Normal and Abnormal Puberty
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
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.
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).
No pubic hair
Breast bud stage
More growth with appearance of areola
mound of areola and papilla
Adult hair not
reaching medial thighs
recession with projecting papilla
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
- Absent secondary sexual characteristics by the age of 13
- 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
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
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.
Female genital anomalies occur in about 5% of women (Oppelt
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.
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.
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.
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).
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.
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).
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.
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:
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.
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
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
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
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.
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.
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.
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
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
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
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
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.
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):
Tanner JM. Growth at adolescence. 2nd
ed. Oxford: Blackwell, 1962.
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
C and Stanhope R. Delayed puberty. Best Pract Res Clin Endocrinol Metab 2002;
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 -
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.