Chapter 2


Normal and Abnormal Puberty


Normal pubertal development


Normal puberty is the transitional period between childhood and adulthood, characterised by physiological, anatomical and psychological changes denoting physical growth, sexual maturation and readiness for reproductive function. It is also characterised by changes in the relationship of the pubertal girls with her family and peers which reflects her cognitive development, and newly perceived sexuality. In contrast, the climacteric is the period of time, denoting a decline and finally cessation of reproductive function ending with the menopause. To be able to deal with abnormal pubertal problems, one should understand the different stages leading to a full functioning hypothalamo-pituitary-ovarian (HPO) axis with intact and functioning genital organs.

Four different stages have been recognised leading to full development and maturation of the HPO axis to its adult state. They have already been described in some detail in Chapter 1.


Anatomical maturation

Variations in pubertal development in girls have been documented 40 years ago by Marshall and Tanner in 1969 (1). Accordingly, this section would give the general pattern of such development. Breast development (thelarche) is usually the first visible sign of puberty following activation of the HPO axis, and increased production of oestradiol by the granulosa cell in response to FSH stimulation. Plasma levels of oestradiol during early puberty fluctuate widely, which reflects the growth and atresia of successive groups of follicles without reaching ovulation. Thelarche could occur at different times in different racial groups, but usually by the age of 8-11 years. It might be unilateral to start with. However, it could follow pubic hair growth in almost 15% of the cases. Racial differences have been reported in this respect, and one report showed that pubic hair growth preceded thelarche in most African-American pubertal girls (Herman-Giddens 1997 (2). The same study showed that 30% of African-American girls had started pubertal development by the age of 7 years, and 50% by the age of 8 years. The corresponding figures for Caucasian girls were 15% by the age of 8 years, and 40% by the age of 9 years. Development of the pubic hair and breasts, during puberty, has been documented by Tanner in 1962 (3) as shown in the table 1.

Table 1 shows the different development stages in Tannersí classification of puberty (Tanner 1962 (3).

 

Stage

Breast development

Pubic hair development

1

No breast development

No pubic hair

2

 Breast bud stage

Sparse labia major hair

3

More growth with appearance of areola

Hair reaching mons pubis

4

Secondary mound of areola and papilla

Adult hair not reaching medial thighs

5

Areola recession with projecting papilla

Hair reaching medial thighs


The increased linear growth, in normal circumstances, is caused by increased levels of oestrogens and growth hormone. The role of adrenal androgens in linear growth in height has been questioned and refuted (Cutler Jr 1997 (4) and Grumbach and Styne 1998 (5). Some linear skeletal growth had already occurred by the time of thelarche, but a peak adolescent growth spurt of 8-9 cm/year usually corresponds to Tanner stage 2-3 breast development (Brook CGD 1993 (5). This usually falls down, thereafter, to 4 cm/year by the onset of menstruation. Complete fusion of the epiphyses usually occurs within 2 years after menarche.

Physiological and anatomical changes in the vulva, vagina and uterus also follow the secretion of oestrogens, increasing their thickness and size in preparation to reproductive function. A noticeable visual sign during vulvar inspection is the change in the vaginal skin from its red prepubertal colour to a well-oestrogenised pink adult colour. Ovarian development has also been tracked, and a volume >1.0 cm3 usually indicates the start of puberty. It increases rapidly in size, and attains an average volume of 4.0 cm3 by the end of puberty. These changes in ovarian volume reflect increased activity, and an increase in oestradiol level. Menarche usually takes place 2-3 years after the start of breast development at an average age of 12-13 years. This could also be affected by familial and racial factors that should be taken into consideration.


Before proceeding to abnormal pubertal development, it is important to remember the following two points:

  • Puberty is an age dependent maturation Pubertal CNS maturation is genetically dependent.
  • Onset of puberty differs in different families and racial groups. 


Abnormal Pubertal Development

 

It has been mentioned already that puberty starts between the ages of 8-14 years, though racial and familial differences do exist. Furthermore, the age of onset of puberty is getting lower and this should be taken into account when comparing information between different reports. Such development could be early or precocious before the age of 8 years. It could also be delayed in different ways including:

  • Absent secondary sexual characteristics by the age of 13 years
  • No menstruation by the age of 16 years with partial or complete development of secondary sexual characteristics
  • Passage of 5 years between thelarche and menarche


Delayed Puberty

To simplify the understanding and management of delayed puberty, a simple diagnostic classification would be used in this manuscript including simple delay, as well as anatomical, hypergonadotropic or hypogondotropic causes. Unlike boys where constitutional delay makes >50% of the cases, more than 80% of girls with delayed puberty have pathological causes (Traggiai and Stanhope 2002 (6); the commonest being Turnerís syndrome.

Anatomical causes could be further divided into mullerian dysgenesis and distal genital tract obstruction. Though they share a common subgrouping, they are very different developmentally.


Mullerian dysgenesis

Female genital anomalies occur in about 5% of women (Oppelt 2007 (7), and could be asymptomatic in many cases. They might be associated with other malformations, especially of the kidneys and skeletal system in about 30% of the patients. Normal development of the fallopian tubes, uterus, cervix and upper two thirds of the vagina depend on normal differentiation, fusion and canalisation of the two mullerian ducts during early fetal life. Such progression of normality could be disrupted at different stages of development leading to different anatomical abnormalities. In the context of menstrual function, the final outcome would be normal secondary sexual characteristics with no menstruation due to partial or complete absence of the uterus, cervix or upper vagina. In cases of cervical or upper vaginal agenesis with an intact uterus, the condition would simulate lower genital tract obstruction with cryptomenorrhoea and accumulation of blood into the uterus (haematometra). This would lead to an enlarged uterus which could be felt abdominally. Obstructive genital tract anomalies could lead to retrograde menstruation, pelvic pain and possibly the development of endometriosis (Sanfilippo JS, 1986 (8). Elevation of the bladder and urethral stretch could lead to retention of urine. Accordingly, continuous suppression of menstruation with an oral contraceptive pill would be indicated in these cases till surgical treatment is arranged.

A specific condition of complete or incomplete uterovaginal dysgenesis is the Mayer-Rokitansky-Küster-Haϋserís (MRKH) syndrome with an incidence of 1:4500 women. An absent vagina might be associated with an absent or very small uterus with normal ovaries and normal 46XX peripheral karyotyping. Accordingly, normal secondary female sexual characteristics would develop without menstrual periods.


Distal genital tract obstruction

Embryologically the lower vagina and vulva are not part of the mullerian system. They develop from the urogenital sinus. The main two anomalies in this group that could prevent discharge of menstrual blood are an imperforate hymen, and transverse vaginal septum.

An exact incidence of imperforate hymen is not confirmed, but has been estimated to occur in one / 1000-10,000 women. In a very small series, Heger et al in 2002 (9) found one case in 147 premenarchal girls with a mean age of 63 months (<1%). It could be diagnosed during the early neonatal examination, but more often after onset of puberty, due to obstruction of the menstrual blood flow. Diagnosis is usually easy, as a bulging mass with blue discolouration would be seen during vulvar inspection. Treatment is easy in this case, with simple incision of the hymen. It should be done under aseptic conditions to prevent ascending infection, as the collected blood is a good media for bacterial growth. However, this should be done only after affirmative diagnosis and exclusion of other causes of vaginal obstruction. Occasionally, a microperforate hymen might form allowing only trickles of menstrual blood loss. This could lead to prolonged menstrual blood loss and accumulation of blood in the vagina on top.

Transverse vaginal septae are less common than the imperforate hymen with an estimated incidence of 1 in 30,000-80,000 women. They could be found at different levels; being 46%, 40% and 14% in the upper, middle and lower parts of the vagina respectively. Upper septae are usually perforate and allow adequate discharge of menstrual blood, but lower ones are usually totally obstructive. The patient could have cryptomenorrhoea, which could lead to vaginal and uterine distension that is palpable abdominally. Diagnosis is usually not difficult. Ultrasound scan examination would reveal a uterus and upper vagina distended with altered fluid of mixed echogenicity. Surgical treatment might be more elaborate in comparison to simple incision of an imperforate hymen. It should involve excision of the septum and the scarred vaginal area surrounding it, to prevent future stricture formations. Accordingly, it should be done by a gynaecologist experienced in adolescent gynaecological surgery. Furthermore, an exact diagnosis is necessary to ascertain the thickness of the septum and the patency of the vagina proximal to it. MRI is the best modality for this purpose. To reduce the risk of scar formation, and hourglass constriction, vaginal dilators should be used after surgery.


Hypogondotropic delayed puberty

Patients with hypogonadotropic hypogonadism usually fail to develop secondary female sexual characteristics and would not start menstruating either. These patients have normal internal and external genital organs, normal peripheral karyotyping and low gonadotrophins and oestradiol blood levels. Constitutional delay makes the most common diagnosis in this diagnostic subgroup. However, other causes include:Isolated gonadotrophin deficiency (Kallmanís Syndrome) could be associated with anosmia as well as other somatic abnormalities. Abnormal development of the olfactory bulbs has been documented with MRI in these cases.

  • Under weight young females and certainly anorexics would have delayed pubertal development, and might not go through this developmental stage at all, depending on the suppression level of the HPO axis. Gymnasts usually fall within this subgroup.
  • Chronic systemic diseases could also interfere with the onset of puberty. Examples of such conditions include Crohnís disease, cystic fibrosis, tuberculosis, thalassaemia major, celiac disease and malnutrition.
  • It is also important to exclude intracranial tumours mainly pituitary adenomas and craniopharyngiomas.
  • Isolated deficiency of GH can lead to pubertal delay. It can reduce gonadal response to gonadotrophins. Normal pubertal growth can be restored by GH administration.
  • Suppression of the HPO axis could also follow hyperprolactinaemia, hyperthyroidism and Cushingís syndrome.Idiopathic panhypopituitarism could be a cause with other tropic hormones also affected, probably to different degrees.

Hypergonadotropic delayed puberty

This is a distressing situation as it usually indicates gonadal failure with little chance of normal recovery. Depending on the onset of the condition, a patient could present with no or partial secondary female sexual characteristics. The most common diagnosis in this group is gonadal dysgenesis. Normal development of the primitive gonads depends on the presence of one X chromosome. Further development depends on the presence or absence of testicular determining factor, encoded in the sex determining region gene (SRY). A primitive gonad would progress into an ovary in the absence of this factor, up to the stage of primary oocytes. Further development of these oocytes would depend on the presence of two X chromosomes; otherwise accelerated atresia of the primary oocytes would take place leading to gonadal dysgenesis with fibrous streak gonads. Accordingly, gonadal dysgenesis could be seen in different forms including 45XO, 46XX, 46XY and in different combinations or mosaics. Further information about this subject would be given in chapter xx. With pure gonadal dysgenesis patients usually fail to develop secondary sexual characteristics, but mosaics could present with variable phenotypes depending on the presence or absence of a Y chromosome.


Androgen insensitivity syndrome

In this diagnostic entity, patients with 46XY chromosomes develop secondary female sexual characteristics mainly in the form of good breasts development and body habitat. They usually have no or sparse axillary and pubic hair with failure to attain menarche. In this group, testicular development is complete with normal production of androgens, but deficiency of androgen receptors prevents masculinization of the urogenital sinus into a male gender. The patient would present with normal looking vulva and short blind vagina. Internal genital organs would be missing due to suppression of the mullerian ducts development. The testicles might be felt in the inguinal canals. The main diagnosis in this group is testicular feminization syndrome that should be differentiated form Mayer-Rokitansky-Kuster-Hauserís (MRKH) syndrome. The latter patients present with vaginal agenesis with absent or very small uterus, normal female 46XX peripheral karyotyping and blood testosterone levels within the normal female range in contradistinction to patients with androgen receptors insensitivity syndrome. On the other hand patients with partial rather than complete androgen receptors insensitivity could present with variable degrees of pubertal masculinization. Despite the involvement of the mammalian HOX genes in urogenital differentiation, a recent study showed that mutations within these genes were not involved as a cause of MRKH syndrome (Burel et al 2006 (10).

Precocious or accelerated puberty

Young girls could start pubertal development before the age of 8 years because of different reasons. Such precocity could be feminine (isosexual) or masculine (heterosexual). Such development could be complete or partial within the different two main groups.


Complete isosexual precocity

Central type

Central complete isosexual precocious puberty could follow premature activation of the HPO axis leading to gonadotrophins dependent oestradiol secretion. This is the most common type and is seen in almost 70% of the cases. It is interesting to note that such patients were found to have a high incidence of polycystic ovaries with clinical and biochemical hyperandrogenisation when examined at least 3 years after attaining menarche (Roberto Franceshi in press (11). The remaining 30% usually have neurogenic causes that are more common in extremely young girls at onset of puberty. Identified lesions include head trauma, hydrocephalus, postinfectious encephalitis, congenital brain defects and tumours including pituitary adenomas, hamartomas and astrocytomas (Cisternino, M 12), hence the need for MRI examination of the brain to exclude such pathology.  A recent study showed that MRI should be performed in all patients with central isosexual precocious puberty, as neither age nor any other clinical parameters could differentiate between patients with or without brain tumours (13).

Peripheral isosexual precocity

In this group, oestrogen production is not related to central activation of the HPO axis. The most common causes are ovarian tumours, especially of the granulosa and theca cell types. Diagnosis would be easy with the help of the relevant endocrine tests and pelvic ultrasound examination. Other causes include hypothyroidism, which could lead to high TRH production resulting in secondary stimulation of gonadotrophins secretion by the pituitary gland. Early diagnosis and treatment of the hypothyroidal state would prevent the development of such precocious puberty. This entails inclusion of thyroid indices in the endocrine testing of such patients. Rarely, oestrogen producing adrenal tumours could be the cause. Yet again, on rare occasions, ingestion of neglected hormones, especially the oral contraceptive pill, could result in such premature development. However, this could lead to abnormal uterine bleeding and might cause some development of the breasts rather than full blown precocious isosexual puberty.

This section would not be complete without mentioning McCune Albright syndrome, which is characterised by autonomous oestrogen secretion by the ovaries with low gonadotrophins blood level. Patients are usually identified with the café-au-lait skin patches characteristic of the syndrome and could have bone fibrous dysplasia as well.

Incomplete isosexual puberty

This is the situation when one secondary sexual characteristic is developed early in isolation, with no other sign of pubertal development. The three main categories are:

1.   Premature development of the breast (thelarche) before the age of 8 years is the most common in this group. It is usually unilateral and no treatment is needed in most cases other than reassurance. A hormone test would show normal oestradiol and LH levels with occasional slight increase in FSH level.

2.   Premature adrenarche entails early development of axillary hair before the age of 8 years. It could also be associated with early change in body odour, due to activation of the apocrine axillary sweat glands. This could be the first sign detectable by the parents. It does not need any treatment if it stayed as an isolated non progressive condition. Follow up would be needed to ascertain no further development.

3.   Premature pubarche indicates growth of pubic hair before the age of 8 years as well. It is more sinister than premature axillary hair development, as it might follow adrenal or organic brain diseases. However, in the majority of cases it follows functional increase in adrenal gland androgen production. This could also lead to a full blown precocious puberty of the heterosexual type.

Heterosexual precocious puberty

Development of secondary male characteristics in an individual brought up as a girl could have devastating effects on the patient and her family as well. Many causes could be involved with such premature hyperandrogenisation, but misuse of drugs should be excluded before starting any further complicated investigations.

The most common cause of such hyperandrogenisation is partial adrenal 21-hydroxylase enzymatic deficiency. Other enzymatic defects could be involved less frequently. Further information would be covered in chapter 4, dealing with the adrenal factor in gynaecology. Ovarian, and less frequently adrenal tumours could be seen in such cases and should be looked for especially in patients with rapid and severe hyperandrogenisation.

It is important to address abnormal production of androgens as early as possible because of its negative impact on the patient and her parents. Early management may prevent disfiguring skin lesions and rapid linear growth that usually results in a final short adult stature

Management of abnormal pubertal development

Only reassurance may be necessary with little or no intervention needed in many cases. Nevertheless, it is important to differentiate patients with underlying pathological conditions from those with mere constitutional or benign pubertal changes. This is especially so in cases of premature thelarche and those with isolated premature growth of axillary hair. However, a more active approach would be needed in cases of early heterosexual precocity, due to the long lasting implications of the condition. Early referral to specialist care will be necessary in such cases, if local expertise is not available. 

Thorough history should be taken including childhood disease especially in cases of delayed puberty. Nutritional history should be ascertained in underweight girls. Similar pubertal developmental abnormality in other family members could point towards a familial tendency. The age the patientís mother and her elder siblings have gone through puberty is also important. Family history of genetic diseases should be sought as well. History of drug misuse, especially neglected oral contraceptive pills is important to elicit, though it is usually denied. 

Clinical assessment of the patientsí phenotype could reveal signs of chromosomal abnormalities and chronic systemic or endocrine diseases. Examination should include height, weight, arms span and blood pressure. Tannersí breasts and pubic hair staging (Tanner 1962 (3) should be documented as well (Table 1). The breast should also be examined in the supine position especially in overweight girls to guard against errors caused by fat. Other somatic and endocrine signs should be noted and included within the total management plan of the case. This is especially so for hyperandrogenic signs, and signs of thyroid dysfunction. Neurological examination should be conducted including examination of the optic fundi, visual fields and sense of smell. However, it is important not to put these young patients into undue stress during examination and they should not be overexposed. The presence of a female member of staff and the mother usually gives reassurance during medical examination. Inspection of the genital organs is important but digital vaginal examination should be done only if the patient is sexually active. It could reveal a blind vagina. Rectal examination should not be attempted as more useful information could be obtained with ultrasound scan examination. Any mass in the groin or anterior abdominal wall should raise the suspicion of a dislocated testicle and should be ascertained with imaging. For follow up purposes, growth velocity should be plotted in a growth chart. This will show the exact growth rate in a fixed time scale, and allow comparison of current height with previous or old records.

Investigations should depend on the patientís age, her mode of presentation and provisional clinical diagnosis. Endocrine tests should include FSH, LH, oestradiol, thyroid indices, 17 hydroxyprogesterone, androgens and prolactin, as indicated. A difference between FSH and LH should be appreciated at this point. Both are produced in pulses with progressive increase in the peak of each hormone during the progressive stages of puberty. However, only the lower limit of LH increases at the same time, but not FSH, and could be used for the diagnosis of precocious puberty (Apter et al 1989 (14) and Brito et al 1999 (15). Pelvic and abdominal ultrasound scan examinations would be necessary to visualise the uterus and ovaries. It would also help in cases of suspected haematometra and haematocolpos. Peripheral karyotyping is also indicated especially in cases of heterosexual precocity, suspected gonadal dysgenesis, hypergonadotropic amenorrhoea and resistant androgen receptors syndrome. Bone age is also important to ascertain with a left hand and wrist X-ray examination against a standard atlas. It should be used to give a meaningful interpretation of hormonal results related to the HPO axis rather than chronological age. It is also useful in differentiating between premature thelarche or adrenarche and true precocious puberty. Furthermore, it is also necessary for deciding when to start treatment in cases of delayed and precocious puberty, and to monitor response. Recently ultrasound examination has been introduced instead, for assessing bone age. If bone age is within one year of chronological age, then puberty has just started, or has not started yet. On the other hand, if the bone age is 2 years more advanced than the chronological age, then puberty has definitely started.

Brain tumours and pituitary adenomas should be investigated with MRI, when indicated. This is especially so in patients with neurological symptoms or signs. Dynamic testing of the pituitary gland with a bolus dose of GnRH analogue may add some helpful information. A brisk response of both gonadotrophins indicates delayed puberty; where as an exaggerated response of only FSH is a marker of partial primary ovarian failure.

Follow up of development and response to treatment should be documented with digital photographs, after seeking consent, and the photographs kept by the family.

Treatment of abnormal puberty

Treatment of abnormal pubertal development depends on the age of the patient, her mode of presentation, bone age and final diagnosis. Delayed puberty should be treated expectantly with regular follow up. This is especially so if there is delay in bone age relative to chronological age, which indicates further growth potential. 

Central isosexual precocity should be treated with GnRH analogues to suppress gonadotrophins and oestrogens production. This is especially important in patients with advanced bone development in relation to chronological age. Patients with heterosexual precocious puberty should be treated similarly as early as possible to prevent masculinization and ultimate short stature. Non-classical congenital adrenal hyperplasia is a good example and should be treated with hydrocortisone replacement therapy. Medication should be monitored and titrated against serial 17 hydroxyprogesterone and androstenedione blood levels. 

It is important to remove dysgenetic gonads in patients with 46XY chromosomes to prevent any chance of malignant transformation. However, it is equally important not to address 46XY chromosomes or the patients with testicular tissue in a male context. They are destined to grow up as females and not as compromised males. Accordingly they should be shielded from this information, at least during their teenage years. Surgical treatment of ovarian and adrenal tumours is should be carried out when indicated.

The scope of surgical treatment of lower vaginal agenesis is beyond the remit of this endocrine oriented chapter. It is definitely more complicated than incision of an imperforate hymen or excision of a transverse vaginal septum. It should be performed in a specialised tertiary unit. Pull-through vaginoplasty could be performed in patients with distended upper vagina, which allows better mobilisation of the upper vagina down to the hymeneal ring. Otherwise, skin grafts should used to bridge the gap between the upper vagina and lower part. In all cases vaginal dilators will be needed to keep the created vaginal canal intact.

 

Summary

This is a very important area of Reproductive Endocrinology that deals with very sensitive issues involving vulnerable young girls. The ultimate objective is to help them to lead satisfying and functional lives, as much as medically possible.  Utmost care should be taken to use simple and reassuring language, without any complicated technicality. Parents should also be involved at all times, and they should understand the exact problems, their management and future implications. A multidisciplinary team including a paediatrician, counsellor, dietician and a clinician experienced in adolescentsí gynaecological surgery should be involved, as and when necessary. 


References

1.  Marshall, W.A. and J.M. Tanner, Variations in pattern of pubertal changes in girls. Arch Dis Child, 1969. 44(235): p. 291-303.

2.  Herman-Giddens ME, Slora EJ, Wasserman RC, Bourdony CG, Bhapkar MV Koch GG and CM Hasemeir CM. Secondary sexual characteristics and menses in young girls seen in office practice: a study from the Paediatric Research in Office Settings network. Pediatrics, 1997. 99(4): 505-12.

3.    Tanner JM. Growth at adolescence. 2nd ed. Oxford: Blackwell, 1962.

4.  Culter Jr GB. The role of oestrogen in bone growth and matuarion during childhood and adolescence The Journal Steroid Biochemistry and Molecular Biology 1997; 61(3-6): 141 Ė 144Brook CGD. A Guide to the Practice of Paediatric Endocrinology; Cambridge University Press, 1993

5.     

6.    Traggi C and Stanhope R. Delayed puberty. Best Pract Res Clin Endocrinol Metab 2002; 16(1): 139-151

7. Oppelt P, von Have M, Paulsen M, Strissel P, Strick R, Brucker S, Wallwiener D and Beckmann M. Female genital malformations and their associated abnormalities. Fertil Steril 2007;87(2):335-42.

8.   Sanfilippo JS Endometriosis in association with uterine anomaly. Am J Obstet Gynecol 1986; 154(1): 39-43.

9.  Heger AH, Ticson L, Guerra L, Lister J, Zaragoza T, McConnell G, Morahan M. Appearance of the genitalia in girls selected for nonabuse: review of hymeneal morphology and nonspecific findings. J Pediatr Adolesc Gynecol 2002; 15(1):27-35.

10. Burel A, Mouchel T, Odent S, Tiker F, Knebelmann B, Pellerin I, Guerrier D.. Role of HOXA7 to HOXA13 and PBX1 genes in various forms of MRKH syndrome (congenital absence of uterus and vagina). J Negat Results Biomed. 2006;5:4

11. Franceschi R. Gaudino R, Marcolongo A, Gallo MC, Rossi L, Antoniazzi F AND Tatò L. Prevalence of polycystic ovary syndrome in young women who had idiopathic central precocious puberty. Fertil Steril 2009; article in press.

12. Cisternino M, Arrigo T, Pasquino AM, Tinelli C, Antoniazzi F, Beduschi L, Bindi G, Borrelli P, De Sanctis V, Farello G, Galluzzi F, Gargantini L, Lo Presti D, Sposito M, Tatò L. Etiology and age incidence of precocious puberty in girls: a multicentric study. J Pediatr Endocrinol Metab, 2000. 13 Suppl 1: p. 695-701.

13. Apter D, Cacciatore B, Alfthan H, Stenman UH. Serum luteinizing hormone concentrations increase 100-fold in females from 7 years to adulthood, as measured by time-resolved immunofluorometric assay. J Clin Endocrinol Metab. 1989; 68: 53 - 57.

14. 

15. Brito VN, Batista MC, Borges MF, Latronico AC, Kohek MB, Thirone AC, Jorge BH, Arnhold IJ, Mendonca BB. Diagnostic value of fluorometric assays in the evaluation of precocious puberty. J Clin Endocrinol Metab. 1999; 84(10): 3539 - 3544.



 
 
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