Hormonal assay in clinical gyn

11/29/2021
You can get this lecture and 480 lectures from:
1.My scientific page on Face book: Aboubakr
Elnashar Lectures.
https://www.facebook.com/groups/2277448840913
51/
2.Slide share web site
3.elnashar53@hotmail.com
4.My clinic: Elthwara St. Mansura
ABOUBAKRMOHAMED ELNASHAR
HORMONAL ASSAY IN CLINICAL
GYNECOLOGY
Prof Aboubakr Elnashar
Benha University Hospital, Egypt
Email: elnashar53@hotmail.com
11/29/2021
1. Prolactin
2. TSH
3. AMH
4. FSH & LH
5. Estrogen
6. Progesterone
7. 17 OH progestrone
8. Androgens
PROLACTIN

11/29/2021
It is secreted by: Mammotropic cells of the anterior
pituitary.
It is necessary for initiation & maintenance of lactation
Reference values: 10–26 ng/ml
 1 ng is equivalent to 21.2 mU/ml
 ng/ml=ug/liter
 Premenopuasal: <20 ng/ml
 Postmenopausal: <12 ng/ml
Forms:
Small:
 mol wt 22,000
 biologically active,
 80%
Big: mol wt 50,000
Big-big: mol wt >100,000
Macro: a big-big form
with reduced bioactivity
{unable to bind to PRL receptors}
exhibits no systemic response.
11/29/2021
Macroprolactinemia
10% of hyperprolactinemia.
Preponderance of the circulating prolactin consists of big and
big big molecules: No effect on fertility: No tt.
In patients with asymptomatic hyperprolactinemia,
assess for macroprolactin (Endocrine Society Clinical Practice Guideline, 2011)
DD between Macroprolactinemia & true hyperprolactinemia
on clinical basis: impossible
Specific serum immunoassays to eliminate the ‘‘big
prolactin molecules with a polyethylene glycol (PEG).
 Hyposecretion: Rare. {Pituitary necrosis or infarction}
 Hyperprolactinaemia:
Ovulatory dysfunction
 Oligo-ovulation
 LPD
 Anovulation
Menstrual troubles
 Oligomenorrhea
 Hypomenorrhea
 Amenorrhea
Osteoporosis
Nervous manifestations (headache)
Visual field defects (Bitemporal Hemianopia)

11/29/2021
Speroff et al, 2020
I. Physiologic factors
 Pain
 Nipple stimulation
 Pregnancy
 Brest feeding
 Pelvic examination
 Exercise
 Sleep
 Stress
II. Drugs
 Dopamine-antagonists
 Dopamine-depleting agents
 Narcotics
III. Pathologic factors
 Hypothalamus
 Pituitary
 Thyroid
IV. Idiopathic
 Causes Of Hyper Prolactinaemia
11/29/2021
II. Drugs: The most frequent cause
of nontumoral
hyperprolactinemia.
 Antidopaminergic drugs
 Anti-psychotics (phenothiazines,
monoamine oxidase inhibitors,
fluoxetine, sulpiride, haloperidol,
butyrophenones, risperidone):
Inhibition of dopamine release
 Anti-emetics (metoclopramide,
domperidone)
 Tricyclic antidepressants
 Opiates: Stimulation of
opioid hypothalamic
receptors
 Strogens:: Stimulation of
lactotrophs
 Verapamil;: Unknown
 Risperidone and
metoclopramide:±
prolactin elevations above
200ng/ml in patients
without evidence of
adenoma

11/29/2021
Indications for Prolactin assay
1. Secondary amenorrhea
2. Galactorrhea
3. Ovulatory dysfunction
4. Unexplained infertility
5. Oligospermic men
 Conditions for detection of PRL
 Late morning, Fasting, After 60 min rest, Not in late follicular
phase
 Endocrine Society Clinical Practice Guideline, 2011
 Avoid excessive venipuncture stress
 can be drawn at any time of the day.
 A single determination is usually sufficient
 Repeat: when in doubt, on a different day at 20 min
intervals {account for possible prolactin pulsatility}
 > 100 ng/ml: 60% pit tumor
 > 300 ng/ml: 100% pit tumor
 Modest elevation can be associated with pit tumor
11/29/2021

11/29/2021
 Hyperprolactinaemia without galactorrhea: 66%
1. Inadequate detection
2. Hypoestrogenic state.
3. Inadequate estrogenic or progetational priming of
the breast
4. High PRL does interact with the breast receptors
TSH
11/29/2021
 It is secreted by the thyrotrophic cells of the anterior
pituitary .
 It stimulates the growth of the thyroid follicular cells &
every step in thyroid hormone synthesis
 American Society for Clinical Pathology2020
 Don’t order multiple tests in the initial evaluation of a patient
with suspected thyroid disease.
 TSH and if abnormal, follow up with additional evaluation or
TT depending on the findings.
 TSH test
 can detect subclinical thyroid disease in patients without
symptoms of thyroid dysfunction.
 value within the reference interval excludes the majority
of cases of primary overt thyroid disease.
 If abnormal, confirm the diagnosis with free thyroxine
(T4). ABOUBAKRMOHAMED ELNASHAR

11/29/2021
TT
Adverse effects
T3
FT4
TSH
Hypothyroidism
Eltroxin
Yes
Overt
Eltroxin if
TPO
±
N
N
Subclinical
Hyperthyroidism TSH FT4 FT3 TT4 TT3
Overt ↓ ↑ ↑ ↑ ↑
Subclinical ↓
No
change
No
change
No
change
No
change
11/29/2021
 Clinical conditions associated with thyroid
dysfunction:
1. Oligomenorhea
2. Amenorrhea
3. Menorrhagia
4. Anovulation.
5. Inadequate corpus luteum.
4. Infertility
Sensitive TSH
High Normal Low
Free T4 Normal thyroid Free T4
Low Normal Normal High
Hypothyroidism Free T3
Subclinical hypothyroidism Normal High
Subclinical hyperthyroidism Hyperthyroidism

11/29/2021
Anti-Mullerian hormone (AMH)
A peptide hormone Glycoprotein
Produced by:
granulosa cells of
Pre-antral
Small antral follicles
Falls linearly with increasing age
11/29/2021
>39 y
34–38 y
24–33y
Parameter
1.1
(0.5–2.3)
1.6
(0.8–2.9)
2.1
(1.1–3.4)
AMH level
(ng/mL)
Median (interquartile range)
7.9
(6.2–10.6)
7.4
(6–9.4)
6.9
(5.5–8.3)
FSH level (IU/L)
7
(4–11)
10
(6–13)
11
(8–16)
AFC
(Imog et al ,2011)
 Limitations:
1. Expensive
2. Assay technical issues
AMH incubated at room temp for upto 7 days increased
progressively in most samples (58% increase overall)
The pre dilution of serum before assay provided AMH levels
up to twice those found in the corresponding undiluted
samples (Rustamov et al, 2012)

11/29/2021
Advantages
1. Not cycle dependant: (Hadlow et al., 2013)
Can be measured any day
Cyclic variation with higher AMH in the late follicular phase
not large enough to warrant a shift in clinical practice towards timing
AMH measurement (Kissell et al., 2014).
2. Less cycle to cycle variation than FSH
3. Not effected by GnRHa: can be measured during
down regulation
4. A single random measurement of AMH has 80%
sensitivity and 93% specificity to predict poor ovarian
response [Seifer et al, 2002].
5. Its levels correlate with the number of oocytes
retrieved
6. Treatment can be individualized for optimal cycle
11/29/2021
 Indications:
 ≥ 35 ys not conceived after 6 months or
 < 35 ys
 Endometriosis
 Unexplained infertility
 Single ovary
 Previous ovarian surgery,
 Poor response to FSH,
 Previous exposure to chemotherapy or
 Radiation (Iii-b) SOGC, 2011
 Ovarian reserve testing
 Woman’s age:
An initial predictor of overall chance of success
through natural conception or with IVF
 Predictors of ovarian response to Gnt stimulation
High response
Low response
16 or more
4 or less
Total AFC
3.5 or more
25
0.8 or less
5.5
AMH
ng/ml
pmol/l
Conversion ratio:7
4 or less
8.9 or more
FSH IU/L

11/29/2021
 Clinical applications
1. Selection of protocol according to ovarian reserve
Reserve ‘Low’ ‘Average’ ‘High’
AFC <7 7-14 >14
AMH <1.1 ng/ml 1.1-3.5 >3.5
Starting FSH
dose IU
Amp
375
5
225
3
150
2
Protocol - Antagonist
-Microdose flare
-Agonist stop
-GH
-Natural
-Modified
natural
-Long
protocol
-Antagonist
-Long
protocol
-Antagonist
Specific marker of ovarian response to
gonadotrophins.
Although no established cutoff for normal& abnormal
AMH exists, it is generally accepted that AMH> 0.8–1.0
ng/ml are suggestive (Wang et al, 2018)
11/29/2021
2. ACOG, 2020: AMH
 Not part of the accepted diagnostic criteria for PCOS
 As a predictor of the
 Pregnancy loss is not recommended.
 Onset of menopause is unsuitable for clinical practice
at this time
 Post chemotherapy fertility and to guide fertility
counseling in these patients: more data are needed
FSH &LH

11/29/2021
 They are secreted by the anterior pituitary.
 The alpha subunit is identical for all glycoprotein hormones (TSH, HCG, LH
& FSH), but the beta subunit differs.
 The peak of FSH is coincident with the peak of LH, but it is of lesser
magnitude & briefer duration. Following the midcycle surge of LH & FSH, there
is drop in both.
 Normal values:
FSH LH
Adult 5-10 mIU/ml 5-20 mIU/ml
Mid cycle peak 2 times the basal level 3 times the basal level
 Clinical uses:
FSH LH
1. Hypogonadotrophic state e.g.
prepubertal
< 5 mIU/ml < 5 mIU/ml
2. Hypergonadotropic state e.g.
postmenopuse
Ovarian failure
>40 mIU/ml >40 mIU/ml
3. PCOS
Follicular phase ratio
normal or decreased
1
High
2
11/29/2021
4. Testing for ovarian function:
 Day 3 FSH
 < 4 = high response
 < 10 IU/L = normal
 > 25 IU/L ( or age >44) is independently
associated with near zero chance of pregnancy

11/29/2021
5. Detection of ovulation
Follicular rupture occurs:
36 h after the onset of serum LH surge
12 H after LH peak.
A positive urine result is often found only 12 h after
the onset of serum LH. (around the point of LH
peak).
So ovulation is expected to occur 24 h after the
urine LH surge
 LH surge in urine:
 Quick, sensitive, relatively inexpensive,
 pinpoint the day of ovulation &
 has reduced the uncertainty in interpretation of
progesterone levels by better-identifying the time
of peak progestrone secretion at which to obtain
serum
11/29/2021
6. Diagnosis of the cause of precocious puberty:
(Breast development <8 y or menstruation <9 y.)
X ray of the lower ends of radius & ulna: bone age
a. Retarded: hypothyroidism
b. Normal: Partial
c. Advanced:
 FSH: <2 IU/ml: pseudo
> 2 mIU/ml: true:
 CT or MRI: Normal: idiopathic
Abnormal: CNS lesion
7. Diagnosis of the cause of amenorrhea
Primary: absence of menstruation by the age of 16 yr
regardless of SSC or by the age of 14 yr in absence of SSC
Secondary: Cessation of menstruation > 6 months

11/29/2021
1. Pregnancy test.
2. TSH &PRL.
3. Progestin challenge test: (MPA 5mgX2X5d)
+ve: Anovulation
-ve: E + P :
 -ve: outflow or uterine failure  HSG, hysteroscopy, IVP
& laparoscopy.
 +ve: Ovarian failure or pituitary-hypothalamic
dysfunction.
4. FSH:
high: Ovarian failure.
If 1ry: Karyotyping.
If 2ndry: premature menopause
Low or Normal: CT of Pituitary-hypothalamic region.
 Abnormal: pituitary disease
 Normal: hypothalamic dysfunction.
 Limitations.
1. Women with high androgen levels, (such as occurs with
PCOS and CAH,) may have an atrophic endometrium and
may fail to bleed (Rarick, 1990)
2. Estrogen levels may fluctuate both in hypothalamic
amenorrhea & in the early stages of ovarian failure: patients
with these disorders may have at least some bleeding after
progesterone withdrawal (Nakamura, 1996).
 Use of this test is best restricted to those situations in which
accurate serum hormone measurements are unavailable.
11/29/2021
IHH = idiopathic hypogonadotropic hypogonadism
ESTROGENS

11/29/2021
 More than 30 estrogens have been identified, but only 3
estrogens are used in cl practice: E1, E2, E3
 E1, E2 & E3 are bound to SHBG.
 E1
 Highest concentration in postmenopausal females
 Most E1 is derived from peripheral conversion of
androstenedione & from E2 metabolism.
 E2
 secreted almost entirely by the ovary
 The most abundant E in premenopausal females
 The most potent E
 E2 & not total E is used for clinical purposes.
 Normal values of E2 (pg/ml)
 Follicular phase: 25-27
 Midcycle peak: 200-600
 Luteal phase: 100-300
 Postmenopausal: 5-25
11/29/2021
 E2 rises during 2nd half of the follicular phase & reach peak 24
h before LH surge & 36 h before ovulation.
 Following LH surge E2 drops to preovulatory levels, but then
rises slightly to 100-300 pg/ml during luteal phase
 Clinical applications:
1. E increases in E secreting tumors e.g. granulosa theca cell
tumors
2. To classify hypogonadism: E is usually interpreted with
gonadotropin measurements
3. Test for ovarian reserve:
 Low D3 E2 (<75 pg/ml) combined with normal FSH:
good ovarian reserve
 Evaluation of both E2 & FSH is better predictor of
ovarian reserve than using either measurement alone.

11/29/2021
4. An indication of down regulation in the long protocol
for superovulation in ART. E2: < 50 pg/ml
5. Monitoring COS in ART:
 The goal is an E2 level of 200 pg/ml per large
(>14mm) follicle
 The risk of OHSS is significant if E2 is >4000 pg/ml
(Sperof,2020)
 The number of follicles & the type of patient should
be considered.
6. Monitoring of induction of ovulation with HMG
(Sperof,2018).
 E2: 1000-1500 pg/ml is optimal
 1500-2000 pg/ml: increase risk of OHSS
 >2000 pg/ml: high risk of OHSS, consider cycle
cancellation
11/29/2021
PROGESTERONE
In the serum:
 18% is bound to cortisol binding globulin
 79% is bound to albumin
 3% is free

11/29/2021
Normal values (ng/ml):
 Low prior to the mid cycle GnT surge.
 Shortly after that, P begin to rise rapidly reaching peak levels during the
middle of the luteal phase (8days after LH peak).
 Thereafter, a progressive fall occurs with barely detectable P levels reached
prior to menses.
 Follicular phase: <1
 Luteal phase: 5-20
 Post menopause: <1
Clinical applications
1. Diagnosis of ovulation: in cases of infertility & DUB
midluteal phase serum level of 5 ng/ml
2. Diagnosis of corpus luteal dysfunction:
 Midluteal phase level of 10 ng/ml.
 Sum of 3 progesterone levels from D11-4
before menses: 15 ng/ml
3. P on the day of trigger: Elevated 1.5 ng/ml
11/29/2021
17 OH PROGESTERONE
 It is an intermediate metabolite in steroidogenesis in
the adrenals
 It is used for diagnosis of enzymatic deficiency in the
adrenals.
 Increased 17 OH progesterone indicates congenital
adrenal hyperplasia
 Clinical application
1. Hirsutism
2. Ambigous genitalia

11/29/2021
ANDROGENS
11/29/2021
 Sources of androgens
 Androgen in the blood
Male Normal
female
Hirsute
female
Free 3% 1% 2%
Albumin 19% 19% 19%
SHBG 78% 80% 79%

11/29/2021
 Normal values (ng/dl):
Premenopause Postmenopause
Testosterone 20-80 15-70
Androstenedione 60-300 30-150
 Indications:
1. PCOS: Biochemical hyperandrogenism
Most useful when cl hyperandrogenism is unclear.
Assay:
High quality assays needed for most accurate
assessment: chromatography–mass spectrometry(LCMS)/mass
spectrometry& extraction/chromatography immunoassays
Direct free testosterone assays
Radiometric or enzyme-linked assays
Not preferred, should not be used
{poor sensitivity, accuracy and precision}.
11/29/2021
Use
 Free androgen index= TX 100 / SHBG if > 4.5: PCOS
Not done routinely in presence of hirsutism
calculated bioavailable testosterone.
Calculated Free testosterone
Androstenedione & DHEAS
 could be considered if total or FT are not elevated
 However, have limited role in PCOS diagnosis.
 Clinical application In PCOS:
 DHEAS > 2ug/ml: CC + Corticosteroid (ACOG,2002)
2. In hirsutism
 Total testosterone measures the ovarian & adrenal activity
(Speroff et al, 2018)

11/29/2021
 Free testosterone
 Good correlation with total production rate (= secretion rate
+ peripheral conversion rate) which correlate well with
degree of virilization
 Normal level: 1.5-11.4 pg/ml
 Not done routinely in presence of hirsutism
•Free androgen index (FAI)=
TX 100 / SHBG if > 4.5 : PCOS
•Dehydoepiandrosterone sulphate (DHEAS)
 The principal contribution of 17 ketosteroids (KS) is from
DHES.
 It correlates with urinary 17 KS.
 It is more reliable indicator of adrenal androgen than 24 h
17 KS.
 In hirsutism: DHEAS: >2 ug/ml: COCs + Corticosteroids
11/29/2021
3. Evaluation of infant with ambiguous genitalia
Karyotype, Androgens, 17 OHP
XY
Normal androgens Normal androgens
signs of adrenal failure Normal 17OHP
normal 17 OHP
CAH with 3B IAIS, 5reductase def, true hph, . Dehydogenase
mixed gonadal dysgenesis,
block in male. abnormal androgen synthesis
Gonadectomy

Hormonal assay in clinical gyn

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Hormonal assay in clinical gyn

Similar to Hormonal assay in clinical gyn (20)

More from Aboubakr Elnashar

More from Aboubakr Elnashar (20)

Recently uploaded

Recently uploaded (20)

Hormonal assay in clinical gyn