help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1583
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/4/1953    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, P. Y.
Right arrow Articles by Khosla, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, P. Y.
Right arrow Articles by Khosla, S.
Related Collections
Right arrow Calcium and Bone Metabolism
Right arrow Female Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 1953-1960
Copyright © 2005 by The Endocrine Society

A Randomized Placebo-Controlled Trial of Short-Term Graded Transdermal Estradiol in Healthy Gonadotropin-Releasing Hormone Agonist-Suppressed Pre- and Postmenopausal Women: Effects on Serum Markers of Bone Turnover, Insulin-Like Growth Factor-I, and Osteoclastogenic Mediators

Peter Y. Liu, Kelley A. Hoey, Kristi L. Mielke, Johannes D. Veldhuis and Sundeep Khosla

Endocrine Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota, 55905

Address all correspondence and requests for reprints to: Sundeep Khosla, Endocrine Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota 55905. E-mail: Khosla.Sundeep{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The acute effects of estradiol on procollagen type 1 formation in pre- and postmenopausal women are controversial. Twenty-three premenopausal women and 13 postmenopausal women received two consecutive im injections of 3.75 mg leuprolide acetate 3 wk apart to block endogenous ovarian steroidogenesis. Transdermal estradiol therapy commenced on the night of the second leuprolide injection in all subjects, except five pre- and two postmenopausal women who were randomized to receive placebo patches. Estradiol therapy was applied incrementally, with each dose of 0.05, 0.10, 0.15, and 0.20 mg/d administered for 4 consecutive days, to mimic the estradiol changes typifying the follicular phase of the menstrual cycle. Blood aminoterminal propeptide of type I procollagen (PINP), intact osteocalcin (OC), carboxyterminal telopeptide of type I collagen (CTx), IGF-I, and estradiol were measured before and at the end of each estradiol increment. Potential mediators such as osteoprotegerin and receptor activator of nuclear factor-{kappa}B ligand (RANKL) were also measured. Despite comparable increases in serum estradiol, PINP increased more in postmenopausal compared with premenopausal women (between-group P = 0.03) and occurred at a time when CTx and OC did not change. CTx and IGF-I changed minimally and inconsistently, whereas OC, RANKL, and osteoprotegerin were stable. Repeated measures linear regression disclosed a significant negative association between increases in estradiol and PINP in premenopausal women (P = 0.0006) only. This suggests that lower dose estradiol should greatly increase PINP. Analogous regressions also showed significant negative relationships between changes in estradiol and RANKL in both pre- (P = 0.04) and postmenopausal (P = 0.002) women. Changes in serum markers of bone formation (PINP or OC) did not correlate with those of IGF-I.

We conclude that lower dose estradiol rapidly increases osteoblastic collagen synthesis in women at a time when collagen degradation is stable and that this response differs between pre- and postmenopausal women. The effect of estradiol on bone formation does not appear to be mediated by IGF-I. In contrast, RANKL is likely to mediate the effect of estradiol on osteoclastogenesis.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE EFFECTS OF lower dose estradiol on biochemical markers of bone turnover in postmenopausal women are controversial and increasingly important. The widespread concern regarding the safety of unopposed (1) and combined (2) estrogen/progestin therapy, despite established fracture efficacy (3), has led to the desire to investigate lower dose therapies that may prove to have comparable efficacy but improved safety. Critically, oral estradiol therapy at doses lower than previously investigated for cardiovascular protection is now known to increase bone mineral density (4), and additional trials examining the longer-term effects of such therapy on fracture incidence, as well as acute mechanistic effects on bone turnover, are needed. Whereas the longer-term antiresorptive effects of estradiol in postmenopausal women are well recognized, the acute effects of estradiol on bone turnover, particularly on osteoblastic collagen formation, are much less studied. Only one study, in 10 postmenopausal women, has previously investigated the effect of estradiol therapy on markers of bone turnover within the first month of therapy, but putative mechanistic mediators such as IGF-I, osteoprotegerin (OPG), or receptor activator of nuclear factor-{kappa}B ligand (RANKL) were not examined (5).

The impact of estradiol therapy on bone turnover in healthy premenopausal women is also little studied. Such explorations may bolster the often-quoted, but largely untested, hypothesis that therapeutically important responses to estradiol in key tissues vary with age (6, 7). Nevertheless, ancillary data are highly suggestive that rapid alterations in bone turnover occur in healthy premenopausal women during the menstrual cycle and that estradiol is responsible. Serum markers of both bone formation and resorption have been reported to change during the menstrual cycle, particularly during the follicular phase when serum estradiol concentrations are steadily rising (8, 9, 10, 11, 12). These studies show that if bone turnover changes during the follicular phase, bone formation increases, whereas bone resorption may increase or decrease (see Discussion). Furthermore, serum estradiol correlates negatively with markers of bone resorption deoxypyridinoline (DPD) and pyridinoline] (12) and positively with formation markers [intact osteocalcin (OC) and bone-specific alkaline phosphatase (BSAP)] (9). Although highly suggestive, these significant correlational data cannot prove that estradiol is responsible for these rapid changes in bone turnover. Such a causal role requires an interventional study.

For these reasons, we performed a randomized placebo-controlled study of escalating transdermal estradiol, designed to mimic the initially low, but rapidly increasing, serum estradiol concentrations typifying the follicular phase of the menstrual cycle in healthy pre- and postmenopausal women. To obtain comparable estrogen repletion, and to minimize possible confounding by unequal testosterone and progesterone concentrations, a GnRH agonist was administered first to down-regulate the gonadal axis (see Subjects and Methods). Such a strategy also removes the considerable confounding effects of endogenous estradiol production, even when examining postmenopausal women (7). Furthermore, this investigative paradigm allows dissection of differing age-related responsiveness to estradiol. Because IGF-I (13) and OPG/RANKL (14) are presumptive mediators of the effects of estradiol on bone formation and resorption, respectively, these serum markers were also measured.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Healthy premenopausal (n = 23, five randomized to receive placebo) and postmenopausal (n = 13, two randomized to receive placebo) women completed the protocol (below), after providing written informed consent approved by the Mayo Institutional Review Board. Volunteers were nonsmokers without cardiac, cerebral, or peripheral arterial or venous thromboembolic disease, breast cancer, or untreated gallstones. They were healthy, nonobese (body mass index, <30 kg/m2), weight-stable, community-dwelling women with normal menarchal and menstrual history and without acute or chronic disease, psychiatric illness, or substance abuse. None received neuroactive medications or commenced medications known to alter calcium metabolism. Women were excluded for any bisphosphonate use. The last exposure to hormone replacement therapy was in excess of 12 months for all postmenopausal women, except one who ceased 6 wk before study enrolment. Each subject had an unremarkable medical history and physical examination, and normal screening laboratory tests of hepatic, renal, endocrine, metabolic, and hematological function. In postmenopausal women, ovariprival status was confirmed by concentrations of FSH more than 50 IU/liter, LH more than 20 IU/liter, and estradiol less than 20 pg/ml (SI unit, <74 pmol/liter).

Study design

This was designed as a randomized placebo-controlled study weighted toward estradiol rather than placebo treatment, because a priori data supporting the bone effects of estradiol are extensive. Each woman received two consecutive im injections of 3.75 mg leuprolide acetate 3 wk apart to block endogenous steroidogenesis. In premenopausal women, leuprolide was given in the early follicular phase (within 7 d of menses onset) after a negative blood pregnancy test. Commencing on the night of the second leuprolide injection, transdermal estradiol was administered in an incremental stepwise schema, with each dose of 0.05, 0.10, 0.15, and 0.20 mg/d administered for 4 consecutive days in 18 pre- and 12 postmenopausal women. The intent was to mimic the estradiol changes typifying the follicular phase of the menstrual cycle. Blood was sampled at 0730 h, with the subject fasting before and every 4 d during estradiol therapy. Plasma was separated on ice and frozen at –70 C within 30 min. On study completion, progesterone was administered (100 mg orally for 12 d) to women with an intact uterus, according to good standards of clinical practice.

In addition to the 29 women who received estradiol, five pre- and two postmenopausal women underwent the same procedure, but they were randomized to receive placebo instead of estradiol patches. This control group was used to examine whether Lupron treatment alone altered bone turnover, particularly in premenopausal women who would have been rendered acutely hypogonadal.

Blood bone turnover marker and hormone assays

All samples from the same subject were assayed together. Serum propeptide of type I procollagen (PINP) was measured by RIA (DiaSorin, Stillwater, MN). Intraassay coefficients of variation (CV) were 2.3% at 45 µg/liter, rising to 12.7% at 100 µg/liter. Interassay CV were 6.8% at 28 µg/liter, rising to 9.2% at 165 µg/liter. Serum carboxyterminal telopeptide of type I collagen (CTx) was measured by one-step ELISA (Osteometer BioTech, Herlev, Denmark). Intraassay CV were 2.7–3.7% at 0.36–0.52 ng/ml. Interassay CV were 7–14% at 0.34–0.47 ng/ml. Intact OC was measured by two-site immunoradiometric assay (ELSA-Osteo, CisBio, Bedford, MA). Intraassay CV were 3.8% at 22 ng/ml and 3.9% at 184 ng/ml. Interassay CV were 7% at 27 ng/ml and 8% at 72 ng/ml. OPG was measured by an in-house ELISA, using a mouse monoclonal antibody as capture antibody and a rabbit polyclonal antibody for detection (Amgen, Thousand Oaks, CA), as described previously (14). This assay detects both monomeric and dimeric forms of OPG, as well as OPG bound to RANKL. Intraassay and interassay CV are less than 15%. Soluble RANKL was measured by a sandwich ELISA (BioNet, Inc., Southbridge, MA). The detection limit for the RANKL assay is 0.08 pmol/liter. Intraassay and interassay CV were less than 5 and 9%, respectively.

Serum estradiol and testosterone were quantitated by automated competitive chemiluminescent immunoassay (Bayer, Tarrytown, NY). Intraassay CV were 4.1% at 173 pg/ml and 3.9% at 371 pg/ml. Interassay CV were 7% at 71.2 pg/ml and 4% at 261 pg/ml (estradiol), and intra- and interassay CV were 6.8% and 8.3%, with an assay sensitivity of 8 ng/dl (testosterone). Total IGF-I concentrations were measured by immunoradiometric assay after extraction (Diagnostic Systems Laboratories, Webster, TX). Interassay CV were 9% at 64 µg/liter and 6.2% at 157 µg/liter. Intraassay CV were 3.4% at 9.4, 3% at 55.4, and 1.5% at 264 µg/liter.

Other statistical comparisons

Data were examined for normality by the Kolmogorov-Smirnov test and appropriately transformed before analysis. However, data are shown as the arithmetic mean ± SEM of the untransformed data. All data were analyzed by a means model repeated measures analysis of absolute changes from baseline with SAS Proc Mixed (version 8.02, SAS Institute, Inc., Cary, NC), thereby providing separate estimates of the means by day of study (d 1, 5, 9, 13, and 17) and menopausal status. An unstructured variance-covariance form among the repeated measurements was assumed, and estimates of the SE values of parameters were used to perform statistical tests and construct 95% confidence intervals. When significant main effects (group, time, and group by time interaction) were detected, post hoc Student’s t tests (with Bonferroni adjustment) were used to compare the pairwise differences between the model-based treatment means (least-squares means) at each time point. The model-based means are unbiased with unbalanced and noninformative missing data. In addition, repeated measures linear regression was applied to explore the relationship between changes in serum markers of bone turnover with changes in estradiol, using a homogeneity of slopes model to test whether a significant linear relationship was present, and then whether the slope of the relationship differed between pre- and postmenopausal women. Effectively, separate linear regressions are performed for each time point, and these are combined to obtain a cohort linear regression, which allows for correlation between repeated measures in the same individual. In this way, the model accounts for time and repeated measures. To explore significant relationships disclosed by this method additionally, separate post hoc linear regressions were performed at each time point. The significance of these relationships was assessed by Pearson’s or Spearman’s test for normal or nonnormal data, respectively. Because IGF-I, RANKL, and OPG are presumptive mediators, linear repeated measures regressions on these variables and post hoc linear regressions at each time point were performed analogously. All tests were two-sided, and P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Table 1Go summarizes baseline subject characteristics. Estradiol administration caused a sense of abdominal bloating, breast tenderness, headache, or mild pedal edema in several volunteers. The mean (±SEM) age was 27.3 ± 1.0 and 64.5 ± 2.5 yr in pre- and postmenopausal volunteers, respectively. Corresponding body mass index was comparable, and it averaged 24.3 ± 0.8 and 24.7 ± 0.8 kg/m2, respectively.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Baseline subject characteristics

 
Figure 1AGo illustrates the absolute change in efficacy parameters, namely blood hormone (estradiol) and bone turnover marker (intact OC, PINP, and CTx) concentrations in pre- and postmenopausal women who received estradiol therapy. This verifies that the treatment regimen increased estradiol comparably in pre- and postmenopausal women [between group, P = not significant (NS)], as expected. In absolute terms, estradiol increased from 16.2 ± 3.0 (SEM) to 146 ± 11 pg/ml and from 7.1 ± 0.9 to 150 ± 25 pg/ml, respectively (data not shown). Furthermore, the increase in serum estradiol over time was highly significant (P < 0.0001).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 1. A, Cohort means (±SEM) in change from baseline in OC (top), PINP, CTx, and estradiol (bottom) concentration measured on d 1 (baseline), 5, 9, 13, and 17 in premenopausal (n = 18, {circ}), and postmenopausal (n = 11, •) women treated with escalating doses (0.05, 0.10, 0.15, then 0.20 mg/d) of transdermal estradiol. A significant group difference between pre- and postmenopausal women is indicated by the asterisk next to the name of the parameter. The asterisk in the figure indicates a significant (P < 0.05, Bonferroni adjusted) difference from zero, within each group, with the double asterisk indicating P < 0.001. The dotted line is the line of no change. B, Cohort means (±SEM) in change from baseline in IGF-I (top), OPG, and RANKL (bottom) concentration measured on d 1 (baseline), 5, 9, 13, and 17 in premenopausal (n = 18, {circ}) and postmenopausal (n = 11, •) women treated with escalating doses (0.05, 0.10, 0.15, then 0.20 mg/d) of transdermal estradiol. #, Significant (Bonferroni adjusted) difference from zero, after combining both groups. The dotted line is the line of no change.

 
The changes in PINP significantly differed between pre- and postmenopausal women despite equivalent changes in estradiol. As illustrated, the increase in PINP was greater in postmenopausal, compared with premenopausal, women (between group, P = 0.03). A highly significant time effect (P < 0.0001) was also detected. This was due to significant increases from baseline after d 9 in postmenopausal women and after d 13 in premenopausal women (P < 0.05, by post hoc Bonferroni-adjusted Student’s t test). The change in PINP from d 13 to d 17 in postmenopausal women approached significance (P = 0.06, with Bonferroni correction). Although PINP increased, CTx and OC did not change (P = NS, time effect). Furthermore, the changes in CTx and OC did not differ by menopausal status (between group, P = NS). However, a highly significant group by time interaction was observed for CTx (P = 0.003). By post hoc testing, this was explained by a significant increase in CTx on d 17 in the premenopausal women, as illustrated.

Figure 1BGo illustrates the absolute change in putative mechanistic intermediaries, namely IGF-I, OPG, and RANKL concentrations in pre- and postmenopausal women who received estradiol therapy. To examine mechanistic intermediaries, we performed the same analysis on change in IGF-I, OPG, and RANKL. A significant time effect (P = 0.04) was detected for IGF-I. This decrease was significant at d 17 by post hoc testing only in the combined pre- and postmenopausal group (as shown). Between-group differences and group by time interactions were not present. The changes in OPG or RANKL did not differ by menopausal status (between group, P = NS) or with time. Approximately 10% of all RANKL measurements were undetectable.

Figure 2Go is analogous to Fig. 1Go, A and B, except that serum changes in women randomized to receive placebo patches are illustrated. To facilitate comparison with Fig. 1Go, the vertical scales for corresponding plots are identical. Hormones (estradiol and IGF-I) and markers of bone turnover (intact OC, PINP, and CTx) did not significantly change from baseline in premenopausal, postmenopausal, or all women. When comparing Figs. 1Go and 2Go, the mean changes in PINP were much less than those observed in the estradiol-treated group. In contrast, CTx and IGF-I changes were comparable in magnitude and highly variable. These data show that the effect of estradiol on PINP cannot be explained by Lupron-only therapy. Hence, although the duration of Lupron therapy was only 6 wk, the changes in PINP and estradiol were relatively stable from wk 3–6, in comparison with the much larger changes in those who received estradiol patch therapy. Importantly, this study was not designed to exclude subtle changes in PINP; however, these presumptive changes are of much smaller magnitude to the changes detected in the estradiol-treated group. Furthermore, the possibility that the effect of estradiol on CTx does not differ from the effect of Lupron alone cannot be excluded. This is consistent with other studies showing particularly profound effects of hypoestrogenemia on bone resorption.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 2. Cohort means (±SEM) in change from baseline in IGF-I, OC, PINP, CTx, and estradiol concentration measured on d 1 (baseline), 5, 9, 13, and 17 in premenopausal (n = 5, {circ}) and postmenopausal (n = 2, •) women treated with placebo. The vertical scale for each plot is identical with Fig. 1Go. No significant differences from zero, within each group and by pooling groups, are present. The dotted line illustrates no change.

 
Figure 3Go presents the linear regression analysis of the absolute changes in RANKL (top), PINP (middle), and CTx (bottom) on the absolute change in blood estradiol in pre- and postmenopausal women. This analysis was performed to directly assess any linear relationship with estradiol and to determine whether menopausal status altered these relationships. Change in estradiol was significantly associated with changes in RANKL (P = 0.005), PINP (P = 0.0006), and CTx (P = 0.03). Changes in estradiol did not correlate with changes in OC, OPG, or IGF-I, and the changes are not illustrated. Furthermore, the slopes of these regressions were significantly steeper in premenopausal women (P = 0.01 and P = 0.0005 for PINP and CTx, respectively), whereas slopes did not significantly differ from zero in postmenopausal women. The linear regression equation relating PINP and estradiol in premenopausal women shows that for changes in estradiol up to 150 pg/ml, the change in PINP is positive (i.e. more than zero), but the magnitude of this change in PINP diminishes with larger changes in estradiol (i.e. the slope is negative). With estradiol changes greater than 150 pg/ml, the change in PINP is negative. These data suggest that estradiol may only promote osteoblastic collagen synthesis with lower dose estradiol therapy and complements the repeated measures ANOVA data (shown in Fig. 1AGo) by relating bone turnover changes directly with estradiol changes and disclosing the dose-response relationship. Post hoc linear regressions in premenopausal women disclosed significant associations between change in estradiol and change in PINP (P = 0.03; R = –0.55) on d 5 and change in estradiol and change in CTx (P = 0.03; R = 0.52) on d 13 (regressions not shown, but primary data shown in Fig. 3Go).



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 3. Comparison by menopausal status of linear regression analysis of change from baseline in RANKL, PINP, and CTx on change from baseline in estradiol in premenopausal (n = 18, open symbols) and postmenopausal (n = 11, closed symbols) women measured at d 5 (triangle), 9 (square), 13 (hexagon), and 17 (circle). Regression lines are continuous (postmenopausal) and dotted (premenopausal). Significantly different regression between pre- and postmenopausal women are indicated by the asterisk next to the name of the parameter.

 
The slopes of the regressions of change in RANKL on change in estradiol were significantly negative (and nonzero) for both pre- (P = 0.04) and postmenopausal (P = 0.002) women. This is consistent with the known antiresorptive effects of estradiol, but it also shows that the effect of estradiol on RANKL may be more pronounced in postmenopausal women. Post hoc linear regressions disclosed significant associations between change in estradiol and change in RANKL on d 9 (P = 0.03; R = –0.40) and d 17 (P = 0.002; R = –0.57) (regressions not shown, but primary data shown in Fig. 3Go).

To further explore mechanistic relationships, we analogously regressed changes in markers of bone turnover (CTx, PINP, and OC) with changes in presumptive mediators (IGF-I, OPG, and RANK-L). Changes in IGF-I were not related to changes in any bone turnover marker (data not shown). In addition, linear repeated measures regression did not disclose any significant relationship between changes in OPG or changes in RANKL with changes in CTx.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study shows that transdermal estradiol acutely increases osteoblastic collagen synthesis and that this effect differs between pre- and postmenopausal women despite comparable increases in systemic estradiol exposure. The time course of these changes was slower and less pronounced in premenopausal women, and direct regression analysis unveiled a dose-responsive relationship with estradiol, but only in these premenopausal women. Furthermore, the stimulatory effect of estradiol on osteoblastic collagen synthesis was most prominent with lower dose therapy. In contrast, dosage does not seem to modify response in older women. The physiological importance of these relationships is highlighted by the blood concentrations of estradiol obtained (increasing from 40 to 150 pg/ml), which closely mimic the estradiol changes typifying the follicular phase of the normal menstrual cycle (10, 11, 12, 15, 16, 17). Because our regression analysis unveiled a direct relationship between changes in estradiol and PINP by repeated measures (which accounts for correlation between measurements taken on the same individual at different times) as well as simple linear regression (which does not require correlational analyses, because only a single measurement for each individual is used) at d 5, we therefore believe that these changes in PINP are predominately due to changes in estradiol. However, because escalating estradiol and lengthening time occurred simultaneously, our study design, which did not randomize the order of estradiol dose therapy, does not allow us to entirely exclude the possibility that the changes in PINP reflect time course rather than estradiol dose.

Outside of this report, interventional studies showing a stimulatory effect of estradiol on osteoblastic collagen synthesis in healthy premenopausal women are lacking. To the contrary, decreased carboxyterminal propeptide of type I procollagen (PICP), OC, and BSAP were reported in a study of 45 young women with hypothalamic amenorrhea and osteopenia who were randomized to receive triphasic norgestimate and ethinyl estradiol or placebo for 3 months (18). However, this apparent inconsistency may be explained by the supraphysiological doses employed, longer duration of treatment, or specific population characteristics. Indeed, our regression analysis shows that PINP may decrease with greater increases in serum estradiol. Furthermore, longitudinal evaluation of the follicular phase of the menstrual cycle has revealed an increase in either serum PICP or BSAP (9, 12), although not all studies concur (8, 9). Together, these studies indirectly strengthen the plausibility of our otherwise novel finding that osteoblastic collagen synthesis is stimulated by estradiol in premenopausal women.

Interventional studies examining postmenopausal women are more widely available, and these confirm the stimulatory effect of estradiol on osteoblastic collagen formation over a wide range of estradiol doses. Together, these data additionally support our finding that estradiol dose does not seem to modify osteoblastic response in postmenopausal women. In the most similar but smaller study, lower dose (0.05 mg/d) transdermal estradiol increased PINP and PICP during the first 1–2 wk before returning to baseline at 4 wk (5). In another study from the same investigators, 21 hysterectomised postmenopausal women were randomized to receive sc estradiol therapy (25 mg every 6 months) or sham procedure (19). Treatment significantly increased markers of bone formation (PINP, BSAP, and OC) at 1 month, with the effect diminishing significantly over the subsequent 5 months (19). Interestingly, this study also reported that within postmenopausal women, the response of PINP was at least marginally (P = 0.06) positively related to age, consistent with our general findings. Uncontrolled reports using larger estradiol doses (0.1 mg/d transdermally or 50–75 mg sc) confirm increases in serum markers of bone formation (13, 20) or mean wall thickness (an index of osteoblastic activity) by bone histomorphometry (21, 22). Interventional data implicating estradiol in osteoblastic collagen synthesis are also available in older men (23, 24) and in rodents (25, 26, 27).

Our finding of no significant change in OC is also supported by these same studies. Serum OC does not change during the follicular phase of the menstrual cycle (8, 9, 11) and inconsistently increases or does not change in randomized interventional studies (5, 13, 19, 24). In our study, estradiol therapy tended to increase OC, although not significantly. It is possible that OC changes require more time to evolve, compared with changes in collagen synthesis that occur earlier. It is also possible that the various OC assays employed, each of which detects different fragments, may partly explain these inconsistencies among studies.

After 17 d of escalating estradiol treatment, CTx marginally increased in premenopausal women but remained unchanged in postmenopausal women. Although not statistically significant, a change of similar magnitude was observed in our placebo-treated groups. The importance of these minor changes is therefore questionable. Nevertheless, cross-sectional studies of normally menstruating premenopausal women support the possibility that the effect of estradiol on bone resorption may differ in these women. Whereas urinary DPD (12) and serum cross-linked carboxyterminal telopeptide of type I collagen (8) decrease, other markers of collagen degradation, such as urinary CTx (9) or urinary type I collagen cross-linked N-telopeptide (10), increase. However, some studies have reported no change in urinary DPD (8, 9), urinary pyridinoline (8), or serum cross-linked carboxyterminal telopeptide of type I collagen (9), as well as other bone resorption markers such as urinary hydroxy-DPD and DPD (10). This is despite comparable power (each examining approximately 10 women) and subjects (normally cycling premenopausal women) (8, 9, 10, 11, 12). Empiric data from interventional studies in healthy premenopausal women are not otherwise available, and hence, our preliminary data must be interpreted cautiously. In contrast, the antiresorptive effect of estradiol, particularly with prolonged therapy, in postmenopausal women is long established by multiple therapeutic studies (4, 28, 29, 30, 31, 32, 33).

We also examined whether the IGF-I system might mediate these changes in bone turnover, particularly on bone formation. Our data disclosed a small decrease in IGF-I, which was apparent only at d 17 when all women were analyzed, and no significant linear relationship between IGF-I and any parameters of bone turnover. The lack of relationship between estradiol and IGF-I may reflect potentially slow evolution of IGF-I changes, which may not have been apparent within 17 d. Therefore, our data contrast with some studies of transdermal estradiol, reporting increased IGF-I and osteoblastic function after 8 wk (13), but not others showing decreased IGF-I after 2 wk (34) or no change in IGF-I at all (35). Together, these data suggest the possibility that the effect of IGF-I may be time, hepatic dose, or delivery method dependent (13, 34). Although we emphasize potential mediators of bone formation, estradiol is likely to influence bone turnover directly because osteoblasts exhibit estrogen receptors and estrogens can enhance collagen synthesis in vitro (36, 37).

In contrast, OPG and RANKL are important, known determinants of osteoclastogenesis, and, increasingly, their role in mediating the antiresorptive effects of estradiol are being recognized (38). Whereas circulating RANKL levels may not necessarily reflect changes occurring in the bone microenvironment (38), our data illustrating a negative relationship between changes in estradiol and changes in RANKL extend the existing literature by confirming a similar relationship in healthy pre- and postmenopausal women. Although estradiol has been shown to stimulate OPG production in vitro (39, 40) and we previously found that estrogen increased (whereas testosterone decreased) circulating OPG levels in acutely hypogonadal men (41), we were unable to detect any clear effects of estradiol on circulating OPG levels in either pre- or postmenopausal women. Perhaps the major explanation for the difference between our previous in vivo findings in men and the present findings in women is that the previous study examined the effects of estradiol treatment in the setting of withdrawal of testosterone treatment, which represents a very different experimental paradigm than used here.

Several caveats should be noted. First, this study was not designed to examine long-term effects on bone turnover. Indeed, in postmenopausal women, longer-term randomized placebo-controlled studies of low-dose (0.25 mg/d orally, or 7.5 µg/d transdermally) estradiol show that serum markers of bone formation and bone resorption decrease by 3–6 months (4, 30, 31, 32, 33). Second, the observed differences in pre- and postmenopausal women may not persist with prolonged hypoestrogenemia. However, extending estrogen deprivation for longer than 3 wk in otherwise healthy premenopausal women becomes increasingly unethical. Third, the increase in PINP may be due to nonosteoblastic collagen synthesis, notably from the uterus. However, interventional studies implicating estradiol in collagen synthesis and PINP production in men, where a uterine source can be excluded, are available (23, 24). In addition, although PINP is produced by a number of tissues, these tissues generally have a much lower rate of turnover than bone, and it is currently believed that changes in circulating PINP levels largely reflect changes in bone collagen synthesis (42). These data support our contention that PINP is being released by the osteoblast, although we cannot entirely exclude a nonosteoblastic source, nor can we exclude the possibility that the effects of estradiol are due to changes in the degradation and/or release, rather than the synthesis, of PINP. And finally, the effects of other gonadal hormones, such as testosterone, on bone turnover (11) were not examined. Additional studies should investigate the molecular basis for the altered response to estradiol with age, which may include direct mechanisms such as changes in estrogen-receptor expression arising from long-term hypoestrogenemia (43, 44) as well as indirect changes in important mediators.

We conclude that lower dose estradiol rapidly increases osteoblastic collagen synthesis in premenopausal as well as postmenopausal women at a time when collagen degradation is stable and that this response is blunted in premenopausal women. The effects of estradiol on bone formation are most likely direct and do not appear to be mediated by IGF-I. However, RANKL appears to be a major mediator of estradiol’s antiresorptive effects. Whether the short-term stimulatory effect of estradiol on osteoblastic collagen synthesis can be harnessed therapeutically remains speculative and requires confirmation with appropriate studies of cyclical estradiol therapy.


   Acknowledgments
 
We thank the Mayo Immunochemical Laboratory for assay assistance and the Mayo research nursing staff for conduct of the protocol.


   Footnotes
 
This work was supported by fellowships from the National Health and Medical Research Council of Australia (Grant ID 262025 to P.Y.L.) and the Royal Australasian College of Physicians (Vincent Fairfax Family Fellowship to P.Y.L.), the General Clinical Research Center Grant MO1 RR00585 [to the Mayo Clinic and Foundation from the National Center for Research Resources (Rockville, MD)], and Grants R01 NIA AG 14799 and P01 NIA AG004875 from the National Institutes of Health (Bethesda, MD).

First Published Online December 28, 2004

Abbreviations: BSAP, Bone-specific alkaline phosphatase; CTx, carboxyterminal telopeptide of type I collagen; CV, coefficient(s) of variation; DPD, D-pyridinoline; OC, osteocalcin; OPG, osteoprotegerin; PICP, propeptide of type I carboxyterminal procollagen; PINP, propeptide of type I procollagen.

Received August 9, 2004.

Accepted December 16, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H, Bonds D, Brunner R, Brzyski R, Caan B, Chlebowski R, Curb D, Gass M, Hays J, Heiss G, Hendrix S, Howard BV, Hsia J, Hubbell A, Jackson R, Johnson KC, Judd H, Kotchen JM, Kuller L, LaCroix AZ,et al. 2004 Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial. JAMA 291:1701–1712[Abstract/Free Full Text]
  2. Manson JE, Hsia J, Johnson KC, Rossouw JE, Assaf AR, Lasser NL, Trevisan M, Black HR, Heckbert SR, Detrano R, Strickland OL, Wong ND, Crouse JR, Stein E, Cushman M 2003 Estrogen plus progestin and the risk of coronary heart disease. N Engl J Med 349:523–534[Abstract/Free Full Text]
  3. Cauley JAD, Robbins JMD, Chen ZP, Cummings SRMD, Jackson RDMD, LaCroix AZPMPH, LeBoff MMD, Lewis CEMDM, McGowan JP, Neuner JMDMPH, Pettinger MMS, Stefanick MLP, Wactawski-Wende JP, Watts NBMD, for the Women’s Health Initiative I 2003 Effects of estrogen plus progestin on risk of fracture and bone mineral density: the Women’s Health Initiative randomized trial. JAMA 290:1729–1738[Abstract/Free Full Text]
  4. Prestwood KM, Kenny AM, Kleppinger A, Kulldorff M 2003 Ultralow-dose micronized 17ß-estradiol and bone density and bone metabolism in older women: a randomized controlled trial. JAMA 290:1042–1048[Abstract/Free Full Text]
  5. Hannon R, Blumsohn A, Naylor K, Eastell R 1998 Response of biochemical markers of bone turnover to hormone replacement therapy: impact of biological variability. J Bone Miner Res 13:1124–1133[CrossRef][Medline]
  6. Hulley SB, Grady D 2004 The WHI estrogen-alone trial—do things look any better? JAMA 291:1769–1771[Free Full Text]
  7. Turgeon JL, McDonnell DP, Martin KA, Wise PM 2004 Hormone therapy: physiological complexity belies therapeutic simplicity. Science 304:1269–1273[Abstract/Free Full Text]
  8. Schlemmer A, Hassager C, Risteli J, Risteli L, Jensen SB, Christiansen C 1993 Possible variation in bone resorption during the normal menstrual cycle. Acta Endocrinol (Copenh) 129:388–392[Medline]
  9. Gorai I, Taguchi Y, Chaki O, Kikuchi R, Nakayama M, Yang BC, Yokota S, Minaguchi H 1998 Serum soluble interleukin-6 receptor and biochemical markers of bone metabolism show significant variations during the menstrual cycle. J Clin Endocrinol Metab 83:326–332[Abstract/Free Full Text]
  10. Gorai I, Chaki O, Nakayama M, Minaguchi H 1995 Urinary biochemical markers for bone resorption during the menstrual cycle. Calcif Tissue Int 57:100–104[CrossRef][Medline]
  11. Massafra C, De Felice C, Agnusdei DP, Gioia D, Bagnoli F 1999 Androgens and osteocalcin during the menstrual cycle. J Clin Endocrinol Metab 84:971–974[Abstract/Free Full Text]
  12. Zittermann A, Schwarz I, Scheld K, Sudhop T, Berthold HK, von Bergmann K, van der Ven H, Stehle P 2000 Physiologic fluctuations of serum estradiol levels influence biochemical markers of bone resorption in young women. J Clin Endocrinol Metab 85:95–101[Abstract/Free Full Text]
  13. Ho KK, Weissberger AJ 1992 Impact of short-term estrogen administration on growth hormone secretion and action: distinct route-dependent effects on connective and bone tissue metabolism. J Bone Miner Res 7:821–827[Medline]
  14. Khosla S, Arrighi HM, Melton 3rd LJ, Atkinson EJ, O’Fallon WM, Dunstan C, Riggs BL 2002 Correlates of osteoprotegerin levels in women and men. Osteoporos Int 13:394–399[CrossRef][Medline]
  15. Burger HG, Dudley EC, Hopper JL, Groome N, Guthrie JR, Green A, Dennerstein L 1999 Prospectively measured levels of serum follicle-stimulating hormone, estradiol, and the dimeric inhibins during the menopausal transition in a population-based cohort of women. J Clin Endocrinol Metab 84:4025–4030[Abstract/Free Full Text]
  16. Wihlback AC, Sundstrom Poromaa I, Bixo M, Allard P, Mjorndal T, Spigset O 2004 Influence of menstrual cycle on platelet serotonin uptake site and serotonin 2A receptor binding. Psychoneuroendocrinology 29:757–766[CrossRef][Medline]
  17. Randolph Jr JF, Sowers M, Bondarenko IV, Harlow SD, Luborsky JL, Little RJ 2004 Change in estradiol and follicle-stimulating hormone across the early menopausal transition: effects of ethnicity and age. J Clin Endocrinol Metab 89:1555–1561[Abstract/Free Full Text]
  18. Grinspoon SK, Friedman AJ, Miller KK, Lippman J, Olson WH, Warren MP 2003 Effects of a triphasic combination oral contraceptive containing norgestimate/ethinyl estradiol on biochemical markers of bone metabolism in young women with osteopenia secondary to hypothalamic amenorrhea. J Clin Endocrinol Metab 88:3651–3656[Abstract/Free Full Text]
  19. Pereda CA, Hannon RA, Naylor KE, Eastell R 2002 The impact of subcutaneous oestradiol implants on biochemical markers of bone turnover and bone mineral density in postmenopausal women. BJOG 109:812–820[CrossRef][Medline]
  20. Sands RH, Studd JW, Jones J, Alaghband-Zadeh J 2000 Comparison of the biochemical effects of testosterone and estrogen on bone markers in surgically menopausal women. Gynecol Endocrinol 14:382–387[Medline]
  21. Khastgir G, Studd J, Holland N, Alaghband-Zadeh J, Fox S, Chow J 2001 Anabolic effect of estrogen replacement on bone in postmenopausal women with osteoporosis: histomorphometric evidence in a longitudinal study. J Clin Endocrinol Metab 86:289–295[Abstract/Free Full Text]
  22. Khastgir G, Studd J, Holland N, Alaghband-Zadeh J, Sims TJ, Bailey AJ 2001 Anabolic effect of long-term estrogen replacement on bone collagen in elderly postmenopausal women with osteoporosis. Osteoporos Int 12:465–470[CrossRef][Medline]
  23. Falahati-Nini A, Riggs BL, Atkinson EJ, O’Fallon WM, Eastell R, Khosla S 2000 Relative contributions of testosterone and estrogen in regulating bone resorption and formation in normal elderly men. J Clin Invest 106:1553–1560[Medline]
  24. Meier C, Liu PY, Ly LP, de Winter-Modzelewski J, Jimenez M, Handelsman DJ, Seibel MJ 2004 Recombinant human chorionic gonadotropin, but not dihydrotestosterone alone stimulates osteoblastic collagen synthesis in older men with partial age-related androgen deficiency. J Clin Endocrinol Metab 89:3033–3041[Abstract/Free Full Text]
  25. Chow JW, Lean JM, Chambers TJ 1992 17 ß-estradiol stimulates cancellous bone formation in female rats. Endocrinology 130:3025–3032[Abstract]
  26. Edwards MW, Bain SD, Bailey MC, Lantry MM, Howard GA 1992 17ß Estradiol stimulation of endosteal bone formation in the ovariectomized mouse: an animal model for the evaluation of bone-targeted estrogens. Bone 13:29–34[Medline]
  27. Takano-Yamamoto T, Rodan GA 1990 Direct effects of 17 ß-estradiol on trabecular bone in ovariectomized rats. Proc Natl Acad Sci USA 87:2172–2176[Abstract/Free Full Text]
  28. Chesnut 3rd CH, Bell NH, Clark GS, Drinkwater BL, English SC, Johnson Jr CC, Notelovitz M, Rosen C, Cain DF, Flessland KA, Mallinak NJ 1997 Hormone replacement therapy in postmenopausal women: urinary N-telopeptide of type I collagen monitors therapeutic effect and predicts response of bone mineral density. Am J Med 102:29–37[CrossRef][Medline]
  29. Bjarnason NH, Byrjalsen I, Hassager C, Haarbo J, Christiansen C 2000 Low doses of estradiol in combination with gestodene to prevent early postmenopausal bone loss. Am J Obstet Gynecol 183:550–560[CrossRef][Medline]
  30. Field CS, Ory SJ, Wahner HW, Herrmann RR, Judd HL, Riggs BL 1993 Preventive effects of transdermal 17 ß-estradiol on osteoporotic changes after surgical menopause: a two-year placebo-controlled trial. Am J Obstet Gynecol 168:114–121[Medline]
  31. Giske LE, Hall G, Rud T, Landgren BM 2002 The effect of 17ß-estradiol at doses of 0.5, 1 and 2 mg compared with placebo on early postmenopausal bone loss in hysterectomized women. Osteoporos Int 13:309–316[CrossRef][Medline]
  32. Naessen T, Berglund L, Ulmsten U 1997 Bone loss in elderly women prevented by ultralow doses of parenteral 17ß-estradiol. Am J Obstet Gynecol 177:115–119[CrossRef][Medline]
  33. Cooper C, Stakkestad JA, Radowicki S, Hardy P, Pilate C, Dain MP, Delmas PD 1999 Matrix delivery transdermal 17ß-estradiol for the prevention of bone loss in postmenopausal women. The International Study Group. Osteoporos Int 9:358–366[CrossRef][Medline]
  34. Bellantoni MF, Vittone J, Campfield AT, Bass KM, Harman SM, Blackman MR 1996 Effects of oral versus transdermal estrogen on the growth hormone/insulin-like growth factor I axis in younger and older postmenopausal women: a clinical research center study. J Clin Endocrinol Metab 81:2848–2853[Abstract]
  35. Friend KE, Hartman ML, Pezzoli SS, Clasey JL, Thorner MO 1996 Both oral and transdermal estrogen increase growth hormone release in postmenopausal women—a clinical research center study. J Clin Endocrinol Metab 81:2250–2256[Abstract]
  36. Komm BS, Terpening CM, Benz DJ, Graeme KA, Gallegos A, Korc M, Greene GL, O’Malley BW, Haussler MR 1988 Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells. Science 241:81–84[Abstract/Free Full Text]
  37. Eriksen EF, Colvard DS, Berg NJ, Graham ML, Mann KG, Spelsberg TC, Riggs BL 1988 Evidence of estrogen receptors in normal human osteoblast-like cells. Science 241:84–86[Abstract/Free Full Text]
  38. Eghbali-Fatourechi G, Khosla S, Sanyal A, Boyle WJ, Lacey DL, Riggs BL 2003 Role of RANK ligand in mediating increased bone resorption in early postmenopausal women. J Clin Invest 111:1221–1230[CrossRef][Medline]
  39. Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Spelsberg TC, Riggs BL 1999 Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology 140:4367–4370[Abstract/Free Full Text]
  40. Saika M, Inoue D, Kido S, Matsumoto T 2001 17ß-estradiol stimulates expression of osteoprotegerin by a mouse stromal cell line, ST-2, via estrogen receptor-{alpha}. Endocrinology 142:2205–2212[Abstract/Free Full Text]
  41. Khosla S, Atkinson EJ, Dunstan CR, O’Fallon WM 2002 Effect of estrogen versus testosterone on circulating osteoprotegerin and other cytokine levels in normal elderly men. J Clin Endocrinol Metab 87:1550–1554[Abstract/Free Full Text]
  42. Szulc P, Delmas PD 2001 Biochemical markers of bone turnover in men. Calcif Tissue Int 69:229–234[CrossRef][Medline]
  43. Lim SK, Won YJ, Lee HC, Huh KB, Park YS 1999 A PCR analysis of ER{alpha} and ERß mRNA abundance in rats and the effect of ovariectomy. J Bone Miner Res 14:1189–1196[CrossRef][Medline]
  44. Hoyland JA, Baris C, Wood L, Baird P, Selby PL, Freemont AJ, Braidman IP 1999 Effect of ovarian steroid deficiency on oestrogen receptor {alpha} expression in bone. J Pathol 188:294–303[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
90/4/1953    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, P. Y.
Right arrow Articles by Khosla, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, P. Y.
Right arrow Articles by Khosla, S.
Related Collections
Right arrow Calcium and Bone Metabolism
Right arrow Female Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals