels were compared by t test. ANOVA for repeated measures was performed to examine the effect of age and hormone replacement on mean levels of FSH, LH, FAS, FAS pulse frequency, and FAS pulse amplitude individually. Where appropriate, posthoc analysis was performed, using Newman-Keuls testing, to determine the age and gonadal steroid effects for each variable. For technical reasons, data for one subject (young PMW) is available only for baseline and E + P and she was therefore excluded from the ANOVA component of the analysis. Values are expressed as mean ± SEM, unless otherwise indicated and P values less than 0.05 are considered significant.
Results
Baseline characteristics
The mean body mass index of the participants was 24.7 ± 0.8 kg/m2 (mean ± SEM; range 19.2–27.7) and was not different between the younger and older subjects. At baseline, menopausal status was confirmed in all subjects by low E2 and elevated gonadotropin and FAS levels (Table 1
). There were no apparent differences in hormonal levels between the older women with and without ovaries, although the numbers in each subgroup are small (E2 20 ± 1 and 20 ± 0.5 pg/ml; FSH 91.4 ± 10.3 and 98.0 ± 22.3 IU/liter; LH 41.8 ± 3.0 and 63.7±8.0 IU/liter; FAS 318 ± 126 and 606 ± 55 ng/liter; for older women with and without ovaries, respectively).
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With E and E + P administration, mean E2 and P levels increased to physiologic levels in both young and old PMW (E2 72.7 ± 4.8 and 78.4 ± 7.3 pg/ml with E and E + P, respectively, and P 7.7 ± 0.9 ng/ml with E + P). There was a significant independent effect of both age (P < 0.003) and hormonal status (P < 0.0001) on LH, with lower levels observed throughout in the older PMW (Fig. 1). There was a significant decrease in LH from baseline with E alone (P < 0.005) and a further decrease in the presence of E + P (P < 0.0001). There was no significant interaction of age and hormonal status on mean LH indicating that the negative feedback effect of E and E + P was similar in older and younger PMW. When expressed as a percent decrease from baseline, there was a 26 ± 8 and 38 ± 10% change in LH in young and old PMW with E and a 57 ± 7 and 77 ± 9% change in young and old PMW with E + P. The differences in percent change were not significant between young and old.
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). There was a decrease in FSH with E alone (P < 0.0001) and a further decline with E + P (P < 0.0001). The negative feedback effects of gonadal steroid replacement on FSH were similar in young and old PMW. Expressed as a percent decrease from baseline, there was a 38 ± 5 and 39 ± 11% change in FSH in young and old PMW with E and a 56 ± 6 and 69 ± 9% change in young and old PMW with E + P.
FAS was also lower in older compared with younger PMW (P < 0.001) and there was a significant effect of HRT (P < 0.0001) (Fig. 1). There was a decrease in FAS with E alone (P < 0.005) and a further decline with E + P (P < 0.001). For FAS, there was an interaction of hormone replacement with age (P < 0.005), suggesting that the negative feedback effect of E and E + P was attenuated in the older PMW when changes are expressed in absolute terms. However, expressed as percent decrease from baseline, there was no difference in the effect of E or E + P as a function of age (26 ± 6 and 19 ± 23% change in FAS in young and old PMW with E and a 65 ± 7 and 52 ± 12% change in young and old PMW with E + P).
Effect of age and gonadal steroid negative feedback on FAS pulse characteristics
FAS pulse frequency was slower in older compared with younger PMW at baseline (P < 0.05) (Table 1, Figs. 2 and 3). FAS pulse amplitude was also significantly lower in the older compared with the younger PMW in the absence of gonadal hormone replacement (P < 0.05) (Table 1, Figs. 2 and 3). FAS pulse amplitude was decreased by both age (P < 0.005) and gonadal steroid administration (P < 0.05) (Figs. 2 and 3). There was no interaction between age and hormonal treatment indicating that the negative feedback effect of E and E + P on FAS pulse amplitude is not altered with aging. Although there was an overall effect of hormone replacement on FAS pulse frequency (P < 0.002), this was due entirely to the effect of P (Figs. 2 and 3) as there was no effect of E alone on FAS pulse frequency in young or old PMW. The effect of E + P on pulse frequency was not attenuated with age.
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There is ample evidence for negative feedback regulation of gonadotropin secretion with low doses of E (24, 25, 26) and with P (27, 28, 29, 30); however, it is unclear whether this effect it is altered with aging (11, 12, 30). With loss of ovarian function, postmenopausal women provide a unique model in which it is possible to investigate the independent effects of aging and controlled gonadal steroid feedback on the hypothalamic and pituitary components of the reproductive axis. In this study, we have examined the impact of aging on the negative feedback effects of E and P. These studies have shown that the negative feedback effects of E2 and P on mean levels of LH, FSH, and FAS are maintained with aging. In addition, FAS pulse frequency is unchanged with E replacement despite decreases in mean FAS and FAS pulse amplitude in both young and old postmenopausal women. Addition of P is associated with a marked decrease in FAS pulse frequency with no loss of responsiveness to P negative feedback with age.
The marked suppression of serum levels of LH, FSH, and FAS with low dose E replacement in the current studies is consistent with previous studies in E-deficient women who have shown a decrease in LH, FSH, and FAS in association with E replacement (9, 11, 12, 15, 31, 32, 33). These negative feedback effects in women are also consistent with studies in a variety of animal species (24, 25, 26). However, it is unclear to what degree these pituitary changes are mediated by negative feedback effects on hypothalamic GnRH secretion (26, 34).
Using LH as a marker of pulsatile GnRH, Rossmanith et al. (11) found a greater decrease in pulse frequency in response to clomiphene citrate in younger compared with older postmenopausal women. In contrast, Santoro (12) found a greater decrease in LH pulse frequency in naturally menopausal compared with prematurely menopausal women in response to E administration. The current studies using FAS as a better marker of GnRH secretion indicate that there is no change in pulse frequency with replacement of physiologic levels of E in postmenopausal women. We have also shown that the overall quantity of GnRH, assessed using incomplete GnRH receptor blockade, is decreased in postmenopausal women with the same low levels of E replacement as in the current study (15). Taken together, these data suggest that E negative feedback is accompanied by a decrease in the amount of GnRH secreted with each pulse.
Results of the current study are consistent with the majority of studies in animal models that indicate that E negative feedback is associated with a decrease in GnRH pulse amplitude, but not frequency and that the hypothalamus is a major site of E negative feedback (35, 36). These data are supported by autopsy studies in the human in which an increase in GnRH expression in GnRH-containing neurons in the medial basal hypothalamus is seen in PMW compared with premenopausal women (17). However, there is also evidence that the pituitary may be an additional site of E negative feedback (see Refs. 24, 25, 26 for review). In the current study, we cannot exclude an additional pituitary site of action of E negative feedback.
Responsiveness to the negative feedback effects of E on LH and FSH secretion is maintained with aging as there was no interaction between aging and hormone effect and no difference in the absolute or percent change in LH or FSH in response to E administration between old and young PMW. These findings are similar to the studies of Santoro et al. (12), who demonstrated comparable responsiveness to E negative feedback in women with early and normal menopause. In contrast, in studies in which clomiphene citrate (which acts as an E agonist in E-deficient women) was given to old and young postmenopausal women, suppression of LH and FSH was attenuated with age (11). In the current studies, mean FAS levels were markedly lower at baseline, in the absence of E, in older compared with younger PMW. The effect of E negative feedback was less pronounced in older compared with younger PMW when expressed in absolute terms, but not different when expressed as a percent decrease from baseline. Although there was no effect of E on FAS pulse frequency in either younger or older PMW, there was a marked decrease in FAS pulse amplitude with E replacement. However, the decrease in FAS pulse amplitude with E treatment was not affected by age, whether expressed as an absolute difference or as a percent change from baseline.
Addition of P resulted in a marked decrease in mean levels of LH and FSH as previously seen in postmenopausal women using either P (31, 32) or medroxyprogesterone acetate (9). There is ample evidence that P results in slowing of the GnRH pulse generator in reproductive aged women (27, 28), but that this effect is not seen in the absence of E (29). We have now shown that addition of P results in a marked slowing of FAS pulse frequency, confirming the effects of P on the GnRH pulse generator in postmenopausal women previously suggested by studies in which LH was used as a marker of GnRH pulse frequency (9, 31). This decrease in pulse frequency is accompanied by a decrease in the overall amount of GnRH secreted (15). From these and other studies, it appears that P has a predominate, if not exclusive, hypothalamic site of negative feedback on the neuroendocrine components of the reproductive axis (24, 25).
We have now shown that the negative feedback effect of P on mean LH and FSH is not attenuated with age. While the effect of E + P on absolute levels of FAS was decreased with aging, there was no difference between young and old PMW when values are expressed as percent change from baseline. In the current studies, we have further shown that the negative feedback effect of P on the frequency and amplitude of FAS pulses is maintained with aging in postmenopausal women.
In summary, these studies indicate that both age and gonadal steroids independently decrease mean LH, FSH, and FAS. The effect of steroid negative feedback on LH, FSH, and FAS is preserved with aging. FAS pulse frequency is unchanged with E2 administration but decreases dramatically with addition of P in both old and young postmenopausal women. Taken together, these results suggest that gonadal steroid negative feedback effects on the hypothalamic and pituitary components of the reproductive axis are maintained through the eighth decade of life in normal women.
Acknowledgments
We gratefully acknowledge the technicians of the RIA Core Laboratory of the Reproductive Endocrine Unit, under the direction of Patrick Sluss, Ph.D., for their superb technical contributions to this study; the nurses from the General Clinical Research Unit for their excellent work; and the contributions of our research assistants. We also thank the women who participated in this study for their cooperation and commitment to this research.
Footnotes
This work was supported by Grants R01-AG-13241 and M01-RR-1066. S.G. received fellowship support from the British Columbia Endocrine Research Foundation and Parke-Davis Canada. H.B.L. received fellowship support from the Samuel R. McLaughlin Foundation, the Royal College of Physicians of Canada (Detweiler Award), and Ferring Pharmaceuticals Ltd. Canada, Germany. Y.B.-A. was received fellowship support from the government of Kuwait.
Abbreviations: CV, Coefficient of variation; E, estrogen; FAS, free -subunit; HRT, hormone replacement therapy; P, progesterone; PMW, postmenopausal women.
Received May 31, 2001.
Accepted February 15, 2002.
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