Hypothalamic-Pituitary-Gonadal Axis

Hypothalamic-Pituitary-
Gonadal Axis
By
OLUKOYEJO O.E.
January 23, 2024 OLUKOYEJO O.E. 1

OUTLINE
• Introduction
• Male reproductive system & disorders
• Female reproductive system & disorders
• Infertility – General Considerations
• Endocrine Investigation of the Infertile Couple
• Analytical Methods for Reproductive Hormones
• Conclusion
• References

INTRODUCTION
The hormones of the hypothalamic-pituitary-gonadal axis as well as the
adrenal glands are crucial for reproductive function and include
• gonadotropin-releasing hormone (GnRH),
• luteinizing hormone (LH),
• follicle-stimulating hormone (FSH), and
• a multitude of sex steroids.

MALE REPRODUCTIVE SYSTEM
• The mature testes synthesize both sperm and androgens.
• The testes contain a structured network of tightly packed

seminiferous Tubules lined by maturing germ cells and Sertoli cells.
• Sertoli cells play a crucial role in sperm maturation and secrete

inhibin, a glycoprotein that inhibits the pituitary secretion of FSH.

MALE REPRODUCTIVE SYSTEM……..
• Surrounding the seminiferous tubules are the interstitial Leydig cells,
the primary site of androgen production.
• The principal androgen in man is testosterone,
• Testosterone is required for sexual differentiation, spermatogenesis,

and promotion and maintenance of sexual maturity at puberty.
• Testicular function is under the control of the hypothalamic-pituitary-

gonadal axis.

The hypothalamic-pituitary-gonadal axis
• Gonadotropin-releasing hormone (GnRH) is a decapeptide
synthesized in the hypothalamus and transported to the anterior
pituitary gland, where it stimulates the release of both FSH and LH.
• LH acts on Leydig cells to stimulate the conversion of cholesterol to

pregnenolone.
• FSH acts on Sertoli cells and spermatocytes and is central to the

initiation (in puberty) and maintenance (in adulthood) of
spermatogenesis.
HYPOTHALAMO – PITUITARY GONADAL HORMONES MALE)
Higher Centre Neurotransmitter

Adrenaline, Dopamine, Serotonin
Acuate Nucleus Hypothalamus GnRH Pulsatile Release
Anterior Pituitary
LH
FSH
Free Testosterone
LH FSH
TESTICULAR Leydig Cells Sertoli Cells
Testosterone Spermatogenesis
Hypothalamic-pituitary-gonadal axis…….
• Sex steroids and inhibin provide negative feedback control of LH and
FSH secretion, respectively.
• INHIBIN- a dimeric glycoprotein. 2 biological forms exist: inhibin A and
B. Secreted by granulosa cells in the ovary in women and sertoli cells
in men.
• Inhibin A- is elevated in the serum of women carrying fetus with
Down syndrome, hence it is included in maternal serum tests for
Down syndrome in 2nd trimester of pregnancy. Also used as
complimentary to CA125 as an ovarian cancer marker.

ANDROGENS
• Androgens are a group of steroids responsible for masculinization of
the genital tract and development and maintenance of male
secondary sex characteristics.
• Testosterone is the principal androgen secreted in men.
• Testosterone is synthesized primarily by the Leydig cells of the testes

(95%) and, to a lesser extent (≈5%) via peripheral conversion from the
precursors dehydroepiandrosterone(DHEA) and androstenedione
(which are synthesized in the zona reticularis of the adrenal glands).

Sex steroids
Produced in ovary, testis and adrenal gland

http://www.sciencedirect.com/science/article/pii/S0197458009004072
Testosterone & DHT
Testosterone
• Male reproductive development (foetal & pubertal)
• Male secondary sex characteristics
• Spermatogenesis
• Bone mass (male & females)
• Also effects on erythropoiesis, lipids
Dihydrotestosterone
• Peripheral conversion from testosterone by 5α-reductase (prostate, skin)
• Testosterone has direct & indirect effects
• In some tissues testosterone is a prohormone for DHT
• Male reproductive development requires both
• DHT measurement & Testosterone measurement

ANDROGENS…….
• Testosterone and DHT circulate in plasma freely (≈2 to 3%) or bound
to plasma proteins.
• Binding proteins include the specific sex hormone–binding globulin

(SHBG) and nonspecific proteins such as albumin.

SHBG
• Sex Hormone Binding Globulin
• Glycoprotein produced in the liver
• High affinity for testosterone & DHT

• Testosterone:
• 44-65% bound in males
• 66-78% in females
• Not important for DHEA/DHEA-S
• N.B. Albumin also binding protein:

• DHEA & DHEA-S primarily bound to albumin
• Testosterone & DHT: ~33-50% bound in males, ~20-30% in females (‘bioavailable’)
Causes of a high SHBG
• High oestrogen (female sex, pregnancy, HRT, OCP)

• Hyperthyroidism
• Liver disease
• Anorexia (?insulin/IGF-1)
• Anticonvulsant drugs phenytoin and phenobarbitone (hepatic enzyme induction)
Causes of a low SHBG
• High androgen (male sex, androgen use)

• Hypothyroidism
• Insulin resistance
• Obesity
• Diabetes
• Cushing’s disease
Effect of Age
• high in childhood, decrease in puberty (male>female),
• increase in elderly male, decrease in post-menopausal female
Male Reproductive Abnormalities
A wide variety of abnormalities affect the male reproductive system
before birth, in childhood, or in adulthood and they can be categorized
into 5 main groups
(1) hypogonadotropic hypogonadism,

(2) hypergonadotropic hypogonadism,
(3) defects in androgen action
(4) erectile dysfunction, and
(5) gynecomastia
Hypogonadism
• Male hypogonadism is a condition caused by decreased function of
the testes, which can lead to retardation of sexual development if
manifested early in life.
• The disorder is classified as hypogonadotropic or hypergonadotropic

Hypogonadotropic Hypogonadism
• Hypogonadotropic hypogonadism occurs when defects in the
hypothalamus or pituitary prevent normal gonadal stimulation.
• Causative factors include congenital or acquired panhypopituitarism,

hypothalamic syndromes, GnRH deficiency, hyperprolactinemia,
malnutrition or anorexia, and iatrogenic causes.

Kallmann syndrome
• This is the most common form of hypogonadotropic hypogonadism,
results from a deficiency of GnRH in the hypothalamus during
embryonic development.
• It is characterized by hypogonadism and anosmia (loss of the sense of

smell) in male or female patients,
• It is inherited as an autosomal dominant trait.

Kallmann Syndrome…….
• This syndrome arises from a defect in the migration of GnRH neurons
to the hypothalamus.
• The pituitary disorders are characterized by isolated gonadotropin

deficiency with or without growth hormone deficiency.
• Patients with isolated gonadotropin deficiency display sexual

infantilism and long arms and legs;
• those with combined deficiency do not have long arms and legs.

Kallman Syndrome…….
• In all of these patients, LH, FSH, and testosterone concentrations are
lower than normal.
• However, heterogeneity exists in the degree of gonadotropin
deficiency;
• hence concentrations of LH, FSH, and testosterone have been shown
to differ among affected patients.

Common causes of hypogonadotropic
gonadism

Hypergonadotropic Hypogonadism
• Hypergonadotropic hypogonadism results from a primary gonadal
disorder.
• Patients with primary testicular failure have increased concentrations

of LH and FSH and decreased concentrations of testosterone.

Klinefelter’s syndrome
• Occurs in 1 in 400 men and is caused by the presence of an extra
chromosome.
• Men with this disorder have small firm Testes and gynaecomastia at
the time of diagnosis due to reduced production of testosterone.
• LH levels are elevated due to deficient seminiferous tubule mass.
• FSH levels are also elevated due to inhibin underproduction

Klinefelter’s Syndrome
• Both LH and FSH levels induce increased aromatase activity leading to

increased Estrogen levels.
• Other causes of hypergonadotropic hypogonadism includes:

• Testicular feminization syndrome
• Sertoli cell only syndrome

Investigations
• Hypergonadotropic Hypogonadism
• Measurement of the concentration of FSH is indicated in men with
sperm count lower than 5 to 10 million/mL.
• Elevated concentrations of FSH indicate Sertoli cell dysfunction and,

in azoospermic men, primary germinal cell failure, Sertoli cell–only
syndrome, or genetic conditions such as Klinefelter syndrome.

Investigations……
• Elevated FSH (>120 mIU/mL) in the setting of decreased testosterone
(<200 ng/dL) and oligospermia indicate primary testicular failure

FEMALE REPRODUCTIVE BIOLOGY
• The ovaries produce ova and secrete the sex hormones progesterone
and estrogen.
• Every healthy female neonate possesses approximately 400,000

primordial follicles,
• During the reproductive life span of an adult woman, 300 to 400
follicles will reach maturity
Hypothalamic-Pituitary-Gonadal Axis
• In adult women, a tightly coordinated feedback system exists
between hypothalamus, anterior pituitary, and ovaries to orchestrate
menstruation. FSH serves to stimulate follicular growth, and LH
stimulates ovulation and progesterone secretion from the developing
corpus luteum.

Female reproductive axis
Hypothalamus
- -
GnRH
Pituitary
- -
FSH LH
Ovary
Oestradiol Progesterone
• Oestrogen usu –ve feedback inhibition of FSH and LH but positive feedback to produce LH surge & ovulation
Estrogens
• Estrogens are responsible for the development and maintenance of
female sex organs and female secondary sex characteristics.
• In conjunction with progesterone, they participate in regulation of the

menstrual cycle and of breast and uterine growth, and in the
maintenance of pregnancy.

• More than 97% of circulating E2 is bound to plasma proteins. It is
bound specifically and with high affinity to SHBG, and nonspecifically
to albumin

Progesterone
• Progesterone, similar to the estrogens, is a female sex hormone. In
conjunction with estrogens, it helps to regulate accessory organs
during the menstrual cycle
• Biosynthesis of progesterone in ovarian tissues follows the same path

from acetate to cholesterol through pregnenolone as it does in the
adrenal cortex

Progesterone……
• Progesterone does not have a specific plasma-binding protein but,
similar to cortisol, is found bound to corticosteroid binding globulin.
• Reported concentrations for plasma free progesterone vary from 2%

to 10% of total concentration, and the percentage of unbound
progesterone remains constant throughout the normal menstrual
cycle

Oestrogen & Progesterone
Oestradiol
• Maintains function of reproductive tract
• Endometrial thickening
• Mucus secretion at ovulation
• Secondary sexual characteristics
• Systemic effects
• Bone density
• Cardioprotective
Progesterone
• Prepares endometrium for implantation
• Essential for maintenance of early pregnancy
• Day 21 progesterone – indicates ovulation
Prolactin
• Produced by lactotrophs of anterior pituitary
• Negative control by dopamine
• Positive feedback by oestrogens and TRH
• Direct effects
• Milk production
• Measure in females with:

• Oligo or amenorrhoea
• Galactorrhoea
• Subfertility
• Measure in males with

• Hypogonadotrophic hypogonadism with unknown cause
Normal Menstrual Cycle
• During a normal menstrual cycle, a closely coordinated interplay of
feedback effects occurs between the hypothalamus, the anterior lobe
of the pituitary gland, and the ovaries.
• In addition cyclic hormone changes lead to functional and structural

changes in the ovaries , uterus , and vagina.

• The menstrual cycle is measured beginning on day 1 as the first day of
menstrual bleeding. Each cycle consists of a follicular phase followed
by ovulation and then a luteal phase.
• Follicular Phase. The follicular phase, that is, the initiation of follicular
growth, During the early part of the follicular phase, concentrations of
FSH are elevated, but they decline up until ovulation.

• LH secretion begins to increase around the middle of the follicular
phase.
• Just before ovulation, estrogen secretion by the follicle increases

dramatically; this positively stimulates the hypothalamus and triggers
the LH surge.
• The LH surge is a reliable predictor of ovulation.
• Ovulation occurs around day 14 of the menstrual cycle in a 28 day

cycle; about 14 days to the next menstrual cycle.

• Luteal Phase. The luteal phase, the last half of the cycle, is
characterized by increasing production of progesterone and estrogen
from the corpus luteum with consequent gradual lowering of LH and
FSH concentrations.
• The concentration of progesterone reaches a peak at about 8 days

post ovulation
• If ovulation does not occur, the corpus luteum fails to form, and a
cyclic rise in progesterone is subnormal.
• If ovulation and pregnancy occurs, hCG maintains the corpus luteum

and progesterone continues to rise.
Normal Menstrual Cycle……
• In the absence of conception, the corpus luteum resolves, resulting in
a decrease in estrogen and progesterone concentrations and a
breakdown of the endometrium,
• The average duration of menstrual flow is 4 to 6 days, and average

menstrual blood loss is 30 mL-50ml

HORMONE PROFILE IN THE NORMAL MENSTRUAL CYCLE
…
…LH,
LH,
__
__FSH
FSH
----
----PROG,
PROG,
E2
***** E2
*****
DAY;O 14 28

Female Reproductive Abnormalities
• A wide variety of abnormalities affect the female reproductive
system and have been classified into;
• (1) pseudohermaphroditism,
• (2) precocious puberty,
• (3) irregular menses, and
• (4) menopause

Female Pseudohermaphroditism
• In pseudohermaphroditism, the gonadal sex varies from the genetic
sex.
• The female pseudohermaphrodite is an individual who is genetically

female, but whose phenotypic characteristics are, to varying degrees,
male.
• In neonates with a 46,XX karyotype and ambiguous genitalia,

congenital adrenal hyperplasia(CAH) should be considered.

• CAH is a family of autosomal recessive disorders of adrenal
steroidogenesis resulting in deficiency or excess of androgens.
• Only deficiencies of 21-hydroxylase and 11β-hydroxylase are

predominantly virilizing disorders.
• In female fetuses, exposure to androgens before the 12th week of

gestation causes ambiguous genitalia; after 13 weeks, it results in
clitoral enlargement.

• Diagnosis of 21-hydroxylase deficiency is made in infants and children
with excess excretion of urinary 17-KS and pregnanetriol (a
metabolite of 17-hydroxyprogesterone and elevated concentrations
of plasma 17-hydroxyprogesterone and androstenedione.
• However, sick and premature infants may have elevated

concentrations 17-hydroxyprogesterone and androstenedione.
• Elevation of 17-hydroxyprogesterone concentrations in early infancy

(>3000 ng/dL) confirms the diagnosis of this disorder.

Precocious Puberty
• Precocious puberty is the development of secondary sexual characteristics
in girls younger than 8 years old and boys younger than 9 years old.
• it is important to distinguish between benign advanced pubertal conditions

and true precocious puberty.
• Early puberty is manifested by the appearance of secondary sexual

characteristics such as premature thelarche (premature breast
development), premature adrenarche (premature sexual hair
development), or phallic enlargement.

Precocious Puberty……
• When presented as isolated cases, these secondary sexual
characteristics are not considered to be pathologic, as none
progresses to full-blown puberty, nor are they associated with
increased rates of bone growth and maturation.
• However, if a child has at least two signs of puberty and also

demonstrates increased rates of bone growth and maturation, then
many causes of true precocious puberty must be considered

Precocious Puberty……
• Precocious puberty has been classified as GnRH dependent or
independent.
• GnRH-dependent precocious puberty (also called central precocious

puberty) is due to precocious activation of the hypothalamic-pituitary-
gonadal axis.
• GnRH-independent precocious puberty (also called pseudoprecocious
puberty) refers to precocious sex steroid secretion that is independent
of pituitary gonadotropin release

Infertility – General Considerations
• Defined as failure to conceive after 12 months of regular sexual intercourse without

contraception
• Causes:
• unexplained infertility (25%)
• ovulatory disorders (25%)
• tubal damage (20%)
• uterine or peritoneal disorders (10%)
• factors in the male causing infertility (30%)
• ~40% of cases are found in both the man & woman

Infertility - female
• Role for biochemical investigations:
• Confirming ovulation (regular & irregular cycles): progesterone
• Investigating irregular menstrual cycles: FSH & LH
• Ovulatory disorders: prolactin
• Predicting response to IVF stimulation protocol: AMH & FSH
• Women with regular monthly menstrual cycles are likely to be ovulating

• In women with prolonged irregular menstrual cycles measurement timing of
progesterone may need to be adjusted e.g. day 28 of a 35-day cycle & repeated weekly
thereafter until the next menstrual cycle starts

Infertility - male
• Semen analysis
• Endocrine:
• Testosterone
• LH
• FSH

Hyperprolactinaemia
Common causes:
• Dopamine antagonists (dopamine negative feedback)
• Stress
• Pregnancy
Macroprolactin
• IgG complex
• Low bioactivity
• Should be screened to avoid unnecessary investigations
Oligo- or amenorrhoea
• Oligomenorrhoea = infrequent menstruation that occurs fewer than 9

times per year.
• Amenorrhoea = absence of menstruation or cycle length >6 months
• Ovulation is infrequent

Ovulation disorders
• Hypothalamic pituitary failure
• hypothalamic amenorrhoea or hypogonadotrophic hypogonadism
• E.g. low BMI, excessive exercise
• Hypothalamic-pituitary-ovarian dysfunction
• predominately polycystic ovary syndrome
• Hyperprolactinaemia
• Hypothyroidism (TRH  prolactin)
• Ovarian failure
• Primary – cause often unknown
• Secondary e.g. post radiotherapy, surgery
• “Premature” if <40y (definition can vary)
PCOS
• Two of the following:
• Oligo- or an-ovulation
• Clinical &/or biochemical evidence of hyperandrogenism
• Polycystic ovaries (ultrasound scan)
• And other causes excluded (e.g. late onset CAH, adrenal / ovarian tumours, Cushing’s syndrome)
•  therefore biochemical tests usually performed
• Testosterone & androstenedione often raised, DHEA-S may also be raised

• LH may be raised, FSH normal
• Similar presentation possible with late onset CAH:

• measure 17α-hydroxyprogesterone concentrations (SST)
PCOS
• Management dependent upon the desired outcome e.g. promote
fertility, reduce hirsutism etc
• Weight loss
• OCP
• Metformin

Female: excess androgens
• Hirsutism
• Excessive androgen-dependent hair growth
• Note difference with hypertrichosis
• Normal/mildly raised androgens
• Virilisation
• usually marked increase in androgens
• manifestations include temporal hair recession, clitoromegaly, increased muscle mass,
breast atrophy, deepening of voice, oligo/amenorrhoea
• Grossly elevated testosterone (>5 nmol/L) with sudden onset hirsutism/virilisation more
worrying
Male hypogonadism
The diagnosis of hypogonadism is based upon
• Appropriate symptoms
• Measurement of testosterone in the morning on >1 occasion
• Ideally 9am, in practice 7-11am
• Prepubertal testicular failure

• Lack of sexual maturation
• Increased arm span (delayed epiphyseal closure)
• Post-pubertal symptoms less obvious

• Decreased libido, impotence, infertility
• Increased oestradiol/testosterone ratio can lead to gynaecomastia
Male hypogonadism
Primary (i.e. testicular dysfunction)
• Genetic
• Klinefelter’s syndrome (most commonly 46,XXY)
• Cryptorchidism
Secondary (i.e. pituitary or hypothalamic dysfunction)

• Congenital
• Kallman’s syndrome
• Acquired
• N.B. Can see both testicular and pituitary dysfunction e.g. haemochromatosis
ENDOCRINE INVESTIGATION OF THE INFERTILE COUPLE
REQUESTS FOR HORMONAL ASSAY

Reasons:
A. To make a diagnosis of the cause of infertility
B. To plan treatment of infertility
C. To monitor treatment.
(A), and (B) are often done together.
(A), should be based on provisional diagnosis of possible cause

of infertility.
(C), is commonly neglected, but is very important.

Investigation
• LH & FSH
• differentiate primary or secondary (hyper- & hypo-gonadotropic respectively)
• Prolactin
• If testosterone <5.2 nmol/L
• or when secondary hypogonadism suspected

A To Make a Diagnosis of the Cause of Infertility
1. Amenorrhoea
i. Do Pregnancy Test
ii. Measure Plasma LH, FSH and Oestradiol
(Pulsatile release: pooled samples x 3: 15mins interval, 8am to 11am).
Interpretation
(a) Normal LH, FSH with Low Oestradiol
- in Primary Amenorrhoea – consider Testicular Feminization –
Do Testosterone; (Increased, i e. Male range = Defective androgen Action:
conversion to DHT or most commonly mutation in androgen receptor gene on Xq11 to Xq12.)
(b) Raised LH & FSH (especially FSH) with low oestradiol

Indicative of ovarian failure.
(c) Low LH & FSH with low oestradiol

Indicative of pituitary or hypothalamic failure.
iii Measure Plasma Prolactin

Very high levels – support pituitary or hypothalamic cause.
iv Perform GnRH (100μg I.V) Challenge Test.

Measure LH, FSH at 0min 30min & 60min Normal pituitary if >5 i.u/L.
Then Clomid (Clomiphine) may be useful in Treatment of hypo function.

A. (Continuation of Investigation of cause of Infertility)
Patient has Normal Regular Menstrual Cycle and Husband has normal
semen analysis.
Determine if she is ovulating.
Measure; Day 21 or day 22 progesterone i.e. 7 days to the next expected menstrual
cycle.
-Progesterone > 38nmol/L (10ng/ml) indicate ovulation had occurred.
- Progesterone < 19nmol/L (5ng/l)

Repeat assay in another cycle.
- Progesterone < 7.6nmol/L (2ng/ml) indicative of anovulatory cycle.
Then investigation for P.C.O. and ovarian failure:

Request Follicular phase FSH, LH and Oestradiol and Testosterone.

B. Requesting Hormone for Planning Treatment
For Super Ovulation:

· Measure Basal Follicular Hormone profile
FSH, LH & Oestradiol (E2 )
For Artificial Insemination:

· Measure Basal profile
· Mid cycle profile

Serial E2 or LH, to time ovulation
Previous and present cycle.
(Plasma E2 or urinary oestriol, or Plasma LH, or
Urinary LH/Creatinine Ratio)
For In-vitro fertilization:

· Measure Basal LH, FSH & E2

C. Requesting Hormone to Monitor Treatment:
* To Monitor ovarian suppression Treatment (i.e. GNRH agonist, to

suppress ovary).
- Measure Daily E2 (Plasma) or Daily urinary oestriol.
*To Monitor Response to ovarian stimulation;

- Measure Daily E2 (Plasma) or Daily urinary oestriol.
(can predict premature LH surge and premature
ovulation, Measure Urine LH/Creatinine ratio to confirm)
* To assess success or failure of treatment:

- Measure Mid-Follicular to mid-cycle plasma E 2 or
urinary oestriol.

Summary of timing of hormonal assay
… LH,
… LH,
__
FSH
__
FSH
---- PROG,
---- PROG,
***** E2
***** E2
DAY;O 14 21 28
FOLLICULAR
PHASE MID-CYCLE LUTEAL PHASE
Day 12 to 16
REQUEST: Plasma LH, FSH, E2, SERIAL Plasma E2, or Day 21 FROM
DAY 25
+ TESTO, DHEAS , PROL. SERIAL Urine Oestriol, or Plasma Progest
(Early, i.e., Day 2 – 5) SERIAL Urine LH/Creatinine Beta-HCG
Clinical Conditions:
Menstrual irregularity Determine response to treatm. Determine if Determine
Androgen problems eg PCOS. Time ovulation patient ovulated Pregnancy &
Peri-menopausal: Ovarian Failure Assess prognosis Bioch. pregn.
Plan treatment, Baseline levels

Dynamic function testing
• hCG OR hMG stimulation test
• Distinguish primary testicular failure from gonadotrophin deficiency
• Confirm presence of testicular tissue (cryptorchidism)
• Combined pituitary and testicular dysfunction e.g. haemochromatosis
• GnRH stimulation test

• Same principle
• Rarely used

ANALYTICAL METHODS FOR REPRODUCTIVE
HORMONES
• A variety of methods are available for measuring sex steroids
in body fluids.
Currently, the most common method is non-isotopic immunoassay
• However, use of mass spectrometry to measure sex steroids is

increasing.

Methods for Determination of Total
Testosterone in Blood
Circulating testosterone comprises three different forms or pools:
• (1) a non–protein-bound or “free” form,
• (2) a weakly bound form, and
• (3) a tightly bound form.
• Gas chromatography combined with mass spectrometry (GC-MS)

remains the reference method for testosterone measurement and is
often used to assess the bias of routine immunoassay methods

Methods of Determination of Total
Testosterone in Blood……
Specimen Collection and Storage
• Serum or heparinized plasma is used to measure total testosterone.
Testosterone is subject to diurnal variation, reaching a peak
concentration at between 0400 and 0800 hours. Therefore, morning
specimens are preferred. Serum/plasma samples are stable for up to
24 hours at room temperature, up to 1 week refrigerated, and up to 1
year frozen at −20 °C.
• DHEA supplementation should be avoided before testing

• Various methods are available for determining the concentrations of
free or bioavailable forms of testosterone in serum or plasma. They
include the following:
• 1. Estimation of the free testosterone fraction by equilibrium dialysis
or ultrafiltration.
• 2. Estimation of free hormone using a direct (analog tracer)
radioimmunoassay.
• 3. Estimation of combined free and weakly bound (bioavailable)
testosterone fractions by selective precipitation of the tightly bound
form.
• 4. Calculation of the androgen index using indices that reflect the
ratios of testosterone pools.
• 5. Calculation of free and weakly bound testosterone concentrations
by mathematical modeling
Methods for the Detection of Anabolic
Steroids
• The ratio of testosterone to epitestosterone (17 α-epimer) in urine
has been used as a screening test for the detection of anabolic steroid
abuse. A ratio of testosterone to epitestosterone greater than 6 : 1
suggests exogenous steroid use

Methods for the Determination
of Estradiol in Blood
• Both chromatography-mass spectrometry and immunoassay based
methods are used to measure estrogens in blood.
• GC-MS methods utilizes isotope dilution, and provides the most

accurate and reliable measurement of E2

Methods for the Determination of Estradiol in
Blood……
• Specimen Collection and Storage
• Serum and plasma (with EDTA or heparin as anticoagulant) have been
used. Specimens should be centrifuged and separated within 24
hours. Serum/plasma specimens may be stored at room temperature
for 1 day, refrigerated for 3 days, or frozen for up to 1 year.
• Oral contraceptives have been known to alter E2 concentrations

Methods for the Determination of Progesterone
in Blood
• Initial immunoassays for serum progesterone measurement used
organic solvents to remove the steroid from endogenous binding
proteins such as corticosteroid-binding globulin and albumin.
• Direct (nonextraction) measurement of progesterone in serum or

plasma is considered the method of choice for routine applications.

CONCUSION
The Hypothalamic-pituitary-gonadal axis is very
critical to the regulation of hormonal functions
and changes associated with male and female
reproductive functions and is responsible for
normal ovulation, menstrual cycles and testicular
functions, without which, the resultant effect
would be the various male and female gonadal
disorders.
REFERENCES
• Clinical Biochemistry and Metabolic Medicine by
Martin A. Crooks, Eight Edition.
• Clinical Chemistry by Lawrence A. Kaplan et al, Fourth
Edition.
• Tietz Fundamentals of Clinical Chemistry and
Molecular Diagnostics, Seventh Edition.
• WebMD

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Hypothalamic-Pituitary-

January 23, 2024 OLUKOYEJO O.E. 1

January 23, 2024 OLUKOYEJO O.E. 2

January 23, 2024 OLUKOYEJO O.E. 3

• The testes contain a structured network of tightly packed

• Sertoli cells play a crucial role in sperm maturation and secrete

January 23, 2024 OLUKOYEJO O.E. 4

• Testosterone is required for sexual differentiation, spermatogenesis,

• Testicular function is under the control of the hypothalamic-pituitary-

January 23, 2024 OLUKOYEJO O.E. 5

• LH acts on Leydig cells to stimulate the conversion of cholesterol to

• FSH acts on Sertoli cells and spermatocytes and is central to the

Higher Centre Neurotransmitter

Acuate Nucleus Hypothalamus GnRH Pulsatile Release

TESTICULAR Leydig Cells Sertoli Cells

January 23, 2024 OLUKOYEJO O.E. 8

• Testosterone is synthesized primarily by the Leydig cells of the testes

January 23, 2024 OLUKOYEJO O.E. 9

Produced in ovary, testis and adrenal gland

January 23, 2024 OLUKOYEJO O.E. 12

• Binding proteins include the specific sex hormone–binding globulin

January 23, 2024 OLUKOYEJO O.E. 13

• High affinity for testosterone & DHT

• N.B. Albumin also binding protein:

• High oestrogen (female sex, pregnancy, HRT, OCP)

Causes of a low SHBG

• High androgen (male sex, androgen use)

(1) hypogonadotropic hypogonadism,

• The disorder is classified as hypogonadotropic or hypergonadotropic

January 23, 2024 OLUKOYEJO O.E. 17

• Causative factors include congenital or acquired panhypopituitarism,

January 23, 2024 OLUKOYEJO O.E. 18

• It is characterized by hypogonadism and anosmia (loss of the sense of

January 23, 2024 OLUKOYEJO O.E. 19

• The pituitary disorders are characterized by isolated gonadotropin

• Patients with isolated gonadotropin deficiency display sexual

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• Patients with primary testicular failure have increased concentrations

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• LH levels are elevated due to deficient seminiferous tubule mass.

• FSH levels are also elevated due to inhibin underproduction

• Both LH and FSH levels induce increased aromatase activity leading to

• Other causes of hypergonadotropic hypogonadism includes:

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• Elevated concentrations of FSH indicate Sertoli cell dysfunction and,

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• Every healthy female neonate possesses approximately 400,000

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• In conjunction with progesterone, they participate in regulation of the

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• Biosynthesis of progesterone in ovarian tissues follows the same path

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• Reported concentrations for plasma free progesterone vary from 2%

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• Measure in females with:

• Measure in males with

• In addition cyclic hormone changes lead to functional and structural

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