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Endocrine Care |
Göteborg Pediatric Growth Research Center, Institute for the Health of Women and Children, Göteborg University (C.A.-L., M.E., K.A.W., E.N.), S-416 85 Göteborg; and Department of Pediatrics, Lund University (M.E.), 221 85 Lund, Sweden
Address all correspondence and requests for reprints to: Dr. Ensio Norjavaara, Queen Silvia Children’s Hospital, Göteborg University, Institute for the Health of Women and Children, Göteborg Pediatric Growth Research Center, S-416 85 Göteborg, Sweden. E-mail: ensio.norjavaara{at}astrazeneca.com
Abstract
The objective of pubertal induction in children with hypogonadism is to mimic spontaneous puberty in terms of physical and psychological development. In a clinical observation study, we induced puberty in 15 girls with hyper- or hypogonadotropic hypogonadism using low doses of transdermal estradiol patches attached only during the night and compared the estradiol concentrations obtained with those in healthy girls. Pubertal induction was started between the ages of 12.3 and 18.1 yr. A transdermal matrix patch of 17ß-estradiol (25 µg/24 h; Evorel, Janssen Pharmaceuticals-Cilag) was cut into pieces corresponding to 3.1, 4.2, or 6.2 µg/24 h initially and attached to the buttock. After 4–14 months, the dose was increased gradually. Serum 17ß-estradiol concentrations were measured every 2 h by RIA (detection limit, 6.0 pmol/L; 1.6 pg/mL).
The results show that it is possible to mimic the spontaneous levels as well as the diurnal pattern of serum 17ß-estradiol in early puberty, by cutting a transdermal 17ß-estradiol matrix patch and attaching a part of it, corresponding to 0.08–0.12 µg estradiol/kg BW, to the buttock nocturnally. In most of the girls, breast development occurred within 3–6 months of the start of treatment.
THE AIM OF pubertal induction in children with hypogonadism is to mimic the pubertal development in children with spontaneous puberty. The induction of pubertal development in girls has, until now, been achieved mainly by using oral synthetic estrogens (1, 2, 3), ethinyl estradiol followed by the addition of progestogen after 1–2 yr. Although this treatment is effective, it has many disadvantages. Oral estrogens have variable bioavailability due to intestinal and hepatic first passage metabolism (4, 5), which also affects hepatic activity and the clotting system (6, 7). Furthermore, studies of girls with Turner syndrome have shown that once treatment has started with commonly used doses of oral estrogens, linear growth may be affected (8, 9). To avoid this negative impact on final height, pubertal induction has to be started at a relatively late age compared with that of girls undergoing spontaneous puberty, which may have negative psychological effects. This delay of pubertal induction may prevent the attainment of a normal peak bone mass (10, 11).
During the past decade, transdermal patches containing natural estrogen (17ß-estradiol) have become available for treatment of postmenopausal women and have also been used for pubertal induction (12, 13, 14). The major advantage of transdermal estrogens compared with oral estrogens is that liver passage is avoided and, therefore, the influence on liver metabolism is minimized (6, 12, 14). The new generation of transdermal patches with a matrix composition also makes it possible to cut the patches into pieces (15) and thereby to start induction at very low doses. The development of sensitive methods for measuring 17ß-estradiol means that it is now possible to attempt to mimic the normal levels of estradiol during childhood (16, 17) and puberty (18). With a patch, it is also possible to monitor the duration of attachment (that is the duration of hormonal treatment), which makes it possible to mimic the normal diurnal variations in 17ß-estradiol. The present investigation is a clinical observation study of patients in Sweden whose puberty has been induced with nocturnal transdermal estradiol patches. We describe a simple method by which to mimic the diurnal pattern of 17ß-estradiol levels seen during spontaneous early puberty.
Subjects and Methods
Study patients
The study group consisted of 15 girls with hyper- or
hypogonadotropic hypogonadism, whose blood samples were analyzed at the
Göteborg Pediatric Growth Research Center, Queen Silvia
Children’s Hospital (Göteborg, Sweden). Patient characteristics
at the start of induction are given in Table 1
. Pubertal induction was started between
the ages of 12.3 and 18.1 yr, and none of the patients had previously
been treated with any other type of estrogen. The girls were
investigated at five different hospitals in Sweden (The Children’s
Clinics in Falun, Gällivare, Göteborg, Lund, and
Ume
). Informed consent was obtained from all children and their
parents.
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Prepubertal girls were initially given a matrix patch of 17ß-estradiol (Evorel, known as Systen in some countries, Janssen Pharmaceuticals-Cilag, Beerse, Belgium; 25 µg/24 h), cut into quarters (6.2 µg/24 h) and attached to the buttock. In some girls, the initial quarter patch dose had to be reduced to 4.2 or 3.1 µg/24 h, because of high serum concentrations of 17ß-estradiol (maximum peak, >40 pmol/L). In the later part of the study this was the initial dose if the girls’ weight was below 55 kg.
The girls were maintained on the initial low dose 17ß-estradiol matrix patch for 4–14 months (median, 8 months), with the aim of mimicking the 17ß-estradiol levels of girls in early puberty, and to induce breast development to stage 2. Thereafter, the dose was increased in an attempt to obtain midpubertal levels of 17ß-estradiol after 15–24 months after the initiation of treatment and, as a result, development to breast stage 3. As a first step, the dose was increased to one half patch (12.5 µg/24 h) for the girls who started with one quarter patch (6.2 µg/24 h) or to one quarter patch (6.2 µg/24 h) for the girls who started with one eighth to one sixth patches (3.1- 4.2 µg/24 h). For two girls, the dose was increased a second time, to a three quarter patch (18.8 µg/24 h). Progestogen was added to the estradiol treatment within 2 yr of the start of induction.
The patch was attached at bedtime and removed the next morning. On most occasions (n = 14), the attachment was standardized to be between 2200 and 0800 h during serum sampling. On nine occasions during collection of serum profiles, the patch had been attached before 2200 h and removed before or after 0800 h.
Breast development was assessed according to the method of Tanner (19).
Study protocol
The girls stayed in the hospital for 1–1.5 days. A heparinized
needle was inserted and serum samples (2 mL) were taken every 2 h
for 16–18 h, starting at 2000 h and with an interruption at 1200
(n = 5) or 1400 (n = 18) h for measurements of
17ß-estradiol. Eight of the girls had 1 serum 17ß-estradiol profile
taken, but as the dose was adjusted over time, 6 of the girls had 2
profiles taken, and 1 girl had 3 profiles taken. In total, 10 serum
profiles were collected during a patch dose of 6.2 µg
17ß-estradiol/24 h, 8 profiles were collected during a dose of 12.5
µg/24 h, and 2 profiles were collected during a dose of 18.8 µg/24
h (Table 1
). Serum profiles were also attained from 3 girls who either
after an initial profile had their dose of transdermal 17ß-estradiol
reduced to 3.1 or 4.2 µg/24 h or had started with this low dose
because their weight was below 55 kg. Serum levels of 17ß-estradiol
were compared with values from healthy girls in early puberty (breast
stage 2, n = 18; age range, 9.9–13.3 yr) and midpuberty (breast
stage 3–4, premenarche, n = 17; age range, 11.5–14.6 yr)
(18).
The serum profile collections were carried out in parallel with the
initial patch attachment (n = 7) or after 1–2 months (n =
4), and in parallel with dose changes or after 2–4 months
(n = 3). However, in four of the girls the serum profile was not
collected with the initial dose (Table 1).
17ß-Estradiol measurements
Serum 17ß-estradiol concentrations were determined in duplicate by a modified RIA using coated tube technology (Spectria estradiol, Orion Diagnostica, Espoo, Finland). To increase the sensitivity of the assay, 700 µL serum were purified and concentrated by extraction with 5 mL diethyl ether, giving a detection limit of 6 pmol/L (1.6 pg/mL). The interassay coefficient of variation (CV) was 13.8% for a mean value of 36.7 pmol/L and 28.8% for a mean value of 11.2 pmol/L. The intraassay CV was 17.2% for a mean value of 10.0 pmol/L, calculated from 20 diethyl ether extracted replicates. The method has been described in detail previously (18).
Statistical methods
P < 0.05 was considered significant. For comparison, medians with 95% confidence intervals are used for serum 17ß-estradiol concentrations during puberty in healthy girls. Linear regression analyses are given with 95% prediction intervals. As the serum 17ß-estradiol levels were not normally distributed, linear regression analyses were performed on log-transformed data.
Results
The descriptive results obtained from the 17ß-estradiol
measurements in 13 girls given one eighth, one sixth, or one quarter of
an estradiol matrix patch, corresponding to 3.1, 4.2, or 6.2 µg/24 h,
are shown in Fig. 1, A and B. In 9 of the
13 girls, the serum 17ß-estradiol levels produced by these doses were
similar to the concentrations seen in girls undergoing spontaneous
early puberty. In addition, nocturnal application of transdermal
estradiol produced a diurnal variation similar to that seen in
spontaneous early puberty (Table 2). Even
though the patch attachment was not standardized in all girls, the
diurnal variation did not show any difference between different
durations of attachment (10, 11.25, 12, or 13 h; Table 2).
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and Table 2). All three girls weighed less than 55 kg at the time of
sampling, and the dose was subsequently reduced in two of the girls
(girls 1 and 14).
In one girl (girl 15), pubertal induction was started with one eighth
patch, and the dose was increased after 1 yr to one quarter patch. Her
17ß-estradiol concentrations were within the early pubertal range
with the initial dose (Fig. 1A and Table 2) and within the midpubertal
range with one quarter patch (Fig. 1B and Table 2).
Two 18-h samples were collected on consecutive days in girl 11. There was a good concordance between the two serum concentration curves; the CV for the area under the curve was 6.4%, and the mean serum 17ß-estradiol concentrations over the 12 h of patch attachment had a CV of 4.1%.
Serum concentrations of 17ß-estradiol in 8 girls given one half patch
(12.5 µg/24 h) and 2 girls given a three quarters patch (18.8 µg/24
h) are shown in Fig. 1, C and D. In 3 of the 10 girls, maximum levels
of 17ß-estradiol were within the expected limits for girls in
midpuberty, although the maximum 17ß-estradiol levels were reached
about 4 h earlier during the night than those in spontaneous
puberty (Fig. 1, C and D, and Table 2). Furthermore, the samples taken
between 1200 and 2200 h contained lower concentrations than those
in healthy girls in midpuberty. Thus, just 5–7 of the 9 or 10 samples
contained 17ß-estradiol concentrations within the range for
midpuberty (Table 2). In 7 of the 10 girls, the serum estradiol
concentrations were lower than those in spontaneous midpuberty (Fig. 1, C and D, and Table 2). One of these girls had levels in the early
pubertal range (girl 11, Fig. 1C); in the others, the concentrations
were between the early and midpubertal reference levels.
Linear regression analysis between the 17ß-estradiol patch dose and serum 17ß-estradiol
The maximum and mean serum 17ß-estradiol levels correlated
significantly with the 17ß-estradiol matrix patch dose applied per
weight of the patient (micrograms per kg; r = 0.72;
P < 0.001 and r = 0.70; P <
0.001 for the maximum and mean values, respectively). A similar result
was seen when the maximum and mean serum levels of 17ß-estradiol were
analyzed against the applied dose per body surface area (micrograms per
m2; r = 0.61; P < 0.01, for
both the maximum and mean value). Figure 2 shows the prediction interval for the
linear regression analysis between the dose of 17ß-estradiol
matrix patches and the maximum serum 17ß-estradiol
concentrations.
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Maximum serum levels of 17ß-estradiol occurred between 4 h and over 10 h after attachment of the patch; in nine serum profiles, maximal levels were probably not reached in the 10–12 h during which the patch was attached, because the concentrations continued to increase until the patch was removed.
The half-lives of elimination of serum 17ß-estradiol after removal of the patch were calculated as the time from removal of the patch to the time at which the half concentration was reached. In 12 of the 23 profiles, the half-life was shorter than 2 h (first sampling time after removal of the patch). The half-lives of elimination were between 2 and 4 h in 7 profiles, and between 4 and 6 h in 1 profile (girl 3). In 3 profiles, the half-lives of elimination were more than 6 h (girl 5, both profiles, and girl 12).
Clinical observations
Breast development (corresponding to breast stage 2) had occurred by the first or second visit to the clinic, that is 3–6 months from first administration of transdermal 17ß-estradiol matrix patches, in 12 of the 15 girls. However, girl 12 was not followed regularly at the clinic. It is therefore only possible to state that breast development had occurred at the 10-month visit. Girls 4 and 15 were observed clinically every 3 or 4 months and did not develop to breast stage 2 during the first 4 or 9 months on the initial dose (0.08 µg/kg). However, after dose increases to 0.11 or 0.14 µg/kg, breast stage 2 was reached within the next 3 months in both girls.
Seven girls developed to breast stage 3 (girls 7, 8, 9, 10, 11, 13, and 14) during the observation time. The time from the first clinic visit when breast stage 2 was observed to the clinic visit when breast stage 3 was observed was 3.5–29 months (median, 10 months). Girls 8, 9, 10, 11, and 13 were on doses between 0.17 and 0.34 (median, 0.24) µg estradiol/kg BW at the clinic visit when breast stage 3 was observed. However, girls 7 and 14 started pubertal induction with transdermal 17ß-estradiol patch doses that produced 17ß-estradiol concentrations exceeding the early pubertal range. Both of these girls developed to breast stage 2 within 3 months and to breast stage 3 within 10 months on the initial treatment dose (0.13 and 0.17 µg/kg). However, in this limited number of girls and with the differences in frequency of patient examination, no correlation was found between the given dose/body weight and the rate of progression of breast development.
Discussion
Puberty is a physical and emotionally disturbing milestone in life and is a prerequisite for a normal adult life. The average duration of puberty is between 4 and 4.5 yr in healthy girls (20, 21). Therefore, the induction of puberty in hypogonadal girls needs to be carefully performed. Preferably, puberty should be induced with low doses of hormone replacement therapy, which should be increased slowly as puberty progresses.
This clinical observation study has shown that it is possible to normalize estradiol concentrations and the diurnal pattern of 17ß-estradiol in hypogonadal girls. Dividing the 17ß-estradiol matrix patches and attaching the cut patches to the buttock overnight provides an important tool for individualizing the induction of puberty by estrogen in girls. A good correlation was obtained between serum levels of 17ß-estradiol and the applied dose of 17ß-estradiol per body weight and surface area. It is therefore possible to predict the correct initiating dose of estrogen.
All girls in whom samples were taken during a transdermal estradiol
dose of 0.08–0.12 µg/kg had serum 17ß-estradiol levels in the
early pubertal range (peak value between 10 and 40 pmol/L; Fig. 1A and
Table 2). This range of 17ß-estradiol concentrations probably not
only promotes breast development, but also increases growth velocity,
as it is well known that the peak height velocity in girls is seen in
early puberty (20, 21). Particular interest in this
context is that the maximal growth velocity in boys is associated with
serum estradiol concentrations between 11 and 15 pmol/L, whereas
epiphyseal maturation and fusion, with a consequent reduction in growth
velocity, is associated with serum estradiol concentrations above 40
pmol/L (22, 23). Therefore, if transdermal estradiol
patches are used for pubertal induction in clinics, with or without
sensitive methods for determining 17ß-estradiol, we would recommend
starting with a one eighth (3.1 µg/24 h) patch if the girl’s weight
is below 40 kg, a one sixth patch (4.2 µg/24 h) if the girl’s weight
is between 40 and 54 kg, and a one fourth patch (6.2 µg/24 h) if the
girl’s weight is 55 kg or more, and careful follow-up during
development. If sensitive methods are used for measurements of
17ß-estradiol, and the girl has estradiol levels in the early
pubertal range but has no breast development, we would recommend that
the dose should be maintained for 9 months before it is increased. The
aim of this cautious approach to treatment is to mimic the progress of
spontaneous pubertal development (19, 20, 21) and to avoid any
possible adverse effects. It is obvious from this study that there is a
risk of rapid breast development if excessive doses are used. Two of
the three girls in whom the initial dose produced levels in excess of
the early pubertal range (maximum peaks, 54 and 75 pmol/L), developed
to breast stage 3 with the initial dose, in contrast to the other girls
who remained at breast stage 2 until an increase in the dose. However,
it is not clear whether a dose of 0.08 µg/kg is enough to initiate
breast development in the first 9 months.
In one third of the studied patients, transdermal 17ß-estradiol matrix patches (12.5–18.8 µg/24 h) attached nocturnally, reproduced the morning peak levels (75–130 pmol/L) seen in spontaneous midpuberty, although the basal levels during the day declined to below the desired levels. Thus, further studies are required to optimize estrogen replacement therapy in midpubertal girls. This could be done either by prolonging the time during which the patch is attached, e.g. from bedtime until the afternoon, about 1600–1800 h, or by taking off only 50% of the patch in the morning and leaving the remaining patch attached until bedtime, when a new patch is applied. Dividing the divided patch into two parts before attaching them at bedtime could achieve this latter regimen. Further studies are also required to optimize complementary hormonal induction of puberty, e.g. by using progestogen and androgens, and also to optimize the tempo of pubertal development.
This original clinical observation study has shown that if pubertal
induction is started with a low dose of transdermal 17ß-estradiol
(
0.12 µg/kg), the dose can be increased in two steps before
midpubertal levels are reached. We recommend maintaining the starting
dose (e.g. 4.2 µg) for 9 months and the second dose
(e.g. 6.2 µg) for 6 months before increasing to the third
dose (e.g. 12.5 µg). The aim is to achieve serum
17ß-estradiol concentrations in the early pubertal range for at least
9 months and to have achieved the midpubertal range at 15 months.
However, treatment for the midpubertal period has to be improved as
described above.
It is clear from this study of a small number of girls that there is interindividual variation in serum 17ß-estradiol concentrations after transdermal 17ß-estradiol patch attachment and removal. The variation is probably due to the heterogeneity of diagnosis, the flux of drugs across the skin, the thickness of adipose tissue, the metabolism of 17ß-estradiol, and the variation in the cut size of the patch and the position of attachment (15, 24). Therefore, we recommend that 17ß-estradiol levels are checked after the start of treatment and after dose changes. Even if the treating physician has no access to a laboratory with a sensitive method for 17ß-estradiol measurements, a value above 40 pmol/L just before the patch is removed must be considered as indicating an excessive starting dose.
In previous studies of postmenopausal women, it has been shown that the time to reach peak levels of estradiol is more than 12 h (15, 25). In our study, 61% of the girls reached their peak levels within 10 h, probably due to differences in body composition compared with postmenopausal women. The calculated half-lives of elimination after removal of the transdermal 17ß-estradiol patch differ markedly from the biological 17ß-estradiol half-life of 22 min (26). Adipose tissue under the patch functions as a reservoir of 17ß-estradiol. Thus, when the patch is removed, the serum 17ß-estradiol level is a reflection of diffusion of stored 17ß-estradiol and its metabolism.
We conclude that it is possible to mimic the spontaneous levels as well as the diurnal pattern of serum 17ß-estradiol in early puberty by cutting a transdermal 17ß-estradiol matrix patch and attaching a part of it, corresponding to 0.08–0.12 µg estradiol/kg BW, to the buttock nocturnally. However, further studies are required before we can mimic the spontaneous concentrations of 17ß-estradiol seen in midpuberty. The patches were well tolerated, and nocturnal application makes them more cosmetically acceptable. It is hoped that this method will make it possible to induce puberty in hypogonadal girls at a similar age as those with spontaneous puberty without a negative impact on final height.
Acknowledgments
We are grateful to the staff at the children’s clinic in Falun
(Gällivare, Göteborg, Lund, and Ume, Sweden) for
taking care of the children.
Footnotes
1 This work was supported by grants from Swedish Medical Research
Council (no. 7509), Wilhelm and Martina Lundgren’s Foundation,
the Free Mason Foundation, Göteborg University, and
Pharmacia & Upjohn, Inc. 
Received September 9, 2000.
Revised January 24, 2001.
Revised March 26, 2001.
Accepted March 30, 2001.
References
This article has been cited by other articles:
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  C. Ankarberg-Lindgren and E. Norjavaara A purification step prior to commercial sensitive immunoassay is necessary to achieve clinical usefulness when quantifying serum 17 -estradiol in prepubertal children Eur. J. Endocrinol., January 1, 2008; 158(1): 117 - 124. [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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R. L. Rosenfield, N. Devine, J. J. Hunold, N. Mauras, T. Moshang Jr., and A. W. Root Salutary Effects of Combining Early Very Low-Dose Systemic Estradiol with Growth Hormone Therapy in Girls with Turner Syndrome J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6424 - 6430. [Abstract] [Full Text] [PDF]
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L. Soriano-Guillen, J. Coste, E. Ecosse, J. Leger, M. Tauber, S. Cabrol, M. Nicolino, R. Brauner, the StaTur Study Group, J.-L. Chaussain, et al. Adult Height and Pubertal Growth in Turner Syndrome after Treatment with Recombinant Growth Hormone J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5197 - 5204. [Abstract] [Full Text] [PDF]
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S. Piippo, H. Lenko, P. Kainulainen, and I. Sipila Use of Percutaneous Estrogen Gel for Induction of Puberty in Girls with Turner Syndrome J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3241 - 3247. [Abstract] [Full Text] [PDF]
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), one sixth (4.2 µg; 
), one quarter (6.2 µg;
), one half (12.5 µg; 
), and three quarters (18.8 µg; 
) of
a patch, and serum 17ß-estradiol concentrations were measured.
Shaded areas represent the 95% confidence interval for
the median of serum 17ß-estradiol concentrations in healthy girls in
early puberty (A and lower area in B) and midpuberty (C,
D, and upper area in B). A and B, Serum profiles
obtained in girls at breast stage 1: girls 1–6, 11 and 15, given a
dose of 0.08–0.12 µg/kg (A); and girls 1, 7, 12, 14, and 15, given a
dose of 0.13–0.16 µg/kg (B). C, Serum profiles obtained in girls at
breast stage 2, given a dose of 0.17–0.26 µg/kg: girls 5, 6, 8, 9,
11, and 13. D, Serum profiles obtained in girls at breast stage 3,
given a dose of 0.23–0.34 µg/kg: girls 7, 8, 10, and 11.