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Estrogen levels in girls with premature thelarche compared with normal prepubertal girls as determined by an ultrasensitive recombinant cell bioassay
E
Estrogen levels in girls with premature thelarche compared with normal prepubertal girls as determined by an ultrasensitive recombinant cell bioassay
Karen Oerter Klein, MD, Veronica Mericq, MD, Jacquelyn M. Brown-Dawson, Kimberly A. Larmore, Patricia Cabezas, and Amanda Cortinez, MD
Objective: Estradiol levels in girls with premature thelarche have not previously been well defined because of the lack of adequate sensitivity of previously available estradiol assays. The ultrasensitive recombinant cell bioassay for estradiol has made the study of estradiol levels in premature thelarche possible. We hypothesized that girls with premature thelarche have higher estradiol levels than normal prepubertal girls. Study design: We used an ultrasensitive recombinant cell bioassay to study estradiol levels in 20 girls with premature thelarche and 15 normal prepubertal girls less than 3 years of age. The 2 groups were compared by Student t test. Results: Estradiol levels were significantly greater in the girls with premature thelarche (8.4 ± 4.5 pmol/L estradiol equivalents) than in the normal prepubertal girls (3.3 ± 3.5 pmol/L estradiol equivalents; P < .01). The estradiol level was not significantly correlated with age, height, weight, body mass index, age at onset of thelarche, or the presence or absence of ovarian cysts. Conclusion: Girls with premature thelarche have significantly higher estradiol levels than normal prepubertal girls. This is consistent with the hypothesis that the mechanism of premature thelarche involves increased estradiol levels rather than increased sensitivity of breast tissue to normal estradiol levels. (J Pediatr 1999;134:190-2)
Premature thelarche is defined as isolated breast development with no other clinical signs of sexual maturation in girls before the age of 8 years
and is most commonly seen in the first 2 years of life.1 It is considered a benign condition that rarely progresses to central precocious puberty.2 The
From the Department of Clinical Science, duPont Hospital for Children, Wilmington, Delaware, and the Institute of Maternal and Child Research, University of Chile, Santiago, Chile.
Submitted for publication June 2, 1998; revision received Oct. 5, 1998; accepted Nov. 4, 1998.
Reprint requests: Karen Oerter Klein, MD, Department of Clinical Science, duPont Hospital for Children, PO Box 269, 1600 Rockland Rd, Wilmington, DE 19899. Copyright © 1999 by Mosby, Inc. 0022-3476/99/$8.00 + 0 9/21/95640
190
mechanism of premature thelarche is still unknown. It may be caused by increased estradiol levels or by increased sensitivity of breast tissue to normal prepubertal estradiol levels, or a combination of both. BMI
Body mass index
Estradiol levels in girls with premature thelarche are not well defined because of the lack of adequate sensitivity of available estradiol assays. They are usually reported as undetectable3,4 or as close to the detection limit of the assay used.5-8 We used an ultrasensitive recombinant cell bioassay for estradiol to study estradiol levels in girls with premature thelarche compared with normal prepubertal girls. We hypothesized that the increased sensitivity of the recombinant cell bioassay would allow us to determine whether there is a difference in estradiol levels between girls with premature thelarche and normal prepubertal girls, and whether the presence or absence of a difference in estradiol levels would help us understand the cause of early breast development in girls with premature thelarche.
METHODS Study Design We studied 20 girls with premature thelarche (1.4 ± 0.5 years of age) and 15 normal prepubertal girls (1.5 ± 0.5
KLEIN ET AL
THE JOURNAL OF PEDIATRICS VOLUME 134, NUMBER 2 years). Girls with premature thelarche were referred for evaluation of early breast development to one of our pediatric endocrinology clinics, either The duPont Children’s Hospital, Wilmington, Del; The Children’s Hospital of Orange County, Calif; the Nemours Institute, Jacksonville, Fla; or the Institute of Maternal and Child Research, University of Chile, Santiago. Normal prepubertal girls were recruited for the study at The duPont Children’s Hospital, The Children’s Hospital of Orange County, and The Institute of Maternal and Child Research at the University of Chile. A single blood collection was obtained for determination of the estradiol level from each girl in the morning. Pelvic ultrasonography was performed in 13 of the 20 girls with premature thelarche. Pubertal stage was determined by the same two observers (K.O.K. in the United States; A.C. in Chile) in all but 3 patients. Breast stage was determined according to Tanner.9 Girls with premature thelarche had breast tissue but no pubic hair, no advanced bone maturation (bone age within 2 SD in girls studied), and no increased growth velocity. Height, weight, and body mass index were normal in all girls (79.8 ± 7.6 cm, 10.3 ± 2.0 kg, and 16.1 ± 1.2 kg/m2, respectively, for girls with premature thelarche; 81.1 ± 6.8 cm, 12.2 ± 5.8 kg, and 18.0 ± 5.8 kg/m2, respectively, for normal prepubertal girls). Normal prepubertal girls had no breast tissue and no pubic hair. All girls were healthy, had normal findings on general physical examination, and were receiving no medications. All girls had no known exposure to any preparations containing estrogens and had no other signs or symptoms of puberty. The study was approved by the appropriate review committee at each institution. Consent was given by one parent of each subject.
Estrogen Assay The bioassay for estradiol was recently described in detail.10 Briefly, it
Figure. Estradiol level in girls with premature thelarche and in normal prepubertal girls. Individual data points are shown.The bars represent mean ± SD. *P < .01 versus normal.
used a strain of Saccharomyces cerevisiae that was transformed with 2 plasmids. One plasmid contained the human estrogen receptor complementary DNA, and the other contained an estrogen response element upstream of the yeast iso-1-cytochrome c promoter fused to the structural gene for β-galactosidase. The transformed yeast cultures were grown in selective media in the presence of estradiol extracted from serum. β-Galactosidase activity was assayed and converted to estradiol equivalent units by linear interpolation from a standard curve constructed by adding known amounts of estradiol to charcoal-stripped plasma samples. The sensitivity of the bioassay was 0.07 to 0.7 pmol/L (0.02-0.2 pg/mL) during the time frame of the study. The intraassay and interassay coefficients of variation at 0.7 pmol/L (0.2 pg/mL) ranged from 10% to 50% during the time frame of the study.
Statistics BMI was calculated as weight in kilograms divided by height in meters squared. All data are expressed as the mean ± SD. Values below the detection limit were set equal to the detection
limit for analysis. Comparisons between groups were made by means of the 2-tailed Student t test. Differences were considered significant at P < .05. Correlations were made by using simple linear regression analysis.
RESULTS Estradiol levels were significantly higher in the girls with premature thelarche (8.4 ± 4.5 pmol/L estradiol equivalents (2.3 ± 1.2 pg/mL) ) compared with the normal prepubertal girls (3.3 ± 3.5 pmol/L estradiol equivalents [0.9 ± 1.0 pg/mL]; P < .01), although there was a large overlap in the range for the 2 groups (Figure). There were no significant differences in age, height, weight, or BMI between the 2 groups. Estradiol level was not significantly correlated with age, height, weight, BMI, or age at onset of thelarche (data not shown). Five girls had persistent thelarche since birth. There was no correlation between estradiol level and the presence of thelarche since birth. In the 13 girls with premature thelarche who underwent pelvic ultrasonography, 5 girls had small 191
KLEIN ET AL
(<7 mm) bilateral ovarian cysts. There was no correlation between estradiol level and the presence or absence of ovarian cysts. The variability for repeated measures in the same individual ranged from 13% to 50%. A subset of samples from each group was rerun in a single assay and the results did not change.
DISCUSSION The cause of premature thelarche is still unknown. It has been postulated by others to result from increased breast sensitivity of estradiol,11 from transient estradiol secretion by ovarian cysts,12-14 from increased production of estradiol from precursors of adrenal origin,7 from increased dietary estrogen,15 and from transient partial activation of the hypothalamic-pituitarygonadal axis with excessive secretion of follicle-stimulating hormone.3,16 Previous reports of estradiol levels in premature thelarche include descriptions of undetectable estradiol levels or of estradiol levels measured by assays with detection limits much greater than the levels measured in our report—and are thus inappropriate for adequately addressing this condition.2-6 Fluctuations in estradiol level by time of day17 may contribute to the wide range of values seen in each group and hence the large overlap between groups. More extensive studies, including serial sampling of estradiol in these groups, may further define the differences between the groups. Clinically the girls in this study had simple premature thelarche and, on the basis of the girls’ normal growth velocities or normal bone maturation, or both, did not appear to be at risk of having a precocious puberty. Because there was a large overlap in estradiol levels between the 2 groups, measurement of an ultrasensitive estradiol level would not change the medical management of these girls at this time. However, further longitudinal studies, in192
THE JOURNAL OF PEDIATRICS FEBRUARY 1999 cluding those of girls more likely to be at risk of precocious puberty, are needed to determine the outcome of premature thelarche and whether the ultrasensitive estradiol assay will help in distinguishing girls with simple nonprogressive premature thelarche from girls at risk of precocious puberty. Further studies are also needed to determine the source of the increased estradiol levels. Ovarian cysts are not the source of increased estradiol in this group, because those with and those without cysts had similar values. This study did not evaluate gonadotropin levels or address the possibility of dietary or environmental estrogens contributing to the development of premature thelarche or cross-reactivity in the bioassay.
7.
8.
9.
10.
We thank Dr Nelly Mauras and Dr Kenneth Copeland for contributing several patients to this study.
11.
REFERENCES
12.
1. Underwood LE, Van Wyk JJ. Normal and aberrant growth. In: Wilson JD, Foster DW, editors. Textbook of endocrinology. Philadelphia: WB Saunders; 1985. p. 195. 2. Pasquino AM, Pucarelli I, Passeri F, Segni M, Mancini MA, Municchi G. Progression of premature thelarche to central precocious puberty. J Pediatr 1995;126:11-4. 3. Reiter EO, Kaplan SL, Conte FA, Grumbach MM. Responsivity of pituitary gonadotropins to luteinizing hormone-releasing factor in idiopathic precocious puberty, precocious thelarche, precocious adrenarche, and in patients treated with medroxyprogesterone acetate. Pediatr Res 1975;9:11-116. 4. Garibaldi LR, Aceto T Jr, Weber C. The pattern of gonadotropin and estradiol secretion in exaggerated thelarche. Acta Endocrinol (Copenh) 1993;128: 345-50. 5. Pescovitz OH, Hench KD, Barnes KM, Loriaux DL, Cutler GB. Premature thelarche and central precocious puberty: the relationship between clinical presentation and the gonadotropin response to luteinizing hormone-releasing hormone. J Clin Endocrinol Metab 1988;67:474-9. 6. Jenner MR, Kelch RP, Kaplan SL,
13.
14.
15.
16.
17.
Grumbach MM. Hormonal changes in puberty. IV. Plasma estradiol, LH, and FSH in prepubertal children, pubertal females, and in precocious puberty, premature thelarche, hypogonadism, and in a child with a feminizing ovarian tumor. J Clin Endocrinol Metab 1972;34:521-30. Dumic M, Tajic M, Mardesic D, Kalafatic Z. Premature thelarche: a possible adrenal disorder. Arch Dis Child 1982;57:200-3. Freedman SM, Kreitzer PM, Elkowitz SS, Soberman N, Leonidas JC. Ovarian microcysts in girls with isolated premature thelarche. J Pediatr 1993;122: 246-9. Marshall WA, Tanner JM. Variations in pattern of pubertal changes in girls. Arch Dis Child 1969;44:291-303. Klein KO, Baron J, Colli MJ, McDonnell DP, Cutler GB Jr. Estrogen levels in childhood determined by an ultra-sensitive recombinant cell bioassay. J Clin Invest 1994;94:2475-80. Ilicki A, Prager Lewin R, Kauli R, Kaugman H, Schachter A, Laron Z. Premature thelarche: natural history and sex hormone secretion in 68 girls. Acta Paediatr Scand 1984;73:756-62. Sizonenko PC. Preadolescent and adolescent endocrinology: physiology and physiopathology. II. Hormonal changes during abnormal pubertal development. Am J Dis Child 1978;132: 797-805. Collett-Solberg PR, Grumbach MM. A simplified procedure for evaluating estrogenic effects and the sex chromatin pattern in exfoliated cells in urine: studies in premature thelarche and gynecomastia of adolescence. J Pediatr 1965;66:883-90. Silver HK, Sami D. Premature thelarche: precocious development of the breast. Pediatrics 1964;34:107-11. Saenz de Rodriguez CA, Bongiovanni AM, Conde de Borrego L. An epidemic of precocious development in Puerto Rican children. J Pediatr 1985;107: 393-6. Pasquino AM, Piccolo F, Scalamandre A, Malvaso M, Ortolani R, Boscherini B. Hypothalamic-pituitary-gonadotropic function in girls with premature thelarche. Arch Dis Child 1980;55:941-4. Klein KO, Larmore KA, deLancey E, Brown JM, Considine RV, Hassink SG. Effect of obesity on estradiol level, and its relationship to leptin, bone maturation, and bone mineral density in children. J Clin Endocrinol Metab 1998;83:3469-75.
Estrogen levels in girls with premature thelarche compared with normal prepubertal girls as determined by an ultrasensitive recombinant cell bioassay
Karen Oerter Klein, MD, Veronica Mericq, MD, Jacquelyn M. Brown-Dawson, Kimberly A. Larmore, Patricia Cabezas, and Amanda Cortinez, MD
Objective: Estradiol levels in girls with premature thelarche have not previously been well defined because of the lack of adequate sensitivity of previously available estradiol assays. The ultrasensitive recombinant cell bioassay for estradiol has made the study of estradiol levels in premature thelarche possible. We hypothesized that girls with premature thelarche have higher estradiol levels than normal prepubertal girls. Study design: We used an ultrasensitive recombinant cell bioassay to study estradiol levels in 20 girls with premature thelarche and 15 normal prepubertal girls less than 3 years of age. The 2 groups were compared by Student t test. Results: Estradiol levels were significantly greater in the girls with premature thelarche (8.4 ± 4.5 pmol/L estradiol equivalents) than in the normal prepubertal girls (3.3 ± 3.5 pmol/L estradiol equivalents; P < .01). The estradiol level was not significantly correlated with age, height, weight, body mass index, age at onset of thelarche, or the presence or absence of ovarian cysts. Conclusion: Girls with premature thelarche have significantly higher estradiol levels than normal prepubertal girls. This is consistent with the hypothesis that the mechanism of premature thelarche involves increased estradiol levels rather than increased sensitivity of breast tissue to normal estradiol levels. (J Pediatr 1999;134:190-2)
Premature thelarche is defined as isolated breast development with no other clinical signs of sexual maturation in girls before the age of 8 years
and is most commonly seen in the first 2 years of life.1 It is considered a benign condition that rarely progresses to central precocious puberty.2 The
From the Department of Clinical Science, duPont Hospital for Children, Wilmington, Delaware, and the Institute of Maternal and Child Research, University of Chile, Santiago, Chile.
Submitted for publication June 2, 1998; revision received Oct. 5, 1998; accepted Nov. 4, 1998.
Reprint requests: Karen Oerter Klein, MD, Department of Clinical Science, duPont Hospital for Children, PO Box 269, 1600 Rockland Rd, Wilmington, DE 19899. Copyright © 1999 by Mosby, Inc. 0022-3476/99/$8.00 + 0 9/21/95640
190
mechanism of premature thelarche is still unknown. It may be caused by increased estradiol levels or by increased sensitivity of breast tissue to normal prepubertal estradiol levels, or a combination of both. BMI
Body mass index
Estradiol levels in girls with premature thelarche are not well defined because of the lack of adequate sensitivity of available estradiol assays. They are usually reported as undetectable3,4 or as close to the detection limit of the assay used.5-8 We used an ultrasensitive recombinant cell bioassay for estradiol to study estradiol levels in girls with premature thelarche compared with normal prepubertal girls. We hypothesized that the increased sensitivity of the recombinant cell bioassay would allow us to determine whether there is a difference in estradiol levels between girls with premature thelarche and normal prepubertal girls, and whether the presence or absence of a difference in estradiol levels would help us understand the cause of early breast development in girls with premature thelarche.
METHODS Study Design We studied 20 girls with premature thelarche (1.4 ± 0.5 years of age) and 15 normal prepubertal girls (1.5 ± 0.5
KLEIN ET AL
THE JOURNAL OF PEDIATRICS VOLUME 134, NUMBER 2 years). Girls with premature thelarche were referred for evaluation of early breast development to one of our pediatric endocrinology clinics, either The duPont Children’s Hospital, Wilmington, Del; The Children’s Hospital of Orange County, Calif; the Nemours Institute, Jacksonville, Fla; or the Institute of Maternal and Child Research, University of Chile, Santiago. Normal prepubertal girls were recruited for the study at The duPont Children’s Hospital, The Children’s Hospital of Orange County, and The Institute of Maternal and Child Research at the University of Chile. A single blood collection was obtained for determination of the estradiol level from each girl in the morning. Pelvic ultrasonography was performed in 13 of the 20 girls with premature thelarche. Pubertal stage was determined by the same two observers (K.O.K. in the United States; A.C. in Chile) in all but 3 patients. Breast stage was determined according to Tanner.9 Girls with premature thelarche had breast tissue but no pubic hair, no advanced bone maturation (bone age within 2 SD in girls studied), and no increased growth velocity. Height, weight, and body mass index were normal in all girls (79.8 ± 7.6 cm, 10.3 ± 2.0 kg, and 16.1 ± 1.2 kg/m2, respectively, for girls with premature thelarche; 81.1 ± 6.8 cm, 12.2 ± 5.8 kg, and 18.0 ± 5.8 kg/m2, respectively, for normal prepubertal girls). Normal prepubertal girls had no breast tissue and no pubic hair. All girls were healthy, had normal findings on general physical examination, and were receiving no medications. All girls had no known exposure to any preparations containing estrogens and had no other signs or symptoms of puberty. The study was approved by the appropriate review committee at each institution. Consent was given by one parent of each subject.
Estrogen Assay The bioassay for estradiol was recently described in detail.10 Briefly, it
Figure. Estradiol level in girls with premature thelarche and in normal prepubertal girls. Individual data points are shown.The bars represent mean ± SD. *P < .01 versus normal.
used a strain of Saccharomyces cerevisiae that was transformed with 2 plasmids. One plasmid contained the human estrogen receptor complementary DNA, and the other contained an estrogen response element upstream of the yeast iso-1-cytochrome c promoter fused to the structural gene for β-galactosidase. The transformed yeast cultures were grown in selective media in the presence of estradiol extracted from serum. β-Galactosidase activity was assayed and converted to estradiol equivalent units by linear interpolation from a standard curve constructed by adding known amounts of estradiol to charcoal-stripped plasma samples. The sensitivity of the bioassay was 0.07 to 0.7 pmol/L (0.02-0.2 pg/mL) during the time frame of the study. The intraassay and interassay coefficients of variation at 0.7 pmol/L (0.2 pg/mL) ranged from 10% to 50% during the time frame of the study.
Statistics BMI was calculated as weight in kilograms divided by height in meters squared. All data are expressed as the mean ± SD. Values below the detection limit were set equal to the detection
limit for analysis. Comparisons between groups were made by means of the 2-tailed Student t test. Differences were considered significant at P < .05. Correlations were made by using simple linear regression analysis.
RESULTS Estradiol levels were significantly higher in the girls with premature thelarche (8.4 ± 4.5 pmol/L estradiol equivalents (2.3 ± 1.2 pg/mL) ) compared with the normal prepubertal girls (3.3 ± 3.5 pmol/L estradiol equivalents [0.9 ± 1.0 pg/mL]; P < .01), although there was a large overlap in the range for the 2 groups (Figure). There were no significant differences in age, height, weight, or BMI between the 2 groups. Estradiol level was not significantly correlated with age, height, weight, BMI, or age at onset of thelarche (data not shown). Five girls had persistent thelarche since birth. There was no correlation between estradiol level and the presence of thelarche since birth. In the 13 girls with premature thelarche who underwent pelvic ultrasonography, 5 girls had small 191
KLEIN ET AL
(<7 mm) bilateral ovarian cysts. There was no correlation between estradiol level and the presence or absence of ovarian cysts. The variability for repeated measures in the same individual ranged from 13% to 50%. A subset of samples from each group was rerun in a single assay and the results did not change.
DISCUSSION The cause of premature thelarche is still unknown. It has been postulated by others to result from increased breast sensitivity of estradiol,11 from transient estradiol secretion by ovarian cysts,12-14 from increased production of estradiol from precursors of adrenal origin,7 from increased dietary estrogen,15 and from transient partial activation of the hypothalamic-pituitarygonadal axis with excessive secretion of follicle-stimulating hormone.3,16 Previous reports of estradiol levels in premature thelarche include descriptions of undetectable estradiol levels or of estradiol levels measured by assays with detection limits much greater than the levels measured in our report—and are thus inappropriate for adequately addressing this condition.2-6 Fluctuations in estradiol level by time of day17 may contribute to the wide range of values seen in each group and hence the large overlap between groups. More extensive studies, including serial sampling of estradiol in these groups, may further define the differences between the groups. Clinically the girls in this study had simple premature thelarche and, on the basis of the girls’ normal growth velocities or normal bone maturation, or both, did not appear to be at risk of having a precocious puberty. Because there was a large overlap in estradiol levels between the 2 groups, measurement of an ultrasensitive estradiol level would not change the medical management of these girls at this time. However, further longitudinal studies, in192
THE JOURNAL OF PEDIATRICS FEBRUARY 1999 cluding those of girls more likely to be at risk of precocious puberty, are needed to determine the outcome of premature thelarche and whether the ultrasensitive estradiol assay will help in distinguishing girls with simple nonprogressive premature thelarche from girls at risk of precocious puberty. Further studies are also needed to determine the source of the increased estradiol levels. Ovarian cysts are not the source of increased estradiol in this group, because those with and those without cysts had similar values. This study did not evaluate gonadotropin levels or address the possibility of dietary or environmental estrogens contributing to the development of premature thelarche or cross-reactivity in the bioassay.
7.
8.
9.
10.
We thank Dr Nelly Mauras and Dr Kenneth Copeland for contributing several patients to this study.
11.
REFERENCES
12.
1. Underwood LE, Van Wyk JJ. Normal and aberrant growth. In: Wilson JD, Foster DW, editors. Textbook of endocrinology. Philadelphia: WB Saunders; 1985. p. 195. 2. Pasquino AM, Pucarelli I, Passeri F, Segni M, Mancini MA, Municchi G. Progression of premature thelarche to central precocious puberty. J Pediatr 1995;126:11-4. 3. Reiter EO, Kaplan SL, Conte FA, Grumbach MM. Responsivity of pituitary gonadotropins to luteinizing hormone-releasing factor in idiopathic precocious puberty, precocious thelarche, precocious adrenarche, and in patients treated with medroxyprogesterone acetate. Pediatr Res 1975;9:11-116. 4. Garibaldi LR, Aceto T Jr, Weber C. The pattern of gonadotropin and estradiol secretion in exaggerated thelarche. Acta Endocrinol (Copenh) 1993;128: 345-50. 5. Pescovitz OH, Hench KD, Barnes KM, Loriaux DL, Cutler GB. Premature thelarche and central precocious puberty: the relationship between clinical presentation and the gonadotropin response to luteinizing hormone-releasing hormone. J Clin Endocrinol Metab 1988;67:474-9. 6. Jenner MR, Kelch RP, Kaplan SL,
13.
14.
15.
16.
17.
Grumbach MM. Hormonal changes in puberty. IV. Plasma estradiol, LH, and FSH in prepubertal children, pubertal females, and in precocious puberty, premature thelarche, hypogonadism, and in a child with a feminizing ovarian tumor. J Clin Endocrinol Metab 1972;34:521-30. Dumic M, Tajic M, Mardesic D, Kalafatic Z. Premature thelarche: a possible adrenal disorder. Arch Dis Child 1982;57:200-3. Freedman SM, Kreitzer PM, Elkowitz SS, Soberman N, Leonidas JC. Ovarian microcysts in girls with isolated premature thelarche. J Pediatr 1993;122: 246-9. Marshall WA, Tanner JM. Variations in pattern of pubertal changes in girls. Arch Dis Child 1969;44:291-303. Klein KO, Baron J, Colli MJ, McDonnell DP, Cutler GB Jr. Estrogen levels in childhood determined by an ultra-sensitive recombinant cell bioassay. J Clin Invest 1994;94:2475-80. Ilicki A, Prager Lewin R, Kauli R, Kaugman H, Schachter A, Laron Z. Premature thelarche: natural history and sex hormone secretion in 68 girls. Acta Paediatr Scand 1984;73:756-62. Sizonenko PC. Preadolescent and adolescent endocrinology: physiology and physiopathology. II. Hormonal changes during abnormal pubertal development. Am J Dis Child 1978;132: 797-805. Collett-Solberg PR, Grumbach MM. A simplified procedure for evaluating estrogenic effects and the sex chromatin pattern in exfoliated cells in urine: studies in premature thelarche and gynecomastia of adolescence. J Pediatr 1965;66:883-90. Silver HK, Sami D. Premature thelarche: precocious development of the breast. Pediatrics 1964;34:107-11. Saenz de Rodriguez CA, Bongiovanni AM, Conde de Borrego L. An epidemic of precocious development in Puerto Rican children. J Pediatr 1985;107: 393-6. Pasquino AM, Piccolo F, Scalamandre A, Malvaso M, Ortolani R, Boscherini B. Hypothalamic-pituitary-gonadotropic function in girls with premature thelarche. Arch Dis Child 1980;55:941-4. Klein KO, Larmore KA, deLancey E, Brown JM, Considine RV, Hassink SG. Effect of obesity on estradiol level, and its relationship to leptin, bone maturation, and bone mineral density in children. J Clin Endocrinol Metab 1998;83:3469-75.