BIOCHEMICAL INVESTIGATION

OF INFERTILE COUPLE

BY
DR ODUNAFOLABI O.O
 OUTLINE

• INTRODUCTION/DEFINITION
• AETIOLOGY
• CLASSIFICATION
• EVALUATION OF THE INFERTILE COUPLE
• OVULATION ASSESSMENT
• POLYCYSTIC OVARIAN SYNDROME
• ANALYTICAL METHODS FOR REPRODUCTIVE HORMONES
• ASSISTED REPRODUCTIVE TECHNIQUES (ARTs) & OVARIAN RESERVE TESTING
• BRIEF OTHER NON-BIOCHEMICAL CONTRIBUTIONS TO INFERTILITY
• CONCLUSION
• REFERENCES
 Introduction
• Nigeria embraces reproduction of children
• Infertility is becoming more prevalent in this
environment.
• Increasing demand for infertility investigations
during assisted reproductive techniques
• Infertility is defined as a disease of the reproductive
system characterized by the failure to achieve
successful pregnancy after twelve months or more of
regular unprotected sexual intercourse
• 50% of couple achieve pregnancy in 3 months; 75%
in 6 months; and 80 – 85% in 1 year.
• After one year of sub fertility, there is need for
investigation
 Aetiology
• -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
 Unexplained Infertility

Sequencing of the human genome has identified subtle

genetic defects on the long arm of the Y chromosome
which had previously been considered as idiopathic

infertility.
• In adult men, GnRH and thus LH and FSH are secreted
in pulsatile patterns.
• A circadian rhythm is present, with higher
concentrations found in the early morning hours and
lower concentrations in the late evening.
• Two gonadotrophins, two germ cells theory;
– Luteinizing hormone LH acts on Leydig cells to stimulate
the conversion of cholesterol to pregnenolone. Rate
limiting step in testicular steriodogenesis.
– FSH acts on Sertoli cells and spermatocytes and is central
to the initiation (in puberty) and maintenance (in
adulthood).
• Gonadal steroids and inhibin , provide
negative feedback control of LH and FSH
secretion, respectively.
• LH secretion is also inhibited by testosterone
and by its metabolites, estradiol and DHT.
• Positive feedback; During the menstrual cycle,
estrogens provide a positive influence on
GnRH effects on LH and FSH secretion, and the
rise in estrogen during the follicular phase is
the stimulus for the LH and FSH ovulatory
surge.
• Negative feedback; In women, primary
gonadal failure or menopause results in
elevations of LH and FSH.
 Evaluation of Infertile couple
• History and physical examination
• Semen analysis
• Endocrine evaluation
– Determination of gonadal function
– Determination of ovulation
– Measurement of Prolactin and Thyroid hormones
 Male Infertility Factors
-Endocrine Disorders
•-Hypothalamic dysfunction (Kallmann syndrome)
-Pituitary failure, Hyperprolactinemia,
Exogenous androgens, Thyroid disorders,
Adrenal hyperplasia, Testicular failure.
-Anatomic Abnormalities, Abnormal motility
-Abnormal Spermatogenesis
-Idiopathic
 SEMEN ANALYSIS
The results of semen analysis conducted as part of an
initial assessment should be compared to the following
WHO reference values

• volume:  2.0 ml
• liquefaction time: within 60 minutes
• pH:  7.2
• sperm concentration:  20 million per ml
• total sperm number:  40 million per ejaculate
• motility:  50% (grades a and b) or 25% or more with
progressive motility (grade a) within 60 minutes of ejaculation
• vitality : 75% or more live
• white blood cells: fewer than 1 million per ml
• normal morphology: 30% or 15% (based on strict morphological
criteria)
 Semen analysis cont’d
• If the result of the first semen analysis is abnormal,
a repeat confirmatory test should be offered.)
• Repeat confirmatory tests should ideally be
undertaken 3 months after the initial analysis to
allow time for the cycle of spermatozoa formation to
be completed. However, if a gross spermatozoa
deficiency (azoospermia or severe oligozoospermia)
has been detected the repeat test should be
undertaken as soon as possible.
Algorithm of Male Infertility
 Cont’d
• Semen analysis; with oligospermia or
azoospermia, measure serum testosterone, LH,
and FSH concentrations, ± prolactin and TSH.
• Hypergonadotropic hypogonadism.
– ↑ FSH indicate Sertoli cell dysfunction, primary
germinal cell failure, Sertoli cell syndrome,
Klinefelter syndrome.105

– ↑FSH (>120 mIU/mL), ↓testosterone (<200 ng/dL)

and oligospermia indicate primary testicular failure.
 Male Evaluation
Hypogonadotropic Hypogonadism;
↓testosterone (<200 ng/dL) and ↓FSH (<10
mIU/mL).
GnRH stimulation test is administered to
distinguish between gonadal insufficiencies
caused by pituitary versus hypothalamic failure
• Prolactin assay: Hyperprolactinemia is a cause of
secondary testicular dysfunction, by impairing GnRH
release it causes hypogonadism, under
androgenisation and erectile dysfunction.
• TSH Hyperthyroidism and hypothyroidism alter
spermatogenesis.
– Hyperthyroidism alters the secretion of releasing
hormones and increases conversion of androgens to
estrogens.
– In hypothyroidism, ↑TRH results in hyperprolactinaemia
Evaluation of immunologic parameters

• Measuring antisperm antibodies-

– Not always recommended
– Techniques available include agglutination
test,enzyme linked immunosorbent assays,
radioimmunoassays
• Molecular and Genetic Evaluation: using fluorescence
in situ hybridization (FISH) and Polymerase chain
reaction (PCR)
• X chromosome abmormality: X-linked Kallmann
syndrome, DAX1 gene mutation
• Y chromosome abnormalities: (AZF1and
AZF2) on the long arm of the Y chromosome
are associated with an inability to make
sperm. In addition, genes such as SRY (sex-
determining region Y) are on the short arm of
chromosome Y. Deletion of these regions is
associated with azoospermia or, less
frequently, oligospermia.
• The utility of testing for Y-chromosome
microdeletions is to provide additional
information as to whether or not sperm might
be retrieved on a testicular sperm extraction
procedure
 Female Infertility Factors
Structural problems
Tubal pathology
Uterine abnormalities
Disorders of Ovulation
Anovulation – PCOS
Luteal phase deficiency
Hypogonadism – primary or secondary
Tubal pathology
Uterine abnormalities
Functional problems
Suboptimal cervical mucus
Autoimmunity –antisperm antibody
Algorithm of Female Infertility
• Ovarian / hormonal-
– metabolic (thyroid, liver, obesity, PCOS,
androgenism,)
– Hypergonadotropic hypogonadism: menopause,
luteal phase deficiency, gonadal dysgenesis,
premature ovarian failure (cytotoxic,
autoimmune, tumour)
– Hypogonadotropic hypogonadism:
hyperprolactinemia, pituitary insufficiency,
hypothalamic insufficiency
 EVALUATION OF OVULATION
Disorders of ovulation account for about 18% of all
infertility problems.
-Basal body temperature – unreliable
 -Endometrial biopsy – invasive.
 -Mid luteal phase progesterone ; An increase in
progesterone concentration indicates that a
corpus luteum has been formed, but does not
confirm ovulation.
 -Measurement of the LH surge – urinary LH
assay.
EVALUATION OF ENDOCRINE ASSESSMENT

Assay of progesterone
-Primary test of ovulation
-Increase indicate formation of corpus luteum but
not confirm egg release
-Mid luteal progesterone >10ng/ml indicate
normal ovulation. If lower value then probably
anovulation, inadequate luteal phase progesterone
or inappropriate timing of sample collection
• 6-10ng/ml or 30nmol/l (likely ovulated)
• <6ng/ml ( luteal phase deficiency);
< the detection limit for the kit
(anovulation
 Hypergonadotropic Hypogonadism:
-↑ FSH (>30 IU/L) in three consecutive cycles
or
 >40 IU/L once
-↓Estradiol (<20 IU/L)
Causes include; primary ovarian insufficiency,
gonadal dysgenesis, resistant ovary syndrome,
menopause, and luteal phase deficiency.
-In women aged below 40 years – premature
ovarian failure.
 Hypogonadotropic Hypogonadism
↓FSH (<10 IU/L),↓LH (<10 IU/L)
↓Serum Estradiol (<40pg/mL)
Prolactin: ↑Prolactin inhibits GnRH, FSH and LH→
hypogonadotropic hypogonadism.
TSH: ↓ and ↑ thyroidism contribute directly and
indirectly to infertility
Anti-Mullerian hormone (AMH) is a marker of ovarian
reserve and decline in reproductive capacity. Low
levels are associated with low follicular antral cell
count.
• IN FEMALES • IN MALES
• Amenorrhea • Gynaecomastia
• Hirsutism • Infertility*
• Virilism
• Infertility*
HYPERPROLACTINEMIA
The classical manifestations of PRL excess :
-amenorrhea and galactorrhea
-The gonadal dysfunction can produce any menstrual
cycle dysfunction(amenorrhea, oligomenorrhea
with anovulation, infertility)
-Estrogen deficiencymay result in
-decreased vaginal lubrication
-decreased libido
-osteopenia
CAUSES OF HYPERPROLACTINAEMIA
Physiological
Pregnancy
Nursing
Nipple stimulation
Stress (physical, psychological, hypoglycemia)
Exercise
Food intake
Sleep
Pharmacological
Numerous drugs stimulate PRL
Antihypertensive drugs:-reserpine, a-methyldopa, verapamil
Neuroleptics & antidepressants:-phenothiazines,
butyrophenones, IMAO,
benzamide, imipramine
Antiemetics:metoclopramide,domperidone
Hormones:estrogens (high dosage), TRH
Opiates
Anti-histaminic :cimetidine
Anti-TB:isoniazide
Pathological
•Prolactinomas
•Mixed pituitary adenomas : GH + PRL
•Defective hypothalamic dopamine secretion or transportto
the lactotroph:
-Hypothalamic tumors
-Pituitary tumors (pseudoprolactinoma)
-Trauma (stalk section)
-Radiotherapy sequellae
INVESTIGATION OF HYPREPROLACTINEMIA
When a hyperprolactinaemia is suspected, before
further and expensive investigations are proposed,
it is necessary to
-Obtain a careful history of drug intake
-Eliminate a primary hypothyroidism
-Control kidney and liver functions
-In women with recent onset of amenorrhea or
galactorrhea : pregnancy test
Basal PRL levels

Values >400ng/mlare virtually diagnostic of

prolactinoma

Values between 100 and 300 ng/mlare usually caused

by a prolactinoma which is radiological evident

If PRL values < 100 ng/ml :can be difficult !

There is generally a good correlation between PRL

levels and the size of the adenoma
Human chorionic gonadotrophin
-Produced by the chorion and developing placenta
-It prevents the involution of the corpus luteum as
LH and FSH fall after pregnancy is achieved
-Plasma and urine concentrations, 1 or 2 weeks after
the missed menstrual pregnancy are most commonly
used to diagnose pregnancy
-Measurement is of the B- subunit
1. Patient has Normal Regular Menstrual Cycle and
Husband has normal semen analysis: Determine if she is
ovulating.
Anovulatory infertility is probably the commonest form of
female infertility
Measure MID-LUTEAL progesterone i.e. 7 days to the next
expected menstrual cycle (some say Day 21).
 -Progesterone > 38nmol/L (10ng/ml) indicate ovulation had
occurred.
-Progesterone < 19nmol/L (5ng/l) – impaired luteal function
-Progesterone < 7.6nmol/L (2ng/ml) indicative of anovulatory
cycle. 
This test should be repeated on > 1 occasion.
Commonest cause of low Progesterone result is
inaccurate timing
. Irregular Menstrual cycle with Suspected
Androgen Hypersecretion
Investigate for:
-PCOS
-Simple Hirsutism,
-Ovarian tumour,
-Adrenal tumour,
-Congenital adrenal hyperplasia (CAH)
POLYCYSTIC OVARIAN SYNDROME
A condition of hyperandrogenism with anovulation
and abnormal ovarian morphology.
Presenting features- hirsutism, menstrual disturbances,
enlarged polycystic ovaries, infertility.
Associated with obesity, insulin resistance,
hyperinsulinaemia. Also glucose intolerance,
hypertension, hyperlipidaemia (Metabolic syndrome).
Lab features:
-Increased serum LH
-Normal serum FSH
-LH/FSH Ratio > 2 (usually 0.5 – 1.0),
-Increased serum Testosterone and Androstenedione
-Serum DHEAS – usually normal
-Multiple ovarian cysts on ultrasound
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
•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 E2 or urinary

oestriol.
 Analytical methods for the reproductive
hormone
 -Radioimmunoassay (RIA)
 -Immunoenzymometric assay (IEMA)
 Chemiluminescent Immunoassay (CLIA)
 Non-isotopic immunoassay
 Mass spectrometry
•PRE ART EVALUATION
•It is pertinent to ascertain the ovarian reserve, of a
woman undergoing ART, as this will determine the
appropriate therapy to institute.Hormonal
investigations to assess ovarian reserve include;
• Serum FSH and Estradiol
• Serum Anti-Mullerian Hormone (AMH)
• Serum Inhibin B
 0VARIAN RESERVE
 Ovarian reserve is a function of the number and
quality of remaining oocytes.
 Decreased ovarian reserve (DOR) describes women
having regular menstrual cycles, but reduced
response to ovarian stimulation when compared
women to women of similar age. The cause is
unknown, and is not fully explained by ageing. DOR
might represents a pathologic condition
 -DOR might represents a pathologic condition resulting from
abnormally rapid atresia in a normal pool of oocytes, from
normal atresia of an abnormally small pool of oocytes, or the
extreme end of a normal bell-shaped population distribution
of oocytes.
 -Ovarian reserve testing identify assisted reproductive
technique (ART) clients at risk for DOR, who are more likely
to exhibit a ‘‘poor’’ response to gonadotropin stimulation
and lesser chance of achieving conception
Ovarian Reserve Testing
Basal measurements;
• FSH
• Oestradiol
• Inhibin B
• Anti- mϋllerian hormone
Dynamic function testing
•Clomiphene citrate challenge test
•Exogenous FSH ovarian reserve test.
OVARIAN RESERVE –FSH
•FSH- high values have been associated with, but do
not predict, poor ovarian stimulation nor failure to
conceive.
• Consistently elevated FSH concentrations confer a
poor prognosis, a single elevated FSH value in women >
40 years may not predict a poor response to
stimulation.
OVARIAN RESERVE TEST-ESTRADIOL
•It is used only as an aid to interpret a basal serum
FSH value.
•When the basal FSH concentration is ‘‘normal’’ but
the estradiol level is elevated (>60–80 pg/mL) in the
early follicular phase, there is a lower risk of poor
response, and higher pregnancy rates.
OVARIAN RESERVE;INHIBIN B
•Inhibin B is not a reliable measure of ovarian reserve,
levels rise with gonadotropin-releasing hormone
(GnRH) or FSH stimulation, and exhibit high intra-cycle
variability.
•Inhibin B is lower in poor responders than in women
with a normal ovarian response to stimulation in the
general IVF population
OVARIAN RESERVE TEST-AMH

• Produced by granulosa cells of early follicles, AMH is

gonadotropin-independent and therefore remain
relatively consistent within and between menstrual
cycles in both normal, ovulating and infertile
women.
• Studies indicate that AMH levels have shown greater
sensitivity to ovarian aging, a stronger relationship
with the number of early antral follicles, and better
cycle-to-cycle reproducibility compared with FSH, E2
and inhibin B levels.
• Lower AMH levels have been associated with, poor
responses to ovarian stimulation, poor embryo
quality, and poor pregnancy outcomes in IVF.
 CLOMIPHENE CITRATE CHALLENGE TEST
 The CCCT involves measurements of serum FSH
before (cycle day 3) and after (cycle day 10)
treatment with clomiphene citrate (100 mg daily,
cycle days 5–9).
 In women with responsive ovaries, rising inhibin B
and oestradiol levels (from growing cohort of
ovarian follicles) will suppress FSH.
 Smaller follicular cohorts recruited in women with
DOR will generate less inhibin B and estradiol,
resulting in decreased negative feedback inhibition
of FSH secretion and higher stimulated FSH
concentrations.
 An elevated FSH concentration after clomiphene
stimulation suggests DOR.
 Studies of CCCT results have observed significant
intercycle variability in stimulated FSH levels and
in the difference between basal and stimulated
estradiol and inhibin B concentrations, which
limits the reliability of the CCCT
OVARIAN STIMULATION
This is achieved by administering the following
hormones;
• GnRH analogues to ‘quieten’ the ovaries (Down
regulation).
• Recombinant FSH analogues to stimulate a
controlled follicular maturation.
OVULATION INDUCTION
With follicular maturation, human chorionic
gonadotrophin is administered as an ovulatory trigger to
mimic the LH surge as the two hormones share greater
than 80% homology and bind the same receptor
Luteinizing Hormone Chorionic Gonadotrophin Receptor
(LHCGR).
EMBRYONAL TRANSFER
Progesterone is administered to offer luteal support
following a successful fertilization and blastocyst
development
 ASSESMENT OF TUBAL FACTORS
The results of semen analysis and assessment of ovulation
should be known before a test for tubal patency is performed.
Women who are not known to have co-morbidities (such as
pelvic inflammatory disease, previous ectopic pregnancy or
endometriosis) should be offered HSG to screen for tubal
occlusion because this is a reliable test for ruling out tubal
occlusion, and it is less invasive and makes more efficient use
of resources than laparoscopy
ASSESSING UTERINE ABNORMALITY
Women should not be offered hysteroscopy on its own
as part of the initial investigation unless clinically
indicated, because the effectiveness of surgical
treatment of uterine abnormalities on improving
pregnancy rates has not been established.
POST COITAL TESTING OF CERVICAL MUCUS
The routine use of post-coital testing of cervical mucus
in the investigation of fertility problems is not
recommended because it has no predictive value on
pregnancy rate.
•The post-coital test(PCT) may be of value in the
diagnosis of sexual dysfunction and ejaculatory
problems.

•Results of post-coital testing may have little influence

on treatment strategy in the light of the widespread
use of IUD for fertility problems associated with
sperm-cervical mucus interaction.

•The lack of effective treatment for anti-sperm

antibodies may render PCT unnecessary.
CONCLUSION
Infertility affects about one in six Nigerian couples and is
the most common complaint at gynaecology clinics in
Nigeria.
It is associated with increased morbidity, psychosocial
complications and increased burden on the health care
system.
However, with thorough evaluation and application of
current treatments 50–60% of infertile couples are
projected to conceive.
•Investigations of infertility should be all encompassing,
taking past medical history, physical examination and
biochemical/ molecular investigations of both partners
into consideration
•Despite all of these, sometimes the cause of the
infertility is not found.
•The treatment of infertility is by treating the underlying
cause.
 REFERENCES
 -CLINICAL BIOCHEMISTRY AND METABOLIC
MEDICINE, BY MARTIN A. CROOK – 8TH EDITION .
 -TIETZ TEXTBOOK OF CLINICAL CHEMISTRY AND
MOLECULAR DIAGNOSTICS, BY NADER RIFAI ET AL –
6TH EDITION .
 -NPMCN UPDATE REVISION COURSE (2017)
THANK YOU