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LABORATORY INVESTIGATION OF
AMENORRHEA:
Dr M du Plessis
Dept of Chemical Pathology
University of Pretoria
23 February 2001
DEFINITIONS:
Primary amenorrhea: Failure to establish spontaneous periodic menstruation by the age of 16
years regardless of whether secondary sex characteristics have developed.
Secondary amenorrhea: Absence of periodic menstruation for at least 6 months in women
who have previously experienced menses.
ESTROGENS AND OVARIAN FUNCTION:
The normal human ovary produces all 3 classes of sex steroids, estrogens (C18-steroids),
progestins (C21-steroids) and androgens (C19-steroids); however, estradiol and
progesterone are its primary secretory products.
The principle and most potent estrogen, derived almost exclusively from the ovaries, is 17estradiol. Its measurement is considered sufficient for evaluation ovarian function.
Two-cell theory of estrogen production by the ovarian follicle:
- Thecal cells: produce androgens (primarily androstenedione), stimulated by LH.
- Granulosa cells: conversion of androgens to estrogens (mainly estradiol) by
aromatisation, regulated by FSH.
Estrogens are also produced by peripheral aromatisation of androgens:
- Important source of postmenopousal estrogens (mainly estrone)
- Source of androgens: adrenal androstenedione
- Major sites of aromatisation: liver and adipose tissue ( with obesity).
Effect on pituitary:
- Slowly rising or sustained high levels of estrogen together with progesterone inhibit
pituitary gonadotrophin (LH and FSH) secretion by NEGATIVE FEEDBACK.
- Rapid rise in estrogen concentration which occurs prior to ovulation stimulates LH
secretion – POSITIVE FEEDBACK.
Figure 1:Three classes of sex steroids:
Figure 2: Estrogen synthesis by aromatisation of androgens:
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PROGESTERONE:
In nonpregnant women, progesterone is secreted mainly by the corpus luteum under the
influence of LH, during the luteal phase of the menstrual cycle.
SUMMARY OF THE ENDOCRINE CONTROL DURING THE
MENSTRUAL CYCLE:
Figure 3:
Follicular phase:
- Estrogen secretion by the ovarian follicle is stimulated by FSH.
- As estrogen concentrations rise, FSH secretion declines until estrogens trigger a
positive feedback mechanism, causing an explosive release of LH (mid-cycle LHsurge) and to a lesser extent FSH.
Ovulation:
- The increase in LH stimulates ovulation and development of the corpus luteum.
Luteal phase:
- Rising levels of estrogens and progesterone (from corpus luteum) then inhibit FSH
and LH secretion. Inhibin (also produced by ovaries) also inhibits FSH secretion.
- If conception does not occur, the corpus luteum regresses, leading to declining levels
of estrogens and progesterone, triggering menstruation and LH and FSH release.
Figure 4: Hormone changes during normal menstrual cycle:
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CAUSES OF AMENORRHEA:
A. Anatomical defects
B. Primary ovarian failure (hypergonadotrophic hypogonadism)
( LH and FSH,  Estradiol)
1. Gonadal agenesis
2. Gonadal dysgenesis (Turner syndrome/pure gonadal dysgenesis)
3. Premature ovarian failure (before age 40 years)
C. Secondary ovarian failure (hypogonadotrophic hypogonadism)
( LH and FSH,  Estradiol)
1. Hypothalamic deficiency of GnRH
a. Organic causes: tumors, infection and other disorders
b. Functional disorders: Stress, weight loss/diet (anorexia
nervosa), exercise, malnutrition, chronic debilitating diseases
(eg end-stage kidney disease, AIDS).
2. Pituitary:
a. Deficiency of LH/FSH
b. Inappropriate seceretion of prolactin: Drugs, hypothyroidism,
prolactinoma.
D. Chronic anovulation with estrogen present:
1. PCOS
2. Adrenal disease
a. Cushing syndrome
b. Adult-onset adrenal hyperplasia
3. Thyroid disease
a. Hyperthyroidism
4. Ovarian tumors
PRIMARY OVARIAN FAILURE (HYPERGONADOTROPHIC
HYPOGONADISM):
Primary defect in the ovaries (absent/destruction):
–  Estradiol-production (< 150 pmol/l) (hypogonadism)
–  Negative feedback -  LH and FSH (> 40 U/l) (HYPERGONADOTROPHIC)
– No withdrawal bleeding following progesterone challenge.
In a patient < 30 years of age:
- Primary amenorrhea
- Sexual infantilism (absence of secondary sexual characteristics)
- Karyotype:
o 45 X: Turner syndrome
o 46 XX/XY: Pure gonadal dysgenesis
o Streak gonad should be removed if Y-chromosome present ( incidence of
malignancy).
Premature ovarian failure:
- Secondary amenorrhea
- Hypergonadotrophic hypogonadism
- Before age 40 years
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SECONDARY OVARIAN FAILURE (HYPOGONADOTROPHIC
HYPOGONADISM):
Ovarian failure is secondary to organic or functional disorders of the CNS-hypothalamicpituitary axis:
-  GnRH (hypothalamic disorder)/ (or inappropriately normal) LH and FSH (pituitary
disorder) (HYPOGONADOTROPHIC)
-  Estradiol (hypogonadism)
– No withdrawal bleeding following progesterone challenge.
Kallmann syndrome:
- Defective hypothalamic release of GnRH   LH/FSH
- Associated with anosmia (agenesis of olfactory bulbs)
Several disorders of the pituitary can lead to hypogonadism:
- Space-occupying lesions that directly inhibit gonadotropin secretion by destruction of
the producing cells or by blocking delivery or secretion of GnRH
- Necrosis of the pituitary (following postpartum hemorrhage – Sheehan syndrome)
- Inappropriate secretion of prolactin (including drugs, other diseases eg
hypothyroidism, prolactinoma)   secretion of LH and FSH.
Primary hypothyroidism (TSH,  free thyroid hormone): decreased negative feedback
causes increased TRH which may stimulate prolactin secretion.
GnRH (gonadorelin) stimulation test may be of use to differentiate between hypothalamic and
pituitary causes of hypogonadism:
- Give 100 ug GnRH intravenously.
- Measure LH and FSH response with samples at 0, 20 and 60 minutes.
- Normal peak LH-response: 3-10 fold increase above baseline, within 15-30 minutes.
- Normal peak FSH-response: 1.5-3 fold increase above baseline, within 15-30
minutes.
- In pituitary disease: absent or blunted response.
- In hypothalamic disease: normal response but repeated stimulation may be needed
(daily infusions of LHRH for 1 week).
Clomiphene (Clomid) stimulation test may be useful to distinguish organic causes of
gonadotrophin deficiency (pituitary or hypothalamic pathology) from functional disorders and
idiopathic delayed puberty.
- In healthy adults clomiphene blocks estrogen feedback mechanisms in the
hypothalamus, thus leading to a rise in GnRH (gonadotrophin-releasing hormone)
and consequently circulating LH and FSH.
- Give Clomid 50 mg bd orally for 7 days.
- Blood samples are collected for LH and FSH at baseline and after clomiphene
stimulation (day 8).
- Normal response: Incresae in LH (> 120% above baseline) and FSH (> 40% above
baseline).
- A normal response essentially rules out organic causes of hypogonadotrophic
hypogonadism and in delayed puberty is an indication that sexual maturity will ensue.
- In hypothalamic-pituitary pathology there is no response.
- The test has little usefulness in prepubertal children and early puberty where
clomiphene suppresses rather than stimulates gonadotrophin release.
The diagnosis of functional disorders is made by exclusion, after some form of imaging of the
brain (CT or MRI) to rule out a tumor.
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CHRONIC ANOVULATION WITH ESTROGEN PRESENT:
These patients experience withdrawal bleeding after progesterone administration due to
acyclic production of estrogen (largely estrone) by extraglandular aromatisation of circulating
androstenedione:
- PCOS
- Adrenal disease
o Cushing syndrome (obesity)
o Adult-onset adrenal hyperplasia ( androstenedione)
- Ovarian tumors ( androstenedione/estrogen,  LH/FSH due to negative feedback)
- Thyroid disease
o Hyperthyroidism: SHBG (sexhormone binding globulin)   levels of sex
hormones, increased peripheral aromatisation.
Polycystic ovarian syndrome (PCOS):
Association between polycystic ovaries and a complex of clinical signs including:
- Amenorrhea
- Obesity
- Hirsutism
Primary defect: Abnormal steroid feedback signals leading to increased secretion of LH (selfperpetuating cycle):
- Increased acyclic production of extraglandular estrogens  positive LH feedback and
negative FSH feedback   LH with normal/FSH  LH/FSH ratio (> 2.5).
- Elevated LH  hyperplasia of ovarian stroma and thecal cells and  ovarian
androgen production (androstenedione,  testosterone).
- Increased adrenal androgen production  hirsutism.
- Obesity and  androgens   peripheral aromatisation and  acyclic estrogen
production  anovulation/amenorrhea.
Laboratory diagnosis:
-  LH/FSH ratio (> 2.5)
- normal estrogen and withdrawal bleeding following progesterone challenge
-  testosterone and androstenedione
-  prolactin (15-20% of patients) (chronic exposure to estrogen/ deficiency of
hypothalamic dopamine).
-  SHBG ( synthesis due to  androgens)
Figure 5: Proposed mechanism for the initiation and perpetuation of chronic anovulation in
PCOS.
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LABORATORY TESTS AVAILABLE FOR EVALUATION OF
PATIENTS WITH AMENORRHEA:
A. Diagnosis of pregnancy: -HCG (on blood sample).
- Any woman of reproductive age with amenorrhea is assumed to be pregnant until
proved otherwise.
B. Prolactin:
- 10% of women with amenorrhea have increased levels
C. Thyroid function tests:
- TSH, if abnormal do a free T4
- Primary hyperthyroidism:  (suppressed) TSH,  free T4
- Primary hypothyroidism:  TSH,  free T4.
D. Hormone profile:
- estradiol : if decreased - hypogonadism
- LH and FSH:
o If : primary gonadal failure (hypergonadatrophic hypogonadism)
o If : secondary gonadal failure (hypogonadotrophic hypogonadism)
E. Progesterone challenge test:
- Determination of relative estrogen status.
- Can be used instead of measurement of S-Estradiol (which fluctuates throughout the
day and menstruation cycle).
- Give additional information regarding the outflow tract.
- Give medroxyprogesterone acetate (Provera) 5 mg bd orally for 5 days.
- If estrogen levels are adequate (> 150 pmol/l) and the outflow tract is intact,
menstrual bleeding should occur within a week of treatment.
- If no response: estrogen priming (Estrogen 1.25 mg/day for 21 days) followed by
another progesterone challenge –
o If bleeding: normal outflow tract, but estrogen deficiency corrected by
administered estrogen: primary or secondary gonadal failure
o If still no bleeding: outflow tract/uterine disease.
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LAB EVALUATION OF AMENORRHEA:
Amenorrhea
Pregnancy test
(B-HCG)
Prolactin
TSH
Positive
Antenatal care
Increased
(> 20 ug/l)
Repeat after
excluding
drugs
Imaging of the pituitary
for prolactinoma
 TSH
Free T4 
Primary
hypothyroidism
 TSH
 Free T4
Primary
hyperthyroidism
Progesterone
challenge test
Withdrawal
bleeding
LH/FSH
ratio > 2.5
No withdrawal
bleeding
Estrogen
priming
LH/FSH
Normal
Bleeding
PCOS
No bleeding
Anovulation
Pituitary or
hypothalamic
disease
FSH and LH
 (< 5U/l)/N
FSH + LH 
(> 40 U/l)
Primary gonadal
failure
GnRHstimulation test
(Clomiphene
stimulation)
Karyotyping
if < 30 years
Negative imaging
Functional cause
Positive imaging
Organic cause
Outflow tract
defect
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Case studies:
Case 1:
A 19-year old woman was referred to an endocrinologist for assessment of secondary
amenorrhea of 1year duration, following a car accident 2 years previously, in which she
sustained a closed head injury.
Clinical examination found a euthyroid woman with some findings suggestive of low estrogen
production.
Lab results:
B-HCG
Prolactin
Estradiol
FSH
LH
Negative
10 ug/l (< 30)
150 pmol/l (200-1500)
2.3 U/l
(1.0-6.5)
3.0 U/l
(1.5-9.0)
No bleeding following progesterone challenge.
Bleeding after estrogen priming and repeat progesterone challenge.
Differential diagnosis and further investigations ??
Case 2:
Obese patient (weight 106 kg) complaining of hirsutism and amenorrhea.
Lab results:
B-HCG Negative
Prolactin 7.0 ug/l (< 30)
TSH
1.6 U/l (0.5-4.0)
Bleeding following progesterone challenge
FSH
2.4 U/l
(1.0-6.5)
LH
10.0 U/l
(1.5-9.0)
Testosterone 2.8 nmol/l (0.6-2.3)
Androstenedione 18 nmol/l (3-10)
SHBG
5.0 nmol/l (15-90)