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Use of Percutaneous Estrogen Gel for Induction of Puberty in Girls with Turner Syndrome

Departments of Obstetrics and Gynecology (S.P.) and Pediatrics (H.L.), Tampere University Hospital, FIN-33521 Tampere, Finland; Orion Pharma (P.K.), 02101 Espoo, Finland; and Department of Pediatrics (I.S.), Helsinki University Hospital, 00029 Hus, Finland

Address all correspondence and requests for reprints to: Saila Piippo, Department of Obstetrics and Gynecology, Tampere University Hospital, P.O. Box 2000, FIN-33521 Tampere, Finland. E-mail: piipot{at}pispala.net.

	Abstract

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The aim of pubertal induction by estrogen in hypogonadic girls is to achieve physical and psychological development similar to that in natural puberty. We investigated the use of percutaneous estradiol gel for induction of puberty in girls with Turner syndrome (TS).

Twenty-three girls with TS and hypogonadism were included in the study. The initial percutaneous dose of 0.1 mg ended as 1.5 mg in the fifth year. The efficacy of the treatment was monitored by measuring height, weight, skeletal age, pubertal status, and serum hormone levels and gynecological ultrasonographic examinations throughout the study. Mean serum estradiol concentrations increased from 22.2 pmol/liter at baseline to 162.2 pmol/liter, and mean FSH levels decreased from 77.4 IU/liter at baseline to 19.2 IU/liter after 5 yr. There were no significant differences between GH users and nonusers with regard to height SD score, weight SD score, bone age acceleration, or adult height. The development of secondary sexual characteristics and uterine growth progressed gradually during the study. All girls reached at least stage B4P4. With percutaneous estradiol gel, the development of secondary sexual characteristics and uterine growth proceeded gradually, mimicking natural puberty. Estradiol gel was safe, easy to use, and well accepted by the subjects and provides an excellent way to individualize pubertal induction.

	Introduction

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FOR GIRLS WITH verified hypogonadism, induction of puberty followed by continuous estrogen replacement therapy is needed for several reasons. The development of secondary sexual characteristics and the maturation of female identity during the induction of puberty are essential for such girls. Sufficient uterine growth and development of the endometrium, with menstrual periods, can be achieved, which is needed later if embryo transfer is considered during adult life (1). GH is a well-documented promoter of growth in girls with Turner syndrome (TS), and estrogen substitution therapy that is started early may also have a positive effect on the final height (2). The addition of low-dose estrogen to a GH regimen has been shown to improve bone deposition and calcium metabolism in girls with TS (3). Estrogen at low doses has also been found to be beneficial with regard to verbal and nonverbal memory in young girls with TS (4).

The dosage and timing of GH and estrogen treatment during the induction of puberty is important. Chernausek et al. (5) used equine estrogen at a dose of 0.3 mg/d for 6 months, followed by 0.6 mg/d, with medroxyprogesterone acetate at 10 mg/d for 1 wk each month for girls with TS, beginning at ages 12 and 15 yr. Using these estrogen doses, they concluded that delaying the initiation of estrogen therapy was advantageous to growth. The number of years of GH therapy before the initiation of estrogen treatment was the most important factor determining final height. Rosenfield et al. (6), on the other hand, found that using very low doses of systemic estradiol, starting with 0.2 mg of depot estradiol before the age of 15 yr, to induce puberty in girls with TS who are also treated with GH, instead of using routine estrogen therapy, can result in increased final height. With transdermal nocturnal application of 17ß-estradiol patches, it has been possible to mimic the appropriate levels and the diurnal pattern of serum 17ß-estradiol in early puberty (7).

The purpose of this study was to investigate the use of percutaneous estradiol gel for the induction of puberty in hypogonadal girls. We wanted to introduce a therapeutic program for induction of puberty starting with low initial percutaneous estrogen doses and a gradual increase of doses to mimic natural puberty, with satisfactory feminization and good compliance. The development of secondary sexual characteristics and uterine growth and the effects on bone maturation, growth, and final height were studied.

	Subjects and Methods

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Subjects

Twenty-three girls with TS (17 XO karyotypes and six mosaics) and hypogonadism were enrolled in an uncontrolled, open, multicenter study, which was carried out between 1992 and 1999. Hypogonadism was verified by elevated gonadotropin and low estrogen levels in the serum. The girls were over 12 yr of age and had a skeletal age over 9 yr at the onset of the study. One girl aged 10.7 yr, who did not fulfill the age criterion, was accepted for treatment. Patients with previous or ongoing estrogen therapy were excluded. All five major pediatric endocrinology centers in Finland participated in the study and in recruitment, and all eligible patients were included in the study. The study was approved by the ethics review boards of each of the hospitals. Written informed consent was obtained from each girl and her legal representative.

Treatment protocol

The study medication, developed by Orion Pharma (Kuopio, Finland) for percutaneous estrogen therapy, consisted of estradiol in hydroalcoholic gel. Single-dose sachets containing 0.1 mg estradiol were prepared specifically for the study. Sachets containing 0.5 mg estradiol in 0.5 g of gel and 1.0 mg in 1 g of gel are commercially available (Divigel; Orion Pharma). Increasing doses of estradiol gel were applied once daily on the skin of the lower trunk or thighs, preferably in the evenings by the girl herself. The girls were instructed to wash their hands after the application. The estradiol doses were as follows: 0.1 mg for the first year, 0.2 mg for the second year, 0.5 mg for the third year, 1.0 mg for the fourth year, and 1.5 mg for the fifth year. The doses used were chosen based on the experiences of estrogen replacement therapy on adults and the bioequivalence of oral estrogens, transdermal patches, or estradiol gel. The starting dose of 0.1 mg of estradiol is equal to 0.13 mg of estradiol valerate or 0.04 mg of conjugated equine estrogen. The studies performed with Divigel have shown that transdermal estradiol in a hydroalcoholic gel is absorbed at a comparable rate and in almost equal amount as oral estradiol valerate tablets. The total duration of the study treatment was 5 yr. Starting from the third year, a progestin test with 10 mg of medroxyprogesterone acetate daily for 10 d to induce menstrual bleeding was performed every 6 months. After a positive test result, cyclic monthly progestin administration using the above dosage was started. If breakthrough bleeding occurred earlier, cyclic progestin was started. Compliance was assessed by asking the girls to return the unused medication.

Clinical follow-up and safety

Serum concentrations of estradiol, FSH, and LH were measured at every yearly visit. Annual assessment also included a GnRH test and assay of dehydroepiandrosterone sulfate and testosterone. To evaluate absorption of the medication, a serum estrogen profile was also obtained yearly; estradiol was not administered during the previous evening, and estradiol concentrations were measured the next morning before administration and 2 and 4 h after administration of the medication. Serum estradiol concentrations were measured using a RIA with an analytical sensitivity of 0.02 nmol/liter. Serum FSH and LH concentrations were measured using a time-resolved fluoroimmunoassay; both assays had the analytical sensitivity of 0.10 IU/liter.

The efficacy of the treatment was assessed by following pubertal development and growth. Height, weight, skeletal age, and pubertal status (according to Tanner) were evaluated every 6 months. Breast development was also evaluated by measuring areolar and mamillary diameters. Heights were measured by standard methods, using a Harpenden stadiometer. Heights were analyzed as SD scores (SDS). Ranke growth standards for girls with TS (8) were used for evaluating height and weight. Bone age x-rays were assessed according to Greulich and Pyle (9). Ten subjects had received GH for at least 2 yr, mainly as part of a GH study protocol, and the GH had always been started after 9 yr of age. Use of GH was not a controlled variable in this study. Abdominal ultrasonography was carried out at baseline and every 6 months after 1 yr of therapy to observe uterine growth and endometrial development. Timing of ultrasonography was not adjusted to any specific time of the menstrual period. Bone age radiographs were centrally read by one of the authors (I.S.), and ultrasonographies were read by another author (S.P.). The interpreters were blinded.

Blood pressure, hematological data, liver enzymes, fasting cholesterol, high-density lipoprotein cholesterol, and triglycerides were investigated to evaluate the safety of the medication. Side effects, such as skin irritation, and any other adverse events were recorded at every visit.

Data processing and statistical methods

Descriptive statistics are presented for all continuous variables as means and SDs. The numbers and percentages of subjects were calculated for categorical variables. All girls who received study medication were included in the efficacy and safety analyses. No imputation of missing data was performed, and all data available were used in the analyses. The statistical analyses were performed with SAS for Windows (SAS Institute, Cary, NC).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The age of the 23 girls at enrollment ranged from 10.7–17.7 yr, and the median age was 13.6 yr. One girl, who was aged 10.7 yr and who did not fulfill the age criterion, was accepted for treatment and taken into account in the analysis. All girls were of Caucasian origin. Compliance was good. The median use of medication was 100% in all but the 6-month (94.4%) and 4.5-yr (93.1%) visits. Administration of the gel was easy. None of the girls had difficulties in using the therapy.

Hormone levels

The mean serum estradiol concentration at baseline was 22.2 pmol/liter, and it increased steadily to 162.2 pmol/liter at 5 yr of treatment. At the same time, the mean FSH concentration decreased from 77.4 IU/liter at baseline to 19.2 IU/liter after 5 yr, and the mean LH concentration decreased from 20.6–6.6 IU/liter. The concentrations of FSH and LH after stimulation with GnRH decreased as the study progressed and estrogen doses were increased. The decline of FSH and FH values at 60 min was statistically significant between every yearly test (P < 0.01). However, with estrogen doses of 1 mg and 1.5 mg, there were only slight elevations in FSH and LH concentrations in the GnRH tests (Fig. 1). Absorption of estradiol from the gel was observed in the estrogen profile as a rise in concentrations after application of the gel (Fig. 2). Individually, the estrogen profile was very variable. An elevation of mean serum dehydroepiandrosterone sulfate values from 4.67–6.53 µmol/liter over the 5-yr period was observed. Serum mean testosterone levels remained constantly low, ranging between 0.59 and 0.86 nmol/liter during the study.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Mean LH and FSH concentrations in GnRH tests.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Serum estradiol concentrations after gel administration.

Ten girls received GH during or before this study. The mean GH dose was 0.1 IU/kg·d, and the mean duration of therapy was 4.4 yr (range, 2.1–6.1 yr). There were no significant differences between the GH users and nonusers with regard to height SDS, weight SDS, bone age acceleration, or adult height. The adult height was 153.1 ± 4.8 cm in the whole group (+1.08 SDS in the GH group and +1.05 SDS in the non-GH group). The mean age, height, weight, height and weight SDS, and bone age at yearly visits is shown in Table 1.

The pubertal stage advanced gradually along with the increasing estrogen doses (Fig. 3). According to our data, information on the pubertal status after 5 yr of treatment was available on 83% of the patients. Of these girls, 79% reached the B5 stage, and 58% reached the P5 stage. All girls reached at least stage B4P4. Mean areolar diameter increased from 20 mm at baseline to 47 mm after 5 yr of treatment. Breast development progressed gradually and symmetrically during the whole study. Treatment with GH did not have an effect on the development of secondary sexual characteristics.

View larger version (42K): [in this window] [in a new window]   FIG. 3. Pubertal development.

Abdominal ultrasonographic data were obtained from 18 girls. Mean uterine length increased from 33 mm (range, 15–66 mm) at baseline to 67 mm (range, 48–91 mm) at 5 yr of therapy. Mean uterine volume increased from 5.5 ml (range, 1.7–12.6 ml) at 1 yr of treatment to 31.5 ml (range, 8.2–82.8 ml) at 5 yr. The mean endometrial thickness at 4–5 yr of therapy was 3.9–2.8 mm (Fig. 4). The maximum endometrial thickness measured during the study was 11 mm. Regular menstrual periods were induced with progestins, according to the protocol, between 2.5 and 3.5 yr of treatment in 19 cases. In three cases, regular progestins were started at 6 months because of spontaneous bleeding, and in one case, regular progestins were started at 1.25 yr.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Development of the uterus and endometrium.

Blood pressure remained normal. Mean fasting total cholesterol levels fell from 4.60 mmol/liter (range, 3.30–6.20 mmol/liter) to 4.27 mmol/liter (range, 2.50–6.10 mmol/liter) at 5 yr, and a similar decline in triglyceride concentrations, from 1.11 mmol/liter at baseline to 0.91 mmol/liter after 5 yr was observed. Mean levels of high-density lipoprotein cholesterol rose at the same time, from 1.29 mmol/liter at baseline to 1.45 mmol/liter after 5 yr. Mean serum alanine transaminase levels remained between 17 and 32 U/liter throughout the study. A single alanine transaminase level of 231 U/liter was found in one case. Routine hematology results remained unaltered.

Side effects and adverse events

Mild skin irritation after application of the medication was reported four times during the study. Spontaneous menstrual bleeding or spotting before the use of cyclic progestins was reported by 11 girls. During the cyclic estrogen/progesterone therapy, nine episodes of irregular bleeding were reported by five subjects. Two girls had transient amenorrhea, and two had menorrhagia. Two girls left the study prematurely at 3.5 yr. One of these girls did not like the gel and changed to another form of estrogen treatment, and the other girl moved abroad.

Some girls had preexisting conditions associated with TS; there were two cases of hypothyreosis, one case of aortic coarctation, and two cases of ear infection. Adverse events reported during the study included three cases of abdominal pain and four cases of upper respiratory tract infection. With regard to serious adverse events, one subject developed anorexia nervosa and recovered from it, one girl with menorrhagia was diagnosed as having von Willebrand disease, and one girl was diagnosed as having aortic aneurysm associated with TS.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In this study, our goal was to modify the induction of puberty in girls with TS toward spontaneous pubertal development by using percutaneous estradiol. The main target of estrogen replacement therapy in such girls has been to induce puberty and feminization sufficiently early without reducing adult height (10). The natural form of estrogen in humans is estradiol. It is relatively ineffective after oral administration because of extensive degradation in first pass metabolism in the liver (6). Transdermal estradiol patches have successfully been used in the induction of puberty (7, 11). Percutaneous estradiol gel is commonly used for postmenopausal estrogen therapy. Administration of estrogen replacement by the percutaneous route in girls with TS may have less deleterious effects on hepatic metabolism than oral estrogen (12).

According to our results, the use of percutaneous estradiol gel in individualized treatment provides an excellent, safe, and physiological means to induce puberty in girls with hypogonadism. The gel provides a good option to administer the very low estrogen doses. Costs of such treatment can be considered reasonable because adult preparations are easy to modify for pediatric use, in contrast to commercial oral preparations containing the adult doses. By reducing the estradiol concentration in the gel, the starting doses can be adjusted to low levels and increased gradually. The use of known dose sachets may be preferable to free dosing from a tube to keep the used dose at the recommended level. Acceptance of the treatment was excellent because the medication was easy to use and not visible. Transmission of estradiol between individuals in skin contacts is unlikely but can be prevented by applying the medication to an area covered by clothing. According to safety parameters and adverse events, the use of percutaneous estradiol was safe for the subjects. However, 11 girls reported menstrual bleeding before the initiation of the cyclic progestin, which means that the initiation of progestin according to protocol was too late. With ultrasonography, the development of endometrium can be followed individually, and cyclic progesterone should be started at an endometrial thickness of 3 mm.

The steady increase of mean estradiol concentrations proves the efficacy of percutaneous estradiol therapy for the induction of puberty. The estradiol concentrations within and between individual subjects showed great variation and cannot be used alone in treatment follow-up. Declines in the basal and stimulated FSH and LH concentrations with increasing estradiol doses are more useful in measuring the efficacy of estradiol therapy. The very small increases in FSH and LH concentrations in GnRH tests with the 1.0-mg and 1.5-mg doses of estradiol indicate that, for some of the subjects, smaller doses would have been sufficient. By following gonadotropin concentrations and by means of ultrasonographic measurement of uterine growth and endometrial thickness, we can tailor individual pubertal induction with percutaneous estradiol gel in hypogonadic subjects.

Percutaneous estradiol therapy resulted in excellent gradual development of secondary sexual characteristics. At 5 yr, all girls had reached Tanner stage B4P4; of the 83% of girls with information available, 79% reached the B5 stage, and 58% reached the P5 stage. According to ultrasonographic examinations of uterine length, normal uterine development was reached at age 16 yr (13). The mean uterine volume reached was 7 ml more than in normal puberty at Tanner stage B4–B5 (14). Achieving normal adult uterine size is important if infertility treatments (embryo transfer) are going to be considered during later life. Endometrial thickness was in the normal range during the menstrual cycle, which indicates endometrial safety of the treatment. Even though liver enzymes may rise during treatment, estrogen should not be considered to be the main cause of elevated liver enzymes in TS (15).

In our study, no significant difference in growth was found between subjects receiving GH and those not receiving GH. The study was not designed to evaluate the effect of estradiol on GH treatment, and the indications to start GH varied in different centers. Therefore, we can only conclude that estradiol treatment did not have a negative effect on the growth of the subjects.

Fluctuation of estradiol concentrations over 24 h might have a positive effect with regard to the natural development of puberty (7). Such an effect could be achieved in our study by means of percutaneous application of the gel in the evenings. In this regard, the use of gel might be preferable to the use of estradiol patches. Percutaneous estrogen has become standard practice for pubertal induction in our clinic. After this study, even more attention has been paid to the very low starting dose and the gradual increase of doses during the therapy. During the use of GH, estradiol concentrations are kept very low, with only a slight decline in FSH concentrations. With estradiol gel, the doses used can be individually adjusted to pubertal Tanner stages, gynecological ultrasonographic data, and circulating FSH concentrations.

Conclusion

The use of percutaneous estradiol gel provided a safe, individual, and well-accepted treatment for the induction of puberty in hypogonadic girls. Secondary sexual characteristics and uterine growth proceeded gradually during the treatment, mimicking natural pubertal development. The regimen of very low initial doses and gradual increases of the dose did not have a negative effect on adult height. With the gel, the estrogen dose can be individually tailored for natural pubertal development. Efficacy of therapy can be followed by means of pubertal signs, ultrasonographic measurement of uterine growth and endometrial thickness, and circulating gonadotropin concentrations.

	Footnotes

 
Abbreviations: SDS, SD score; TS, Turner syndrome.

Received December 2, 2003.

Accepted April 4, 2004.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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