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The University of Chicago Pritzker School of Medicine (R.L.R., N.D.), Chicago, Illinois 60637; Genentech, Inc. (J.J.H.), South San Francisco, California 94080; Nemours Children’s Clinic (N.M.), Jacksonville, Florida 32207; University of Pennsylvania (T.M.), Philadelphia, Pennsylvania 19104; and University of South Florida (A.W.R.), Tampa, Florida 33701
Address all correspondence and requests for reprints to: Robert Rosenfield, M.D., The University of Chicago Comer Children’s Hospital, Section of Pediatric Endocrinology, 5841 South Maryland Avenue, MC-5053, Chicago, Illinois 60637. E-mail: robros{at}peds.bsd.uchicago.edu.
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Abstract |
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Objective: The study objective was to test the hypotheses that physiological estradiol replacement administered early with GH will preserve height potential as much as if administered late and that it will bring about a greater height gain than standard oral estrogen therapy combined with GH.
Design: The study was randomized to early or late estrogen treatment; follow-up was at 3.5 yr or later.
Setting: This was a multicenter outpatient study.
Patients: Turner syndrome girls 12.0–12.9 yr (n = 7) or 14.0–14.9 yr (n = 7) of age who began GH before age 12.0 yr were the patients. The girls were matched to National Cooperative Growth Study registry patients who began GH and oral conjugated estrogen at similar ages and were similarly followed to adult or near-adult height.
Interventions: Depot estradiol, 0.2 mg/month im, was given initially and gradually increased; GH was 0.05 mg/kg daily.
Main Outcome Variable: Adult or near-adult height was the main outcome variable.
Results: Depot estradiol treatment resulted in height significantly taller than predicted at 12 yr of age (P < 0.02). All height potential was gained in the first 2 yr of the study, during which the early group grew 3.5 cm more than the late group, which was receiving GH alone (P < 0.01). The early depot estradiol group also gained 5.9 cm more height after starting estrogen than did the early National Cooperative Growth Study group (P < 0.05). Although feminization proceeded slowly on the lowest dose of estradiol, it advanced normally thereafter.
Conclusions: These results suggest that very low-dose parenteral estradiol permits relatively age-appropriate feminization without interfering with the effect of GH on the enhancement of height potential.
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Introduction |
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Customary oral estrogen replacement therapy clearly is detrimental to growth potential when administered at 12 yr of age. Chernausek et al. (12) added estrogen to GH therapy of girls with Turner syndrome at either 12 or 15 yr of age, and the patients were followed from 12 yr of age until near-adult height. Conjugated equine estrogens were administered as 0.3 mg daily for the first 6 months and subsequently 0.6 mg daily, at which time cyclic medroxyprogesterone acetate was added. The total growth from 12 yr of age averaged 13.0 cm in the group that began estrogen at 12 yr, whereas it averaged 15.8 cm in the group that began estrogen at 15 yr; this difference was highly significant.
We have postulated that estradiol delivered into the systemic circulation in a dosage regimen approximating the low amount of estradiol normally produced during early puberty would provide a physiological form and amount of estrogen that would emulate natural puberty with respect to both feminization and the pubertal growth spurt and so could be started at a normal age without loss of growth potential. If so, it would remove the dilemma faced by Turner syndrome patients—whether to forgo age-appropriate feminization to increase adult height. In 1991 we began enrolling Turner syndrome patients as young as 12 yr of age into a multicenter study to address this possibility in patients receiving recombinant GH treatment. Estradiol was administered in a depot form (estradiol cypionate) as a monthly injection starting at a dose (0.2 mg monthly) well below any previously reported. Preliminary data analysis was performed after nine patients had undergone 1.5–2.0 yr of estradiol therapy (13). This analysis suggested that height potential was promoted by combining early pubertal amounts of estradiol with GH treatment for 1.5–2.0 yr, supporting the concept that early, very low replacement doses of estrogen might preserve height potential while allowing adequate, timely feminization. Therefore, we continued to enroll patients into the study and have now followed them to near-adult or adult height. These results, reported in this study, support the hypothesis that very low doses of systemically administered estradiol promote growth in response to GH when given as young as 12–13 yr of age, while simultaneously stimulating a generally normal pace of pubertal development. Thus, they indeed seem to be physiological.
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Subjects and Methods |
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The institutional review boards of each of the four participating centers approved these studies. Subjects with Turner syndrome were recruited after giving informed written consent. Their clinical characteristics are summarized in Table 1
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Fourteen patients were recruited to undergo site-balanced randomization to begin estradiol "early" (12.0–12.9 yr of age) or "late" (14.0–14.9 yr of age) during their 12th year of age (Fig. 1). These two groups had been receiving GH since 10.0 ± 1.2, SEM, and 9.5 ± 0.4 yr of age, respectively. Natural sequence recombinant GH (Nutropin) was provided by Genentech on a "no cost to patient basis" through a combination of insurance and a grant program. Dosage was maintained throughout the study at 0.05 mg/kg daily sc, divided into six or seven daily doses. A baseline height velocity was calculated for the preceding 6 months. If possible, patients were maintained on the protocol until they achieved adult or near-adult height; these were respectively defined as an annualized growth velocity less than 1.25 cm/yr and 1.25–2.5 cm/yr in the preceding 6-month period.
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Prospective BA determinations by the pediatric endocrinologist at each study site, using the method of Greulich and Pyle, were used for computations of height prediction, after the principal investigator determined that the results did not differ significantly from his blind readings of all available BA films before therapy (14). Height predictions were determined from the fraction of height achieved at each of these BA, according to the method of Bayley and Pinneau (15). Serum was frozen at –70 C until it was assayed in one batch at the University of Chicago Hospital Endocrinology Laboratory upon completion of the study. Estradiol was assayed by a sensitive immunoassay kit (Pantex, Santa Monica, CA) (16), LH and FSH by immunometric assay kits specific for the gonadotropin ß subunits (Delfia, PerkinElmer, Boston, MA) (17), and IGF-I by an immunometric assay kit (Nichols, San Clemente, CA). Complete blood count, urinalysis, thyroid function tests, and blood sugar were monitored locally.
Statistical analysis
Within-group comparisons were made by paired Student’s t test, and comparisons between the early and late estradiol treatment groups were made by unpaired Student t test. P values are two-tailed; P < 0.05 was considered statistically significant.
We secondarily compared the growth of these patients to that of Turner syndrome patients on oral estrogen treatment, as recorded in the National Cooperative Growth Study (NCGS) registry. We matched our subjects to all registry patients who had started GH treatment for a similar lead-in time before 12.0 yr of age, had received conjugated equine estrogen replacement for at least 1.5 yr commencing at either 12.0–12.9 or 14.0–14.9 yr of age, and had been followed to adult or near-adult height. Conjugated equine estrogen was the most commonly specified form of estrogen; eight had started it early and 11 late; the starting dose was specified as 0.3 mg daily in 80% of each group. The outcomes for the early and late NCGS groups were compared with the corresponding depot estradiol groups by two-sample, two-tailed t test.
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Results |
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). Six of the seven in the early estradiol treatment group were followed until attainment of adult height, and three of the four remaining in the late estradiol treatment group did so. Three randomized to the late estradiol treatment group dropped out of the study before achieving near-adult or adult height; two (L and M in Table 1) terminated participation at 14.8 and 15.6 yr of age after 6 and 12 months of estradiol treatment, respectively, when breast development had not occurred or was minimal (stage 2), respectively, so their data were available for some analyses; the other dropped out at 12.4 yr of age immediately after learning of assignment to the late estradiol group, so there were no data to be evaluated. Sufficient height was achieved for three subjects in the early treatment group to discontinue GH treatment after 3 yr, including one who had not yet attained adult height (C in Table 1). All but one study subject discontinued the GH treatment during their 15th or 16th year, even the two who still had not attained adult height. As a whole, the subjects treated with this estradiol replacement regimen reached adult or near-adult heights significantly greater than predicted at 12 yr of age (P < 0.02). They achieved all of this increase in height potential by the end of the second year of the study, whether they received GH alone or GH plus estradiol. Their height at the end of the study was similar to their height predictions at 14 yr of age.
The early estradiol treatment group grew 17.3 ± 0.9 cm, SEM, from the start of the study at 12.0–12.9 yr of age until adult or near-adult height (from 136.7 ± 2.0 cm in the beginning to 154.0 ± 1.7 cm by the end of the study). Although they were 1.3 cm shorter at the beginning of the study, they grew 2.3 cm more than those treated with GH alone over this same period of time. Although the difference in overall growth between early and late estrogen-treated groups during the study was not statistically significant, the early treatment group tended to exceed their 12-yr height prediction by 1.6 cm more than the late treatment group (P < 0.07).
Most of the growth of the early-estrogen-treated patients took place during the first 2 yr of adding estradiol to GH therapy. During this time, they grew 3.5 cm more than those on GH alone: 12.7 ± 0.4 cm, SEM, vs. 9.2 ± 0.6 cm; P < 0.01 (Fig. 2). The bulk of this increase in growth occurred on the lowest doses of estradiol, which induced significant increases in height velocity within the first 1.5 yr of estradiol replacement, and particularly within the first 6 months (Fig. 2). Coincidentally, their height potential increased from 150.3 ± 1.5 to 154.6 ± 2.0 cm in the first 2 yr of the study because their BA did not advance disproportionately. Consequently, their height potential increased by 4.3 cm, from 150.3 ± 1.5 to 154.6 ± 2.0 cm at 14 yr of age, which proved to be similar to their height at the conclusion of the study.
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). Those followed to the end of the study grew 8.9 ± 0.8 cm on GH treatment alone during the first 2 yr of the study, and then grew 6.1 ± 1.3 cm during the remainder of the study after estradiol treatment was added to GH. Their growth on estradiol was significantly less (P < 0.01) than that experienced by the early estradiol treatment group during their first 2 yr of estradiol therapy, but not significantly different than the 4.6 ± 1.0 cm after 14 yr of age by the early estradiol group. Height potential of subjects treated with GH alone from 12–14 yr of age increased from 152.1 ± 2.7 to 155.2 ± 1.7 cm, and the height in those followed to the end of the study was similar to that predicted at 14 yr of age (Table 1). The 3.1-cm increase in height potential while on GH therapy alone, before estradiol was begun, was not significantly different from the increase in height potential experienced simultaneously by the study group treated with the combination of GH and early estradiol during that time.
The group treated late with estradiol did not experience a clear pubertal growth spurt (Fig. 2). The only hint of a growth-promoting effect of estradiol in these patients was a slight upward deflection from their previously steadily waning response to GH over time. This suggestion of increased growth occurred 1.0–1.5 yr after estradiol was begun, on the 0.4- to 0.6-mg dosage of depot estradiol, not on the initial, lowest estradiol dose on which peak height velocity of the early treatment group occurred.
The group treated early with estradiol also gained significantly more height after the institution of estrogen (17.3 ± 0.9 cm) than did the NCGS comparison group treated early with oral conjugated estrogen (11.4 ± 1.5 cm) (Fig. 3). The gain in height after the institution of estrogen was not significantly different between the two forms of estrogen replacement in those whose treatment was begun late (14.0–14.9 yr of age), however.
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). Nearly half of the study subjects did not experience breast budding (stage 2) during the first 6 months of therapy. Thereafter, breast development advanced to maturity by an average of one stage in response to each incremental increase in estradiol dose. By the end of 2 yr of treatment, breast development was present in all, and ranged from stage 2–5, with most patients attaining stage 3 or 4; the girl with the slowest pace of breast development experienced the slowest BA advancement (case G). Five of the eight study subjects who received estradiol for 2.5 yr menstruated during the study period; menarche occurred at an average of 2.0 yr (between 1.4 and 2.5 yr) after the initiation of estradiol therapy in all of these, but did not occur within 4 yr otherwise. Notably, two girls in the early treatment group did not experience menarche during the second 2-yr study period although they had been at stage 4 of breast development for 2.5 yr and were on an adult dose of depot estradiol (3.0 mg monthly).
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shows that there was a linear relationship between estradiol dose and plasma level. The correlation, although weaker than expected (r = 0.59), is significant and contrasts with the lack of evidence for a suppressive effect of estradiol on plasma IGF-I levels (Fig. 5B). Figure 5C depicts a significant biphasic response of FSH levels to depot estradiol throughout the course of the study. All patients were euthyroid throughout the study period. No adverse reactions occurred.
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Discussion |
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These data support the notion that, in conjunction with GH, very low-dose, systemic estradiol administered early (12.0–12.9 yr of age) preserves height potential as much as when administered late (14.0–14.9 yr). Furthermore, the overall growth of the early treatment group from 12.0 yr to adult height tended to be greater than in the group in which estradiol treatment began late (P = 0.07), and those treated with estradiol early tended to reach a slightly greater adult height. Notably, the early treatment group grew significantly more between 12.0 and 13.5 yr of age while receiving the combination of 0.2–0.6 mg depot estradiol monthly and GH than did the other group, which was only on GH during this time. Although both groups, taken as a whole, reached an adult height significantly greater than predicted 4 yr earlier, during their 12th year of life, their adult height was virtually identical to their predicted adult height during their 14th year of age. This was the case whether they received GH plus estradiol or GH alone throughout this time (early or late estradiol treatment group, respectively). These data indicate that the low doses of depot estradiol, less than 0.8 mg/month, used during the first 1.5 yr of treatment did not interfere with GH-induced growth enhancement and stimulated growth without deleteriously affecting height potential and possibly promoting it. This suggests that this form of estradiol treatment is particularly efficacious when administered at an age (in the 12th year) and BA (about 11 yr) appropriate for early puberty. Conversely, after 14.0 yr of age (at BA about 12.75 yr) there appears to be no advantage to using such a low dosage of estradiol compared with 1.0–1.5 mg monthly, which we previously reported to be growth-stimulatory at that age without compromising adult height potential and which brings about the more rapid tempo of pubertal change that is desirable at that age (1, 19).
These conclusions are buttressed by the secondary comparison of overall growth of those treated early with depot estradiol to a matched group of NCGS subjects who were treated with conjugated estrogen at a similar age and followed to near-adult or adult height. Those treated with depot estradiol grew significantly more over the same span of time, by an average of 5.9 cm.
A significant linear relationship was found between depot estradiol dose and serum estradiol levels 3–9 d after depot estradiol injection, at which time unconjugated estradiol levels peak as they are hydrolyzed from the cyclopentylpropionate ester (18). However, the blood levels of estradiol were lower by half than predicted from the earlier study; this may be related to the high variance of the responses, which in turn may be related to technical problems in local administration of the small injection volume. No such evidence of a suppressive effect of these estradiol doses on IGF-I levels was found. A biphasic effect of estradiol dose on serum FSH levels was observed, such that the low doses of estradiol used early in treatment were more FSH-suppressive than were the higher doses employed as the girls aged. This may be related to the previously documented decrease of sensitivity of the "gonadostat" to negative feedback by estrogen with advancing age and neuroendocrine puberty (1).
Our experience also suggests that feminization issues become as important as height issues for 14-yr-old girls with Turner syndrome, contrary to the expectation of most U.S. pediatric endocrinologists (20). The desire for a faster pace of feminization was the sole reason for drop-out from the group randomized to start estradiol late; three of the seven subjects assigned to this group left the study before achieving adult or near-adult height. The tempo of feminization on this estradiol replacement regimen was slightly slower than average (21); this was primarily because breast budding did not occur in one third of the patients on the starting dose (0.2 mg monthly) of depot estradiol. After 2 yr of estradiol therapy, one of the 11 patients had still only achieved stage 2 breast development; nevertheless, eight of the 11 patients had attained stages 3 or 4 and one had achieved stage 5. This variability is compatible with marked individual differences in target organ sensitivity. Two of the 11 experienced menarche during this time. By the end of 3 yr of therapy, 80% had experienced menarche, but those who had not done so did not experience periods in the subsequent one or more years of follow-up. The delayed menarche in these patients is likely to be due to the study design, which did not call for progestin replacement before 4 yr, because these patients both had been in breast stage 4 for over 2 yr and were on a full adult replacement dose of estradiol. Not only does feminization become increasingly important by 14 yr of age, no further growth is gained by delaying it beyond the 12th year of age when using this estradiol treatment regimen, for these doses of estrogen clearly did not interfere with the response to GH therapy.
These studies indicate that a very low level of estradiol administered systemically promotes optimal pubertal growth, which was the rationale for the study. The mechanisms for these observations are not fully characterized. An abundant body of data supports the concept that oral and systemically administered estrogen have differential effects in vivo (11, 13). Oral estrogens, by first passage through the portal circulation may impair the expression of the GH receptor and IGF-I generation, essentially functioning as GH antagonists. These effects are not observed with physiological doses of systemically administered estrogen.
Our study is also consistent with the concept that the various forms of estrogen may differ in their effects. The growth-stimulatory dose of estradiol appears to be quite broad: this study demonstrates that depot estradiol doses in the range of 0.2–0.6 mg monthly stimulate growth beyond that attributable to GH alone, whereas our earlier study, in the absence of GH supplementation, showed that a dosage of 1.0–1.5 mg monthly is likewise growth-stimulatory (1). Because one third of depot estradiol consists of the fatty acid ester, this indicates that estradiol is growth-stimulatory over the dose range of 0.13–1.0 mg monthly. This contrasts with the very narrow growth-stimulatory profile of ethinyl estradiol; it stimulates growth at a dosage of about 4 µg daily, i.e. 0.12 mg monthly, but not at a dosage 2- to 4-fold more (6). Even lower doses may accelerate bone maturation disproportionately in young girls (22). These considerations suggest previously unsuspected differences between the effects of estradiol and ethinyl estradiol. Ethinyl estradiol contains an ethinyl group covalently bound to estradiol, and this bond protects it from first-pass hepatic metabolism on oral consumption; furthermore, it is not metabolized to estradiol and instead binds to the estrogen receptor in unmodified form, where it has a more prolonged receptor occupancy time than estradiol itself (23).
Our study also demonstrates that the timing of estrogen is another determinant of its effect. The lowest dose of estrogen was growth-stimulatory only when administered to the younger group of patients, i.e. 12 yr olds. This effect was not seen when the same dose was started in the older group (14 yr olds) after two extra years of GH therapy. This phenomenon is reminiscent of the poor growth spurt observed when puberty is allowed to resume after the discontinuation of gonadotropin suppression treatment of sexual precocity (24). This timing effect is consistent with a model in which growth plate chondrocytes have a finite proliferative potential (8) that is accelerated independently by GH or low doses of estradiol until a point at which senescence curtails the capacity for replication in response to hormonal stimulation.
Unfortunately, optimal vehicles for administering the very low doses of estradiol necessary for physiological estrogen replacement therapy during puberty are not commercially available. The very low estradiol dosage that we have used is inconvenient to administer because the dose is so low that it requires a compounding pharmacist to prepare a sufficiently dilute solution of the commercially available stock solution to permit reasonably accurate dosimetry. However, it may be possible to replicate the pattern of estradiol delivered in this study by using the recently developed transdermal method of delivering estradiol directly into the systemic circulation. A 14-µg estradiol patch and a 25-µg estradiol patch applied for 10 d per month would deliver about the same amount of estradiol over about the same period of time as do the respective 0.2 and 0.4 mg doses of depot estradiol. Thus, it may be possible to administer similarly growth-promoting, very low doses of estradiol by using fractional or intermittent transdermal patch dosing (25).
In conclusion, this study suggests that the dose, route, form, and timing of estrogen are important determinants of estrogen effects on growth. These data demonstrate that very low-dose, systemic estradiol administered early (12th year of age) with GH enhances height velocity more than GH alone, while preserving height potential. Indeed, growth from the 12th year to adult height tended to be greater in the early treatment group and exceeded that of a matched group that received conjugated estrogens over the same period of time. The very low starting dose of estradiol did not bring about feminization in one third of subjects, yet it was the most growth-stimulatory. This raises the possibility that minimally feminizing, very low-dose estradiol replacement could be started at somewhat younger ages without deleterious effects on growth.
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Acknowledgments |
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Footnotes |
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First Published Online September 27, 2005
Abbreviations: BA, Bone age; NCGS, National Cooperative Growth Study.
Received May 16, 2005.
Accepted September 1, 2005.
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  Z. M. Nabhan, L. A. DiMeglio, R. Qi, S. M. Perkins, and E. A. Eugster Conjugated Oral versus Transdermal Estrogen Replacement in Girls with Turner Syndrome: A Pilot Comparative Study J. Clin. Endocrinol. Metab., June 1, 2009; 94(6): 2009 - 2014. [Abstract] [Full Text] [PDF]
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C. A. Bondy and for The Turner Syndrome Consensus Study Group Care of Girls and Women with Turner Syndrome: A Guideline of the Turner Syndrome Study Group J. Clin. Endocrinol. Metab., January 1, 2007; 92(1): 10 - 25. [Abstract] [Full Text] [PDF]
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