Pelvic sonography and uterine artery color Doppler analysis in

Transcription

1 Ultrasound Obstet Gynecol 2002; 19: Pelvic sonography and uterine artery color Doppler analysis in Blackwell Science Ltd the diagnosis of female precocious puberty C. BATTAGLIA*, G. REGNANI, F. MANCINI*, L. IUGHETTI, S. VENTUROLI* and C. FLAMIGNI *Reproductive Medicine Unit and First Department of Obstetrics and Gynecology, University of Bologna, Bologna and Departments of Obstetrics and Gynecology and Pediatrics, University of Modena, Modena, Italy KEYWORDS: Child, Doppler, GnRH, Puberty, Ultrasound ABSTRACT Objectives To evaluate the role of ultrasound and color Doppler analyses in the diagnosis of precocious puberty. Methods Gray-scale sonographic uterine and ovarian evaluation together with color Doppler analysis of the uterine artery were prospectively performed in 29 girls presenting with premature breast development and pubic hair growth. The values were compared with results obtained from the gonodotrophin releasing hormone stimulation test. Excluded from the study were patients with isolated thelarche or isolated pubarche and those patients with gonodotrophin releasing hormoneindependent puberty and with polycystic ovaries. Results According to the Tanner scale, all the girls presented a breast stage of 2 3 and pubic hair stage 2 3. The uterine size was greater in those girls who presented a pubertal response to the gonodotrophin releasing hormone test (Group II; n = 20) (8.07 ± 4.47 ml) than in those who did not (Group I; n = 9) (3.07 ± 1.18 ml; P = 0.001). The ovarian volume and the number of small follicles was not significantly different between the groups. On Doppler analysis, more elevated impedances were observed in Group I (pulsatility index = 3.28 ± 0.37) than in Group II (pulsatility index = 2.29 ± 0.19; P = 0.001) girls. The presence of a low pulsatility index (< 2.5) at the level of the uterine arteries had a high diagnostic value for precocious puberty (sensitivity 86%, specificity 100%). Conclusions Uterine artery Doppler analysis may assist the diagnosis of gonodotrophin releasing hormone-dependent precocious puberty, may be useful for the selection of those girls needing treatment, and may simplify the follow-up of girls treated for precocities. INTRODUCTION Isosexual precocious puberty in girls may be defined as the premature development of secondary sexual characteristics associated with uterine and ovarian maturation. Linear growth acceleration and bone maturation, leading to early epiphyseal fusion and short adult height, may also be present. Clinically, the diagnosis is considered in girls developing pubertal changes prior to 8 years of age 1. Approximately 95% of isosexual precocity is gonodotrophin releasing hormone (GnRH)-dependent and is due to idiopathic activation of the hypothalamic pituitary ovarian axis without evident underlying anatomical causes 2. The causes of GnRH-independent puberty include gonadal (McCune Albright syndrome, ovarian cysts, ovarian secreting tumors), adrenal (congenital adrenal hyperplasia, secreting tumors), ectopic (human chorionic gonadotrophin secreting tumors) and exogenous (iatrogenic) sources of hormone production. Hypo- and hyperthyroidism may also be responsible for GnRH-independent precocious puberty 2,3. The treatment of GnRH-independent puberty is related to the solution of the specific underlying factors, whereas GnRHdependent isosexual precocity requires the GnRH-agonist suppression of the hypothalamic pituitary ovarian axis. However, some studies have described girls with intermediate forms of precocious puberty 4 who were considered to be unsuitable for treatment with GnRH agonists. Thus, it is important to define the limits of an active hypothalamic pituitary ovarian axis in order to determine that true puberty has begun. Pelvic sonography is a rapid, accurate and non-invasive method for evaluating the female pelvis in infancy and childhood. Several investigators have documented increases in uterine and ovarian volume during childhood, with an increase in the number and size of the developing follicles in the years leading up to puberty 3,5,6. The changes observed with ultrasound agree with those observed at postmortem 7. Many studies have described pelvic sonographic variations in pubertal abnormalities, some of them reporting that sonography is useful to differentiate central precocious puberty from isolated premature thelarche or premature adrenarche, others describing wide overlap in quantitative and qualitative findings in the above pathological conditions 1,5,8,9. The use of color Doppler ultrasound facilitates the detection of small vessels in the utero-ovarian circulation and the Correspondence: Dr C. Battaglia, Reproductive Medicine Unit, University of Bologna, Via Massarenti, Bologna, Italy ( battaglia@med.unibo.it) Accepted ORIGINAL PAPER

2 measurement of impedance to flow in this vascular tree. However, to the best of our knowledge, only one paper has analyzed the influence, during puberty, of hormonal changes and subsequent internal genitalia transformations on uterine vascular modifications 10. The aim of our study was to evaluate the role of gray-scale ultrasound and color Doppler analyses in improving the diagnosis of GnRH-dependent isosexual precocity suitable for treatment with GnRH agonists. MATERIALS AND METHODS Study population The study protocol was in accordance with the Helsinky II declaration and was approved by the Hospital Research Review Committee. Twenty-nine girls were referred to Modena Hospital Auxological and Paediatric Endocrine Clinic for the evaluation of premature breast development and pubic hair growth. By definition, both symptoms developed before the age of 8 years. Girls participated in the study after both informed consent from parents and agreement from the minors had been obtained. On the basis of history, physical examination, basal sonography and laboratory data, excluded from the study were patients with: chronic disease, Cushing s syndrome, hyperprolactinemia, ovarian cysts (> 10 mm in maximum diameter), and polycystic ovaries (> five subcortical follicles 2 10 mm in maximum diameter, increased ovarian volume and increased ovarian stroma echogenicity) 11. Cases of GnRH-independent puberty (i.e. hypo/hyperthyroidism, sex steroid-secreting tumors, congenital adrenal hyperplasia) were also excluded, as were patients with isolated premature pubarche and isolated premature thelarche. No patients had received hormonal therapy before the study. The pubertal development in all girls was staged by a single examiner (L.I.) according to the classification of Tanner and Marshall 12,13. Staging of the breast was as follows: stage I, preadolescent; stage II, elevation of breast and papilla as a small mound, enlargement of areola diameter; stage III, further enlargement of breast and areola with no separation of contours; stage IV, projection of areola and papilla to form a secondary mound above the level of the breast; stage V, projection of a mature papilla and areola part of the general breast contour. Staging for pubic hair was as follows: stage I, preadolescent; stage II, sparse, slightly pigmented, straight; stage III, darker, coarser, beginning to curl, increased amount; stage IV, adult in type, but less area covered; stage V, adult in quantity and type, distributed as an inverse triangle, spread to medial surface of thighs. Standing height was measured using a Harpenden stadiometer (Holtain Ltd, Crymych, UK) to the nearest 0.1 cm; weight was measured on a digital scale with a precision of 0.1 kg (SECA 707, HH, Modena, Italy). The body mass index (BMI = weight (kg)/(height 2 (m 2 )) was calculated in all patients. Skeletal maturation was staged according to Greulich and Pyle 14. All the girls were further submitted to basal hormonal assay, GnRH stimulation test and ovarian and uterine gray-scale sonographic and color Doppler evaluation. On the basis of the GnRH stimulation test, the patients were subdivided into girls presenting serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) prepubertal response (Group I, n = 9) or pubertal response (Group II, n = 20). The first group was submitted to an adequate follow-up program (careful examination of the rate of progression of physical changes, linear growth and bone maturation, and of hormonal variations), whereas the latter was submitted to hypothalamic pituitary ovarian axis suppression with GnRH agonists and periodical auxological, clinical and hormonal evaluations. Ultrasound and Doppler examination Uterine and ovarian sonographic examinations were performed with the use of a 3.5-MHz convex transducer (AU4 Idea; Esaote, Milan, Italy). The ultrasound scans were performed transabdominally when the participants had a full bladder, obtained by voluntary urine retention and oral administration of fluids. Uterine and ovarian volumes, endometrial halo and thickness, number, diameter and distribution of the follicles were recorded. Volumes were calculated by measuring length, width and depth assuming the forms to be ellipsoid, using the formula based on a prolate ellipsoid: V = π/6 D1 D2 D3, where D1, D2, and D3 are the maximal longitudinal, anteroposterior and transverse diameters. The maximum diameter of each follicle was reported. No significant differences between left and right ovaries were observed; therefore the average value of both ovaries was used for statistical analysis. The presence of a midline endometrial echo was checked and endometrial thickness was measured as the distance between the two internal sides of the myometrium. Doppler flow measurements of the uterine vessels was performed transabdominally with a 3.5-MHz color Doppler system (AU4 Idea color Doppler, Esaote, Milan, Italy). All the patients were in a recumbent position and were evaluated between and h to exclude the effects of circadian rhythmicity on utero-ovarian blood flow 15. Furthermore, they rested in a waiting room for at least 15 min before being scanned in order to minimize external effects on pelvic blood flow 16. A 50-Hz filter was used to eliminate low-frequency signals originating from vessel wall movements. Color flow images of the ascending branches of the uterine arteries were sampled laterally to the cervix in a longitudinal plane 17. The angle of insonation was always adjusted to obtain maximum color intensity. When good signals were obtained, blood flow velocity waveforms were recorded by placing the sample volume across the vessel and activating the pulsed Doppler mode. The pulsatility index (PI), defined as the difference between the peak systolic and end-diastolic flow divided by the mean maximum flow velocity, was calculated for the uterine arteries. For each examination, the mean value of three consecutive waveforms was obtained. No significant differences between the PIs of the left and right uterine arteries were observed and therefore the average value of both arteries was used. The correlation between PI and heart rate was not tested 11. Ultrasound and color Doppler analyses were performed by a single examiner (C.B.) who was unaware of the response to the GnRH test. Ultrasound in Obstetrics and Gynecology 387

3 Hormonal assay Peripheral blood was obtained from all patients between and h, after an overnight fast, on the same day that sonographic and Doppler examinations took place, and different hormonal parameters were analyzed. Plasma concentrations of LH, FSH, estradiol (E 2 ), and testosterone (T) were assayed as previously described 18. Dehydroepiandrosterone sulfate (DHEAS), and 17-hydroxy progesterone (17-OH-Pg) were determined by radioimmunoassay using the Coat-A- Count kit (DPC; Los Angeles, CA, USA). Androstenedione (A) was measured by RIA with the Quantitative Measurement of Androstenedione in Serum and Plasma kit (DSL Inc., Webster, TX, USA). All hormone analyses were performed in duplicate. The GnRH stimulation test was performed using a standard dose of 100 µg GnRH administered as an intravenous bolus. Serum LH and FSH concentrations were measured at 0, 30, 60 and 90 min. For defining a pubertal GnRH test the following criteria had to be fulfilled: a baseline LH value > 0.3 IU/L, a peak LH level > 15 IU/L, a LH/FSH peak ratio > 0.66 and a LH -value (peak basal value) > 7 IU/L All samples were stored at 20 C until they were assayed. Results of hormonal values were converted to SI units using the following conversion factors: LH (IU/L) = miu/ml 1.0; FSH (IU/L) = miu/ml 1.0; E 2 (pmol/l) = pg/ml 3.761; T (nmol/ L) = ng/ml 3.467; A (nmol/ L) = ng/dl ; 17-OH-Pg (nmol/ L) = ng/dl ; DHEAS (µmol/ L) = µg/ml Statistical analysis Statistical analysis (SPSS software; SPSS Inc, Chicago IL, USA) was performed using the Mann Whitney test and one-way analysis of variance. The relationship between the parameters analysed was assessed using the stepwise linear regression method. A probability (P) of < 0.05 was considered as statistically significant. Data are presented as mean ± standard deviation, unless otherwise indicated. RESULTS All the patients completed the study. The basal plasma LH, FSH, E 2, T, A, 17-OH-Pg and DHEAS concentrations and the value of the GnRH stimulation test are reported in Table 1. The chronological age (CA), although lower in girls with prepubertal response to the GnRH test (Group I), was not significantly different between the groups (Table 2). However, the bone age (BA) was significantly higher in Group II than in Group I girls (Table 2). The BA/CA ratio was not significantly different among the whole studied population; however, in girls with a pubertal response to the GnRH test, the BA was always > 2 years above the CA. The basal height and BMI were significantly lower in Group I than in Group II (Table 2). None of the studied patients was hirsute. According to the Tanner scale, all the girls presented a breast stage of 2 3 and pubic hair stage of 2 3. No significant differences were noted among the two groups. Axillary hair was present in 5/20 in Group II and 1/9 in Group I. The ultrasound assessment allowed measurement of the uterine volume in 100% of the cases. The uterine size was greater in those girls who presented a pubertal response to the GnRH test (Group II, 8.07 ± 4.47 ml) than in those who presented a prepubertal response (Group I, 3.07 ± 1.18 ml; P = 0.001). In addition, the uterine volume exceeded our own normal reference range (1.5 4 ml) for prepubertal stage in 90% and 22% of the cases in Group II and Group I patients, Table 1 Hormonal value at baseline and after the GnRH test in girls with sexual precocity Pubertal response (n = 20) (Mean (SD)) Prepubertal response (n = 9) (Mean (SD)) P LH (IU/L) 1.81 (1.42) 0.20 (0.18) < FSH (IU/L) 4.33 (1.5) 2.05 (1.87) E 2 (pmol/l) 132 (39) 43 (9) < T (nmol/l) 128 (60) 42 (19) A (nmol/l) 4.9 (1.4) 2.1 (0.6) OH-Pg (nmol/l) 4.6 (1.6) 3.2 (1.2) DHEAS (µmol/l) 4.8 (1.6) 2.4 (1.5) GnRH-test LH peak (IU/L) 19.7 (2.7) 2.7 (2.2) < FSH peak (IU/L) 11.0 (6.0) 9.9 (8.2) LH peak/fsh peak 1.94 (1.14) 0.31 (0.12) < LH, luteinizing hormone; FSH, follicle stimulating hormone; E 2, estradiol; T, testosterone; A, androstenedione; 17-OH-Pg, 17-hydroxy progesterone; DHEAS, dehydroepiandrosterone sulfate; GnRH, gonodotrophin releasing hormone. Table 2 Clinical and auxological data in girls with sexual precocity Pubertal response (n = 20) (Mean (SD)) Prepubertal response (n = 9) (Mean (SD)) P Chronological age (years) 7.3 (0.5) 6.6 (0.6) Bone age (years) 9.8 (0.7) 8.0 (0.4) 0.01 Height (cm) 130 (8) 119 (9) Body mass index (kg/m 2 ) 18.9 (2) 17.1 (1.9) Ultrasound in Obstetrics and Gynecology

4 respectively. Endometrial echo was observed in 80% and 44% and endometrial thickness was 3.0 ± 1.1 mm and 2.1 ± 0.8 mm, in Group II and Group I girls, respectively. In 25 of 29 (86%) participants, both ovaries were visualized. Only one ovary could be visualized in three girls in Group II and in one girl in Group I. The ovarian volume (2.05 ± 1.19 vs ± 1.31) and the number of small (< 1 cm) follicles (2.66 ± 2.91 vs ± 2.80) was not significantly different between girls who presented a prepubertal or a pubertal response to the GnRH test. On Doppler analysis, the uterine arteries were adequately visualized in 100% of the cases and lower impedance was observed in Group II (PI = 2.29 ± 0.19) than in Group I (PI = 3.28 ± 0.37; P = 0.001) (Figure 1). A low PI ( 2.5) was considered an expression of the rapidly growing uterus. The presence of low impedance to flow at the level of the uterine arteries had a generally high diagnostic value for precocious puberty (Table 2). The diagnostic value of Doppler analysis was more accurate than that of uterine volume (cut-off value, 4 ml) and endometrial echo (presence/absence) (Table 3) evaluations. In the total study population, plasma LH concentrations directly correlated with plasma E 2 values (r = 0.545; P = 0.003) and inversely correlated with uterine artery PI (r = 0.600; P = 0.001). In addition, uterine artery PI inversely correlated with both LH peak (r = 0.490; P = 0.007) and LH/FSH ratio (r = 0.571; P = 0.001) after the GnRH stimulation test. Finally, a direct correlation was found between plasma E 2 values and uterine volume (r = 0.535; P = 0.003) and a slight inverse correlation between plasma E 2 values and uterine artery PI (r = 0.410; P = 0.045). DISCUSSION Figure 1 Color Doppler analysis of uterine arteries; transabdominal approach with full bladder technique. Color flow images of ascending branches were sampled laterally to the cervix in a longitudinal plane. (a) True precocious puberty (PI = 1.83). (b) Intermediate form of precocious puberty (PI = 2.71). Puberty is a process whereby, at the age of 9 13 years, a child becomes an adult as a result of the maturation of the hypothalamic pituitary ovarian axis. Sexual precocity is the development of secondary sexual characteristics earlier than normal and includes: isolated premature thelarche (isolated breast development, slight increase of plasma E 2 and FSH concentrations, no advanced bone age, normal height outcome) 22, isolated premature adrenarche (isolated development of pubic hair in response to the secretion of adrenal androgens, normal height outcome) 23, and precocious puberty (pubertal maturation, linear growth acceleration and bone maturation with shorter adult height outcome, pubertal response to the GnRH stimulation test). Isolated premature thelarche and premature adrenarche do not require any specific treatment, necessitating only close follow-up. To inhibit pubertal progression and improve adult height, standard treatment of GnRH-dependent precocious puberty involves the suppression of the hypothalamic pituitary ovarian axis with GnRH-agonists 24, whereas GnRH-independent precocious puberty requires the solution of the underlying problem. Although a definitive policy is applied for the treatment of unequivocal pathological entities, an open debate exists on the intermediate forms of precocious puberty. Stanhope and Brook 25 recently described a thelarche variant with features intermediate between isolated premature thelarche and true precocious puberty (persistent breast development without enhanced gonadotrophic secretion and with prepubertal Table 3 Diagnostic value for sexual precocious puberty of uterine artery Doppler analysis (PI 2.5), sonographic uterine volume (4 ml cut-off) and presence of an endometrial echo Doppler Uterine volume Endometrial echo Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Concordance (%) Ultrasound in Obstetrics and Gynecology 389

5 response to GnRH test). Similarly, Palmert et al. 4 reported on girls with early breast development and pubic hair growth, but with a prepubertal response to the GnRH test; and Fontoura et al. 26 described a slow progressive form of precocious puberty characterized by an early but slow progression of the clinical symptoms and with a BA < 2 years above CA. In these studies none of the girls with intermediate forms of precocious puberty was treated and the adult final height was not compromised. From these considerations we can deduce that not all girls with apparently true precocious puberty need medical treatment and that it is important to correctly diagnose the activation of the hypothalamic pituitary ovarian axis in order to avoid unnecessary treatment. In our study, patients with polycystic ovaries, GnRHindependent precocious puberty, and isolated premature thelarche or adrenarche were excluded. Thus the GnRH stimulation test was administered only to distinguish between intermediate forms and true precocious puberty. Although the stimulation test is considered the gold standard, there is no concordance as the criteria to use for the diagnosis. Palmert et al. 4 considered as prepubertal a response to GnRH stimulation with a FSH peak > LH peak and a LH peak < 25 IU/L, while Sklar et al. 21 considered as prepubertal a response characterized by a maximum increase from baseline of < 7 IU/L. Oerter-Klein et al. 19 defined as an expression of true precocious puberty, a response with a LH peak > 15 IU/L and/or a peak LH/peak FSH ratio > In addition, Neely et al. 20 reported that a baseline LH value > 0.3 IU/L or a GnRH stimulated peak > 5 IU/L may be diagnostic of precocity. Even though the criteria used by Oerter-Klein et al. 19 reached an elevated diagnostic value (96% sensitivity, 100% specificity, no false positives), there is no basal or stimulated single level of LH, FSH or E 2 with 100% sensitivity and specificity for precocious puberty. In our study, to improve the diagnostic value of hormonal evaluation and to allow us to consider the GnRH stimulation test as the gold standard, we adopted very strict cumulative criteria. Pelvic ultrasound examination has proved to be an accurate, painless and non-invasive investigation that may give important information about internal genitalia 27. In girls being evaluated for any disturbance in sexual maturation, sonography is generally considered as an adjunctive method in establishing the exact diagnosis of precocious puberty or, as reported, it may replace more invasive procedures such as pelvic transrectal examination, endocrine stimulation tests and laparoscopy 28. In almost all the studies it has been found that subjects with precocities have enlarged ovaries, an increased uterine volume and the development of a midline endometrial echo 2,5,6,8. However, by evaluating the diagnostic value of these sonographic findings it has been found that although associated with a good specificity, their sensitivity is low due either to the transabdominal approach or to the great overlap between normal prepubertal and pathological values. It has been shown that the anatomical structure of the pelvic organs could not be adequately assessed with a transabdominal approach in as many as 42% of cases 29 (due to obesity, limited resolution of low-frequency transducers, troublesome full bladder technique and dilated bowel loops) 30 and that transvaginal sonography may provide a more accurate picture of the uterus and ovaries 31. Although we did not perform transvaginal ultrasound examinations in our patients, owing to their young ages and virginal status, we visualized the uterine structures in 100%, both ovaries in 86% and at least one ovary in 100% of cases. From this we deduced that the low diagnostic value of sonography is not dependent on the use of the transabdominal approach but rather on the operator s skill and the quality of the ultrasound equipment. On the other hand, we showed that the diagnostic value of increased uterine volume (> 4 ml) and the presence of a midline endometrial echo has an adequate specificity (90% and 80%, respectively) but a low sensitivity (78% and 56%, respectively) and a low concordance with the GnRH stimulation test (76% and 69%, respectively). These data agree with those of Griffin et al. 3, but are not in accordance with those of Haber et al. 8. We observed that, for the above findings, a great overlap exists between true precocious puberty and its intermediate forms, making it impossible to define clear cutoff values. Thus, we speculated that only normal endometrial echogenicity and normal uterine volume, reflecting an infantile morphology, are useful sonographic parameters to exclude the activation of the hypothalamic pituitary gonadal axis. Salardi et al. 27 stated that if... ovarian size is within the normal range... it is highly likely that patients have adrenarche or premature thelarche. Our data do not support this: neither increased ovarian volume nor modified ovarian morphology was diagnostic for true precocious puberty. Among the groups, a homogeneous pattern was observed in few ovaries and there were no significant differences in terms of ovarian volume and follicular number and distribution. Although the incidence of small (< 1.0 cm) follicular cysts increases with pubertal development, they are commonly found in the ovaries of prepubertal girls. Many factors (a rise in gonadotrophin secretion or pulsatility and IGF-1 paracrine action) may, in fact, stimulate the follicular selection and development. This limits the usefulness of measurements of follicular number and diameter in the assessment of pubertal status. Contrary to the findings of Mosfeldt Laursen et al. 10, uterine artery flow was found in 100% of our population and measurable diastolic flow was observed in almost three quarters of the studied girls. In addition, we found that color Doppler analysis of the uterine arteries agreed well with the findings of the GnRH stimulation test and was a useful diagnostic tool to differentiate between true precocious puberty and its intermediate forms. Uterine artery PIs, which seem to reflect arterial tone or modifications in resistance to flow in the vascular bed, inversely correlated with plasma LH concentrations. Elevated LH plasma levels may be responsible for increased stromal vascularization by different mechanisms that may act individually or in a cumulative way: neoangiogenesis, catecholaminergic stimulation and leukocyte and cytokine activation The intrinsic interdependence between increased uterine vascularization and premature puberty is further demonstrated by an inverse relationship between uterine artery resistances and LH peak and LH/FSH ratio after the GnRH test. In addition, in girls with an activated hypothalamic pituitary gonadal axis, the effects on uterine vascularization may be enhanced by estrogen activity, as underlined in our study by an inverse correlation between uterine artery PIs and 390 Ultrasound in Obstetrics and Gynecology

6 circulating E 2 levels. Horowitz and Horowitz 35 found receptors for estrogens in the great vessels and the data of other investigators strongly suggest that estrogens have effects on vessel wall physiology 36,37. Estradiol has been shown to decrease vascular resistances either by exerting a direct action on the smooth muscle cells in the media of the uterine artery vessel wall or by indirectly decreasing the calcium-mediated vessel constriction and/or the periarterial sympathetic vasoconstrictor nerve activity. Furthermore, estrogens seem to increase the nitric oxide production and ameliorate the plasma viscosity and prostacyclin/thromboxane balance 38,39. Further longitudinal and extensive studies are necessary for the better understanding of the relationship between the low vascular impedance of uterine arteries and hormonal modifications due to an activated hypothalamic pituitary gonadal axis. However, we conclude that color Doppler analysis of uterine arteries may improve the diagnosis of GnRH-dependent isosexual precocity and may be useful to determine which girls need treatment. REFERENCES 1 Cohen L, Haller J. Pediatric and adolescent genital abnormalities. In Babcock DS, ed. Neonatal and Pediatric Ultrasonography. New York: Churchill Livingstone, 1989: Oerter Klein K. Editorial: precocious puberty: who has it? Who should be treated? J Clin Endocrinol Metab 1999; 84: Griffin IJ, Duncan KA, Hollman AS, Donaldson MDC. Pelvic ultrasound findings in different forms of sexual precocity. Acta Paediatr 1995; 84: Palmert MR, Malin HV, Boepple PA. Unsustained or slowly progressive puberty in young girls: initial presentation and long-term follow-up of 20 untreated patients. J Clin Endocrinol Metab 1999; 84: Buzi F, Pilotta A, Dordoni D, Lombardi A, Zaglio S, Adlard P. Pelvic ultrasonography in normal girls and in girls with pubertal precocity. Acta Pædiatr 1998; 87: Bridges NA, Cooke A, Healey MJR, Hindmarsh PC, Brook CGD. Ovaries in sexual precocity. Clin Endocrinol 1995; 42: Peters H, Himelstein-Braw R, Faber M. The normal development of the ovary in childhood. Acta Endocrinol 1976; 82: Haber HP, Wollman HA, Ranke MB. Pelvic ultrasonography: early differentiation between isolated premature thelarche and central precocious puberty. Eur J Pediatr 1995; 154: Holm K, Mosfeldt Laursen E, Brocks V, Muller J. Pubertal maturation of the internal genitalia: an ultrasound evaluation of 166 healthy girls. Ultrasound Obstet Gynecol 1995; 6: Mosfeldt Laursen E, Holm K, Brocks V, Jarden M, Müller J. Doppler assessment of flow velocity in the uterine artery during pubertal maturation. Ultrasound Obstet Gynecol 1996; 8: Battaglia C, Genazzani AD, Salvatori M, Giulini S, Artini PG, Genazzani AR, Volpe A. Doppler, ultrasonographic and endocrinological environment with regard to the number of small subcapsular follicles in polycystic ovary syndrome. Gynecol Endocrinol 1999; 13: Tanner JM. Growth at adolescence. Oxford: Blackwell and Mott, Marshall WA, Tanner JM. Variations in pattern of pubertal changes in girls. Arch Dis Child 1969; 44: Greulich WW, Pyle SI. Radiographic atlas of skeletal development of the hand and wrist. Stanford: Stanford University Press, Zaidi J, Jurkovic D, Campbell S, Okokon E, Tan SL. Circadian variation in uterine artery blood flow indices during the follicular phase of the menstrual cycle. Ultrasound Obstet Gynecol 1995; 5: Battaglia C, Artini PG, D Ambrogio G, Galli PA, Genazzani AR. Uterine and ovarian blood flow measurement. Does the full bladder modify the flow resistance? Acta Obstet Gynecol Scand 1994; 73: Battaglia C, Artini PG, D Ambrogio G, Genazzani AD, Genazzani AR. The role of color Doppler imaging in the diagnosis of polycystic ovary syndrome. Am J Obstet Gynecol 1995; 172: Battaglia C, Genazzani AD, Artini PG, Salvatori M, Giulini S, Volpe A. Ultrasonographic and color Doppler analysis in the treatment of polycystic ovary syndrome. Ultrasound Obstet Gynecol 1998; 12: Oerter KE, Uriarte MM, Rose SR, Barnes KM, Cutler GB. Gonadotropin secretory dynamics during puberty in normal girls and boys. J Clin Endocrinol Metab 1990; 71: Neely EK, Hintz RL, Wilson DM. Normal ranges for immunochemiluminometric gonadotropin assays. J Pediatr 1995; 127: Sklar C, Rothenberg S, Blumberg D, Oberfield SE, Levine L, David R. Suppression of the pituitary-gonadal axis in children with central precocious puberty: effects on growth, growth hormone, insulinelike growth factor and prolactine secretion. J Clin Endocrinol Metab 1991; 73: Wilkins L. Diagnosis and treatment of endocrine disorders in childhood and adolescence, 3rd edn. Springfield: Thomas, Ibanez L, Virdis R, Potau N, Zampolli M, Ghizzoni L, Albisu MA, Carrascosa A, Bernasconi S, Vicens-Calvet E. Natural history of premature pubarche: an auxological study. J Clin Endocrinol Metab 1992; 74: Comite F, Cassoria F, Barns KM. Luteinizing hormone releasing hormone analogue therapy for central precocious puberty: long-term effect on somatic growth, bone maturation, and predicted height. JAMA 1986; 255: Stanhope R, Brook CGD. Thelarche variant: a new syndrome of precocious sexual maturation? Acta Endocrinol 1990; 123: Fontoura M, Brauner R, Prevot C, Rappaport R. Precocious puberty in girls: early diagnosis of a slow progressing variant. Arch Dis Child 1989; 64: Salardi S, Orsini LF, Cacciari E, Partesotti S, Brondelli L, Cicognani A, Frejaville E, Pluchinotta V, Tonioli S, Bovicelli L. Pelvic ultrasonography in girls with precocious puberty, congenital adrenal hyperplasia, obesity or hirsutism. J Pediatr 1988; 112: Hull MGR. Polycystic ovarian disease: clinical aspects and prevalence. Des Clin Forums 1989; 11: Pache TD, Wladimiroff JW, Hop WCJ, Fauser BCJM. How to discriminate between normal and polycystic ovaries: transvaginal US study. Radiology 1992; 183: Takahashi K, Nishigaki A, Eda Y, Yamasaki H, Yoshino K, Kitao M. Transvaginal ultrasound is an effective method for screening in polycystic ovarian disease: preliminary study. Gynecol Obstet Invest 1990; 30: Findlay JK. Angiogenesis in reproductive tissues. J Endocrinol 1986; 111: Kawakami M, Kubo K, Uemura T, Nagase M, Hyashi R. Involvement of ovarian innervation in steroid secretion. Endocrinol 1981; 109: Brannstrom M, Norman RJ. Involvement of leukocytes and cytokines in the ovulatory process and corpus luteum function. Hum Reprod 1993; 8: Horowitz KB, Horowitz LD. Canine vascular tissues are targets for androgens, estrogens, progestins, and glucocorticoid. J Clin Invest 1982; 69: Bourne TH, Hillard TC, Whitehead MI, Crook D, Campbell S. Oestrogen, arterial status and postmenopausal woman. Lancet 1990; 95: De Ziegler D, Bessis R, Frydman R. Vascular resistance of uterine arteries: physiological effects of oestradiol and progesterone. Fertil Steril 1991; 55: Mijatovic V, Pines A. Menopause-induced changes in cardiovascular functions and HRT. Eur Menopause J 1995; 2: Battaglia C, Salvatori M, Gallinelli A, Artini PG, Gennazzani AR, Volpe A. Color doppler and hormone replacement therapy: the role of thromboxane and plasma viscosity. Gynecol Endocrinol 1999; 13: Ultrasound in Obstetrics and Gynecology 391